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Directed evolution driving the generation of an efficient keratinase variant to facilitate the feather degradation

Directed evolution driving the generation of an efficient keratinase variant to facilitate the... 2021) and B. cereus (Rajesh et  al. 2016) also produce Introduction keratinase to degrade keratin waste. Since most of the Keratin is a class of insoluble hard protein, widely existing Bacillus are food safety strains, keratinases derived from in organism tissues, and composing feathers, hair, wool, these strains have the advantages of food safety and con- nails, horns, hooves and scales (Coulombe and Omary venient for application. It has become a research hotspot 2002; Wang et al. 2016; Wu et al. 1982). It is reported that in the feed (Liang et al. 2021), leather textile (Zhang et al. nearly 10 million tons of feathers are discarded as by- 2016), washing and cleaning (Gong et  al. 2015), medical products every year, and 90% of the discarded feathers (Ye et  al. 2020), and cosmetics (Yeo et  al. 2018). Mean- are excellent protein resources available for feed industry while, studies have shown that the nitrogen source trans- (Choudhury et al. 2020). Traditional feather degradation formed by keratinase degradation of feather waste can be methods, such as physical expanding with high tempera- used as fertilizer and soil amendment to promote plant ture and pressure, or chemical dissolution with strong growth (Bhange et al. 2016). acid and alkali, not only destroy the quality of amino A key step in realizing its industrial applications is to acid and polypeptides, but also consume huge amounts achieve the mass production of keratinase. A variety of of energy and cause serious environmental pollution. expression systems have been used to produce keratinase Enzymatic degradation of feather in an environmentally (Ding et  al. 2020; Dong et  al. 2017). However, the poor friendly way has a high specificity and efficiency, attract - performances of activity and stability still largely limit ing an increasing attention worldwide (Qiu et al. 2020). its commercial interest. Protein engineering methodolo- Keratinase is a specific protease produced by microor - gies, such as directed evolution and rational design have ganisms that exclusively degrades keratin waste, such as been widely used for improving the thermostability and wool, feathers, bovine horn and so on. The keratinase- specific activity of keratinase. Rational design is based producing microorganisms are mainly bacteria, fungi and on analyzing the exact structure of available proteins. actinomycetes, most of which are screened from feather For example, the activity of keratinase KerBp from Bacil- or hair piles (Bokveld et al. 2021; Cao et al. 2021). Among lus pumilus was increased fivefold by using pro-peptide bacteria, the dominant keratinase-producing strain is engineering and saturation site-directed mutation (Su Bacillus, such as Bacillus licheniformis PWD-1 that was et  al. 2017, 2019). Similar results were obtained by Peng firstly reported to be able to secrete keratinase to degrade et al. (2021). The activity of keratinase KerZ1 from Bacil- feathers (Lin et al. 1995). In addition, B. subtilis (De Paiva lus licheniformis BBE11-1 was improved by 86% using et al. 2018), B. pumilus (Jagadeesan et al. 2020; Sun et al. Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 3 of 13 same strategies and the mutant degraded over 90% of double-digested with the restriction enzyme Mlu I and the feather into amino acids and oligopolymer. These Bam H. I. A randomly mutation library was constructed achievements laid the foundation for the degradation according to Zhang et  al. (Zhang and Zhang 2011) with of feathers to make up for the lack of protein resources. modifications by error-prone PCR reaction (5  mM Directed evolution, including error-prone PCR, DNA MgCl , 0.2  mM MnC l , 0.2  mM dATP, 0.2  mM dGTP, 2 2 shuffling, annealing oligonucleotide gene rearrangement, 1  mM dCTP, 1  mM dTTP, 0.05  U/μL polymerase, and cross-extension and non-homologous sequence protein 0.4  mM each of the primers). The error-prone PCR was recombination, has been developed and achieved many conducted by using the NEB Taq DNA polymerase (95 °C successes in recent years. Zhao et  al. enhanced the half- denaturation, 3 min; 29 cycles of 95 °C denaturation, 30 s; life of subtilisin E from B. subtilis at 65 °C more than 200 57  °C annealing, 30  s; and 72  °C extension, 1.5  min, fol- times by using directed evolution technique (Zhao and lowed by 72  °C extension for 5  min). The error-prone Arnold 1999). Among the types of directed evolution, PCR products were gel-purified and connected to the error-prone PCR has great impact on enzyme catalytic plasmid pMA5, which were tramsformed into E. coli specificity, optimal pH, stability, and substrate specific - JM109 competent cells. Plasmids obtained were finally ity and has been widely utilized to screen high-expression expressed in B. subtilis WB600 and the strains with large strains. transparent circles were selected directly. The mutant Due to the lack of knowledge about the structure– strains were cultured at 37  °C in 250-mL flasks contain - function relationship of keratinase, error-prone PCR ing 30 mL TB medium (50 µg/mL Kan ) for 60 h and the was used to introduce random mutations into keratinase supernatant was collected by centrifuging at 4  °C and kerBp gene in this study. Mutant strains with significantly 8000g for 20 min for keratinase activity measurement. improved enzyme activity were obtained through high- throughput screening. Then, the yield of keratinase was Enzymatic properties of mutant keratinase further increased using high-density fermentation strat- The optimum temperature of keratinase was determined egy, laying a favorable foundation for the scale prepara- by measuring enzyme activity at different temperatures tion and application of keratinase. Finally, the prepared (40, 45, 50, 55, 60, 65 and 70 °C). To evaluate thermal sta- keratinase was employed to degrade feather wastes, and bility, the properly diluted keratinase solution was treated the optimal conditions for enzymatic hydrolysis were at series temperatures for 30  min and cooled on ice. explored to provide a theoretical basis for the efficient The residual keratinase activity was determined at 50  °C reuse of feathers and improve the protein utilization in according to the standard enzyme activity method, and poultry farming. the enzyme activity of the untreated enzyme solution was taken as the control. The optimum pH of keratinase was Materials and methods examined with the keratin substrate and enzyme solution Strains, plasmids, and media appropriately diluted in series pH buffer (pH 6.0, 7.0, 8.0, The original keratinase gene kerBp was mined and 9.0, 10.0, 11.0, 12.0). expressed in our  previous studies (Su et  al. 2017). B. subtilis WB600, Escherichia.coli JM109, and the vector Homology modeling of keratinase KerBp pMA5 used in this experiment were all preserved in our The 3D structure of keratinase KerBp was homologous laboratory. modeled with the crystal structure of subtilisin NAT The seed medium composition for E.coli and B. subtilis (3VYN) from Bacillus subtilis (76% sequence similar- (g/L): tryptone 10, yeast extract 5, NaCl: 10. The fermen - ity), subtilisin BPN (1TO2) from Bacillus amyloliquefa- tation medium composition for B. subtilis (g/L): glycerin ciens (76% sequence similarity), and subtilisin DY (1BH6) 5, yeast extract 24, tryptone 12, K HPO 12.54, KH PO from Bacillus licheniformis (71% sequence similarity) as 2 4 2 4 2.31. The initial pH of the medium was natural pH. The templates using Discovery Studio. Ramanchandran plot optimized medium for B. subtilis (g/L): glucose 10, soya- and profile-3D evaluation model were used to verify the bean 18, soybean cake power 60, K HPO 12.54, K H PO rationality of the protein structure. 