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A multi-component reaction for covalent immobilization of lipases on amine-functionalized magnetic nanoparticles: production of biodiesel from waste cooking oil

A multi-component reaction for covalent immobilization of lipases on amine-functionalized... mono-alkyl esters. Transesterification processes could Introduction be classified based on chemical (alkaline-catalyzed and The exhaustion of fossil fuels is synchronized with an acid-catalyzed) or enzymatic methods (Handayani et al. accelerative rise in oil prices which augments environ- 2016; Budhwani et  al. 2019). The alkaline-catalyzed mental concerns. These challenges led the researches to transesterification as the most used method for indus - explore renewable resources and environment-friendly trial production of biodiesel has several drawbacks fuels (Luna et  al. 2014). In the next few decades, it such as the difficult recovery of glycerol and catalyst, would be required to apply biomass for the applications the necessity of high energy, reaction saponification, that are up to now produced from fossil resources, such and wastewater treatment issues (Hama et  al. 2018). as coal, natural gas, and oil (Franssen et al. 2013). From Enzymatic routes by using lipases (triacylglycerol acyl- an economic point of view, the replacement of fossil- hydrolase, EC 3.1.1.3), on the other hand, are normally based chemicals by biomass sources would be attractive conducted under mild conditions in terms of tempera- since various components of biomass contain molecu- ture, pH, and pressure (Gross et al. 2010). Lipase-cata- lar functionalities that are now presented in the oil- lyzed transesterification can be an excellent substitute derived base chemicals with high costs (Shimada et  al. to produce biodiesel due to the fact that it has been 2002). Biodiesel is mono-alkyl esters of long-chain fatty reported to give high yields, have simple purification acids (Mehde et al. 2018), derived from renewable feed- of products, and need less energy consumption with a stocks such as vegetable oils and animal fats. The key decreased amount of wastewater (Tacias-Pascacio et al. sources of vegetable oil for biodiesel production are 2019; Ali et  al. 2017; Ali et  al. 2017). There have been soybean oil, sunflower oil, palm oil and rapeseed oil very limited commercial lipases applicable in industry (Yücel 2012). Biodiesel has better lubricating properties in their free forms, owing to the high cost and the fact and a much higher cetane value than petroleum diesel that their separation, recovery and reuse are challeng- (Marchetti et al. 2007). Biodiesel is produced in a trans- ing. One of the most cost-effective methods adopted esterification process in which glycerides convert into A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 3 of 15 to eliminate these barriers is immobilization of lipases et  al. 2017). Surface modification is a useful approach to on a solid support and making them more stable het- improve thermal stability of nude magnetic nanoparticles erogeneous biocatalyst (Yushkova et al. 2019; Wu et al. (MNPs) and provide available reaction sites for further 2017). Immobilization of enzymes on solid carriers functionalization. Number of modifications protocols can be achieved by encapsulation, covalent bond and have been reported for functionalization of magnetic adsorption (Barbosa et al. 2015; Garmroodi et al. 2016). nanoparticles (MNPs) surface, for example by cross- The strong binding between the enzyme and its carrier linking with glutaraldehyde, coating with polymers (Jiang matrix in covalent attachment method prevents the et  al. 2007) or coupling with compounds like as agarose problem of leaching and enhance the stability and reus- (He et al. 2014). ability of the enzyme derivatives (Babaki et  al. 2015). In this research superparamagnetic Fe O nanoparticles 3 4 Recently, our group has introduced the use of multi- coated with layer of silica were prepared and subsequently component reactions for covalent immobilization of functionalized by amine functional groups. The prepared lipases on various supports (Mohammadi et  al. 2016; support was then used for the immobilization of Thermomy - Habibi et  al. 2013; Ashjari et  al. 2020b, 2020a; Ahrari ces lanuginosus (TLL), Rhizomucor miehei (RML) as 1,3-spe- et  al. 2022). Enzyme immobilization via multi-compo- cific lipases, and Candida antarctica lipase B (CALB) as a nent reactions offers a very fast process in an extremely non-specific lipase via a multi-component reaction. FT-IR, mild condition with great flexibility of accepting vari - SEM, and XRD were used to characterize the support before ety of functional groups (COOH, N H , and epoxide). and after immobilization. Ultimately, application of the It means that by utilizing this procedure, enzymes can immobilized lipases in the synthesis of biodiesel was evalu- be covalently attached on a large number of functional- ated. Response surface methodology (RSM) that is a power- ized matrixes. Among them, the amine-functionalized ful and efficient mathematical approach was applied for the supports are known as the most widely used carri- process optimization (Yücel 2012; Ghadge and Raheman ers for immobilization of enzymes (Mohammadi et  al. 2006; Jang et al. 2012). A 5-level 5-factor central composite 2018; Sigurdardóttir et  al. 2018; Vashist et  al. 2014). design (CCD) was employed to design the experiments. The Covalent binding of enzymes on these supports is usu- effect of several reaction parameters including water content ally performed via (1) modification of amine groups by (for TLL), water-adsorbent (for RML and CALB), reaction glutaraldehyde as a bifunctional linker which produces time, t-butanol concentration, temperature, and the amount a reversible iminium bonds and requires subsequent of biocatalyst were optimized. reduction by a reducing agent like NaBH4. This 2-step procedure is time-consuming and the chemicals used Materials and methods may have deleterious effect on enzyme activity (Zucca Materials and Sanjust 2014) by using diimide-activated amida- Lipases from Thermomyces lanuginosus (TLL), Rhizomu - tion of carboxylic acids on the surface of enzyme ( Jiang cor miehei (RML), Candida antarctica lipase B (CALB), et  al. 2004). This process is also complicated in prac - FeCl .4HO, FeCl .6H O, p-nitrophenyl butyrate (p- 2 2 3 2 tice and requires multiple and time-consuming steps. NPB), and tetraethyl orthosilicate (TEOS) were pur- Besides, in this approach carboxylic groups are acti- chased from Sigma-Aldrich. Waste cooking oil was also vated by carbodiimides which are quite costly and toxic obtained from a local restaurant and the fatty acids com- reagents (Zucca and Sanjust 2014). These reagents also position of waste cooking oil was determined to be 40.6% cause undesirable side reactions of intermolecular con- oleic acid, 17.58% linoleic acid, 32.24% palmitic acid, and jugation of proteins (Gao and Kyratzis 2008). 5.23% stearic acid. The molecular weight of the waste Nanoparticles have been extensively considered for cooking oil which was determined from the saponifica - enzymes immobilization (Cipolatti et  al. 2014; Ghosh tion value of 196.2  mg KOH/g and acid value of 76  mg et  al. 2012). There are some characteristics related to KOH/g was calculated to be 856.3 g/mol. Water content the nanoparticle-based biocatalysts: (i) they can be eas- in the oil measured by Karl Fischer (Verhoef et al. 1978) ily synthesized in high solid content without surfactants titration method was determined to be 0.01% (w/w). and toxic reagents; (ii) homogeneous and well defined Solvents used including n-hexane, methanol, ethanol, core–shell nanoparticles with a thick enzyme shell can 1-propanol, toluene, t-butanol and blue silica gel were be obtained and (iii) designed according to the research prepared from Merck chemicals. Triethylamine (E t N) necessity (Ansari and Husain 2012). Since the 1970s, was from Titrachem and 3-aminopropyltrimethoxysilane magnetic nanoparticles have progressively been used in (3-APTMS) 97% was purchased from Alfa Aesar. All the the field of bioscience and medicine (Shinkai 2002; Cao other chemicals were accessed commercially. Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 4 of 15 Methods cyclohexyl isocyanide under stirring at room tempera- Preparation of the magnetic Fe O nanoparticle (MNPs) ture. The yield immobilization was determined by meas - 3 4 Magnetic Fe O nanoparticles were prepared by the uring the initial and final concentration of each enzyme 3 4 conventional co-precipitation method (Tang et  al. 2011; in supernatant by using the Bradford’s method (Emami Hu et  al. 2009). First, 1.25  g of F eCl ·4H O and 3.4  g of Bistgani et  al. 2017). Finally, the immobilized lipase was 2 2 FeCl ·6H O were dissolved in 100  ml deionized water removed by an external magnet and washed by deion- 3 2 under nitrogen atmosphere and the reaction temperature ize water. Control experiments were conducted in the was increased to 65 °C. Afterward 9 ml of ammonia solu- absence of cyclohexyl isocyanide. tion (32%) was added by dropping funnel under vigorous stirring at room temperature for 1  h. The black precipi - Enzyme activity assay tates were collected by an external magnetic field and The activities of the soluble and immobilized enzymes washed with absolute ethyl alcohol and deionized water were analyzed spectrophotometrically by measur- several times to reach the pH value of 7.0. Then, the mag - ing the absorbance produced by the release of p-nitro- netic nanoparticles were dried in a vacuum oven at 80 °C phenol in the hydrolysis of p-NPB in 25  mM sodium and used directly for being coated with silica. phosphate buffer at pH 8.0 and 25  °C at 410  nm −1 −1 (ε = 18,400  M  cm ; Verhoef et al. 1978). Briefly, 0.05– 0.2 ml of the lipase suspensions or solutions (without fur- Preparation of silica‑coated magnetic nanoparticles ther dilution) was added to 2.5  ml of substrate solution Fe O @SiO core–shell particles were prepared by the 3 4 2 (0.8  mM) under magnetic stirring. Spontaneous hydrol- modified Stöber sol–gel process (Takeuchi 2017). 1.5  g ysis of p-NPB was measured using 2.5  ml of substrate of Fe O was mixed with 60  ml of ethanol and 10  ml of 3 4 solution (0.8  mM) in the absence of enzyme as control. HPLC water. This suspension was dispersed under ultra- Enzymatic activity is given as 1  μmol of p-nitrophenol sonication for 20  min, then 9  ml of aqueous ammonia released per minute per mg of the enzyme (IU) under the 32% and 4.31  ml of tetraethyl orthosilicate (TEOS) were conditions described above. added to the suspension under nitrogen atmosphere. The mixture was stirred for 5  h at room temperature. The Characterization of the support and the immobilized magnetic Fe O @SiO nanoparticles were separated by 3 4 2 biocatalysts an external magnetic field washed with ethanol and dis - The structure of Fe O @SiO , MNPs-NH , and MNPs-lipase 3 4 2 2 tilled water and dried in a vacuum oven at 80 °C. was characterized by using infrared spectroscopy (IR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). Amine‑functionalization of silica‑coated magnetic Fourier-transform infrared (FT-IR) spectra were performed nanoparticles −1 on a Bruker FT-IR from 4000 to 400  cm using the KBr pel- The dry magnetic Fe O @SiO nanoparticles were 3 4 2 let technique. The X-ray diffraction (XRD) patterns of sam - functionalized with 3-aminopropyltrimethoxysilane ples were obtained on a STOE-STADV Powder Diffraction (3-APTMS). Silica magnetic nanoparticles (1  g) were System. The scanned area was 1° to 80° (2θ value) operated added to a two-necked flask containing 40 ml of dry tolu - at 40 kV and 40 mA using 1.54060 Cu radiation and the size ene and 75  µl of triethylamine. Then, 1.2  ml of (3-ami - and morphology of samples were determined using trans- nopropyl) trimethoxysilane 97% (3-APTMS) was added mission electron microscopy (SEM) analysis with KYKY- to the mixture. The mixture was refluxed under nitrogen EM3200. The images were digitized under the following atmosphere and vigorously stirred at 120 °C for 3 h. The files: voltage 25.0  kV; probe size 3.0  nm and magnification amine-functionalized magnetic nanoparticles (MNPs- 20,000×–80000×. NH ) were washed first with ethanol then with deionized water and the suspension was separated by an external Thermal stability of the free and immobilized lipases magnet. The functionalized MNPs were then dried at To evaluate the thermal stability of the free and immobi- room temperature. lized enzymes, 5 mg of the immobilized derivatives (60 mg lipase/g) were added to 500 μl phosphate buffer (25 mM, pH Immobilization of lipase 7.0) and incubated at different temperatures range between To perform immobilization, 20  mg of amine-functional- 45, 50, 55, 60, 65 and 70 °C for 2 h. The activity was deter - ized nanoparticles was added in vials containing water mined using the p-NPB assay. (pH 7.0) and dispersed under bath ultra-sonication for 20  min. Then, certain amount of enzyme (20–100  mg) Co‑solvent stability of the free and immobilized lipases was added to each sample. Immobilization of the The soluble lipases and 5 mg of the immobilized deriva - enzymes was then started by adding different amount of tives of RML, TLL and CALB (60  mg lipase/g) were A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 5 of 15 added to 500  μl of sodium phosphate solution (25  mM, connected to Zebron capillary column (30  m × 0.25  mm pH 7.0) including different concentrations (10, 20 and i.d.; Phenomenex, USA.) Nitrogen was used as the car- 50% v/v) of the organic solvents (methanol, ethanol, pro- rier gas at a constant flow of 0.8 ml/min. The sample was panol). Samples were taken after 24  h and their residual weighed and mixed with 1000 μl of 0.8  mg/ml methyl activity was evaluated by the p-NPB assay. laurate in n-hexane as an internal standard. Then, 0.5  μl of the diluted sample was injected into the GC. The col - Determination of the optimum pH activity umn temperature was held at 150  °C for 1  min, raised In order to determine the optimum pH activity of lipases, to 210  °C at 25  °C/min, and then increased to 240  °C at the soluble lipase and 5  mg of the immobilized deriva- 10 °C/min and kept for 8 min. The injector and detector tives (60 mg/g) were added in a total volume of 2 ml solu- temperatures were set at 150 and 300  °C, respectively. tion of 25 mM sodium phosphate buffer with various pH Equation (1) was used to calculate biodiesel yield: from 5.0 to 10.5 at 25  °C. The residual activity of each A − A C × V IS IS IS sample was analyzed by the p-NPB assay. C = × × 100%. (1) A m IS Leaching experiment In this equation, (∑A) is the total sub-peak area, ( A ) IS To assess the amount of desorbed enzymes from the is the internal standard sub-peak, (C ) the internal IS surface of the support, 5  mg of the immobilized deriva- standard concentration in (mg/ml), (V ) volume of the IS tives (60  mg/g) was incubated in a solution containing internal standard (ml), and (m) sample mass (mg). NaCl (1 M). The concentration of protein content in the supernatant was evaluated by using both the Bradford’s Experimental design method and enzyme activity assay. Optimization of the transesterification process of waste cooking oil was investigated using a 5-level, 5-factor Biodiesel synthesis Response Surface Central Composite Design (RSCCD) In a typical experiment, the reaction was carried out in of Design Expert Software version 7.0.0 (State Ease Inc. a 5-ml screw-capped vial including 130  mg of waste Minneapolis, USA). The study required 45 experiments, cooking oil and anhydrous methanol at various oil-to- consisting of 32 factorial points, 10 axial points, and 3 methanol molar ratios (1:3, 1:4, 1:5, 1:6, and 1:7). The repetitive tests in central points. In addition, there were mixtures were incubated with the immobilized lipases at