2 4 2 4 2.31. Scale‑up production of keratinase in 7‑L fermenter Error‑prone PCR and expression of keratinase Single colony of Bacillus subtilis with transparent circle The gene kerBp was  amplified with upstream primer was selected on LB-milk solid plate and inoculated with (5′-CGG GAT CCA TGT GCG TTA AAA AGA AAA LB liquid medium for 12  h at 37  °C. 1  mL of the above ATG TTA TGA CAA G-3′) and downstream primer (5′- seed solution was inoculated in 50  mL LB medium and GCA CGC GTT TAA TTT GAT GCT GCT TGC ACA cultured to OD 0.6–0.8. The secondary seeds were TTA ATC-3′). The plasmid pMA5 was extracted and transferred to a 7-L fermenter (Ependorff) with 5% Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 4 of 13 Reducing power analysis of keratinase inoculation for high-density fermentation, and kanamy- Two different methods were used to evaluate the reduc - cin sulfate was added to 50  μg/mL finally. The speed of ibility of keratinase. Reducing power was evaluated as fermenter was set as 500  rpm and the temperature was 3+ 2+ the ability to reduce Fe to Fe , using the potassium 37 °C. 50% glacial acetic acid or 50% ammonia water was ferricyanide reduction method (Clerici et  al. 2021). The used to adjust the pH value around 7.5. The dissolved diluted keratinase (300  μL) was mixed with 0.2  M PBS oxygen concentration at the initial stage of fermrntation (300  μL) buffer solution (pH 6.6) and 300  μL 1% (W/V) is maintained at 20–30% by adjusting the speed. During potassium ferricyanate. After incubation at 50  °C for the fermentation process, samples were taken periodi- 20  min, 300  μL 10% (w/v) TCA was added to stop the cally to detect bacterial concentration and enzyme activ- reaction, and centrifuged at 4000  rpm for 10  min. Then ity. When a sudden increase in dissolved oxygen was 200 μL supernatant was mixed 800 μL 0.01% (W/V) fer- detected, 50% glucose was added at a flow acceleration ric chloride solution and incubated at 30  °C for 10  min. rate of 39.53 mL/h. Finally, the absorbance of the reaction system was detected at 700  nm. The value of the absorbance repre - Feather degradation sents the level of reducing power. The feather waste used for degradation was collected 2,2′-Azo-bis-(3-ethylbenzothiazoline)-6-sulfonic acid from a poultry farm (Wuxi, China). The feather degra - (ABTS) radical scavenging experiment is mainly based dation experiments were carried out in a 500-mL flask on Re et  al. (1999). Add 10  μL of culture supernatant to containing 50  mL enzyme solution and 10  g/L chicken 1 mL of ABTS radical working solution and messure the feather waste. The prepared keratinase was applied to absorbance at 734  nm after 6  min. In the control group, feather degradation, combining with papain, pepsin, 10 μL of distilled water was added to replace the culture trypsin, bromelain, flavor protease, neutral protease and supernatant. The ABTS radical scavenging is calculated alkaline protease. The optimal combination of enzymes as follows: was selected to degrade feathers and the degradation conditions of compound enzymes were optimized, Scavenging (%) = Abs − Abs Abs control sample control including proportion of combined enzyme (The enzyme × 100. activity radio of trypsin and keratinase were 1:3, 1:2, 1:1, 2:1, 3:1, respectively), enzyme content (800, 1200, 1600, 2000, 2400, 2800 U/mL), temperature (30, 35, 40, 45, 50, Analysis of amino acids 55  °C), pH (7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0), degradation The supernatant of degradation products was added with time (8, 16, 24, 32, 40, 48  h) and sulfite content (0.1%, the same volume of TCA. After centrifugation for 30 min, 0.5%, 1%, 1.5%, 2.0%, 2.5%). the supernatant passed through a 0.2-μm membrane fil - ter. The free amino acid composition was determined Analytical methods by high performance liquid chromatography (HPLC, Keratinase assay Agilent 1260, Santa Clara, CA, USA) with o-phthalalde- Add 100  μL 1% keratin substrate solution to 100  μL hyde-9-fluorovinyl methyl chloroformate (OPA-FMOC) appropriately dissoluted keratinase solution, and then pre-column derivatization. An Agilent spectroscopy incubated at 50 °C for 20 min. Immediately after the reac- system was used to calculate the concentration from tion, add 200 μL 5%(W/V) TCA to stop the reaction. The the peak area obtained. The mobile phase is acetoni - control group was added with 200  μL TCA followed by trile–methanol. The detector is VWD, the wavelength is 100  μL keratin substrate solution. Then, the processed 338  nm, and the flow rate is 1  mL/min. The chromato - samples were centrifuged at 12,000 rpm for 5 min. 200 μL graphic column is Hypersil ODS-2 (250 × 4.6 mm, 5 μm), supernatant was mixed with 1  mL 0.4  M N a CO and 2 3 the temperature is 40  °C, and the injection volume is 200 μL folinol solution, and placed in a 40 °C water bath 10 μL. for 20 min. The absorbance value was detected at 660 nm. Feather degradation rate Analysis of soluble peptides The degradation products were filtered to leave the unde - Based on the comparison of peak time and peak area, graded feathers, which then were washed with deionized the molecular weight distribution of soluble peptides water three times to completely remove the soluble sub- was determined by high performance liquid chroma- stances and thallus, and dried at 65  °C for 24  h to con- tography (Agilent 1260, USA). The samples were centri - stant weight. Degradation rate of feathers is defined as fuged at 8000  rpm for 5  min, and then the supernatant the change in dry weight before and after degradation. was collected and passed through a 0.2-μm membrane Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 5 of 13 filter. The molecular weight distribution of soluble kerBp. Moreover, it was found that the efficiency of error- peptides was obtained by gradient elution on TSK gel prone PCR was affected by changing the concentration 2+ 2+ G2000SWXL (7.8 × 300 mm) using phosphate buffer as of Mg (Fig.  1b) and Mn (Fig.  1c). With the increase mobile phase. The detector, wavelength and flow rate of metal ions concentration, the efficiency was decreased were VWD, 214 ηm and 0.8 mL/min, respectively (Peng gradually and less product was obtained; while a low con- et al. 2019). centration of metal ions leading to a low base mutation 2+ rate. According to the experimental results, 5 mM  Mg 2+ and 0.2  mM Mn were selected as the experimental Statistical analysis condition. As a result, a library with more than 8000 All assays in this study were performed in triplicate. Data mutants was constructed after two rounds of error-prone processing in this study was performed by using the PCR and high-throughput screened by fluorescence mean standard deviation (± SD) and analyzed via Graph- chromogenic enzyme activity determination. As expec- Pad Prism 7 (San Diego, CA, USA). tation, nine strains with increased enzyme activity were selected  (Table  1), among which, the highest enzyme Results and discussion activity of the mutant T18 (R72S/F107Y/N291S/N295D) Directed evolution of keratinase by error‑prone PCR was 2382 U/mL, that was 2.1 times of the original kerati- Directed evolution is an efficient tool to generate vari - nase activity (Fig.  1d). The SDS-PAGE results (Fig.  1e) ants with fresh or enhanced properties. Error-prone PCR indicated an expected keratinase protein band. was adopted in this study to construct mutant libraries and provide superior keratinases for better degradation Enzymatic properties of mutant keratinase performance. Figure  1a shows a 1200-bp band of error- Due to the uncertainty of error-prone PCR, not only prone PCR products, consistent with the target gene the enzymatic activity of mutant is altered, but other 2+ Fig. 1 Screening of recombinant strains. a Verification of gene kerBp. Lane M, DNA marker; Lane 1–2, PCR amplification of kerBp. b Mg 2+ 2+ concentration gradient. Lane M, DNA marker; lane 1–10, Mg : 1 mM, 2 Mm, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM. c Mn 2+ concentration gradient. Lane M, DNA marker; lane 1–5, Mn : 0.1 mM, 0.2 Mm, 0.3 mM, 0.4 mM, 0.5 mM. d Rescreening results of shake flasks. e SDS‑PAGE analysis of WT and variant T18. Lane M, protein MW markers; lane 1, supernatant of fermentation broth from B. subtilis WB600 harboring pMA5 as the control; lane 2, WT; lane 3, variant T18 Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 6 of 13 Table 1 Comparison of the performance indexes of mutant strains and WT Enzyme Keratinase activity (U/mL) Specific activity (U/mg) Degradation rate (%) T1 1231.07 ± 91.97 1189.79 ± 77.24 36.84 ± 0.88 T3 1453.13 ± 13.95 1577.74 ± 76.78 24.52 ± 1.71 T5 1248.92 ± 82.58 1215.74 ± 158.08 34.48 ± 1.77 T8 1285.2 ± 142.63 1050.93 ± 101.98 25.78 ± 1.80 T11 1827.93 ± 165.46 1723.44 ± 200.56 40.42 ± 1.60 T12 1800 ± 137.61 1746.34 ± 92.22 42.04 ± 1.34 T14 1866.33 ± 16.58 1515.63 ± 15.90 37.68 ± 0.96 T18 2381.87 ± 13.01 2529.70 ± 53.83 51.42 ± 0.62 T19 2243.87 ± 41.80 1968.43 ± 122.99 40.2 ± 0.38 WT 1150.6 ± 68.11 1468.42 ± 108.18 33.57 ± 2.13 properties such as thermostability and optimum pH pH environment for the complexation of keratinase and may be also affected. Thus, the effects on catalytic prop - other proteases in the later stage. erties were determined. The highest specific activity is In the past 20 years, the directed evolution of enzymes defined as 100%. As shown in Fig.  2a, the specific activ - has gradually become a research hotspot in the field of ity of keratinase was the highest at 55  °C before and bio-catalysis. Compared with natural enzymes, engi- after the mutation, indicating that the optimal tempera- neered enzymes obtained through directed evolution ture of keratinase was not affected by mutation. But the show enhanced properties such as higher activity, higher mutant keratinase activity remained over 70% in the thermostability, better stereoselectivity and better alka- range of 45–55  °C, while the original keratinase activity line or acid stability. Li et  al. (2021) obtained a variant decreased to 52%. In terms of thermal stability (Fig.  2b), (G95P) with ninefold enhancement in specific activity the remaining enzyme activity of the mutant keratinase by error-prone PCR. In addition, the thermostability is higher than the original enzyme activity in the range of and alkaline stability of the alkaline protease were also 40–50 °C. As shown in Fig. 2c, the specific activity of the enhanced. In this study, directed evolution technology mutant enzyme was the highest at pH 9, while the opti- was used to modify keratinase, which not only increased mal pH of the original keratinase was 11, indicating that the activity of keratinase, but also improved the thermo- the optimal pH of the keratinase was changed after muta- stability of keratinase. The improved enzyme activity, tion. The decrease of the optimum pH provides a suitable thermal stability and the decrease of optimal pH make Fig. 2 Enzymatic properties of variant T18. a Optimum temperature; b thermal stability; c optimum pH; d steady‑state kinetic analysis Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 7 of 13 Fig. 3 Three‑ dimensional structural model by homology modeling. a KerBp. b Variant T18 the mutant keratinase more suitable for industrial appli- can modify the catalytic pocket of the enzyme (Laksmi cations, especially for degradation of feathers by com- et al. 2020) and improve its substrate specificity (Dai et al. bining keratinase with other proteases, which would 2021; Zhou et al. 2021). improve the hydrolysis efficiency of feathers. High‑density fermentation Homologous modeling analysis To measure the growth and production of mutant The structure modeling of keratinase KerBp was pre - keratinase and explore its industrial potential, the fer- dicted. The framework was consisted with ten β-folds and mentation was carried out in a 7-L fermenter. Accord- eight α-helixes, as shown in Fig. 3. Two mutation sites of ing to the regular detection of cell density and enzyme R72S/F107Y are located in the non-conserved region of activity as shown in Fig.  4, the mutant strain grew pro-peptide region and the change in amino acid hydro- logarithmically within 0–16 h, while the enzyme activ- phobicity affects spatial configuration of the pro-peptide, ity gradually increased. During 16–60 h, the cells con- leading to folding and degradation of the mature peptide. tinued to grow and tended to be stable. At this stage, Studies have shown that mutations at appropriate site of keratinase was continuously produced and accumu- pro-peptide can change the folding speed of the protein lated, and the enzyme expression level continued to and increase the activity and production of extracellular increase. Subsequently, the cells continued to grow enzyme, so the changes on pro-peptide promoted the through streaming medium and reached the maximum increase of keratinase activity (Grimsby et  al. 2010). In concentration at 92  h with OD value of 51.41. The our previous study, Su et al. (2019) carried out saturation enzyme activity continued to accumulate and reached mutations at six potential sites in the pro-peptide region the maximum with 8448 U/mL at 108 h. of keratinase, and the activity of keratinase increased There are several strategies to improve the activity from 179 to 1114  U/mL. The other two mutation points and yield of recombinant keratinase, such as promoter of N291S/N295D are located on the mature peptide, engineering (Gong et  al. 2020), signal peptide engi- and N291/N295 is close to S1 pocket region. The struc - neering (Tian et al. 2019), heterologous expression ( Jin ture of the S1 and S4 substrate pockets has an important et  al. 2019; Yong et  al. 2020), site-directed mutagen- influence on the substrate specificity of protease (Fang esis (Jaouadi et  al. 2014) and pro-peptide engineering et  al. 2015). The change of amino acids may affect S1 (Su et  al. 2019). Studies have shown that fermentation pocket structure, thus enhancing the substrate specific - optimization is one of the most important strategies ity of keratinase. The application of directed evolution for to improve keratinase activity and yield. The fermen- molecular modification in enzyme catalysis has achieved tation conditions of the neutral protease derived from favorable results (Buller et  al. 2018). Directed evolution B. subtilis were optimized, and the enzyme activity Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 8 of 13 Fig. 4 High‑ density fermentation of the variant T18 and the original strain in a 7‑L fermenter. a WT; b variant T18 was increased by nearly 2.8 times (He et  al. 2021). Feather degradation Using waste feathers as carbon source, the production Although the degradation mechanism of keratinase is of keratinase was increased by five times using deep still not fully understood, it is widely recognized that liquid fermentation optimization method (Jana et  al. reducing power plays an important role in the degra- 2020). dation process (Lange et  al. 2016; Ramnani et  al. 2005). Herein, the reducing ability of keratinase was assured by 3+ reduction of F e (Fig. 5a) and ABTS radicals scavenging Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 9 of 13 Fig. 5 Comparison of reducing power and degradation of feathers. a Reducing power (OD ). 1, pMA5; 2, WT; 3, variant T18. b ATBS radical scavenging. 1, pMA5; 2, WT; 3, variant T18. c Degradation of feathers by combined enzymes. 1, WT; 2, variant T18; 3, papain and variant T18; 4, pepsin and variant T18; 5, trypsin and variant T18; 6, bromelain and variant T18; 7, flavor protease and variant T18; 8, neutral protease and variant T18; 9, alkaline protease and variant T18 test (Fig.  5b). Reducing power measurement is based on higher than those reported previously (Table  3). The Prussian blue production as an indicator. The samples concentrations of histidine (His), glycine (Gly), threo- reduce potassium ferricyanide, and then use ferrous ions nine (Thr), tyrosine (Tyr) and lysine (Lys) increased by to generate Prussian blue. The value of the absorbance 248.7  mg/L, 197.38  mg/L, 911.82  mg/L, 241.41  mg/L at 700  nm represents the level of reducing power with a and 783.42  mg/L, respectively. Threonine increased by positive relationship between them. It was found that the 174 times from the initial value at 12  h of degradation. keratinase of the mutant strain had a stronger reducing Threonine is the second and third limiting amino acid in power than the control. This unique property indicates pig and poultry feed, respectively. It helps to adjust the that the keratinase of the mutant strain has high reduc- amino acid balance, promotes the growth of livestock, ibility and could be used in feather degradation reac- improves the nutritional value and amino acid digest- tion. Compared with the control pMA5 and the original ibility, and reduces the cost