five independent identified variables, contained reaction different temperatures (35, 40, 45, 50 and 55 °C). Metha nolysis reactions were performed with different amounts of t-butanol, water-adsorbent (for RML and CALB) and water (for TLL). To analyze the impact of the solvent on methanolysis, the amount of t-butanol was varied (5, 20, 35, 50 and 65 wt. %) by the substrate weight. Reac tions were performed for 72 h under constant agitation of 500  rpm. Aliquots of the reaction medium was taken at different times, mixed with methyl laurate (as an internal standard) and analyzed by GC. GC analysis The FAME yield in the reaction mixture was analyzed Fig. 1 XRD patterns of a silica core–shell magnetic nanoparticles using gas chromatography (Thermo-Quest-Finnigon) (Fe O @SiO ); b amine-functionalized magnetic nanoparticles 3 4 2 (MNPs-NH ); and c MNPs-TLL equipped with a flame-ionization detector (FID) 2 Scheme 1. Functionalization of MNPs Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 6 of 15 all the terms of interaction, was used to calculate the pre- dicted values: 5 5 Y = β + β X + β X + 0 i i ii i=1 i=1 i=1 (2) β X X + β X , ij i j iii i=1 j=i+1 where (Y) is the yield of biodiesel from waste cook- ing oil, β the offset term, (βi) represents linear effect, (βii) represents squared effect, (βij) represents interac - tion effect, (Xi) represents the i-independent variable, and (Xj) represents the j-independent variable. The data were analyzed using the Design Expert program and Fig. 2 FT-IR spectra of a Fe3O4@SiO2 nanoparticles; b amine-functionalized magnetic nanoparticles (MNPs-NH2); and c the coefficients were calculated by F-test. All statistical immobilized derivative of TLL on magnetic nanoparticles (MNPs-TLL) steps including analysis of variance (ANOVA), regression analysis and plotting of contour plot were used to estab- lish the optimum conditions to obtain high efficiency of temperature (35–55 °C), enzyme amounts (5–25 w/w %), methyl ester. as well as t-butanol concentration (5, 20, 35, 50, and 65 wt %), water for TLL (0–40%) and water absorbance for Results and discussion RML and CALB (20–60%). The stepwise methanol feed - Preparation, modification and characterization ing was applied (oil: methanol ratio 1:3, 1:4, 1:5, 1:6 and of functionalized MNPs 1:7). The level of each independent variable was consid - Recently, MNPs have attracted remarkable atten- ered based on previous researches (Ashjari et  al. 2020b; tion owing to their particular properties, such as Yang et  al. 2005). In addition, the independent variables their  large  surface  area and the presence of plenty of were encoded into low (−1) and high (+ 1) levels. In this hydroxyl groups on their surface which enables their paper, the value of α, the distance from the center, is fixed easy modification. The Fe O magnetic nanoparticles 3 4 at 2 which makes the design rotatble. were prepared by co-precipitation method via iron (II) and iron (III) ions. Surface modification of the activated Statistical analysis MNPs with a layer of silica was performed by using The experimental data obtained from central composite TEOS for improving stability of MNPs and provid- design (CCD) were analyzed by response surface meth- ing more available reaction sites for functionalization. odology. A quadratic equation (Eq.  2), which containing Finally, the reaction of Fe O @SiO with (3-aminopropyl) 3 4 2 Fig. 3 SEM images of a MNPs-NH , and b MNPs-TLL 2 A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 7 of 15 Table 1 Immobilization of the lipases on amine-functionalized magnetic nanoparticles Oer ff ed Yield (%) Immobilized enzyme Immobilization time (h) Specific activity U/mg (recovered enzyme (mg/g support) activity) (mg/g) RML CALB TLL RML CALB TLL RML CALB TLL RML CALB TLL 20 100 99.2 98.5 20.0 19.8 19.7 2 2 2 8.2 (142) 35.0 (157) 26.8 (101) 40 100 98.5 98.7 40.0 39.4 39.5 2 3 3 7.5 (125) 30.1 (112) 23.4 (95) 60 98.5 95.0 98.3 59.1 57.0 58.9 3 5 5 6.6 (90) 24.6 (72) 18.3 (99) 80 96.0 92.32 93.7 76.8 73.8 75.0 12 12 12 5.3 (93) 18.9 (74) 13.7 (100) 100 97.5 94.0 99.5 97.5 94.0 99.5 12 12 12 4.2 (77) 9.3 (56) 9.8 (70) The specific activities for the free forms of RML, CALB and TLL were 10.1 U/mg, 38.5 U/mg, and 27.5 U/mg, respectively trimethoxysilane led to amine-functionalized magnetic Fe O (MNPs-NH ; Scheme  1). Characterization of the 3 4 2 MNPs in various stages was performed via XRD, IR, and SEM. The X-ray diffraction patterns of Fe O @SiO nano- 3 4 2 particles (a), MNPs-NH (b) and one of the immobilized preparations (MNPs-TLL; c) were analyzed by XRD spec- troscopy (Fig. 1). XRD-diagram of the bare MNPs showed spinel ferrites pattern. All the strong peaks observed at 2θ = 30.2°, 36.4°, 43.7°, 53.5°, 56.3° and 62.3° are indexed to the highly crystalline cubic spinel structure of Fe O 3 4 nanoparticles (Yang et  al. 2005). The same sets of char - Fig. 4 Eec ff t of temperature on the activity of free and immobilized acteristic peaks were also observed for Fe O @SiO , 3 4 2 lipases. The incubation at different temperatures was carried out in MNPs-NH and MNPs-TLL indicating the stability of 25 mM sodium phosphate buffer, pH 7.0. The initial activity of each the crystalline phase of F e O nanoparticles during silica derivative before incubation was determined and set as 100% 3 4 coating, amine functionalization and enzyme immobili- zation (Ghasemzadeh et al. 2015). FT-IR spectroscopy was also used to confirm the suc - bunches of spherical shaped in different sizes with uni - cessful functionalization of the support with amine form morphology distribution. By considering SEM groups. FT-IR spectra of F e O @SiO , MNPs-NH 3 4 2 2 images it can be concluded that the modification still and the lipase immobilized preparation are presented preserving the textural properties of the MNPs. in Fig.  2a–c. In Fe O @SiO nanoparticles (Fig.  2a), 3 4 2 −1 the band at 1087.77  cm corresponds to Si–O–Si Immobilization of RML, TLL and CALB on MNPs‑NH symmetric stretching vibrations, is an indication The approach of using isocyanide-based multi-com- of the presence of SiO . Figure  2a shows the pres- ponent reaction provided a single-step, rapid and −1 ence of Fe–O stretching vibration at 609.46  cm and low-cost process for high-density covalent attach- also O–H stretching vibration owing to the physically ment of RML, TLL and CALB on amine-functional- absorbed water and surface hydroxyl groups approxi- ized MNPs. Immobilizations were carried out in water −1 mately at 3400  cm . Decrease in intensity of this band (pH 7.0) at 25 °C, in which high immobilization yields after functionalization together with appearing a dou- were obtained (92–100%). The leaching experiment −1 blet peak located at 3425.32 and 1635.51  cm clearly was conducted to remove those enzyme molecules confirmed successful functionalization of the sup - physically adsorbed on the support. Protein quanti- port by APTMS (Fig.  2b). The IR bands responsible fication in the washing solution was performed with for the lipase immobilized on the magnetic nanopar- both p-NPB assay and the Bradford method show- −1 ticles were observed at 1643.23  cm for amide I and ing no detectable enzyme in the solution. This means −1 at 1388.64  cm for amide II, respectively (Miao et  al. that in the used immobilization method, the reac- 2018; Fig. 2c). tion between enzyme molecules and MNPs is exclu- The morphology and structure of MNPs-NH and sively carried out by covalent binding. Immobilization immobilized TLL were also characterized by SEM. Fig- of lipases on MNPs-NH was investigated in terms ure  3a and b indicates that samples consists of various Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 8 of 15 of immobilization time, immobilization yield, load- observed for the final biocatalysts. Decrease in enzyme ing capacity, and specific activity (Table  1). Various specific activity during covalent immobilization has amounts of RML, TLL and CALB (20, 40, 60, 80, and already been reported in several studies and has been 100 mg/g) was offered to 1 g of MNPs-NH under gen- mostly attributed to the enzyme denaturation caused tle stirring for up to 12  h. Immobilization of 20  mg of during coupling process (Weltz et  al. 2020). Since RML, TLL and CALB on 1 g of the support was carried the specific activity of the immobilized derivatives out shortly after 2  h of incubation, producing 100%, obtained in immobilization with 20  mg of each lipase 98.5%, and 99.2% immobilization yields, respectively. presented a small change in activity compared to the While we have already reported covalent immobiliza- corresponding free enzyme, the remarkable decrease tion of CALB on epoxy-functionalized Fe O @SiO in activity in higher amounts of lipases can be attrib- 3 4 2 within 24  h of incubation with 84% immobilization uted to the diffusion limitation of substrate/product yield (Mehrasbi et  al. 2017). The used strategy was in the immobilized preparations (Mohammadi et  al. based on traditional coupling process and resulted 2014). The activity recovery for the immobilized prep- immobilization of 50  mg of CALB on 1  g of the sup- arations was calculated as the ratio of the activity of port max. With increasing the amount of enzyme to immobilized enzyme to the activity of same amount of 40  mg, almost the same immobilization yields were the corresponding soluble enzyme. The results showed obtained with slight change in immobilization time that with increasing the amount of lipases loaded on of TLL and CALB (3  h). With further increasing the the support the recovered activity decreased particu- amount of suggested enzymes to 100 mg, the immobi- larly for CALB with almost 65% lower recovered activ- lization yields of 97.5%, 99.5%, and 94% were detected ity in 100  mg/g of offered enzyme compared to result for RML, TLL and CALB, respectively, after 12  h of obtained in the process for immobilization of 20 mg/g incubation. We have recently reported immobilization of the enzyme. of RML and TLL on aldehyde-functionalized magnetic nanoparticles via multi-component reactions. Rapid Thermal stability of free and immobilized lipases immobilization processes within 12  h were observed It is well documented that immobilization of enzymes producing 81% and 97% of immobilization yields for can improve their thermal stability. Figure  4 represents RML and TLL, respectively, by offering 100 mg of each the time course of residual activities of the free lipases enzyme per 1 g of the support. The maximum loading and their immobilized derivatives at different tem - capacity of the aldehyde-functionalized support was peratures up to 70  °C. At the lowest temperature tested determined to be 81  mg for RML and 97  mg for TLL (45 °C), the immobilized lipases on amine-functionalized which are lower than the obtained loading amount of MNPs remained completely active. While the free RML TLL and RML in this study at the same conditions. was relatively unstable and lost 40% of its initial activity Table  1 also shows specific activity of the free and at 50  °C, the soluble TLL and CALB showed more sta- immobilized enzymes. With increasing the amount of bility with retaining 80% of their activities. The immo - immobilized enzymes, lower specific activities were bilized forms of all enzymes remained 90% active at the same condition. At 55 °C, the immobilized RML showed 70% residual activity after 2 h of incubation, whereas, the soluble enzyme at the same temperature exhibited rapid inactivation. Increasing temperature to 60  °C showed Fig. 5 The stability of free and MNPs-RML, MNPs-TLL and MNPs-CALB in the presence of 50% of methanol, ethanol and propanol. Incubation of each biocatalyst in 1 ml solution containing 25 mM sodium phosphate buffer (pH 7.0) and 50% of each solvent at 25 °C. The initial activity of each derivative was determined before incubation and set as 100% Fig. 6 Eec ff t of pH on activity of immobilized lipases A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 9 of 15 Table 2 Sequential model sum of squares for immobilized TLL Source Sum of squares df Mean square F value Prob > F Mean vs total 64,883.04 1 64,883.04 Linear vs mean 1904.72 5 380.94 4.37 0.003 2FI vs linear 2441.60 10 244.16 7.42 < 0.0001 Suggested Quadratic vs 2FI 185.30 5 37.06 1.16 0.3591 Cubic vs quadratic 660.28 15 44.02 3.63 0.0281 Aliased Residual 109.06 9 12.12 Total 70,184.00 45 1559.64 20%, 40% and 70% residual activity of free RML, CALB stability of the covalently immobilized preparations and TLL, respectively. Remaining 60% activity of MNPs- compared to the free lipases in the presence of 50% of RML, MNPs-CALB and 80% activity of MNPs-TLL after three used co-solvents. The results showed that immo - 2  h incubation at 60  °C, confirmed the positive effect of bilized derivative of lipases retained their activity (over immobilization on the stability of the lipases. With rais- 70%) in the presence of 50% of methanol while the free ing the temperature to 70  °C, the free RML completely lipases lost their activities in the same condition. In the lost its activity, while its immobilized form retained 45% presence of 10% and 20% ethanol, there were no signifi - of its initial activity. Bearing in mind the important role cant differences between covalently immobilized lipases of aspartic acid residues on the surface of proteins, the and free enzymes. With increasing the percentage of higher stability of TLL derivative can be attributed to the ethanol to 50%, while immobilized RML and CALB kept higher number of exposed Asp residues on the surface of 43% and 72% of their activities, respectively, free CALB TLL (13) compared to RML (10) and CALB (9; Orrego remained only 25% active and free RML was completely et al. 2018). deactivated. Co‑solvent stability of the immobilized and free lipases Optimal pH activity of the immobilized preparations In the most enzymatic reactions, the use of co-solvent The activity of immobilized lipases at pH range between is essential to enhance the solubility of the organic sub- 5.5 and 10.5 was also investigated. As illustrated in Fig. 6, strates which are not soluble in water. To evaluate the sta- the maximum activity was observed at pH 8.0 and 8.5 for bility of free and immobilized preparations of RML, TLL, immobilized lipases. It was concluded that both lower and CALB, 5 mg of the derivatives (60 mg/g) were incu- and higher pH are unfavorable for the enzymatic activ- bated for 24  h in the presence of 10%, 20%, and 50% of ity of the lipases. This could be explained by the changes organic solvents including methanol, ethanol and 1-pro- in the enzyme structure and ionic states of the active site panol (Fig. 5 and Additional file  1: Fig. S1a–c). The results residues. They also showed increased optimal pH activity could be interpreted by the effect of the carbon chain of compared to the corresponding free enzymes which have each solvent on the activity of the enzymes. The results been reported to be 7.5 (Online et al. 2016; Bi et al. 2020; confirmed that using an organic solvent may influence Mehrasbi et al. 2017). Furthermore, the immobilized TLL the structure of the enzyme leads either to change the presented broader optimal pH adaptability ranging from catalytic performance of the enzyme in desired enzy- 8 to 8.5. matic reaction or completely inactivation of the enzyme. This can be raised from the replacement of the crucial Biodiesel production water molecules of enzyme structure (Nawani et al. 2006; Optimization of the reaction conditions Klibanov 1989) which are required for enzyme func- In the present work, five factors such as (A) reaction tion. Organic solvents with log P < 2 have a propensity to temperature, (B) amount of water-adsorbent (RML strip this vital water and diminish the catalytic activity and CALB) or water (TLL), (C) the amount of enzyme, of enzyme (Klibanov 2001). Furthermore, enzymes usu- (D) t-butanol weight percentage and (E) oil: methanol ally tend to form aggregates in organic solvents which molar ratios were considered as effective parameters makes them poorly available for the substrate. Immo- in biodiesel production. The yields of waste cooking oil bilization can remove this detrimental effect of organic methyl ester were from 30–61% for immobilized RML, solvents by individually fixing every single enzyme 13–62% for immobilized TLL and 27–52% for immobi- molecule separately on the support. Figure  5 shows the lized CALB. Process optimization with response surface Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 10 of 15 methodology (RSM) was performed and the effect of between the variables. Values of probability (p) > F less each factor and its interactions were calculated to ascer- than 0.05 emphasized that the model terms were signif- tain the optimum conditions for the reaction. Among icant whereas the values greater than 0.05 indicate that the models that fitted to the response such as linear, two the model terms were not significant (Chen et  al. 2008). factors interaction (2FI), quadratic, and cubic polyno- The terms incorporated in the model F-values of 5.79 mial, the two factors interaction (2FI) was selected as the for the immobilized RML, 8.80 for the immobilized TLL best model. This two-factor interaction (2FI) model was and 8.90 for the immobilized CALB with p-value < 0.0001 suggested by the RSM software as shown in Table  2 for revealed that the model was significant at 95% confidence TLL (Additional file  1: Table S1 for RML and Additional level. The R value of 0.74 for RML, 0.81 for TLL and 0.78 file  1: Table  S2 for CALB). This two-factor interaction for CALB as well as adjusted R values of 0.62 for RML, (2FI) model expressed by Eq.  (3) shows biodiesel yield 0.72 for TLL and 0.70 for CALB showed that the model (Y), reaction temperature (A), water absorbent (RML was significant to predict the response. The predicted R and CALB) or water (TLL) w/w (B), the enzyme weight values of 0.30 for RML, 0.53 for TLL and 0.62 for CALB percentage ratio (C), the t-butanol weight percentage (D) were in reasonable agreement with the adjusted R val- and oil/methanol molar ratios (E). A positive sign in front ues. The model also depicted the statistically non-signif - of the terms indicates the synergistic effect of the rise in icant lack of fit (p 0.39 for RML, p 0.46 for TLL and p FAME yield, while negative sign indicates the antagonis- 0.95 for CALB) which were not significant (p-value > 0.05 tic effect (Babaki et  al. 2017). The results at each point is not significant), indicating that the responses were based on the central composite design (CCD) and their adequate for utilizing in the model (Additional file  1: corresponding predicted values are presented in Addi- Fig. S2a for RML, Additional file  1: Fig. S2(b) for TLL tional file  1: Table  S3 for immobilized RML, Additional and Additional file  1: Fig. S2(c) for CALB). These results file  1: Table S4 for immobilized TLL and Additional file  1: also demonstrated that the model satisfactorily fitted to Table S5 for immobilized CALB. experimental data. Insignificant lack of fit was considered as the significant lack of fit indicating that there might be Y = +40.11 − 1.83 A + 0.24 B RML contribution in the regressor–response relationship that + 0.37 C + 2.42 D + 0.92 E is not accounted for by the model (Noordin et  al. 2004). − 3.22 AB − 1.88 AC + 1.08 AD This analysis was examined using the normal probabil - + 1.58 AE + 2.54 BC − 0.69 BD ity of the residuals (Additional file  1: Fig. S3a–c for TLL, RML and CALB, respectively). The normal probability − 2.59 BE − 2.23 CD plot of the residuals for RML, TLL and CALB showed − 0.042 CE + 0.49 DE that the errors were distributed normally in a straight line Y = +37.97 + 2.97A −2.42B TLL and insignificant. On the other hand, the plot of residuals + 3.95C + 1.13D + 4.14E versus predicted response indicated a structureless plot + 0.51AB − 0.34AC + 2.57 AD (3) suggesting that the model was adequate and that they did + 0.84 AE + 1.80 BC + 2.09BD not show any violation of the independence or constant variance assumption (Lee et al. 2011). − 3.42BE − 6.86CD − 1.40CE − 0.56DE Effect of the reaction parameters on FAME yield Y = +40.30 − 1.71A + 1.20B CALB The effect of 5 parameters on the FAME yield was inves - − 1.00C − 0.75D − 3.63AB tigated for the immobilized lipases. MNPs-TLL was the − 1.08AD + 2.97AE − 0.86BC only derivative that was affected by the reaction tempera - − 1.76BD + 2.04BE − 0.91CD ture and produced higher FAME yield with increasing the temperature. It has been already reported that, improved − 1.94CE − 1.83DE. thermal stability after immobilization and decreasing The results of statistical analysis of variance (ANOVA) mass transfer limitation between the reactants can be which were applied to determine the significance and fit - considered as important parameters to facilitate FAME ness of the two factors interaction (2FI) model and also production (Guo et al. 2020). The negative effect in fatty the effect of significant individual terms and their inter - acid methyl esters yields was observed by increasing the action on the selected responses for RML are presented water for MNPs-TLL, while for MNPs-CALB showed in Additional file  1: Tables S6, S7 for TLL and Additional improved efficiency of biodiesel production. Zaks and file  1: Table  S8 for CALB. The p values were used as a coworkers reported that, the presence of excess water tool to check the importance of each of the coefficients, during biodiesel production might lead to aggregation which in turn may indicate the pattern of the interaction of the enzyme and thus reducing its catalytic activity A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 11 of 15 Fig. 7 Response surface curves showing the interactions for MNPs-TLL. a Water content vs. temperature; b biocatalyst quantity vs. temperature; c t-butanol vs. temperature; d t-butanol vs. water content; e methanol-to-oil ratio vs. water content (Zaks and Klibanov 1988). The results also showed that on biodiesel content for MNPs-TLL while the efficiency increasing the amount of MNPs-RML had no effect on of FAME production was improved for MNPs-RML by biodiesel production while it was effectively improved providing higher contents of t-butanol. Change in the as the amount of MNPs-TLL increased (Tavares et  al. methanol:oil molar ratio showed different results for the 2017). The t-butanol percentage had negligible effect immobilized derivatives. Increasing the methanol-to-oil Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 12 of 15 The interaction between water and methanol-to-oil ratio for MNPs-TLL showed that in a low amount of water (10%), increasing methanol concentration (methanol-to- oil ratio from 4:1 up to 6:1) resulted in higher FAME pro- duction (Fig.  7e), whereas at a high level of water (30%) the presence of higher methanol-to-oil ratio 6:1 caused to decrease in FAME yield. This is most likely because of the fact that with increasing methanol, the produc- tion of methyl ester first improved and then declined as a consequence of decrease in enzyme activity caused by excessive methanol (Yadav and Pawar 2012; Li et  al. 2012). The effect of t-butanol and methanol-to-oil ratio Fig. 8 The effect of repeated use of the immobilized preparations on for MNPs-TLL and MNPs-RML showed that the interac- their activities in biodiesel production at 50 °C tion of both low (20%) and high (50%) level of t-butanol with methanol-to-oil ratio could diminish the destructive effect of methanol on biocatalyst deactivation (Babaki ratio from 1:4 to 1:6 didn’t change the reaction yield for et  al. 2016). The negative effect of methanol on biodiesel MNPs-RML while improved yield of FAME production production has been already reported particularly in was observed for MNPs-TLL. high concentrations (Mohammadi et  al. 2015). Different The interaction of the selected variables and their effect reports can be found on the use of lipases from Ther - on fatty acid methyl ester production were also evalu- momyces lanuginosus lipase, Candida antarctica lipase ated for the immobilized TLL in Fig. 7 (Additional file  1: B and Rhizomucor miehei lipase in transesterification Fig. S4 for RML and Additional file  1: Fig. S5 for CALB). of oils. The different results of each report is due to the Although with increasing temperature the FAME yield fact that the immobilization protocol can effectively alter slightly increased For MNPs-TLL, the response surface the catalytic performance and stability of each individual showed no improvement in FAME content in both levels enzyme (Yousefi et al. 2020). As an example, Ashjari et al. of water (Fig.  7a). The interaction of reaction tempera - have reported transesterification of waste cooking oil by ture and amount of biocatalyst for MNPs-TLL showed using the immobilized RML on aldehyde-functionalized that with the simultaneous increase of temperature and support that producing FAME yield of 57.5% (Ashjari amount of biocatalyst, the reaction efficiency improved et  al. 2020b). The other research has showed encapsula - (Fig.  7b)while for MNPs-RML the result was the oppo- tion of RML in X-shaped zeolitic frameworks and use site. The interaction effect between the temperature and this biocatalyst to produce biodiesel with a conversion t-butanol on biodiesel yield for MNPs-TLL showed that yield of 95.6% (Adnan et  al. 2018). In addition, Cazaban the FAME yield did not change with increasing tem- et al. investigated biodiesel production from canola oil by perature and keeping the amount of t-butanol constant using TLL immobilized on silica nanoparticles producing (Fig. 7c), while with increasing temperature and t-butanol the maximum FAME yield of 88% (Cazaban et al. 2018). at the same time, the efficiency of biodiesel production increased significantly. The interaction between water Reusability of the immobilized derivatives content and t-butanol for MNPs-TLL showed that by The main advantage of immobilization is the reus - increasing the amount of t-butanol and keeping the ability of the immobilized enzyme which decreases the water constant (Fig.  7d), the yield was improved, while final cost of the process. The immobilized derivatives with increasing the amount of water and keeping the of RML, TLL and CALB were investigated in terms of amount of t-butanol constant, the efficiency decreased their operational stability in biodiesel production for 5 significantly. For MNPs-TLL and MNPs-RML by increas - cycles of successive transesterification processes. After ing the amount of TLL and RML in a constant amount each run, the immobilized lipase was recovered, washed of t-butanol, FAME production was increased but for with n-hexane and dried to use in the next batch. The MNPs-CALB, with increasing the amount of biocatalyst results showed that the derivatives have good capability there was no change in FAME yield. Furthermore, the to be repeatedly used up to five cycles with 43%, 68% simultaneous increase in the amount of biocatalyst and and 48% of activity for MNPs-RML, MNPs-TLL, and t-butanol reduced the FAME yield for all three enzymes. MNPs-CALB, respectively (Fig. 8). A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 13 of 15 Conclusion Fig. S4. Response surface curves showing the interactions for RML. Fig. This paper has investigated a facile method for the immo - S5. Response surface curves showing the interactions for CALB. bilization of lipases on amine-functionalized supports. This methodology is based on a three-component reac - Acknowledgements tion including an amine from support, a carboxylic acid This research was financially supported by Shahid Beheshti University of Tehran and the National Institute of Genetic Engineering and Biotechnology from enzyme surface and cyclohexyl isocyanide. RML, (NIGEB) which the authors are thankful. TLL and CALB were successfully immobilized on amine- functionalized magnetic nanoparticles at extremely Author contributions YA contributed to experiments, data analysis, data validation and writing the mild conditions in a relatively short time. The leaching original draft. MS contributed to experimental support, data validation and experiment confirmed that the enzymes were covalently manuscript editing. ZH supervised the project and funding acquisition. MY attached on the support. The immobilized lipases were provided technical support, manuscript editing. MA contributed to study design, data validation and methodology. MM contributed to study design, more active and stable in wide ranges of pH, organic methodology, editing the manuscript. All authors read and approved the final solvents and different temperatures compared to their manuscript. corresponding free forms. MNPs-TLL shows higher sta- Funding bility in comparison to MNPs-RML and MNPs-CALB, Not applicable. retaining 100% of its activity after 24  h of incubation in the presence of organic solvents. MNPs-TLL also showed Availability of data and materials All data generated or analyzed during this study are included in this published broad range of optimum pH activity compared to its article and its additional information files. soluble form and the other immobilized derivatives. The immobilized lipases were then employed in biodiesel Declarations production by transesterification of waste cooking oil with methanol. In an optimization study, the effect of Ethics approval and consent to participate Not applicable. enzyme weight percentage ratio, t-butanol, tempera- ture, methanol: oil ratio, water (for TLL) and the water- Consent for publication adsorbent weight percentage (for CALB and RML) on All authors have read this article and have approved its submission to Biore- sources and Bioprocessing. the FAME yield were evaluated. The maximal conversion to methyl esters of 78% was attained by using MNPs- Competing interests CALB as catalyst and FAME production yield by immo- There is no competing financial interest. bilized TLL reached 81% under optimal conditions while Author details the maximum yield for RML was 74%. Reusability study 1 Department of Organic Chemistry and Oil, Faculty of Chemistry, Shahid showed that the immobilized lipases on MNPs-NH Beheshti University, Tehran, Iran. Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran. Bioprocess Engineering can be easily recovered from the reaction mixture with Department, Institute of Industrial and Environmental Biotechnology, National retaining 43–68% of the initial activities after 5 cycles of Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. the reaction. Received: 3 March 2022 Accepted: 20 May 2022 Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40643- 022- 00552-0. References Adnan M, Li K, Xu L, Yan Y (2018) X-shaped zif-8 for immobilization rhizomucor Additional file 1: Table S1. Sequential model sum of squares (RML). miehei lipase via encapsulation and its application toward biodiesel Table S2. Sequential model sum of squares (CALB). Table S3. Experi- production. Catalysts. https:// doi. org/ 10. 3390/ catal 80300 96 mental design for five-level five-factor surface response design on Ahrari F, Yousefi M, Habibi Z, Mohammadi M (2022) Application of undecan- transesterification of waste cooking oil using immobilized RML. Table S4. edicarboxylic acid to prepare cross-linked enzymes (CLEs) of Rhizomucor Experimental design for five-level five-factor surface response design on miehei lipase (RML); Selective enrichment of polyunsaturated fatty acids. transesterification of waste cooking oil using immobilized TLL. Table S5. Mol Catal 520:112172. https:// doi. org/ 10. 1016/J. MCAT. 2022. 112172 Experimental design for five-level five-factor surface response design on Ali CH, Qureshi AS, Mbadinga SM et al (2017) Biodiesel production from waste transesterification and esterification of waste cooking oil using immobi- cooking oil using onsite produced purified lipase from Pseudomonas lized CALB. Table S6. Analysis of variance (ANOVA) for fitted quadratic pol- aeruginosa FW_SH-1: Central composite design approach. Renew Energy ynominal model for optimization of transesterification parameters (RML). 