of feed materials. Therefore, keratinase, the mutant keratinase scavenged more than hydrolyzed feathers have great potential in the produc- 60% of ABTS radicals within 6  min, showing stronger tion of feed additives and amino acids. Moreover, the sol- antioxidant activity and better degradation performance. uble peptides in the degradation solution were analyzed. Since the mutant keratinase had both reducing and As shown in Fig. 7b, the molecular weight of peptides in hydrolyzing properties, when the keratinase was used the hydrolysate gradually decreased, and the molecular alone to degrade feathers, the degradation rate reached weight of peptides in the hydrolysate was concentrated 49% (Fig. 5c). In order to further improve the degradation below 1  kDa, indicating that the degradation products rate of feathers, the keratinase was compounded with were mainly composed of oligopolymer. These oligopo - trypsin and the degradation conditions were optimized, lymer are easily utilized by animals and have application by which degradation rate increased from 49 to 89% signification in feed industry (Table 3). (Fig.  6). The SEM on the structure of degraded feathers (Fig.  7a) showed that, compared with the blank control Conclusions group, after 16 h of hydrolysis, barbs, barbules and acces- In this study, a keratinase mutant with improved activ- sory pinna were completely separated, and the scapus ity was obtained via directed evolution technique broke irregularly with damage appeared on the surface. employing error-prone PCR and high-throughput The ability of keratinase to degrade feathers significantly screening. Through high-density fermentation, the in a short period provides the basis for its potential enzyme activity increased from 1150 to 8448  U/mL. application in bioconversion of keratin wastes into valu- Moreover, in order to understand the catalytic func- able protein resources. Table  2 indicates the change of tion of the existing keratinase in depth, the homolo- amino acid concentration in feather degradation prod- gous structure of the three-dimensional structure was ucts during feather degradation. After hydrolysis for modeled, and the keratinase was characterized. The 12  h, the total content of amino acids in the degrada- keratinase after mutation had better temperature sta- tion solution reached up to 4972.34  mg/mL, which was bility and optimum pH suitable for compounding. In Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 10 of 13 Fig. 6 Optimization of compound enzyme conditions. a Proportion of compound enzyme (trypsin: keratinase); b compound enzyme addition level; c temperature; d pH; e time; f sulfite addition amount Fig. 7 Scanning electron microscopy and molecular weight distribution of the degradation products. a Scanning electron microscopy; b molecular weight distribution of polypeptide Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 11 of 13 Table 2 The changes of amino acid concentration during feather degradation process by the variant T18 Amino acid Concentration (mg/L) 0 h 12 h 24 h 36 h asp 25.09 ± 0.31 44.05 ± 1.47 28.99 ± 1.80 23.71 ± 4.58 glu 41.12 ± 0.16 122.94 ± 3.16 11.85 ± 2.63 17.32 ± 4.12 ser 0.88 ± 0.80 12.99 ± 0.66 1.92 ± 1.32 0.80 ± 0.59 his 21.03 ± 1.82 269.73 ± 0.45 23.01 ± 1.70 17.31 ± 4.78 gly 22.16 ± 0.13 219.54 ± 6.42 56.85 ± 7.11 3.18 ± 0.27 thr 5.27 ± 0.21 917.09 ± 24.37 272.72 ± 5.64 537.47 ± 12.29 arg 11.31 ± 0.56 25.09 ± 0.43 0.58 ± 0.042 1.06 ± 0.51 ala 32.5 ± 0.62 23.80 ± 0.14 2.61 ± 0.70 3.64 ± 0.42 tyr 146.01 ± 2.09 387.42 ± 5.94 121.41 ± 4.24 114.32 ± 7.87 cys 6.38 ± 0.69 14.56 ± 1.47 4.84 ± 0.67 4.26 ± 0.50 val 165.18 ± 0.83 389.84 ± 22.32 22.86 ± 12.06 22.66 ± 2.60 met 116.92 ± 2.81 370.51 ± 15.36 72.61 ± 6.84 44.6 ± 6.52 phe 199.02 ± 0.95 688.07 ± 14.47 152.44 ± 14.56 154.41 ± 1.88 ile 63.84 ± 0.88 55.39 ± 1.64 1.80 ± 0.30 1.23 ± 0.24 leu 180.32 ± 1.63 349.90 ± 8.14 7.16 ± 1.28 6.57 ± 0.93 lys 282.93 ± 5.09 1066.35 ± 13.87 187.07 ± 6.01 149.06 ± 13.82 pro 231.74 ± 12.78 14.43 ± 1.70 113.69 ± 16.87 111.81 ± 12.49 Table 3 Comparison of amino acids in feather degradation products Amino acid Concentration (mg/L) WT (12 h) Variant T18 (12 h) KerSMD and KerSMF (48 h) Bacillus pumilus AR57 (Peng et al. 2019) keratinase (Jagadeesan et al. 2020) asp 64.66 ± 1.84 44.05 ± 1.47 15.55 ± 1.09 0.612 glu 245.91 ± 6.95 122.94 ± 3.16 20.19 ± 0.13 1.242 ser 10.00 ± 1.08 12.99 ± 0.66 14.61 ± 0.18 9.805 his 44.05 ± 0.39 269.73 ± 0.45 ND 4.794 gly 136.19 ± 7.33 219.54 ± 6.42 14.34 ± 0.24 1.421 thr 464.77 ± 56.77 917.09 ± 24.37 15.74 ± 0.22 3.514 arg 8.8 ± 0.61 25.09 ± 0.43 22.13 ± 0.49 14.110 ala 504.30 ± 5.88 23.80 ± 0.14 10.05 ± 0.28 0.517 tyr 195.87 ± 0.89 387.42 ± 5.94 171.53 ± 0.42 18.789 cys 10.82 ± 1.49 14.56 ± 1.47 ND ND val 148.99 ± 8.97 389.84 ± 22.32 207.51 ± 0.29 8.716 met 187.58 ± 11.44 370.51 ± 15.36 0 ± 0.41 7.684 phe 348.35 ± 1.36 688.07 ± 14.47 183.73 ± 0.21 33.153 ile 18.99 ± 0.94 55.39 ± 1.64 72.96 ± 0.49 4.749 leu 91.94 ± 1.88 349.90 ± 8.14 126.61 ± 0.73 7.083 lys 510.48 ± 5.08 1066.35 ± 13.87 20.94 ± 0.73 3.772 pro 28.14 ± 2.86 14.43 ± 1.70 ND ND addition, the combination of keratinase and trypsin oligopeptides, which provides protein resources for degrades feathers, and the product contains a poultry and has potential applications in the feed large number of amino acids, short peptides and industry. Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 12 of 13 Acknowledgements Dai S, Yao Q, Yu G, Liu S, Yun J, Xiao X, Deng ZJ, Li H (2021) Biochemical charac‑ Not applicable. terization of a novel bacterial laccase and improvement of its efficiency by directed evolution on dye degradation. Front Microbiol 12:9 Authors’ contributions de Paiva DP, de Oliveira SSA, Mazotto AM, Vermelho AB, de Oliveira SS (2018) JZ: investigation, software, methodology, writing—original draft; SC: investiga‑ Keratinolytic activity of Bacillus subtilis Lfb‑fiocruz 1266 enhanced by tion, writing—review and editing; JSG and JSS: supervision, conceptualization, whole‑ cell mutagenesis. 3 Biotech 9:2 methodology, funding acquisition, writing—reviewing and editing; XLK and Ding Y, Yang Y, Ren Y, Xia J, Liu F, Li Y, Tang X‑F, Tang B (2020) Extracellular YLL: investigation, visualization; HL, JQ and ZHX: investigation, visualiza‑ production, characterization, and engineering of a polyextremotoler‑ tion, writing—review and editing. All authors read and approved the final ant subtilisin‑like protease from feather ‑ degrading Thermoactinomyces manuscript. vulgaris strain Cdf. Front Microbiol 11:605771. https:// doi. org/ 10. 3389/ fmicb. 2020. 605771 Funding Dong Y‑Z, Chang W ‑S, Chen PT (2017) Characterization and overexpression of This work was financially supported by the National Key Research and Devel‑ a novel keratinase from Bacillus polyfermenticus B4 in recombinant Bacil- opment Program of China (No. 2021YFC2100900), the National Natural Sci‑ lus subtilis. Bioresour Bioprocess 4:47 ence Foundation of China (No. 21978116), and the Ningxia Hui Autonomous Fang Z, Zhang J, Liu BH, Du GC, Chen J (2015) Insight into the substrate speci‑ Region Key Research & Development Plan (No. 2019BCH01002). ficity of keratinase Kersmd from Stenotrophomonas maltophilia by site ‑ directed mutagenesis studies in the S1 pocket. RSC Adv 5:74953–74960 Availability of data and materials Gong JS, Wang Y, Zhang DD, Li H, Zhang XM, Zhang RX, Lu ZM, Xu ZH, Shi JS All data generated or analyzed during this study are included in the main (2015) A surfactant‑stable Bacillus pumilus K9 alpha‑keratinase and its manuscript file. potential application in detergent industry. Chem Res Chin Univ 31:91–97 Gong JS, Ye JP, Tao LY, Su C, Qin J, Zhang YY, Li H, Li H, Xu ZH, Shi JS (2020) Efficient keratinase expression via promoter engineering strategies for Declarations degradation of feather wastes. Enzyme Microb Tech 137:8 Grimsby JL, Lucero HA, Trackman PC, Ravid K, Kagan HM (2010) Role of lysyl Ethics approval and consent to participate oxidase propeptide in secretion and enzyme activity. J Cell Biochem All authors have read and agreed the ethics for publishing the manuscript. 111:1231–1243 He FM, Jin C, Yang DD, Zhang XQ, Yang CL, Xu ZP, Tian JW, Tian YQ (2021) Consent for publication Optimization of fermentation conditions for production of neutral The authors approved the consent for publishing the manuscript. metalloprotease by Bacillus subtilis Sck6 and its application in goatskin‑ dehairing. Prep Biochem Biotech 9:1995413. https:// doi. org/ 10. 1080/ Competing interests 10826 068. 2021. 19954 13 The authors declare that they have no competing interests. Jagadeesan Y, Meenakshisundaram S, Saravanan V, Balaiah A (2020) Sustain‑ able production, biochemical and molecular characterization of thermo‑ Author details and‑solvent stable alkaline serine keratinase from novel Bacillus pumilus Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry Ar57 for promising poultry solid waste management. 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Appl Microbiol Biotechnol 105:3625–3634 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioresources and Bioprocessing Springer Journals

Directed evolution driving the generation of an efficient keratinase variant to facilitate the feather degradation

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Abstract

2021) and B. cereus (Rajesh et  al. 2016) also produce Introduction keratinase to degrade keratin waste. Since most of the Keratin is a class of insoluble hard protein, widely existing Bacillus are food safety strains, keratinases derived from in organism tissues, and composing feathers, hair, wool, these strains have the advantages of food safety and con- nails, horns, hooves and scales (Coulombe and Omary venient for application. It has become a research hotspot 2002; Wang et al. 2016; Wu et al. 1982). It is reported that in the feed (Liang et al. 2021), leather textile (Zhang et al. nearly 10 million tons of feathers are discarded as by- 2016), washing and cleaning (Gong et  al. 2015), medical products every year, and 90% of the discarded feathers (Ye et  al. 2020), and cosmetics (Yeo et  al. 2018). Mean- are excellent protein resources available for feed industry while, studies have shown that the nitrogen source trans- (Choudhury et al. 2020). Traditional feather degradation formed by keratinase degradation of feather waste can be methods, such as physical expanding with high tempera- used as fertilizer and soil amendment to promote plant ture and pressure, or chemical dissolution with strong growth (Bhange et al. 2016). acid and alkali, not only destroy the quality of amino A key step in realizing its industrial applications is to acid and polypeptides, but also consume huge amounts achieve the mass production of keratinase. A variety of of energy and cause serious environmental pollution. expression systems have been used to produce keratinase Enzymatic degradation of feather in an environmentally (Ding et  al. 2020; Dong et  al. 2017). However, the poor friendly way has a high specificity and efficiency, attract - performances of activity and stability still largely limit ing an increasing attention worldwide (Qiu et al. 2020). its commercial interest. Protein engineering methodolo- Keratinase is a specific protease produced by microor - gies, such as directed evolution and rational design have ganisms that exclusively degrades keratin waste, such as been widely used for improving the thermostability and wool, feathers, bovine horn and so on. The keratinase- specific activity of keratinase. Rational design is based producing microorganisms are mainly bacteria, fungi and on analyzing the exact structure of available proteins. actinomycetes, most of which are screened from feather For example, the activity of keratinase KerBp from Bacil- or hair piles (Bokveld et al. 2021; Cao et al. 2021). Among lus pumilus was increased fivefold by using pro-peptide bacteria, the dominant keratinase-producing strain is engineering and saturation site-directed mutation (Su Bacillus, such as Bacillus licheniformis PWD-1 that was et  al. 2017, 2019). Similar results were obtained by Peng firstly reported to be able to secrete keratinase to degrade et al. (2021). The activity of keratinase KerZ1 from Bacil- feathers (Lin et al. 1995). In addition, B. subtilis (De Paiva lus licheniformis BBE11-1 was improved by 86% using et al. 2018), B. pumilus (Jagadeesan et al. 2020; Sun et al. Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 3 of 13 same strategies and the mutant degraded over 90% of double-digested with the restriction enzyme Mlu I and the feather into amino acids and oligopolymer. These Bam H. I. A randomly mutation library was constructed achievements laid the foundation for the degradation according to Zhang et  al. (Zhang and Zhang 2011) with of feathers to make up for the lack of protein resources. modifications by error-prone PCR reaction (5  mM Directed evolution, including error-prone PCR, DNA MgCl , 0.2  mM MnC l , 0.2  mM dATP, 0.2  mM dGTP, 2 2 shuffling, annealing oligonucleotide gene rearrangement, 1  mM dCTP, 1  mM dTTP, 0.05  U/μL polymerase, and cross-extension and non-homologous sequence protein 0.4  mM each of the primers). The error-prone PCR was recombination, has been developed and achieved many conducted by using the NEB Taq DNA polymerase (95 °C successes in recent years. Zhao et  al. enhanced the half- denaturation, 3 min; 29 cycles of 95 °C denaturation, 30 s; life of subtilisin E from B. subtilis at 65 °C more than 200 57  °C annealing, 30  s; and 72  °C extension, 1.5  min, fol- times by using directed evolution technique (Zhao and lowed by 72  °C extension for 5  min). The error-prone Arnold 1999). Among the types of directed evolution, PCR products were gel-purified and connected to the error-prone PCR has great impact on enzyme catalytic plasmid pMA5, which were tramsformed into E. coli specificity, optimal pH, stability, and substrate specific - JM109 competent cells. Plasmids obtained were finally ity and has been widely utilized to screen high-expression expressed in B. subtilis WB600 and the strains with large strains. transparent circles were selected directly. The mutant Due to the lack of knowledge about the structure– strains were cultured at 37  °C in 250-mL flasks contain - function relationship of keratinase, error-prone PCR ing 30 mL TB medium (50 µg/mL Kan ) for 60 h and the was used to introduce random mutations into keratinase supernatant was collected by centrifuging at 4  °C and kerBp gene in this study. Mutant strains with significantly 8000g for 20 min for keratinase activity measurement. improved enzyme activity were obtained through high- throughput screening. Then, the yield of keratinase was Enzymatic properties of mutant keratinase further increased using high-density fermentation strat- The optimum temperature of keratinase was determined egy, laying a favorable foundation for the scale prepara- by measuring enzyme activity at different temperatures tion and application of keratinase. Finally, the prepared (40, 45, 50, 55, 60, 65 and 70 °C). To evaluate thermal sta- keratinase was employed to degrade feather wastes, and bility, the properly diluted keratinase solution was treated the optimal conditions for enzymatic hydrolysis were at series temperatures for 30  min and cooled on ice. explored to provide a theoretical basis for the efficient The residual keratinase activity was determined at 50  °C reuse of feathers and improve the protein utilization in according to the standard enzyme activity method, and poultry farming. the enzyme activity of the untreated enzyme solution was taken as the control. The optimum pH of keratinase was Materials and methods examined with the keratin substrate and enzyme solution Strains, plasmids, and media appropriately diluted in series pH buffer (pH 6.0, 7.0, 8.0, The original keratinase gene kerBp was mined and 9.0, 10.0, 11.0, 12.0). expressed in our  previous studies (Su et  al. 2017). B. subtilis WB600, Escherichia.coli JM109, and the vector Homology modeling of keratinase KerBp pMA5 used in this experiment were all preserved in our The 3D structure of keratinase KerBp was homologous laboratory. modeled with the crystal structure of subtilisin NAT The seed medium composition for E.coli and B. subtilis (3VYN) from Bacillus subtilis (76% sequence similar- (g/L): tryptone 10, yeast extract 5, NaCl: 10. The fermen - ity), subtilisin BPN (1TO2) from Bacillus amyloliquefa- tation medium composition for B. subtilis (g/L): glycerin ciens (76% sequence similarity), and subtilisin DY (1BH6) 5, yeast extract 24, tryptone 12, K HPO 12.54, KH PO from Bacillus licheniformis (71% sequence similarity) as 2 4 2 4 2.31. The initial pH of the medium was natural pH. The templates using Discovery Studio. Ramanchandran plot optimized medium for B. subtilis (g/L): glucose 10, soya- and profile-3D evaluation model were used to verify the bean 18, soybean cake power 60, K HPO 12.54, K H PO rationality of the protein structure. 