109:93–100. https:// doi. org/ 10. 1016/j. renene. 2017. 03. 018 Table S7. Analysis of variance (ANOVA) for fitted quadratic polynominal Ansari SA, Husain Q (2012) Potential applications of enzymes immobilized on/ model for optimization of transesterification parameters ( TLL). Table S8. in nano materials: A review. Biotechnol Adv 30:512–523. https:// doi. org/ Analysis of variance (ANOVA) for fitted quadratic polynominal model for 10. 1016/j. biote chadv. 2011. 09. 005 optimization of transesterification parameters (CALB). Fig. S1. Stability of Ashjari M, Garmroodi M, Ahrari F et al (2020a) Soluble enzyme cross-linking: immobilized derivatives MNPs-RML (a) and MNPs-TLL (b) in the presence Via multi-component reactions: A new generation of cross-linked of 10, 20 and 50% of various co-solvents. Fig. S2. Predicted fatty acid enzymes. Chem Commun 56:9683–9686. https:// doi. org/ 10. 1039/ d0cc0 methyl ester yield versus experimental fatty acid methyl ester yield for 3429k MNPs-RML (a), MNPs-TLL (b) and MNPs-CALB(c). Fig. S3. Normal probabil- ity plots of residuals for (a) MNPs-TLL (b), MNPs-RML and MNPs-CALB(c). Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 14 of 15 Ashjari M, Garmroodi M, Amiri Asl F et al (2020b) Application of multi- Guo J, Sun S, Liu J (2020) Conversion of waste frying palm oil into biodiesel component reaction for covalent immobilization of two lipases on using free lipase A from Candida antarctica as a novel catalyst. Fuel aldehyde-functionalized magnetic nanoparticles; production of biodiesel 267:117323. https:// doi. org/ 10. 1016/j. fuel. 2020. 117323 from waste cooking oil. Process Biochem 90:156–167. https:// doi. org/ 10. Habibi Z, Mohammadi M, Yousefi M (2013) Enzymatic hydrolysis of racemic 1016/j. procb io. 2019. 11. 002 ibuprofen esters using Rhizomucor miehei lipase immobilized on dif- Babaki M, Yousefi M, Habibi Z et al (2015) Preparation of highly reusable ferent supports. Process Biochem 48:669–676. https:// doi. org/ 10. 1016/j. biocatalysts by immobilization of lipases on epoxy-functionalized silica procb io. 2013. 02. 020 for production of biodiesel from canola oil. Biochem Eng J 101:23–31. Hama S, Noda H, Kondo A (2018) How lipase technology contributes to https:// doi. org/ 10. 1016/j. bej. 2015. 04. 020 evolution of biodiesel production using multiple feedstocks. Curr Opin Babaki M, Yousefi M, Habibi Z et al (2016) Enzymatic production of biodiesel Biotechnol 50:57–64. https:// doi. org/ 10. 1016/j. copbio. 2017. 11. 001 using lipases immobilized on silica nanoparticles as highly reusable Handayani N, Wahyuningrum D, Zulfikar MA et al (2016) The synthesis of biocatalysts: Eec ff t of water, t-butanol and blue silica gel contents. Renew biodiesel catalyzed by Mucor miehei lipase immobilized onto aminated Energy 91:196–206. https:// doi. org/ 10. 1016/j. renene. 2016. 01. 053 polyethersulfone membranes. Bioresour Bioproces 3:22. https:// doi. org/ Babaki M, Yousefi M, Habibi Z, Mohammadi M (2017) Process optimization 10. 1186/ s40643- 016- 0098-4 for biodiesel production from waste cooking oil using multi-enzyme He J, Huang M, Wang D et al (2014) Magnetic separation techniques in systems through response surface methodology. Renew Energy 105:465– sample preparation for biological analysis: a review. J Pharm Biomed Anal 472. https:// doi. org/ 10. 1016/j. renene. 2016. 12. 086 101:84–101. https:// doi. org/ 10. 1016/j. jpba. 2014. 04. 017 Barbosa O, Ortiz C, Berenguer-Murcia Á et al (2015) Strategies for the one-step Hu B, Pan J, Yu HL et al (2009) Immobilization of Serratia marcescens lipase immobilization-purification of enzymes as industrial biocatalysts. Biotech- onto amino-functionalized magnetic nanoparticles for repeated use nol Adv 33:435–456. https:// doi. org/ 10. 1016/j. biote chadv. 2015. 03. 006 in enzymatic synthesis of Diltiazem intermediate. Process Biochem Bi Y, Wang Z, Zhang R et al (2020) Improved catalytic properties of Thermo- 44:1019–1024. https:// doi. org/ 10. 1016/j. procb io. 2009. 05. 001 myces lanuginosus lipase immobilized onto newly fabricated polydopa- Jang MG, Kim DK, Park SC et al (2012) Biodiesel production from crude canola mine-functionalized magnetic Fe3O4 nanoparticles. Processes. https:// oil by two-step enzymatic processes. Renew Energy 42:99–104. https:// doi. org/ 10. 3390/ PR805 0629doi. org/ 10. 1016/j. renene. 2011. 09. 009 Budhwani AAA, Maqbool A, Hussain T, Noman Syed M (2019) Production of Jiang K, Schadler LS, Siegel RW et al (2004) Protein immobilization on carbon biodiesel by enzymatic transesterifcation of non-edible Salvadora persica nanotubes via a two-step process of diimide-activated amidation. J Mater (Pilu) oil and crude coconut oil in a solvent-free system. Bioresources and Chem 14:37–39. https:// doi. org/ 10. 1039/ b3103 59e Bioprocessing 6:41. https:// doi. org/ 10. 1186/ s40643- 019- 0275-3 Jiang XY, Bai S, Sun Y (2007) Fabrication and characterization of rigid magnetic Cao SL, Xu H, Lai LH, Gu WM et al (2017) Magnetic ZIF-8/cellulose/Fe3O4 monodisperse microspheres for protein adsorption. J Chromatogr B Anal nanocomposite: preparation, characterization, and enzyme immobili- Technol Biomed Life Sci 852:62–68. https:// doi. org/ 10. 1016/j. jchro mb. zation. Bioresources and Bioprocessing 4:56. https:// doi. org/ 10. 1186/ 2006. 12. 044 s40643- 017- 0186-0 Klibanov AM (1989) Enzymatic catalysis in monophasic organic solvents. Cazaban D, Illanes A, Wilson L, Betancor L (2018) Bio-inspired silica lipase nano- Enzyme Microb Technol 11:194–211 biocatalysts for the synthesis of fatty acid methyl esters. Process Biochem Klibanov AM (2001) Improving enzymes by using them in organic solvents. 74:86–93. https:// doi. org/ 10. 1016/j. procb io. 2018. 07. 004 Nature 409:241–246. https:// doi. org/ 10. 1038/ 35051 719 Chen X, Du W, Liu D (2008) Response surface optimization of biocatalytic Lee HV, Yunus R, Juan JC, Taufiq-Yap YH (2011) Process optimization design for biodiesel production with acid oil. Biochem Eng J 40:423–429. https:// doi. jatropha-based biodiesel production using response surface methodol- org/ 10. 1016/j. bej. 2008. 01. 012 ogy. Fuel Process Technol 92:2420–2428. https:// doi. org/ 10. 1016/j. fuproc. Cipolatti EP, Silva MJA, Klein M et al (2014) Current status and trends in enzy-2011. 08. 018 matic nanoimmobilization. J Mol Catal B Enzym 99:56–67. https:// doi. Li X, He XY, Li ZL et al (2012) Enzymatic production of biodiesel from Pistacia org/ 10. 1016/j. molca tb. 2013. 10. 019 chinensis bge seed oil using immobilized lipase. Fuel 92:89–93. https:// Emami Bistgani Z, Siadat SA, Bakhshandeh A et al (2017) Interactive effects of doi. org/ 10. 1016/j. fuel. 2011. 06. 048 drought stress and chitosan application on physiological characteristics Luna C, Verdugo C, Sancho DE et al (2014) Production of a biodiesel-like bio- and essential oil yield of Thymus daenensis Celak. Crop J 5:407–415. fuel without glycerol generation, by using Novozym 435, an immobilized https:// doi. org/ 10. 1016/j. cj. 2017. 04. 003 Candida antarctica lipase. Bioresour Bioprocessing 1:11. https:// doi. org/ Franssen MCR, Steunenberg P, Scott EL et al (2013) Immobilised enzymes in 10. 1186/ s40643- 014- 0011-y biorenewables production. Chem Soc Rev 42:6491–6533. https:// doi. org/ Marchetti JM, Miguel VU, Errazu AF (2007) Possible methods for biodiesel 10. 1039/ c3cs0 0004d production. Renew Sustain Energy Rev 11:1300–1311. https:// doi. org/ 10. Gao Y, Kyratzis I (2008) Covalent Immobilization of Proteins on Carbon Nano-1016/j. rser. 2005. 08. 006 tubes Using the. Bioconjug Chem 19:1945–1950 Mehde AA, Mehdi WA, Severgün O et al (2018) Lipase-based on starch mate- Garmroodi M, Mohammadi M, Ramazani A et al (2016) Covalent binding of rial as a development matrix with magnetite cross-linked enzyme aggre- hyper-activated Rhizomucor miehei lipase (RML) on hetero-functional- gates and its application. Int J Biol Macromol 120:1533–1543. https:// doi. ized siliceous supports. Int J Biol Macromol 86:208–215. https:// doi. org/ org/ 10. 1016/j. ijbio mac. 2018. 09. 141 10. 1016/j. ijbio mac. 2016. 01. 076 Mehrasbi MR, Mohammadi J, Peyda M, Mohammadi M (2017) Covalent immo- Ghadge SV, Raheman H (2006) Process optimization for biodiesel production bilization of Candida antarctica lipase on core-shell magnetic nanopar- from mahua (Madhuca indica) oil using response surface methodology. ticles for production of biodiesel from waste cooking oil. Renew Energy Bioresour Technol 97:379–384. https:// doi. org/ 10. 1016/j. biort ech. 2005. 101:593–602. https:// doi. org/ 10. 1016/j. renene. 2016. 09. 022 03. 014 Miao C, Yang L, Wang Z et al (2018) Lipase immobilization on amino-silane Ghasemzadeh MA, Abdollahi-Basir MH, Babaei M (2015) Fe3O4@SiO2–NH2 modified superparamagnetic Fe3O4 nanoparticles as biocatalyst for core-shell nanocomposite as an efficient and green catalyst for the multi- biodiesel production. Fuel 224:774–782. https:// doi. org/ 10. 1016/j. fuel. component synthesis of highly substituted chromeno[2,3-b]pyridines in 2018. 02. 149 aqueous ethanol media. Green Chem Lett Rev 8:40–49. https:// doi. org/ Mohammadi M, Habibi Z, Dezvarei S et al (2014) Improvement of the stability 10. 1080/ 17518 253. 2015. 11071 39 and selectivity of Rhizomucor miehei lipase immobilized on silica nano- Ghosh S, Chaganti SR, Prakasham RS (2012) Polyaniline nanofiber as a novel particles: selective hydrolysis of fish oil using immobilized preparations. immobilization matrix for the anti-leukemia enzyme l-asparaginase. J Mol Process Biochem 49:1314–1323. https:// doi. org/ 10. 1016/j. procb io. 2014. Catal B Enzym 74:132–137. https:// doi. org/ 10. 1016/j. molca tb. 2011. 09. 00904. 012 Gross RA, Ganesh M, Lu W (2010) Enzyme-catalysis breathes new life into Mohammadi M, Ashjari M, Dezvarei S et al (2015) Rapid and high-density polyester condensation polymerizations. Trends Biotechnol 28:435–443. covalent immobilization of Rhizomucor miehei lipase using a multi https:// doi. org/ 10. 1016/j. tibte ch. 2010. 05. 004 component reaction: application in biodiesel production. RSC Adv 5:32698–32705. https:// doi. org/ 10. 1039/ c5ra0 3299g A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 15 of 15 Mohammadi M, Gandomkar S, Habibi Z, Yousefi M (2016) One pot three- immobilization strategies for the selective hydrolysis of fish oil. Molecules component reaction for covalent immobilization of enzymes: Application 25:1–14. https:// doi. org/ 10. 3390/ molec ules2 50305 45 of immobilized lipases for kinetic resolution of: Rac -ibuprofen. RSC Adv Yücel Y (2012) Optimization of biocatalytic biodiesel production from pomace 6:52838–52849. https:// doi. org/ 10. 1039/ c6ra1 1284f oil using response surface methodology. Fuel Process Technol 99:97–102. Mohammadi M, Habibi Z, Gandomkar S, Yousefi M (2018) A novel approach https:// doi. org/ 10. 1016/j. fuproc. 2012. 02. 008 for bioconjugation of Rhizomucor miehei lipase (RML) onto amine- Yushkova ED, Nazarova EA, Matyuhina AV et al (2019) Application of immo- functionalized supports; Application for enantioselective resolution of bilized enzymes in food industry. J Agric Food Chem 67:11553–11567. rac-ibuprofen. Int J Biol Macromol 117:523–531. https:// doi. org/ 10. 1016/j. https:// doi. org/ 10. 1021/ acs. jafc. 9b043 85 ijbio mac. 2018. 05. 218 Zaks A, Klibanov AM (1988) The effect of water on enzyme action in organic Nawani N, Singh R, Kaur J (2006) Immobilization and stability studies of a media. J Biol Chem 263:8017–8021. https:// doi. org/ 10. 1016/ s0021- lipase from thermophilic Bacillus sp: The effect of process parameters on 9258(18) 68435-2 immobilization of enzyme. Electron J Biotechnol 9:559–565. https:// doi. Zucca P, Sanjust E (2014) Inorganic materials as supports for covalent enzyme org/ 10. 2225/ vol9- issue5- fullt ext-9 immobilization: Methods and mechanisms. Molecules 19:14139–14194. Noordin MY, Venkatesh VC, Sharif S et al (2004) Application of response surface https:// doi. org/ 10. 3390/ molec ules1 90914 139 methodology in describing the performance of coated carbide tools when turning AISI 1045 steel. J Mater Process Technol 145:46–58. https:// Publisher’s Note doi. org/ 10. 1016/ S0924- 0136(03) 00861-6 Springer Nature remains neutral with regard to jurisdictional claims in pub- Online VA, Ashjari M, Garmroodi M, Karkhane AA (2016). RSC Adv. https:// doi. lished maps and institutional affiliations. org/ 10. 1039/ C6RA1 4142K Orrego AH, Ghobadi R, Moreno-Perez S et al (2018) Stabilization of immobi- lized lipases by intense intramolecular cross-linking of their surfaces by using aldehyde-dextran polymers. Int J Mol Sci 19:1–16. https:// doi. org/ 10. 3390/ ijms1 90205 53 Shimada Y, Watanabe Y, Sugihara A, Tominaga Y (2002) Enzymatic alcoholy- sis for biodiesel fuel production and application of the reaction to oil processing. J Mol Catal B Enzym 17:133–142. https:// doi. org/ 10. 1016/ S1381- 1177(02) 00020-6 Shinkai M (2002) Functional magnetic particles for medical application. J Biosci Bioeng 94:606–613. https:// doi. org/ 10. 1016/ S1389- 1723(02) 80202-X Sigurdardóttir SB, Lehmann J, Ovtar S et al (2018) Enzyme Immobilization on Inorganic Surfaces for Membrane Reactor Applications: Mass Transfer Challenges, Enzyme Leakage and Reuse of Materials. Adv Synth Catal 360:2578–2607. https:// doi. org/ 10. 1002/ adsc. 20180 0307 Tacias-Pascacio VG, Torrestiana-Sánchez B, Dal Magro L et al (2019) Compari- son of acid, basic and enzymatic catalysis on the production of biodiesel after RSM optimization. Renew Energy 135:1–9. https:// doi. org/ 10. 1016/j. renene. 2018. 11. 107 Takeuchi Y (2017) Sagittal plane spinal mobility is associated with dynamic balance ability of community-dwelling elderly people. J Phys Ther Sci 29:112–114. https:// doi. org/ 10. 1589/ jpts. 29. 112 Tang T, Fan H, Ai S et al (2011) Hemoglobin (Hb) immobilized on amino- modified magnetic nanoparticles for the catalytic removal of bisphenol A. Chemosphere 83:255–264. https:// doi. org/ 10. 1016/j. chemo sphere. 2010. 12. 075 Tavares GR, Gonçalves JE, dos Santos WD, da Silva C (2017) Enzymatic interesterification of crambe oil assisted by ultrasound. Ind Crops Prod 97:218–223. https:// doi. org/ 10. 1016/j. indcr op. 2016. 12. 022 Vashist SK, Lam E, Hrapovic S et al (2014) Immobilization of antibodies and enzymes on 3-aminopropyltriethoxysilane-functionalized bioanalytical platforms for biosensors and diagnostics. Chem Rev 114:11083–11130. https:// doi. org/ 10. 1021/ cr500 0943 Verhoef JC, Cofino WP, Barendrecht E (1978) Mechanism and reaction rate of the Karl-Fischer titration reaction. J Electroanal Chem Interfacial Electro- chem 93:75–80. https:// doi. org/ 10. 1016/ s0022- 0728(78) 80240-x Weltz JS, Kienle DF, Schwartz DK, Kaar JL (2020) Reduced Enzyme Dynam- ics upon Multipoint Covalent Immobilization Leads to Stability-Activity Trade-off. J Am Chem Soc 142:3463–3471. https:// doi. org/ 10. 1021/ jacs. 9b117 07 Wu X, Yang C, Ge J (2017) Green synthesis of enzyme/metal-organic framework composites with high stability in protein denaturing solvents. Bioresources and Bioprocessing 4:24. https:// doi. org/ 10. 1186/ s40643- 017- 0154-8 Yadav GD, Pawar SV (2012) Synergism between microwave irradiation and enzyme catalysis in transesterification of ethyl-3-phenylpropanoate with n-butanol. Bioresour Technol 109:1–6. https:// doi. org/ 10. 1016/j. biort ech. 2012. 01. 030 Yang T, Shen C, Li Z et al (2005) Highly ordered self-assembly with large area of Fe3O 4 nanoparticles and the magnetic properties. J Phys Chem B 109:23233–23236. https:// doi. org/ 10. 1021/ jp054 291f Yousefi M, Marciello M, Guisan JM et al (2020) Fine modulation of the catalytic properties of Rhizomucor miehei lipase driven by different http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioresources and Bioprocessing Springer Journals

A multi-component reaction for covalent immobilization of lipases on amine-functionalized magnetic nanoparticles: production of biodiesel from waste cooking oil

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Abstract

mono-alkyl esters. Transesterification processes could Introduction be classified based on chemical (alkaline-catalyzed and The exhaustion of fossil fuels is synchronized with an acid-catalyzed) or enzymatic methods (Handayani et al. accelerative rise in oil prices which augments environ- 2016; Budhwani et  al. 2019). The alkaline-catalyzed mental concerns. These challenges led the researches to transesterification as the most used method for indus - explore renewable resources and environment-friendly trial production of biodiesel has several drawbacks fuels (Luna et  al. 2014). In the next few decades, it such as the difficult recovery of glycerol and catalyst, would be required to apply biomass for the applications the necessity of high energy, reaction saponification, that are up to now produced from fossil resources, such and wastewater treatment issues (Hama et  al. 2018). as coal, natural gas, and oil (Franssen et al. 2013). From Enzymatic routes by using lipases (triacylglycerol acyl- an economic point of view, the replacement of fossil- hydrolase, EC 3.1.1.3), on the other hand, are normally based chemicals by biomass sources would be attractive conducted under mild conditions in terms of tempera- since various components of biomass contain molecu- ture, pH, and pressure (Gross et al. 2010). Lipase-cata- lar functionalities that are now presented in the oil- lyzed transesterification can be an excellent substitute derived base chemicals with high costs (Shimada et  al. to produce biodiesel due to the fact that it has been 2002). Biodiesel is mono-alkyl esters of long-chain fatty reported to give high yields, have simple purification acids (Mehde et al. 2018), derived from renewable feed- of products, and need less energy consumption with a stocks such as vegetable oils and animal fats. The key decreased amount of wastewater (Tacias-Pascacio et al. sources of vegetable oil for biodiesel production are 2019; Ali et  al. 2017; Ali et  al. 2017). There have been soybean oil, sunflower oil, palm oil and rapeseed oil very limited commercial lipases applicable in industry (Yücel 2012). Biodiesel has better lubricating properties in their free forms, owing to the high cost and the fact and a much higher cetane value than petroleum diesel that their separation, recovery and reuse are challeng- (Marchetti et al. 2007). Biodiesel is produced in a trans- ing. One of the most cost-effective methods adopted esterification process in which glycerides convert into A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 3 of 15 to eliminate these barriers is immobilization of lipases et  al. 2017). Surface modification is a useful approach to on a solid support and making them more stable het- improve thermal stability of nude magnetic nanoparticles erogeneous biocatalyst (Yushkova et al. 2019; Wu et al. (MNPs) and provide available reaction sites for further 2017). Immobilization of enzymes on solid carriers functionalization. Number of modifications protocols can be achieved by encapsulation, covalent bond and have been reported for functionalization of magnetic adsorption (Barbosa et al. 2015; Garmroodi et al. 2016). nanoparticles (MNPs) surface, for example by cross- The strong binding between the enzyme and its carrier linking with glutaraldehyde, coating with polymers (Jiang matrix in covalent attachment method prevents the et  al. 2007) or coupling with compounds like as agarose problem of leaching and enhance the stability and reus- (He et al. 2014). ability of the enzyme derivatives (Babaki et  al. 2015). In this research superparamagnetic Fe O nanoparticles 3 4 Recently, our group has introduced the use of multi- coated with layer of silica were prepared and subsequently component reactions for covalent immobilization of functionalized by amine functional groups. The prepared lipases on various supports (Mohammadi et  al. 2016; support was then used for the immobilization of Thermomy - Habibi et  al. 2013; Ashjari et  al. 2020b, 2020a; Ahrari ces lanuginosus (TLL), Rhizomucor miehei (RML) as 1,3-spe- et  al. 2022). Enzyme immobilization via multi-compo- cific lipases, and Candida antarctica lipase B (CALB) as a nent reactions offers a very fast process in an extremely non-specific lipase via a multi-component reaction. FT-IR, mild condition with great flexibility of accepting vari - SEM, and XRD were used to characterize the support before ety of functional groups (COOH, N H , and epoxide). and after immobilization. Ultimately, application of the It means that by utilizing this procedure, enzymes can immobilized lipases in the synthesis of biodiesel was evalu- be covalently attached on a large number of functional- ated. Response surface methodology (RSM) that is a power- ized matrixes. Among them, the amine-functionalized ful and efficient mathematical approach was applied for the supports are known as the most widely used carri- process optimization (Yücel 2012; Ghadge and Raheman ers for immobilization of enzymes (Mohammadi et  al. 2006; Jang et al. 2012). A 5-level 5-factor central composite 2018; Sigurdardóttir et  al. 2018; Vashist et  al. 2014). design (CCD) was employed to design the experiments. The Covalent binding of enzymes on these supports is usu- effect of several reaction parameters including water content ally performed via (1) modification of amine groups by (for TLL), water-adsorbent (for RML and CALB), reaction glutaraldehyde as a bifunctional linker which produces time, t-butanol concentration, temperature, and the amount a reversible iminium bonds and requires subsequent of biocatalyst were optimized. reduction by a reducing agent like NaBH4. This 2-step procedure is time-consuming and the chemicals used Materials and methods may have deleterious effect on enzyme activity (Zucca Materials and Sanjust 2014) by using diimide-activated amida- Lipases from Thermomyces lanuginosus (TLL), Rhizomu - tion of carboxylic acids on the surface of enzyme ( Jiang cor miehei (RML), Candida antarctica lipase B (CALB), et  al. 2004). This process is also complicated in prac - FeCl .4HO, FeCl .6H O, p-nitrophenyl butyrate (p- 2 2 3 2 tice and requires multiple and time-consuming steps. NPB), and tetraethyl orthosilicate (TEOS) were pur- Besides, in this approach carboxylic groups are acti- chased from Sigma-Aldrich. Waste cooking oil was also vated by carbodiimides which are quite costly and toxic obtained from a local restaurant and the fatty acids com- reagents (Zucca and Sanjust 2014). These reagents also position of waste cooking oil was determined to be 40.6% cause undesirable side reactions of intermolecular con- oleic acid, 17.58% linoleic acid, 32.24% palmitic acid, and jugation of proteins (Gao and Kyratzis 2008). 5.23% stearic acid. The molecular weight of the waste Nanoparticles have been extensively considered for cooking oil which was determined from the saponifica - enzymes immobilization (Cipolatti et  al. 2014; Ghosh tion value of 196.2  mg KOH/g and acid value of 76  mg et  al. 2012). There are some characteristics related to KOH/g was calculated to be 856.3 g/mol. Water content the nanoparticle-based biocatalysts: (i) they can be eas- in the oil measured by Karl Fischer (Verhoef et al. 1978) ily synthesized in high solid content without surfactants titration method was determined to be 0.01% (w/w). and toxic reagents; (ii) homogeneous and well defined Solvents used including n-hexane, methanol, ethanol, core–shell nanoparticles with a thick enzyme shell can 1-propanol, toluene, t-butanol and blue silica gel were be obtained and (iii) designed according to the research prepared from Merck chemicals. Triethylamine (E t N) necessity (Ansari and Husain 2012). Since the 1970s, was from Titrachem and 3-aminopropyltrimethoxysilane magnetic nanoparticles have progressively been used in (3-APTMS) 97% was purchased from Alfa Aesar. All the the field of bioscience and medicine (Shinkai 2002; Cao other chemicals were accessed commercially. Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 4 of 15 Methods cyclohexyl isocyanide under stirring at room tempera- Preparation of the magnetic Fe O nanoparticle (MNPs) ture. The yield immobilization was determined by meas - 3 4 Magnetic Fe O nanoparticles were prepared by the uring the initial and final concentration of each enzyme 3 4 conventional co-precipitation method (Tang et  al. 2011; in supernatant by using the Bradford’s method (Emami Hu et  al. 2009). First, 1.25  g of F eCl ·4H O and 3.4  g of Bistgani et  al. 2017). Finally, the immobilized lipase was 2 2 FeCl ·6H O were dissolved in 100  ml deionized water removed by an external magnet and washed by deion- 3 2 under nitrogen atmosphere and the reaction temperature ize water. Control experiments were conducted in the was increased to 65 °C. Afterward 9 ml of ammonia solu- absence of cyclohexyl isocyanide. tion (32%) was added by dropping funnel under vigorous stirring at room temperature for 1  h. The black precipi - Enzyme activity assay tates were collected by an external magnetic field and The activities of the soluble and immobilized enzymes washed with absolute ethyl alcohol and deionized water were analyzed spectrophotometrically by measur- several times to reach the pH value of 7.0. Then, the mag - ing the absorbance produced by the release of p-nitro- netic nanoparticles were dried in a vacuum oven at 80 °C phenol in the hydrolysis of p-NPB in 25  mM sodium and used directly for being coated with silica. phosphate buffer at pH 8.0 and 25  °C at 410  nm −1 −1 (ε = 18,400  M  cm ; Verhoef et al. 1978). Briefly, 0.05– 0.2 ml of the lipase suspensions or solutions (without fur- Preparation of silica‑coated magnetic nanoparticles ther dilution) was added to 2.5  ml of substrate solution Fe O @SiO core–shell particles were prepared by the 3 4 2 (0.8  mM) under magnetic stirring. Spontaneous hydrol- modified Stöber sol–gel process (Takeuchi 2017). 1.5  g ysis of p-NPB was measured using 2.5  ml of substrate of Fe O was mixed with 60  ml of ethanol and 10  ml of 3 4 solution (0.8  mM) in the absence of enzyme as control. HPLC water. This suspension was dispersed under ultra- Enzymatic activity is given as 1  μmol of p-nitrophenol sonication for 20  min, then 9  ml of aqueous ammonia released per minute per mg of the enzyme (IU) under the 32% and 4.31  ml of tetraethyl orthosilicate (TEOS) were conditions described above. added to the suspension under nitrogen atmosphere. The mixture was stirred for 5  h at room temperature. The Characterization of the support and the immobilized magnetic Fe O @SiO nanoparticles were separated by 3 4 2 biocatalysts an external magnetic field washed with ethanol and dis - The structure of Fe O @SiO , MNPs-NH , and MNPs-lipase 3 4 2 2 tilled water and dried in a vacuum oven at 80 °C. was characterized by using infrared spectroscopy (IR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). Amine‑functionalization of silica‑coated magnetic Fourier-transform infrared (FT-IR) spectra were performed nanoparticles −1 on a Bruker FT-IR from 4000 to 400  cm using the KBr pel- The dry magnetic Fe O @SiO nanoparticles were 3 4 2 let technique. The X-ray diffraction (XRD) patterns of sam - functionalized with 3-aminopropyltrimethoxysilane ples were obtained on a STOE-STADV Powder Diffraction (3-APTMS). Silica magnetic nanoparticles (1  g) were System. The scanned area was 1° to 80° (2θ value) operated added to a two-necked flask containing 40 ml of dry tolu - at 40 kV and 40 mA using 1.54060 Cu radiation and the size ene and 75  µl of triethylamine. Then, 1.2  ml of (3-ami - and morphology of samples were determined using trans- nopropyl) trimethoxysilane 97% (3-APTMS) was added mission electron microscopy (SEM) analysis with KYKY- to the mixture. The mixture was refluxed under nitrogen EM3200. The images were digitized under the following atmosphere and vigorously stirred at 120 °C for 3 h. The files: voltage 25.0  kV; probe size 3.0  nm and magnification amine-functionalized magnetic nanoparticles (MNPs- 20,000×–80000×. NH ) were washed first with ethanol then with deionized water and the suspension was separated by an external Thermal stability of the free and immobilized lipases magnet. The functionalized MNPs were then dried at To evaluate the thermal stability of the free and immobi- room temperature. lized enzymes, 5 mg of the immobilized derivatives (60 mg lipase/g) were added to 500 μl phosphate buffer (25 mM, pH Immobilization of lipase 7.0) and incubated at different temperatures range between To perform immobilization, 20  mg of amine-functional- 45, 50, 55, 60, 65 and 70 °C for 2 h. The activity was deter - ized nanoparticles was added in vials containing water mined using the p-NPB assay. (pH 7.0) and dispersed under bath ultra-sonication for 20  min. Then, certain amount of enzyme (20–100  mg) Co‑solvent stability of the free and immobilized lipases was added to each sample. Immobilization of the The soluble lipases and 5 mg of the immobilized deriva - enzymes was then started by adding different amount of tives of RML, TLL and CALB (60  mg lipase/g) were A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 5 of 15 added to 500  μl of sodium phosphate solution (25  mM, connected to Zebron capillary column (30  m × 0.25  mm pH 7.0) including different concentrations (10, 20 and i.d.; Phenomenex, USA.) Nitrogen was used as the car- 50% v/v) of the organic solvents (methanol, ethanol, pro- rier gas at a constant flow of 0.8 ml/min. The sample was panol). Samples were taken after 24  h and their residual weighed and mixed with 1000 μl of 0.8  mg/ml methyl activity was evaluated by the p-NPB assay. laurate in n-hexane as an internal standard. Then, 0.5  μl of the diluted sample was injected into the GC. The col - Determination of the optimum pH activity umn temperature was held at 150  °C for 1  min, raised In order to determine the optimum pH activity of lipases, to 210  °C at 25  °C/min, and then increased to 240  °C at the soluble lipase and 5  mg of the immobilized deriva- 10 °C/min and kept for 8 min. The injector and detector tives (60 mg/g) were added in a total volume of 2 ml solu- temperatures were set at 150 and 300  °C, respectively. tion of 25 mM sodium phosphate buffer with various pH Equation (1) was used to calculate biodiesel yield: from 5.0 to 10.5 at 25  °C. The residual activity of each A − A C × V IS IS IS sample was analyzed by the p-NPB assay. C = × × 100%. (1) A m IS Leaching experiment In this equation, (∑A) is the total sub-peak area, ( A ) IS To assess the amount of desorbed enzymes from the is the internal standard sub-peak, (C ) the internal IS surface of the support, 5  mg of the immobilized deriva- standard concentration in (mg/ml), (V ) volume of the IS tives (60  mg/g) was incubated in a solution containing internal standard (ml), and (m) sample mass (mg). NaCl (1 M). The concentration of protein content in the supernatant was evaluated by using both the Bradford’s Experimental design method and enzyme activity assay. Optimization of the transesterification process of waste cooking oil was investigated using a 5-level, 5-factor Biodiesel synthesis Response Surface Central Composite Design (RSCCD) In a typical experiment, the reaction was carried out in of Design Expert Software version 7.0.0 (State Ease Inc. a 5-ml screw-capped vial including 130  mg of waste Minneapolis, USA). The study required 45 experiments, cooking oil and anhydrous methanol at various oil-to- consisting of 32 factorial points, 10 axial points, and 3 methanol molar ratios (1:3, 1:4, 1:5, 1:6, and 1:7). The repetitive tests in central points. In addition, there were mixtures were incubated with the immobilized lipases at five independent identified variables, contained reaction different temperatures (35, 40, 45, 50 and 55 °C). Metha nolysis