2 4 2 4 2.31. Scale‑up production of keratinase in 7‑L fermenter Error‑prone PCR and expression of keratinase Single colony of Bacillus subtilis with transparent circle The gene kerBp was  amplified with upstream primer was selected on LB-milk solid plate and inoculated with (5′-CGG GAT CCA TGT GCG TTA AAA AGA AAA LB liquid medium for 12  h at 37  °C. 1  mL of the above ATG TTA TGA CAA G-3′) and downstream primer (5′- seed solution was inoculated in 50  mL LB medium and GCA CGC GTT TAA TTT GAT GCT GCT TGC ACA cultured to OD 0.6–0.8. The secondary seeds were TTA ATC-3′). The plasmid pMA5 was extracted and transferred to a 7-L fermenter (Ependorff) with 5% Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 4 of 13 Reducing power analysis of keratinase inoculation for high-density fermentation, and kanamy- Two different methods were used to evaluate the reduc - cin sulfate was added to 50  μg/mL finally. The speed of ibility of keratinase. Reducing power was evaluated as fermenter was set as 500  rpm and the temperature was 3+ 2+ the ability to reduce Fe to Fe , using the potassium 37 °C. 50% glacial acetic acid or 50% ammonia water was ferricyanide reduction method (Clerici et  al. 2021). The used to adjust the pH value around 7.5. The dissolved diluted keratinase (300  μL) was mixed with 0.2  M PBS oxygen concentration at the initial stage of fermrntation (300  μL) buffer solution (pH 6.6) and 300  μL 1% (W/V) is maintained at 20–30% by adjusting the speed. During potassium ferricyanate. After incubation at 50  °C for the fermentation process, samples were taken periodi- 20  min, 300  μL 10% (w/v) TCA was added to stop the cally to detect bacterial concentration and enzyme activ- reaction, and centrifuged at 4000  rpm for 10  min. Then ity. When a sudden increase in dissolved oxygen was 200 μL supernatant was mixed 800 μL 0.01% (W/V) fer- detected, 50% glucose was added at a flow acceleration ric chloride solution and incubated at 30  °C for 10  min. rate of 39.53 mL/h. Finally, the absorbance of the reaction system was detected at 700  nm. The value of the absorbance repre - Feather degradation sents the level of reducing power. The feather waste used for degradation was collected 2,2′-Azo-bis-(3-ethylbenzothiazoline)-6-sulfonic acid from a poultry farm (Wuxi, China). The feather degra - (ABTS) radical scavenging experiment is mainly based dation experiments were carried out in a 500-mL flask on Re et  al. (1999). Add 10  μL of culture supernatant to containing 50  mL enzyme solution and 10  g/L chicken 1 mL of ABTS radical working solution and messure the feather waste. The prepared keratinase was applied to absorbance at 734  nm after 6  min. In the control group, feather degradation, combining with papain, pepsin, 10 μL of distilled water was added to replace the culture trypsin, bromelain, flavor protease, neutral protease and supernatant. The ABTS radical scavenging is calculated alkaline protease. The optimal combination of enzymes as follows: was selected to degrade feathers and the degradation conditions of compound enzymes were optimized, Scavenging (%) = Abs − Abs Abs control sample control including proportion of combined enzyme (The enzyme × 100. activity radio of trypsin and keratinase were 1:3, 1:2, 1:1, 2:1, 3:1, respectively), enzyme content (800, 1200, 1600, 2000, 2400, 2800 U/mL), temperature (30, 35, 40, 45, 50, Analysis of amino acids 55  °C), pH (7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0), degradation The supernatant of degradation products was added with time (8, 16, 24, 32, 40, 48  h) and sulfite content (0.1%, the same volume of TCA. After centrifugation for 30 min, 0.5%, 1%, 1.5%, 2.0%, 2.5%). the supernatant passed through a 0.2-μm membrane fil - ter. The free amino acid composition was determined Analytical methods by high performance liquid chromatography (HPLC, Keratinase assay Agilent 1260, Santa Clara, CA, USA) with o-phthalalde- Add 100  μL 1% keratin substrate solution to 100  μL hyde-9-fluorovinyl methyl chloroformate (OPA-FMOC) appropriately dissoluted keratinase solution, and then pre-column derivatization. An Agilent spectroscopy incubated at 50 °C for 20 min. Immediately after the reac- system was used to calculate the concentration from tion, add 200 μL 5%(W/V) TCA to stop the reaction. The the peak area obtained. The mobile phase is acetoni - control group was added with 200  μL TCA followed by trile–methanol. The detector is VWD, the wavelength is 100  μL keratin substrate solution. Then, the processed 338  nm, and the flow rate is 1  mL/min. The chromato - samples were centrifuged at 12,000 rpm for 5 min. 200 μL graphic column is Hypersil ODS-2 (250 × 4.6 mm, 5 μm), supernatant was mixed with 1  mL 0.4  M N a CO and 2 3 the temperature is 40  °C, and the injection volume is 200 μL folinol solution, and placed in a 40 °C water bath 10 μL. for 20 min. The absorbance value was detected at 660 nm. Feather degradation rate Analysis of soluble peptides The degradation products were filtered to leave the unde - Based on the comparison of peak time and peak area, graded feathers, which then were washed with deionized the molecular weight distribution of soluble peptides water three times to completely remove the soluble sub- was determined by high performance liquid chroma- stances and thallus, and dried at 65  °C for 24  h to con- tography (Agilent 1260, USA). The samples were centri - stant weight. Degradation rate of feathers is defined as fuged at 8000  rpm for 5  min, and then the supernatant the change in dry weight before and after degradation. was collected and passed through a 0.2-μm membrane Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 5 of 13 filter. The molecular weight distribution of soluble kerBp. Moreover, it was found that the efficiency of error- peptides was obtained by gradient elution on TSK gel prone PCR was affected by changing the concentration 2+ 2+ G2000SWXL (7.8 × 300 mm) using phosphate buffer as of Mg (Fig.  1b) and Mn (Fig.  1c). With the increase mobile phase. The detector, wavelength and flow rate of metal ions concentration, the efficiency was decreased were VWD, 214 ηm and 0.8 mL/min, respectively (Peng gradually and less product was obtained; while a low con- et al. 2019). centration of metal ions leading to a low base mutation 2+ rate. According to the experimental results, 5 mM  Mg 2+ and 0.2  mM Mn were selected as the experimental Statistical analysis condition. As a result, a library with more than 8000 All assays in this study were performed in triplicate. Data mutants was constructed after two rounds of error-prone processing in this study was performed by using the PCR and high-throughput screened by fluorescence mean standard deviation (± SD) and analyzed via Graph- chromogenic enzyme activity determination. As expec- Pad Prism 7 (San Diego, CA, USA). tation, nine strains with increased enzyme activity were selected  (Table  1), among which, the highest enzyme Results and discussion activity of the mutant T18 (R72S/F107Y/N291S/N295D) Directed evolution of keratinase by error‑prone PCR was 2382 U/mL, that was 2.1 times of the original kerati- Directed evolution is an efficient tool to generate vari - nase activity (Fig.  1d). The SDS-PAGE results (Fig.  1e) ants with fresh or enhanced properties. Error-prone PCR indicated an expected keratinase protein band. was adopted in this study to construct mutant libraries and provide superior keratinases for better degradation Enzymatic properties of mutant keratinase performance. Figure  1a shows a 1200-bp band of error- Due to the uncertainty of error-prone PCR, not only prone PCR products, consistent with the target gene the enzymatic activity of mutant is altered, but other 2+ Fig. 1 Screening of recombinant strains. a Verification of gene kerBp. Lane M, DNA marker; Lane 1–2, PCR amplification