reactions were performed with different amounts of t-butanol, water-adsorbent (for RML and CALB) and water (for TLL). To analyze the impact of the solvent on methanolysis, the amount of t-butanol was varied (5, 20, 35, 50 and 65 wt. %) by the substrate weight. Reac tions were performed for 72 h under constant agitation of 500  rpm. Aliquots of the reaction medium was taken at different times, mixed with methyl laurate (as an internal standard) and analyzed by GC. GC analysis The FAME yield in the reaction mixture was analyzed Fig. 1 XRD patterns of a silica core–shell magnetic nanoparticles using gas chromatography (Thermo-Quest-Finnigon) (Fe O @SiO ); b amine-functionalized magnetic nanoparticles 3 4 2 (MNPs-NH ); and c MNPs-TLL equipped with a flame-ionization detector (FID) 2 Scheme 1. Functionalization of MNPs Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 6 of 15 all the terms of interaction, was used to calculate the pre- dicted values: 5 5 Y = β + β X + β X + 0 i i ii i=1 i=1 i=1 (2) β X X + β X , ij i j iii i=1 j=i+1 where (Y) is the yield of biodiesel from waste cook- ing oil, β the offset term, (βi) represents linear effect, (βii) represents squared effect, (βij) represents interac - tion effect, (Xi) represents the i-independent variable, and (Xj) represents the j-independent variable. The data were analyzed using the Design Expert program and Fig. 2 FT-IR spectra of a Fe3O4@SiO2 nanoparticles; b amine-functionalized magnetic nanoparticles (MNPs-NH2); and c the coefficients were calculated by F-test. All statistical immobilized derivative of TLL on magnetic nanoparticles (MNPs-TLL) steps including analysis of variance (ANOVA), regression analysis and plotting of contour plot were used to estab- lish the optimum conditions to obtain high efficiency of temperature (35–55 °C), enzyme amounts (5–25 w/w %), methyl ester. as well as t-butanol concentration (5, 20, 35, 50, and 65 wt %), water for TLL (0–40%) and water absorbance for Results and discussion RML and CALB (20–60%). The stepwise methanol feed - Preparation, modification and characterization ing was applied (oil: methanol ratio 1:3, 1:4, 1:5, 1:6 and of functionalized MNPs 1:7). The level of each independent variable was consid - Recently, MNPs have attracted remarkable atten- ered based on previous researches (Ashjari et  al. 2020b; tion owing to their particular properties, such as Yang et  al. 2005). In addition, the independent variables their  large  surface  area and the presence of plenty of were encoded into low (−1) and high (+ 1) levels. In this hydroxyl groups on their surface which enables their paper, the value of α, the distance from the center, is fixed easy modification. The Fe O magnetic nanoparticles 3 4 at 2 which makes the design rotatble. were prepared by co-precipitation method via iron (II) and iron (III) ions. Surface modification of the activated Statistical analysis MNPs with a layer of silica was performed by using The experimental data obtained from central composite TEOS for improving stability of MNPs and provid- design (CCD) were analyzed by response surface meth- ing more available reaction sites for functionalization. odology. A quadratic equation (Eq.  2), which containing Finally, the reaction of Fe O @SiO with (3-aminopropyl) 3 4 2 Fig. 3 SEM images of a MNPs-NH , and b MNPs-TLL 2 A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 7 of 15 Table 1 Immobilization of the lipases on amine-functionalized magnetic nanoparticles Oer ff ed Yield (%) Immobilized enzyme Immobilization time (h) Specific activity U/mg (recovered enzyme (mg/g support) activity) (mg/g) RML CALB TLL RML CALB TLL RML CALB TLL RML CALB TLL 20 100 99.2 98.5 20.0 19.8 19.7 2 2 2 8.2 (142) 35.0 (157) 26.8 (101) 40 100 98.5 98.7 40.0 39.4 39.5 2 3 3 7.5 (125) 30.1 (112) 23.4 (95) 60 98.5 95.0 98.3 59.1 57.0 58.9 3 5 5 6.6 (90) 24.6 (72) 18.3 (99) 80 96.0 92.32 93.7 76.8 73.8 75.0 12 12 12 5.3 (93) 18.9 (74) 13.7 (100) 100 97.5 94.0 99.5 97.5 94.0 99.5 12 12 12 4.2 (77) 9.3 (56) 9.8 (70) The specific activities for the free forms of RML, CALB and TLL were 10.1 U/mg, 38.5 U/mg, and 27.5 U/mg, respectively trimethoxysilane led to amine-functionalized magnetic Fe O (MNPs-NH ; Scheme  1). Characterization of the 3 4 2 MNPs in various stages was performed via XRD, IR, and SEM. The X-ray diffraction patterns of Fe O @SiO nano- 3 4 2 particles (a), MNPs-NH (b) and one of the immobilized preparations (MNPs-TLL; c) were analyzed by XRD spec- troscopy (Fig. 1). XRD-diagram of the bare MNPs showed spinel ferrites pattern. All the strong peaks observed at 2θ = 30.2°, 36.4°, 43.7°, 53.5°, 56.3° and 62.3° are indexed to the highly crystalline cubic spinel structure of Fe O 3 4 nanoparticles (Yang et  al. 2005). The same sets of char - Fig. 4 Eec ff t of temperature on the activity of free and immobilized acteristic peaks were also observed for Fe O @SiO , 3 4 2 lipases. The incubation at different temperatures was carried out in MNPs-NH and MNPs-TLL indicating the stability of 25 mM sodium phosphate buffer, pH 7.0. The initial activity of each the crystalline phase of F e O nanoparticles during silica derivative before incubation was determined and set as 100% 3 4 coating, amine functionalization and enzyme immobili- zation (Ghasemzadeh et al. 2015). FT-IR spectroscopy was also used to confirm the suc - bunches of spherical shaped in different sizes with uni - cessful functionalization of the support with amine form morphology distribution. By considering SEM groups. FT-IR spectra of F e O @SiO , MNPs-NH 3 4 2 2 images it can be concluded that the modification still and the lipase immobilized preparation are presented preserving the textural properties of the MNPs. in Fig.  2a–c. In Fe O @SiO nanoparticles (Fig.  2a), 3 4 2 −1 the band at 1087.77  cm corresponds to Si–O–Si Immobilization of RML, TLL and CALB on MNPs‑NH symmetric stretching vibrations, is an indication The approach of using isocyanide-based multi-com- of the presence of SiO . Figure  2a shows the pres- ponent reaction provided a single-step, rapid and −1 ence of Fe–O stretching vibration at 609.46  cm and low-cost process for high-density covalent attach- also O–H stretching vibration owing to the physically ment of RML, TLL and CALB on amine-functional- absorbed water and surface hydroxyl groups approxi- ized MNPs. Immobilizations were carried out in water −1 mately at 3400  cm . Decrease in intensity of this band (pH 7.0) at 25 °C, in which high immobilization yields after functionalization together with appearing a dou- were obtained (92–100%). The leaching experiment −1 blet peak located at 3425.32 and 1635.51  cm clearly was conducted to remove those enzyme molecules confirmed successful functionalization of the sup - physically adsorbed on the support. Protein quanti- port by APTMS (Fig.  2b). The IR bands responsible fication in the washing solution was performed with for the lipase immobilized on the magnetic nanopar- both p-NPB assay and the Bradford method show- −1 ticles were observed at 1643.23  cm for amide I and ing no detectable enzyme in the solution. This means −1 at 1388.64  cm for amide II, respectively (Miao et  al. that in the used immobilization method, the reac- 2018; Fig. 2c). tion between enzyme molecules and MNPs is exclu- The morphology and structure of MNPs-NH and sively carried out by covalent binding. Immobilization immobilized TLL were also characterized by SEM. Fig- of lipases on MNPs-NH was investigated in terms ure  3a and b indicates that samples consists of various Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 8 of 15 of immobilization time, immobilization yield, load- observed for the final biocatalysts. Decrease in enzyme ing capacity, and specific activity (Table  1). Various specific activity during covalent immobilization has amounts of RML, TLL and CALB (20, 40, 60, 80, and already been reported in several studies and has been 100 mg/g) was offered to 1 g of MNPs-NH under gen- mostly attributed to the enzyme denaturation caused tle stirring for up to 12  h. Immobilization of 20  mg of during coupling process (Weltz et  al. 2020). Since RML, TLL and CALB on 1 g of the support was carried the specific activity of the immobilized derivatives out shortly after 2  h of incubation, producing 100%, obtained in immobilization with 20  mg of each lipase 98.5%, and 99.2% immobilization yields, respectively. presented a small change in activity compared to the While we have already reported covalent immobiliza- corresponding free enzyme, the remarkable decrease tion of CALB on epoxy-functionalized Fe O @SiO in activity in higher amounts of lipases can be attrib- 3 4 2 within 24  h of incubation with 84% immobilization uted to the diffusion limitation of substrate/product yield (Mehrasbi et  al. 2017). The used strategy was in the immobilized preparations (Mohammadi et  al. based on traditional coupling process and resulted 2014). The activity recovery for the immobilized prep- immobilization of 50  mg of CALB on 1  g of the sup- arations was calculated as the ratio of the activity of port max. With increasing the amount of enzyme to immobilized enzyme to the activity of same amount of 40  mg, almost the same immobilization yields were the corresponding soluble enzyme. The results showed obtained with slight change in immobilization time that with increasing the amount of lipases loaded on of TLL and CALB (3  h). With further increasing the the support the recovered activity decreased particu- amount of suggested enzymes to 100 mg, the immobi- larly for CALB with almost 65% lower recovered activ- lization yields of 97.5%, 99.5%, and 94% were detected ity in 100  mg/g of offered enzyme compared to result for RML, TLL and CALB, respectively, after 12  h of obtained in the process for immobilization of 20 mg/g incubation. We have recently reported immobilization of the enzyme. of RML and TLL on aldehyde-functionalized magnetic nanoparticles via multi-component reactions. Rapid Thermal stability of free and immobilized lipases immobilization processes within 12  h were observed It is well documented that immobilization of enzymes producing 81% and 97% of immobilization yields for can improve their thermal stability. Figure  4 represents RML and TLL, respectively, by offering 100 mg of each the time course of residual activities of the free lipases enzyme per 1 g of the support. The maximum loading and their immobilized derivatives at different tem - capacity of the aldehyde-functionalized support was peratures up to 70  °C. At the lowest temperature tested determined to be 81  mg for RML and 97  mg for TLL (45 °C), the immobilized lipases on amine-functionalized which are lower than the obtained loading amount of MNPs remained completely active. While the free RML TLL and RML in this study at the same conditions. was relatively unstable and lost 40% of its initial activity Table  1 also shows specific activity of the free and at 50  °C, the soluble TLL and CALB showed more sta- immobilized enzymes. With increasing the amount of bility with retaining 80% of their activities. The immo - immobilized enzymes, lower specific activities were bilized forms of all enzymes remained 90% active at the same condition. At 55 °C, the immobilized RML showed 70% residual activity after 2 h of incubation, whereas, the soluble enzyme at the same temperature exhibited rapid inactivation. Increasing temperature to 60  °C showed Fig. 5 The stability of free and MNPs-RML, MNPs-TLL and MNPs-CALB in the presence of 50% of methanol, ethanol and propanol. Incubation of each biocatalyst in 1 ml solution containing 25 mM sodium phosphate buffer (pH 7.0) and 50% of each solvent at 25 °C. The initial activity of each derivative was determined before incubation and set as 100% Fig. 6 Eec ff t of pH on activity of immobilized lipases A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 9 of 15 Table 2 Sequential model sum of squares for immobilized TLL Source Sum of squares df Mean square F value Prob > F Mean vs total 64,883.04 1 64,883.04 Linear vs mean 1904.72 5 380.94 4.37 0.003 2FI vs linear 2441.60 10 244.16 7.42 < 0.0001 Suggested Quadratic vs 2FI 185.30 5 37.06 1.16 0.3591 Cubic vs quadratic 660.28 15 44.02 3.63 0.0281 Aliased Residual 109.06 9 12.12 Total 70,184.00 45 1559.64 20%, 40% and 70% residual activity of free RML, CALB stability of the covalently immobilized preparations and TLL, respectively. Remaining 60% activity of MNPs- compared to the free lipases in the presence of 50% of RML, MNPs-CALB and 80% activity of MNPs-TLL after three used co-solvents. The results showed that immo - 2  h incubation at 60  °C, confirmed the positive effect of bilized derivative of lipases retained their activity (over immobilization on the stability of the lipases. With rais- 70%) in the presence of 50% of methanol while the free ing the temperature to 70  °C, the free RML completely lipases lost their activities in the same condition. In the lost its activity, while its immobilized form retained 45% presence of 10% and 20% ethanol, there were no signifi - of its initial activity. Bearing in mind the important role cant differences between covalently immobilized lipases of aspartic acid residues on the surface of proteins, the and free enzymes. With increasing the percentage of higher stability of TLL derivative can be attributed to the ethanol to 50%, while immobilized RML and CALB kept higher number of exposed Asp residues on the surface of 43% and 72% of their activities, respectively, free CALB TLL (13) compared to RML (10) and CALB (9; Orrego remained only 25% active and free RML was completely et al. 2018). deactivated. Co‑solvent stability of the immobilized and free lipases Optimal pH activity of the immobilized preparations In the most enzymatic reactions, the use of co-solvent The activity of immobilized lipases at pH range between is essential to enhance the solubility of the organic sub- 5.5 and 10.5 was also investigated. As illustrated in Fig. 6, strates which are not soluble in water. To evaluate the sta- the maximum activity was observed at pH 8.0 and 8.5 for bility of free and immobilized preparations of RML, TLL, immobilized lipases. It was concluded that both lower and CALB, 5 mg of the derivatives (60 mg/g) were incu- and higher pH are unfavorable for the enzymatic activ- bated for 24  h in the presence of 10%, 20%, and 50% of ity of the lipases. This could be explained by the changes organic solvents including methanol, ethanol and 1-pro- in the enzyme structure and ionic states of the active site panol (Fig. 5 and Additional file  1: Fig. S1a–c). The results residues. They also showed increased optimal pH activity could be interpreted by the effect of the carbon chain of compared to the corresponding free enzymes which have each solvent on the activity of the enzymes. The results been reported to be 7.5 (Online et al. 2016; Bi et al. 2020; confirmed that using an organic solvent may influence Mehrasbi et al. 2017). Furthermore, the immobilized TLL the structure of the enzyme leads either to change the presented broader optimal pH adaptability ranging from catalytic performance of the enzyme in desired enzy- 8 to 8.5. matic reaction or completely inactivation of the enzyme. This can be raised from the replacement of the crucial Biodiesel production water molecules of enzyme structure (Nawani et al. 2006; Optimization of the reaction conditions Klibanov 1989) which are required for enzyme func- In the present work, five factors such as (A) reaction tion. Organic solvents with log P < 2 have a propensity to temperature, (B) amount of water-adsorbent (RML strip this vital water and diminish the catalytic activity and CALB) or water (TLL), (C) the amount of enzyme, of enzyme (Klibanov 2001). Furthermore, enzymes usu- (D) t-butanol weight percentage and (E) oil: methanol ally tend to form aggregates in organic solvents which molar ratios were considered as effective parameters makes them poorly available for the substrate. Immo- in biodiesel production. The yields of waste cooking oil bilization can remove this detrimental effect of organic methyl ester were from 30–61% for immobilized RML, solvents by individually fixing every single enzyme 13–62% for immobilized TLL and 27–52% for immobi- molecule separately on the support. Figure  5 shows the lized CALB. Process optimization with response surface Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 10 of 15 methodology (RSM) was performed and the effect of between the variables. Values of probability (p) > F less each factor and its interactions were calculated to ascer- than 0.05 emphasized that the model terms were signif- tain the optimum conditions for the reaction. Among icant whereas the values greater than 0.05 indicate that the models that fitted to the response such as linear, two the model terms were not significant (Chen et  al. 2008). factors interaction (2FI), quadratic, and cubic polyno- The terms incorporated in the model F-values of 5.79 mial, the two factors interaction (2FI) was selected as the for the immobilized RML, 8.80 for the immobilized TLL best model. This two-factor interaction (2FI) model was and 8.90 for the immobilized CALB with p-value < 0.0001 suggested by the RSM software as shown in Table  2 for revealed that the model was significant at 95% confidence TLL (Additional file  1: Table S1 for RML and Additional level. The R value of 0.74 for RML, 0.81 for TLL and 0.78 file  1: Table  S2 for CALB). This two-factor interaction for CALB as well as adjusted R values of 0.62 for RML, (2FI) model expressed by Eq.  (3) shows biodiesel yield 0.72 for TLL and 0.70 for CALB showed that the model (Y), reaction temperature (A), water absorbent (RML was significant to predict the response. The predicted R and CALB) or water (TLL) w/w (B), the enzyme weight values of 0.30 for RML, 0.53 for TLL and 0.62 for CALB percentage ratio (C), the t-butanol weight percentage (D) were in reasonable agreement with the adjusted R val- and oil/methanol molar ratios (E). A positive sign in front ues. The model also depicted the statistically non-signif - of the terms indicates the synergistic effect of the rise in icant lack of fit (p 0.39 for RML, p 0.46 for TLL and p FAME yield, while negative sign indicates the antagonis- 0.95 for CALB) which were not significant (p-value > 0.05 tic effect (Babaki et  al. 2017). The results at each point is not significant), indicating that the responses were based on the central composite design (CCD) and their adequate for utilizing in the model (Additional file  1: corresponding predicted values are presented in Addi- Fig. S2a for RML, Additional file  1: Fig. S2(b) for TLL tional file  1: Table  S3 for immobilized RML, Additional and Additional file  1: Fig. S2(c) for CALB). These results file  1: Table S4 for immobilized TLL and Additional file  1: also demonstrated that the model satisfactorily fitted to Table S5 for immobilized CALB. experimental data. Insignificant lack of fit was considered as the significant lack of fit indicating that there might be Y = +40.11 − 1.83 A + 0.24 B RML contribution in the regressor–response relationship that + 0.37 C + 2.42 D + 0.92 E is not accounted for by the model (Noordin et  al. 2004). − 3.22 AB − 1.88 AC + 1.08 AD This analysis was examined using the normal probabil - + 1.58 AE + 2.54 BC − 0.69 BD ity of the residuals (Additional file  1: Fig. S3a–c for TLL, RML and CALB, respectively). The normal probability − 2.59 BE − 2.23 CD plot of the residuals for RML, TLL and CALB showed − 0.042 CE + 0.49 DE that the errors were distributed normally in a straight line Y = +37.97 + 2.97A −2.42B TLL and insignificant. On the other hand, the plot of residuals + 3.95C + 1.13D + 4.14E versus predicted response indicated a structureless plot + 0.51AB − 0.34AC + 2.57 AD (3) suggesting that the model was adequate and that they did + 0.84 AE + 1.80 BC + 2.09BD not show any violation of the independence or constant variance assumption (Lee et al. 2011). − 3.42BE − 6.86CD − 1.40CE − 0.56DE Effect of the reaction parameters on FAME yield Y = +40.30 − 1.71A + 1.20B CALB The effect of 5 parameters on the FAME yield was inves - − 1.00C − 0.75D − 3.63AB tigated for the immobilized lipases. MNPs-TLL was the − 1.08AD + 2.97AE − 0.86BC only derivative that was affected by the reaction tempera - − 1.76BD + 2.04BE − 0.91CD ture and produced higher FAME yield with increasing the temperature. It has been already reported that, improved − 1.94CE − 1.83DE. thermal stability after immobilization and decreasing The results of statistical analysis of variance (ANOVA) mass transfer limitation between the reactants can be which were applied to determine the significance and fit - considered as important parameters to facilitate FAME ness of the two factors interaction (2FI) model and also production (Guo et al. 2020). The negative effect in fatty the effect of significant individual terms and their inter - acid methyl esters yields was observed by increasing the action on the selected responses for RML are presented water for MNPs-TLL, while for MNPs-CALB showed in Additional file  1: Tables S6, S7 for TLL and Additional improved efficiency of biodiesel production. Zaks and file  1: Table  S8 for CALB. The p values were used as a coworkers reported that, the presence of excess water tool to check the importance of each of the coefficients, during biodiesel production might lead to aggregation which in turn may indicate the pattern of the interaction of the enzyme and thus reducing its catalytic activity A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 11 of 15 Fig. 7 Response surface curves showing the interactions for MNPs-TLL. a Water content vs. temperature; b biocatalyst quantity vs. temperature; c t-butanol vs. temperature; d t-butanol vs. water content; e methanol-to-oil ratio vs. water content (Zaks and Klibanov 1988). The results also showed that on biodiesel content for MNPs-TLL while the efficiency increasing the amount of MNPs-RML had no effect on of FAME production was improved for MNPs-RML by biodiesel production while it was effectively improved providing higher contents of t-butanol. Change in the as the amount of MNPs-TLL increased (Tavares et  al. methanol:oil molar ratio showed different results for the 2017). The t-butanol percentage had negligible effect immobilized derivatives. Increasing the methanol-to-oil Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 12 of 15 The interaction between water and methanol-to-oil ratio for MNPs-TLL showed that in a low amount of water (10%), increasing methanol concentration (methanol-to- oil ratio from 4:1 up to 6:1) resulted in higher FAME pro- duction (Fig.  7e), whereas at a high level of water (30%) the presence of higher methanol-to-oil ratio 6:1 caused to decrease in FAME yield. This is most likely because of the fact that with increasing methanol, the produc- tion of methyl ester first improved and then declined as a consequence of decrease in enzyme activity caused by excessive methanol (Yadav and Pawar 2012; Li et  al. 2012). The effect of t-butanol and methanol-to-oil ratio Fig. 8 The effect of repeated use of the immobilized preparations on for MNPs-TLL and MNPs-RML showed that the interac- their activities in biodiesel production at 50 °C tion of both low (20%) and high (50%) level of t-butanol with methanol-to-oil ratio could diminish the destructive effect of methanol on biocatalyst deactivation (Babaki ratio from 1:4 to 1:6 didn’t change the reaction yield for et  al. 2016). The negative effect of methanol on biodiesel MNPs-RML while improved yield of FAME production production has been already reported particularly in was observed for MNPs-TLL. high concentrations (Mohammadi et  al. 2015). Different The interaction of the selected variables and their effect reports can be found on the use of lipases from Ther - on fatty acid methyl ester production were also evalu- momyces lanuginosus lipase, Candida antarctica lipase ated for the immobilized TLL in Fig. 7 (Additional file  1: B and Rhizomucor miehei lipase in transesterification Fig. S4 for RML and Additional file  1: Fig. S5 for CALB). of oils. The different results of each report is due to the Although with increasing temperature the FAME yield fact that the immobilization protocol can effectively alter slightly increased For MNPs-TLL, the response surface the catalytic performance and stability of each individual showed no improvement in FAME content in both levels enzyme (Yousefi et al. 2020). As an example, Ashjari et al. of water (Fig.  7a). The interaction of reaction tempera - have reported transesterification of waste cooking oil by ture and amount of biocatalyst for MNPs-TLL showed using the immobilized RML on aldehyde-functionalized that with the simultaneous increase of temperature and support that producing FAME yield of 57.5% (Ashjari amount of biocatalyst, the reaction efficiency improved et  al. 2020b). The other research has showed encapsula - (Fig.  7b)while for MNPs-RML the result was the oppo- tion of RML in X-shaped zeolitic frameworks and use site. The interaction effect between the temperature and this biocatalyst to produce biodiesel with a conversion t-butanol on biodiesel yield for MNPs-TLL showed that yield of 95.6% (Adnan et  al. 2018). In addition, Cazaban the FAME yield did not change with increasing tem- et al. investigated biodiesel production from canola oil by perature and keeping the amount of t-butanol constant using TLL immobilized on silica nanoparticles producing (Fig. 7c), while with increasing temperature and t-butanol the maximum FAME yield of 88% (Cazaban et al. 2018). at the same time, the efficiency of biodiesel production increased significantly. The interaction between water Reusability of the immobilized derivatives content and t-butanol for MNPs-TLL showed that by The main advantage of immobilization is the reus - increasing the amount of t-butanol and keeping the ability of the immobilized enzyme which decreases the water constant (Fig.  7d), the yield was improved, while final cost of the process. The immobilized derivatives with increasing the amount of water and keeping the of RML, TLL and CALB were investigated in terms of amount of t-butanol constant, the efficiency decreased their operational stability in biodiesel production for 5 significantly. For MNPs-TLL and MNPs-RML by increas - cycles of successive transesterification processes. After ing the amount of TLL and RML in a constant amount each run, the immobilized lipase was recovered, washed of t-butanol, FAME production was increased but for with n-hexane and dried to use in the next batch. The MNPs-CALB, with increasing the amount of biocatalyst results showed that the derivatives have good capability there was no change in FAME yield. Furthermore, the to be repeatedly used up to five cycles with 43%, 68% simultaneous increase in the amount of biocatalyst and and 48% of activity for MNPs-RML, MNPs-TLL, and t-butanol reduced the FAME yield for all three enzymes. MNPs-CALB, respectively (Fig. 8). A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 13 of 15 Conclusion Fig. S4. Response surface curves showing the interactions for RML. Fig. This paper has investigated a facile method for the immo - S5. Response surface curves showing the interactions for CALB. bilization of lipases on amine-functionalized supports. This methodology is based on a three-component reac - Acknowledgements tion including an amine from support, a carboxylic acid This research was financially supported by Shahid Beheshti University of Tehran and the National Institute of Genetic Engineering and Biotechnology from enzyme surface and cyclohexyl isocyanide. RML, (NIGEB) which the authors are thankful. TLL and CALB were successfully immobilized on amine- functionalized magnetic nanoparticles at extremely Author contributions YA contributed to experiments, data analysis, data validation and writing the mild conditions in a relatively short time. The leaching original draft. MS contributed to experimental support, data validation and experiment confirmed that the enzymes were covalently manuscript editing. ZH supervised the project and funding acquisition. MY attached on the support. The immobilized lipases were provided technical support, manuscript editing. MA contributed to study design, data validation and methodology. MM contributed to study design, more active and stable in wide ranges of pH, organic methodology, editing the manuscript. All authors read and approved the final solvents and different temperatures compared to their manuscript. corresponding free forms. MNPs-TLL shows higher sta- Funding bility in comparison to MNPs-RML and MNPs-CALB, Not applicable. retaining 100% of its activity after 24  h of incubation in the presence of organic solvents. MNPs-TLL also showed Availability of data and materials All data generated or analyzed during this study are included in this published broad range of optimum pH activity compared to its article and its additional information files. soluble form and the other immobilized derivatives. The immobilized lipases were then employed in biodiesel Declarations production by transesterification of waste cooking oil with methanol. In an optimization study, the effect of Ethics approval and consent to participate Not applicable. enzyme weight percentage ratio, t-butanol, tempera- ture, methanol: oil ratio, water (for TLL) and the water- Consent for publication adsorbent weight percentage (for CALB and RML) on All authors have read this article and have approved its submission to Biore- sources and Bioprocessing. the FAME yield were evaluated. The maximal conversion to methyl esters of 78% was attained by using MNPs- Competing interests CALB as catalyst and FAME production yield by immo- There is no competing financial interest. bilized TLL reached 81% under optimal conditions while Author details the maximum yield for RML was 74%. Reusability study 1 Department of Organic Chemistry and Oil, Faculty of Chemistry, Shahid showed that the immobilized lipases on MNPs-NH Beheshti University, Tehran, Iran. Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran. Bioprocess Engineering can be easily recovered from the reaction mixture with Department, Institute of Industrial and Environmental Biotechnology, National retaining 43–68% of the initial activities after 5 cycles of Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. the reaction. Received: 3 March 2022 Accepted: 20 May 2022 Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40643- 022- 00552-0. References Adnan M, Li K, Xu L, Yan Y (2018) X-shaped zif-8 for immobilization rhizomucor Additional file 1: Table S1. Sequential model sum of squares (RML). miehei lipase via encapsulation and its application toward biodiesel Table S2. Sequential model sum of squares (CALB). Table S3. Experi- production. Catalysts. https:// doi. org/ 10. 3390/ catal 80300 96 mental design for five-level five-factor surface response design on Ahrari F, Yousefi M, Habibi Z, Mohammadi M (2022) Application of undecan- transesterification of waste cooking oil using immobilized RML. Table S4. edicarboxylic acid to prepare cross-linked enzymes (CLEs) of Rhizomucor Experimental design for five-level five-factor surface response design on miehei lipase (RML); Selective enrichment of polyunsaturated fatty acids. transesterification of waste cooking oil using immobilized TLL. Table S5. Mol Catal 520:112172. https:// doi. org/ 10. 1016/J. MCAT. 2022. 112172 Experimental design for five-level five-factor surface response design on Ali CH, Qureshi AS, Mbadinga SM et al (2017) Biodiesel production from waste transesterification and esterification of waste cooking oil using immobi- cooking oil using onsite produced purified lipase from Pseudomonas lized CALB. Table S6. Analysis of variance (ANOVA) for fitted quadratic pol- aeruginosa FW_SH-1: Central composite design approach. Renew Energy ynominal model for optimization of transesterification parameters (RML). 109:93–100. https:// doi. org/ 10. 1016/j. renene. 2017. 03. 018 Table S7. Analysis of variance (ANOVA) for fitted quadratic polynominal Ansari SA, Husain Q (2012) Potential applications of enzymes immobilized on/ model for optimization of transesterification parameters ( TLL). Table S8. in nano materials: A review. Biotechnol Adv 30:512–523. https:// doi. org/ Analysis of variance (ANOVA) for fitted quadratic polynominal model for 10. 1016/j. biote chadv. 2011. 09. 005 optimization of transesterification parameters (CALB). Fig. S1. Stability of Ashjari M, Garmroodi M, Ahrari F et al (2020a) Soluble enzyme cross-linking: immobilized derivatives MNPs-RML (a) and MNPs-TLL (b) in the presence Via multi-component reactions: A new generation of cross-linked of 10, 20 and 50% of various co-solvents. Fig. S2. Predicted fatty acid enzymes. Chem Commun 56:9683–9686. https:// doi. org/ 10. 1039/ d0cc0 methyl ester yield versus experimental fatty acid methyl ester yield for 3429k MNPs-RML (a), MNPs-TLL (b) and MNPs-CALB(c). Fig. S3. Normal probabil- ity plots of residuals for (a) MNPs-TLL (b), MNPs-RML and MNPs-CALB(c). Amini et al. Bioresources and Bioprocessing (2022) 9:60 Page 14 of 15 Ashjari M, Garmroodi M, Amiri Asl F et al (2020b) Application of multi- Guo J, Sun S, Liu J (2020) Conversion of waste frying palm oil into biodiesel component reaction for covalent immobilization of two lipases on using free lipase A from Candida antarctica as a novel catalyst. Fuel aldehyde-functionalized magnetic nanoparticles; production of biodiesel 267:117323. https:// doi. org/ 10. 1016/j. fuel. 2020. 117323 from waste cooking oil. Process Biochem 90:156–167. https:// doi. org/ 10. Habibi Z, Mohammadi M, Yousefi M (2013) Enzymatic hydrolysis of racemic 1016/j. procb io. 2019. 11. 002 ibuprofen esters using Rhizomucor miehei lipase immobilized on dif- Babaki M, Yousefi M, Habibi Z et al (2015) Preparation of highly reusable ferent supports. Process Biochem 48:669–676. https:// doi. org/ 10. 1016/j. biocatalysts by immobilization of lipases on epoxy-functionalized silica procb io. 2013. 02. 020 for production of biodiesel from canola oil. Biochem Eng J 101:23–31. Hama S, Noda H, Kondo A (2018) How lipase technology contributes to https:// doi. org/ 10. 1016/j. bej. 2015. 04. 020 evolution of biodiesel production using multiple feedstocks. Curr Opin Babaki M, Yousefi M, Habibi Z et al (2016) Enzymatic production of biodiesel Biotechnol 50:57–64. https:// doi. org/ 10. 1016/j. copbio. 2017. 11. 001 using lipases immobilized on silica nanoparticles as highly reusable Handayani N, Wahyuningrum D, Zulfikar MA et al (2016) The synthesis of biocatalysts: Eec ff t of water, t-butanol and blue silica gel contents. Renew biodiesel catalyzed by Mucor miehei lipase immobilized onto aminated Energy 91:196–206. https:// doi. org/ 10. 1016/j. renene. 2016. 01. 053 polyethersulfone membranes. Bioresour Bioproces 3:22. https:// doi. org/ Babaki M, Yousefi M, Habibi Z, Mohammadi M (2017) Process optimization 10. 1186/ s40643- 016- 0098-4 for biodiesel production from waste cooking oil using multi-enzyme He J, Huang M, Wang D et al (2014) Magnetic separation techniques in systems through response surface methodology. Renew Energy 105:465– sample preparation for biological analysis: a review. J Pharm Biomed Anal 472. https:// doi. org/ 10. 1016/j. renene. 2016. 12. 086 101:84–101. https:// doi. org/ 10. 1016/j. jpba. 2014. 04. 017 Barbosa O, Ortiz C, Berenguer-Murcia Á et al (2015) Strategies for the one-step Hu B, Pan J, Yu HL et al (2009) Immobilization of Serratia marcescens lipase immobilization-purification of enzymes as industrial biocatalysts. Biotech- onto amino-functionalized magnetic nanoparticles for repeated use nol Adv 33:435–456. https:// doi. org/ 10. 1016/j. biote chadv. 2015. 03. 006 in enzymatic synthesis of Diltiazem intermediate. Process Biochem Bi Y, Wang Z, Zhang R et al (2020) Improved catalytic properties of Thermo- 44:1019–1024. https:// doi. org/ 10. 1016/j. procb io. 2009. 05. 001 myces lanuginosus lipase immobilized onto newly fabricated polydopa- Jang MG, Kim DK, Park SC et al (2012) Biodiesel production from crude canola mine-functionalized magnetic Fe3O4 nanoparticles. Processes. https:// oil by two-step enzymatic processes. Renew Energy 42:99–104. https:// doi. org/ 10. 3390/ PR805 0629doi. org/ 10. 1016/j. renene. 2011. 09. 009 Budhwani AAA, Maqbool A, Hussain T, Noman Syed M (2019) Production of Jiang K, Schadler LS, Siegel RW et al (2004) Protein immobilization on carbon biodiesel by enzymatic transesterifcation of non-edible Salvadora persica nanotubes via a two-step process of diimide-activated amidation. J Mater (Pilu) oil and crude coconut oil in a solvent-free system. Bioresources and Chem 14:37–39. https:// doi. org/ 10. 1039/ b3103 59e Bioprocessing 6:41. https:// doi. org/ 10. 1186/ s40643- 019- 0275-3 Jiang XY, Bai S, Sun Y (2007) Fabrication and characterization of rigid magnetic Cao SL, Xu H, Lai LH, Gu WM et al (2017) Magnetic ZIF-8/cellulose/Fe3O4 monodisperse microspheres for protein adsorption. J Chromatogr B Anal nanocomposite: preparation, characterization, and enzyme immobili- Technol Biomed Life Sci 852:62–68. https:// doi. org/ 10. 1016/j. jchro mb. zation. Bioresources and Bioprocessing 4:56. https:// doi. org/ 10. 1186/ 2006. 12. 044 s40643- 017- 0186-0 Klibanov AM (1989) Enzymatic catalysis in monophasic organic solvents. Cazaban D, Illanes A, Wilson L, Betancor L (2018) Bio-inspired silica lipase nano- Enzyme Microb Technol 11:194–211 biocatalysts for the synthesis of fatty acid methyl esters. Process Biochem Klibanov AM (2001) Improving enzymes by using them in organic solvents. 74:86–93. https:// doi. org/ 10. 1016/j. procb io. 2018. 07. 004 Nature 409:241–246. https:// doi. org/ 10. 1038/ 35051 719 Chen X, Du W, Liu D (2008) Response surface optimization of biocatalytic Lee HV, Yunus R, Juan JC, Taufiq-Yap YH (2011) Process optimization design for biodiesel production with acid oil. Biochem Eng J 40:423–429. https:// doi. jatropha-based biodiesel production using response surface methodol- org/ 10. 1016/j. bej. 2008. 01. 012 ogy. Fuel Process Technol 92:2420–2428. https:// doi. org/ 10. 1016/j. fuproc. Cipolatti EP, Silva MJA, Klein M et al (2014) Current status and trends in enzy-2011. 08. 018 matic nanoimmobilization. J Mol Catal B Enzym 99:56–67. https:// doi. Li X, He XY, Li ZL et al (2012) Enzymatic production of biodiesel from Pistacia org/ 10. 1016/j. molca tb. 2013. 10. 019 chinensis bge seed oil using immobilized lipase. Fuel 92:89–93. https:// Emami Bistgani Z, Siadat SA, Bakhshandeh A et al (2017) Interactive effects of doi. org/ 10. 1016/j. fuel. 2011. 06. 048 drought stress and chitosan application on physiological characteristics Luna C, Verdugo C, Sancho DE et al (2014) Production of a biodiesel-like bio- and essential oil yield of Thymus daenensis Celak. Crop J 5:407–415. fuel without glycerol generation, by using Novozym 435, an immobilized https:// doi. org/ 10. 1016/j. cj. 2017. 04. 003 Candida antarctica lipase. Bioresour Bioprocessing 1:11. https:// doi. org/ Franssen MCR, Steunenberg P, Scott EL et al (2013) Immobilised enzymes in 10. 1186/ s40643- 014- 0011-y biorenewables production. Chem Soc Rev 42:6491–6533. https:// doi. org/ Marchetti JM, Miguel VU, Errazu AF (2007) Possible methods for biodiesel 10. 1039/ c3cs0 0004d production. Renew Sustain Energy Rev 11:1300–1311. https:// doi. org/ 10. Gao Y, Kyratzis I (2008) Covalent Immobilization of Proteins on Carbon Nano-1016/j. rser. 2005. 08. 006 tubes Using the. Bioconjug Chem 19:1945–1950 Mehde AA, Mehdi WA, Severgün O et al (2018) Lipase-based on starch mate- Garmroodi M, Mohammadi M, Ramazani A et al (2016) Covalent binding of rial as a development matrix with magnetite cross-linked enzyme aggre- hyper-activated Rhizomucor miehei lipase (RML) on hetero-functional- gates and its application. Int J Biol Macromol 120:1533–1543. https:// doi. ized siliceous supports. Int J Biol Macromol 86:208–215. https:// doi. org/ org/ 10. 1016/j. ijbio mac. 2018. 09. 141 10. 1016/j. ijbio mac. 2016. 01. 076 Mehrasbi MR, Mohammadi J, Peyda M, Mohammadi M (2017) Covalent immo- Ghadge SV, Raheman H (2006) Process optimization for biodiesel production bilization of Candida antarctica lipase on core-shell magnetic nanopar- from mahua (Madhuca indica) oil using response surface methodology. ticles for production of biodiesel from waste cooking oil. Renew Energy Bioresour Technol 97:379–384. https:// doi. org/ 10. 1016/j. biort ech. 2005. 101:593–602. https:// doi. org/ 10. 1016/j. renene. 2016. 09. 022 03. 014 Miao C, Yang L, Wang Z et al (2018) Lipase immobilization on amino-silane Ghasemzadeh MA, Abdollahi-Basir MH, Babaei M (2015) Fe3O4@SiO2–NH2 modified superparamagnetic Fe3O4 nanoparticles as biocatalyst for core-shell nanocomposite as an efficient and green catalyst for the multi- biodiesel production. Fuel 224:774–782. https:// doi. org/ 10. 1016/j. fuel. component synthesis of highly substituted chromeno[2,3-b]pyridines in 2018. 02. 149 aqueous ethanol media. Green Chem Lett Rev 8:40–49. https:// doi. org/ Mohammadi M, Habibi Z, Dezvarei S et al (2014) Improvement of the stability 10. 1080/ 17518 253. 2015. 11071 39 and selectivity of Rhizomucor miehei lipase immobilized on silica nano- Ghosh S, Chaganti SR, Prakasham RS (2012) Polyaniline nanofiber as a novel particles: selective hydrolysis of fish oil using immobilized preparations. immobilization matrix for the anti-leukemia enzyme l-asparaginase. J Mol Process Biochem 49:1314–1323. https:// doi. org/ 10. 1016/j. procb io. 2014. Catal B Enzym 74:132–137. https:// doi. org/ 10. 1016/j. molca tb. 2011. 09. 00904. 012 Gross RA, Ganesh M, Lu W (2010) Enzyme-catalysis breathes new life into Mohammadi M, Ashjari M, Dezvarei S et al (2015) Rapid and high-density polyester condensation polymerizations. Trends Biotechnol 28:435–443. covalent immobilization of Rhizomucor miehei lipase using a multi https:// doi. org/ 10. 1016/j. tibte ch. 2010. 05. 004 component reaction: application in biodiesel production. RSC Adv 5:32698–32705. https:// doi. org/ 10. 1039/ c5ra0 3299g A mini et al. Bioresources and Bioprocessing (2022) 9:60 Page 15 of 15 Mohammadi M, Gandomkar S, Habibi Z, Yousefi M (2016) One pot three- immobilization strategies for the selective hydrolysis of fish oil. Molecules component reaction for covalent immobilization of enzymes: Application 25:1–14. https:// doi. org/ 10. 3390/ molec ules2 50305 45 of immobilized lipases for kinetic resolution of: Rac -ibuprofen. RSC Adv Yücel Y (2012) Optimization of biocatalytic biodiesel production from pomace 6:52838–52849. https:// doi. org/ 10. 1039/ c6ra1 1284f oil using response surface methodology. Fuel Process Technol 99:97–102. Mohammadi M, Habibi Z, Gandomkar S, Yousefi M (2018) A novel approach https:// doi. org/ 10. 1016/j. fuproc. 2012. 02. 008 for bioconjugation of Rhizomucor miehei lipase (RML) onto amine- Yushkova ED, Nazarova EA, Matyuhina AV et al (2019) Application of immo- functionalized supports; Application for enantioselective resolution of bilized enzymes in food industry. J Agric Food Chem 67:11553–11567. rac-ibuprofen. Int J Biol Macromol 117:523–531. https:// doi. org/ 10. 1016/j. https:// doi. org/ 10. 1021/ acs. jafc. 9b043 85 ijbio mac. 2018. 05. 218 Zaks A, Klibanov AM (1988) The effect of water on enzyme action in organic Nawani N, Singh R, Kaur J (2006) Immobilization and stability studies of a media. J Biol Chem 263:8017–8021. https:// doi. org/ 10. 1016/ s0021- lipase from thermophilic Bacillus sp: The effect of process parameters on 9258(18) 68435-2 immobilization of enzyme. Electron J Biotechnol 9:559–565. https:// doi. Zucca P, Sanjust E (2014) Inorganic materials as supports for covalent enzyme org/ 10. 2225/ vol9- issue5- fullt ext-9 immobilization: Methods and mechanisms. Molecules 19:14139–14194. Noordin MY, Venkatesh VC, Sharif S et al (2004) Application of response surface https:// doi. org/ 10. 3390/ molec ules1 90914 139 methodology in describing the performance of coated carbide tools when turning AISI 1045 steel. J Mater Process Technol 145:46–58. https:// Publisher’s Note doi. org/ 10. 1016/ S0924- 0136(03) 00861-6 Springer Nature remains neutral with regard to jurisdictional claims in pub- Online VA, Ashjari M, Garmroodi M, Karkhane AA (2016). RSC Adv. https:// doi. lished maps and institutional affiliations. org/ 10. 1039/ C6RA1 4142K Orrego AH, Ghobadi R, Moreno-Perez S et al (2018) Stabilization of immobi- lized lipases by intense intramolecular cross-linking of their surfaces by using aldehyde-dextran polymers. Int J Mol Sci 19:1–16. https:// doi. org/ 10. 3390/ ijms1 90205 53 Shimada Y, Watanabe Y, Sugihara A, Tominaga Y (2002) Enzymatic alcoholy- sis for biodiesel fuel production and application of the reaction to oil processing. J Mol Catal B Enzym 17:133–142. https:// doi. org/ 10. 1016/ S1381- 1177(02) 00020-6 Shinkai M (2002) Functional magnetic particles for medical application. J Biosci Bioeng 94:606–613. https:// doi. org/ 10. 1016/ S1389- 1723(02) 80202-X Sigurdardóttir SB, Lehmann J, Ovtar S et al (2018) Enzyme Immobilization on Inorganic Surfaces for Membrane Reactor Applications: Mass Transfer Challenges, Enzyme Leakage and Reuse of Materials. Adv Synth Catal 360:2578–2607. https:// doi. org/ 10. 1002/ adsc. 20180 0307 Tacias-Pascacio VG, Torrestiana-Sánchez B, Dal Magro L et al (2019) Compari- son of acid, basic and enzymatic catalysis on the production of biodiesel after RSM optimization. Renew Energy 135:1–9. https:// doi. org/ 10. 1016/j. renene. 2018. 11. 107 Takeuchi Y (2017) Sagittal plane spinal mobility is associated with dynamic balance ability of community-dwelling elderly people. J Phys Ther Sci 29:112–114. https:// doi. org/ 10. 1589/ jpts. 29. 112 Tang T, Fan H, Ai S et al (2011) Hemoglobin (Hb) immobilized on amino- modified magnetic nanoparticles for the catalytic removal of bisphenol A. Chemosphere 83:255–264. https:// doi. org/ 10. 1016/j. chemo sphere. 2010. 12. 075 Tavares GR, Gonçalves JE, dos Santos WD, da Silva C (2017) Enzymatic interesterification of crambe oil assisted by ultrasound. Ind Crops Prod 97:218–223. https:// doi. org/ 10. 1016/j. indcr op. 2016. 12. 022 Vashist SK, Lam E, Hrapovic S et al (2014) Immobilization of antibodies and enzymes on 3-aminopropyltriethoxysilane-functionalized bioanalytical platforms for biosensors and diagnostics. Chem Rev 114:11083–11130. https:// doi. org/ 10. 1021/ cr500 0943 Verhoef JC, Cofino WP, Barendrecht E (1978) Mechanism and reaction rate of the Karl-Fischer titration reaction. J Electroanal Chem Interfacial Electro- chem 93:75–80. https:// doi. org/ 10. 1016/ s0022- 0728(78) 80240-x Weltz JS, Kienle DF, Schwartz DK, Kaar JL (2020) Reduced Enzyme Dynam- ics upon Multipoint Covalent Immobilization Leads to Stability-Activity Trade-off. J Am Chem Soc 142:3463–3471. https:// doi. org/ 10. 1021/ jacs. 9b117 07 Wu X, Yang C, Ge J (2017) Green synthesis of enzyme/metal-organic framework composites with high stability in protein denaturing solvents. Bioresources and Bioprocessing 4:24. https:// doi. org/ 10. 1186/ s40643- 017- 0154-8 Yadav GD, Pawar SV (2012) Synergism between microwave irradiation and enzyme catalysis in transesterification of ethyl-3-phenylpropanoate with n-butanol. Bioresour Technol 109:1–6. https:// doi. org/ 10. 1016/j. biort ech. 2012. 01. 030 Yang T, Shen C, Li Z et al (2005) Highly ordered self-assembly with large area of Fe3O 4 nanoparticles and the magnetic properties. J Phys Chem B 109:23233–23236. https:// doi. org/ 10. 1021/ jp054 291f Yousefi M, Marciello M, Guisan JM et al (2020) Fine modulation of the catalytic properties of Rhizomucor miehei lipase driven by different

Journal

Bioresources and Bioprocessing – Springer Journals

Published: May 30, 2022

Keywords: Biodiesel; Lipase; Multi-component reaction; Covalent immobilization; Magnetic nanoparticles

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