of kerBp. b Mg 2+ 2+ concentration gradient. Lane M, DNA marker; lane 1–10, Mg : 1 mM, 2 Mm, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM. c Mn 2+ concentration gradient. Lane M, DNA marker; lane 1–5, Mn : 0.1 mM, 0.2 Mm, 0.3 mM, 0.4 mM, 0.5 mM. d Rescreening results of shake flasks. e SDS‑PAGE analysis of WT and variant T18. Lane M, protein MW markers; lane 1, supernatant of fermentation broth from B. subtilis WB600 harboring pMA5 as the control; lane 2, WT; lane 3, variant T18 Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 6 of 13 Table 1 Comparison of the performance indexes of mutant strains and WT Enzyme Keratinase activity (U/mL) Specific activity (U/mg) Degradation rate (%) T1 1231.07 ± 91.97 1189.79 ± 77.24 36.84 ± 0.88 T3 1453.13 ± 13.95 1577.74 ± 76.78 24.52 ± 1.71 T5 1248.92 ± 82.58 1215.74 ± 158.08 34.48 ± 1.77 T8 1285.2 ± 142.63 1050.93 ± 101.98 25.78 ± 1.80 T11 1827.93 ± 165.46 1723.44 ± 200.56 40.42 ± 1.60 T12 1800 ± 137.61 1746.34 ± 92.22 42.04 ± 1.34 T14 1866.33 ± 16.58 1515.63 ± 15.90 37.68 ± 0.96 T18 2381.87 ± 13.01 2529.70 ± 53.83 51.42 ± 0.62 T19 2243.87 ± 41.80 1968.43 ± 122.99 40.2 ± 0.38 WT 1150.6 ± 68.11 1468.42 ± 108.18 33.57 ± 2.13 properties such as thermostability and optimum pH pH environment for the complexation of keratinase and may be also affected. Thus, the effects on catalytic prop - other proteases in the later stage. erties were determined. The highest specific activity is In the past 20 years, the directed evolution of enzymes defined as 100%. As shown in Fig.  2a, the specific activ - has gradually become a research hotspot in the field of ity of keratinase was the highest at 55  °C before and bio-catalysis. Compared with natural enzymes, engi- after the mutation, indicating that the optimal tempera- neered enzymes obtained through directed evolution ture of keratinase was not affected by mutation. But the show enhanced properties such as higher activity, higher mutant keratinase activity remained over 70% in the thermostability, better stereoselectivity and better alka- range of 45–55  °C, while the original keratinase activity line or acid stability. Li et  al. (2021) obtained a variant decreased to 52%. In terms of thermal stability (Fig.  2b), (G95P) with ninefold enhancement in specific activity the remaining enzyme activity of the mutant keratinase by error-prone PCR. In addition, the thermostability is higher than the original enzyme activity in the range of and alkaline stability of the alkaline protease were also 40–50 °C. As shown in Fig. 2c, the specific activity of the enhanced. In this study, directed evolution technology mutant enzyme was the highest at pH 9, while the opti- was used to modify keratinase, which not only increased mal pH of the original keratinase was 11, indicating that the activity of keratinase, but also improved the thermo- the optimal pH of the keratinase was changed after muta- stability of keratinase. The improved enzyme activity, tion. The decrease of the optimum pH provides a suitable thermal stability and the decrease of optimal pH make Fig. 2 Enzymatic properties of variant T18. a Optimum temperature; b thermal stability; c optimum pH; d steady‑state kinetic analysis Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 7 of 13 Fig. 3 Three‑ dimensional structural model by homology modeling. a KerBp. b Variant T18 the mutant keratinase more suitable for industrial appli- can modify the catalytic pocket of the enzyme (Laksmi cations, especially for degradation of feathers by com- et al. 2020) and improve its substrate specificity (Dai et al. bining keratinase with other proteases, which would 2021; Zhou et al. 2021). improve the hydrolysis efficiency of feathers. High‑density fermentation Homologous modeling analysis To measure the growth and production of mutant The structure modeling of keratinase KerBp was pre - keratinase and explore its industrial potential, the fer- dicted. The framework was consisted with ten β-folds and mentation was carried out in a 7-L fermenter. Accord- eight α-helixes, as shown in Fig. 3. Two mutation sites of ing to the regular detection of cell density and enzyme R72S/F107Y are located in the non-conserved region of activity as shown in Fig.  4, the mutant strain grew pro-peptide region and the change in amino acid hydro- logarithmically within 0–16 h, while the enzyme activ- phobicity affects spatial configuration of the pro-peptide, ity gradually increased. During 16–60 h, the cells con- leading to folding and degradation of the mature peptide. tinued to grow and tended to be stable. At this stage, Studies have shown that mutations at appropriate site of keratinase was continuously produced and accumu- pro-peptide can change the folding speed of the protein lated, and the enzyme expression level continued to and increase the activity and production of extracellular increase. Subsequently, the cells continued to grow enzyme, so the changes on pro-peptide promoted the through streaming medium and reached the maximum increase of keratinase activity (Grimsby et  al. 2010). In concentration at 92  h with OD value of 51.41. The our previous study, Su et al. (2019) carried out saturation enzyme activity continued to accumulate and reached mutations at six potential sites in the pro-peptide region the maximum with 8448 U/mL at 108 h. of keratinase, and the activity of keratinase increased There are several strategies to improve the activity from 179 to 1114  U/mL. The other two mutation points and yield of recombinant keratinase, such as promoter of N291S/N295D are located on the mature peptide, engineering (Gong et  al. 2020), signal peptide engi- and N291/N295 is close to S1 pocket region. The struc - neering (Tian et al. 2019), heterologous expression ( Jin ture of the S1 and S4 substrate pockets has an important et  al. 2019; Yong et  al. 2020), site-directed mutagen- influence on the substrate specificity of protease (Fang esis (Jaouadi et  al. 2014) and pro-peptide engineering et  al. 2015). The change of amino acids may affect S1 (Su et  al. 2019). Studies have shown that fermentation pocket structure, thus enhancing the substrate specific - optimization is one of the most important strategies ity of keratinase. The application of directed evolution for to improve keratinase activity and yield. The fermen- molecular modification in enzyme catalysis has achieved tation conditions of the neutral protease derived from favorable results (Buller et  al. 2018). Directed evolution B. subtilis were optimized, and the enzyme activity Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 8 of 13 Fig. 4 High‑ density fermentation of the variant T18 and the original strain in a 7‑L fermenter. a WT; b variant T18 was increased by nearly 2.8 times (He et  al. 2021). Feather degradation Using waste feathers as carbon source, the production Although the degradation mechanism of keratinase is of keratinase was increased by five times using deep still not fully understood, it is widely recognized that liquid fermentation optimization method (Jana et  al. reducing power plays an important role in the degra- 2020). dation process (Lange et  al. 2016; Ramnani et  al. 2005). Herein, the reducing ability of keratinase was assured by 3+ reduction of F e (Fig. 5a) and ABTS radicals scavenging Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 9 of 13 Fig. 5 Comparison of reducing power and degradation of feathers. a Reducing power (OD ). 1, pMA5; 2, WT; 3, variant T18. b ATBS radical scavenging. 1, pMA5; 2, WT; 3, variant T18. c Degradation of feathers by combined enzymes. 1, WT; 2, variant T18; 3, papain and variant T18; 4, pepsin and variant T18; 5, trypsin and variant T18; 6, bromelain and variant T18; 7, flavor protease and variant T18; 8, neutral protease and variant T18; 9, alkaline protease and variant T18 test (Fig.  5b). Reducing power measurement is based on higher than those reported previously (Table  3). The Prussian blue production as an indicator. The samples concentrations of histidine (His), glycine (Gly), threo- reduce potassium ferricyanide, and then use ferrous ions nine (Thr), tyrosine (Tyr) and lysine (Lys) increased by to generate Prussian blue. The value of the absorbance 248.7  mg/L, 197.38  mg/L, 911.82  mg/L, 241.41  mg/L at 700  nm represents the level of reducing power with a and 783.42  mg/L, respectively. Threonine increased by positive relationship between them. It was found that the 174 times from the initial value at 12  h of degradation. keratinase of the mutant strain had a stronger reducing Threonine is the second and third limiting amino acid in power than the control. This unique property indicates pig and poultry feed, respectively. It helps to adjust the that the keratinase of the mutant strain has high reduc- amino acid balance, promotes the growth of livestock, ibility and could be used in feather degradation reac- improves the nutritional value and amino acid digest- tion. Compared with the control pMA5 and the original ibility, and reduces the cost of feed materials. Therefore, keratinase, the mutant keratinase scavenged more than hydrolyzed feathers have great potential in the produc- 60% of ABTS radicals within 6  min, showing stronger tion of feed additives and amino acids. Moreover, the sol- antioxidant activity and better degradation performance. uble peptides in the degradation solution were analyzed. Since the mutant keratinase had both reducing and As shown in Fig. 7b, the molecular weight of peptides in hydrolyzing properties, when the keratinase was used the hydrolysate gradually decreased, and the molecular alone to degrade feathers, the degradation rate reached weight of peptides in the hydrolysate was concentrated 49% (Fig. 5c). In order to further improve the degradation below 1  kDa, indicating that the degradation products rate of feathers, the keratinase was compounded with were mainly composed of oligopolymer. These oligopo - trypsin and the degradation conditions were optimized, lymer are easily utilized by animals and have application by which degradation rate increased from 49 to 89% signification in feed industry (Table 3). (Fig.  6). The SEM on the structure of degraded feathers (Fig.  7a) showed that, compared with the blank control Conclusions group, after 16 h of hydrolysis, barbs, barbules and acces- In this study, a keratinase mutant with improved activ- sory pinna were completely separated, and the scapus ity was obtained via directed evolution technique broke irregularly with damage appeared on the surface. employing error-prone PCR and high-throughput The ability of keratinase to degrade feathers significantly screening. Through high-density fermentation, the in a short period provides the basis for its potential enzyme activity increased from 1150 to 8448  U/mL. application in bioconversion of keratin wastes into valu- Moreover, in order to understand the catalytic func- able protein resources. Table  2 indicates the change of tion of the existing keratinase in depth, the homolo- amino acid concentration in feather degradation prod- gous structure of the three-dimensional structure was ucts during feather degradation. After hydrolysis for modeled, and the keratinase was characterized. The 12  h, the total content of amino acids in the degrada- keratinase after mutation had better temperature sta- tion solution reached up to 4972.34  mg/mL, which was bility and optimum pH suitable for compounding. In Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 10 of 13 Fig. 6 Optimization of compound enzyme conditions. a Proportion of compound enzyme (trypsin: keratinase); b compound enzyme addition level; c temperature; d pH; e time; f sulfite addition amount Fig. 7 Scanning electron microscopy and molecular weight distribution of the degradation products. a Scanning electron microscopy; b molecular weight distribution of polypeptide Zhang  et al. Bioresources and Bioprocessing (2022) 9:38 Page 11 of 13 Table 2 The changes of amino acid concentration during feather degradation process by the variant T18 Amino acid Concentration (mg/L) 0 h 12 h 24 h 36 h asp 25.09 ± 0.31 44.05 ± 1.47 28.99 ± 1.80 23.71 ± 4.58 glu 41.12 ± 0.16 122.94 ± 3.16 11.85 ± 2.63 17.32 ± 4.12 ser 0.88 ± 0.80 12.99 ± 0.66 1.92 ± 1.32 0.80 ± 0.59 his 21.03 ± 1.82 269.73 ± 0.45 23.01 ± 1.70 17.31 ± 4.78 gly 22.16 ± 0.13 219.54 ± 6.42 56.85 ± 7.11 3.18 ± 0.27 thr 5.27 ± 0.21 917.09 ± 24.37 272.72 ± 5.64 537.47 ± 12.29 arg 11.31 ± 0.56 25.09 ± 0.43 0.58 ± 0.042 1.06 ± 0.51 ala 32.5 ± 0.62 23.80 ± 0.14 2.61 ± 0.70 3.64 ± 0.42 tyr 146.01 ± 2.09 387.42 ± 5.94 121.41 ± 4.24 114.32 ± 7.87 cys 6.38 ± 0.69 14.56 ± 1.47 4.84 ± 0.67 4.26 ± 0.50 val 165.18 ± 0.83 389.84 ± 22.32 22.86 ± 12.06 22.66 ± 2.60 met 116.92 ± 2.81 370.51 ± 15.36 72.61 ± 6.84 44.6 ± 6.52 phe 199.02 ± 0.95 688.07 ± 14.47 152.44 ± 14.56 154.41 ± 1.88 ile 63.84 ± 0.88 55.39 ± 1.64 1.80 ± 0.30 1.23 ± 0.24 leu 180.32 ± 1.63 349.90 ± 8.14 7.16 ± 1.28 6.57 ± 0.93 lys 282.93 ± 5.09 1066.35 ± 13.87 187.07 ± 6.01 149.06 ± 13.82 pro 231.74 ± 12.78 14.43 ± 1.70 113.69 ± 16.87 111.81 ± 12.49 Table 3 Comparison of amino acids in feather degradation products Amino acid Concentration (mg/L) WT (12 h) Variant T18 (12 h) KerSMD and KerSMF (48 h) Bacillus pumilus AR57 (Peng et al. 2019) keratinase (Jagadeesan et al. 2020) asp 64.66 ± 1.84 44.05 ± 1.47 15.55 ± 1.09 0.612 glu 245.91 ± 6.95 122.94 ± 3.16 20.19 ± 0.13 1.242 ser 10.00 ± 1.08 12.99 ± 0.66 14.61 ± 0.18 9.805 his 44.05 ± 0.39 269.73 ± 0.45 ND 4.794 gly 136.19 ± 7.33 219.54 ± 6.42 14.34 ± 0.24 1.421 thr 464.77 ± 56.77 917.09 ± 24.37 15.74 ± 0.22 3.514 arg 8.8 ± 0.61 25.09 ± 0.43 22.13 ± 0.49 14.110 ala 504.30 ± 5.88 23.80 ± 0.14 10.05 ± 0.28 0.517 tyr 195.87 ± 0.89 387.42 ± 5.94 171.53 ± 0.42 18.789 cys 10.82 ± 1.49 14.56 ± 1.47 ND ND val 148.99 ± 8.97 389.84 ± 22.32 207.51 ± 0.29 8.716 met 187.58 ± 11.44 370.51 ± 15.36 0 ± 0.41 7.684 phe 348.35 ± 1.36 688.07 ± 14.47 183.73 ± 0.21 33.153 ile 18.99 ± 0.94 55.39 ± 1.64 72.96 ± 0.49 4.749 leu 91.94 ± 1.88 349.90 ± 8.14 126.61 ± 0.73 7.083 lys 510.48 ± 5.08 1066.35 ± 13.87 20.94 ± 0.73 3.772 pro 28.14 ± 2.86 14.43 ± 1.70 ND ND addition, the combination of keratinase and trypsin oligopeptides, which provides protein resources for degrades feathers, and the product contains a poultry and has potential applications in the feed large number of amino acids, short peptides and industry. Zhang et al. Bioresources and Bioprocessing (2022) 9:38 Page 12 of 13 Acknowledgements Dai S, Yao Q, Yu G, Liu S, Yun J, Xiao X, Deng ZJ, Li H (2021) Biochemical charac‑ Not applicable. terization of a novel bacterial laccase and improvement of its efficiency by directed evolution on dye degradation. Front Microbiol 12:9 Authors’ contributions de Paiva DP, de Oliveira SSA, Mazotto AM, Vermelho AB, de Oliveira SS (2018) JZ: investigation, software, methodology, writing—original draft; SC: investiga‑ Keratinolytic activity of Bacillus subtilis Lfb‑fiocruz 1266 enhanced by tion, writing—review and editing; JSG and JSS: supervision, conceptualization, whole‑ cell mutagenesis. 3 Biotech 9:2 methodology, funding acquisition, writing—reviewing and editing; XLK and Ding Y, Yang Y, Ren Y, Xia J, Liu F, Li Y, Tang X‑F, Tang B (2020) Extracellular YLL: investigation, visualization; HL, JQ and ZHX: investigation, visualiza‑ production, characterization, and engineering of a polyextremotoler‑ tion, writing—review and editing. All authors read and approved the final ant subtilisin‑like protease from feather ‑ degrading Thermoactinomyces manuscript. vulgaris strain Cdf. Front Microbiol 11:605771. https:// doi. org/ 10. 3389/ fmicb. 2020. 605771 Funding Dong Y‑Z, Chang W ‑S, Chen PT (2017) Characterization and overexpression of This work was financially supported by the National Key Research and Devel‑ a novel keratinase from Bacillus polyfermenticus B4 in recombinant Bacil- opment Program of China (No. 2021YFC2100900), the National Natural Sci‑ lus subtilis. Bioresour Bioprocess 4:47 ence Foundation of China (No. 21978116), and the Ningxia Hui Autonomous Fang Z, Zhang J, Liu BH, Du GC, Chen J (2015) Insight into the substrate speci‑ Region Key Research & Development Plan (No. 2019BCH01002). ficity of keratinase Kersmd from Stenotrophomonas maltophilia by site ‑ directed mutagenesis studies in the S1 pocket. 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Bioresources and BioprocessingSpringer Journals

Published: Apr 4, 2022

Keywords: Keratinase; Error-prone PCR; Feather wastes; Biodegradation; Feed additive

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