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Cellobiose dehydrogenase from the agaricomycete Coprinellus aureogranulatus and its application for the synergistic conversion of rice straw

Cellobiose dehydrogenase from the agaricomycete Coprinellus aureogranulatus and its application... From the biotechnological viewpoint, the enzymatic disintegration of plant lignocellulosic biomass is a promising goal since it would deliver fermentable sugars for the chemical sector. Cellobiose dehydrogenase (CDH) is a vital com- ponent of the extracellular lignocellulose-degrading enzyme system of fungi and has a great potential to improve catalyst efficiency for biomass processing. In the present study, a CDH from a newly isolated strain of the agaricomy- −1 cete Coprinellus aureogranulatus (CauCDH) was successfully purified with a specific activity of 28.9 U mg . This pure enzyme (MW = 109 kDa, pI = 5.4) displayed the high oxidative activity towards β-1–4-linked oligosaccharides. Not least, CauCDH was used for the enzymatic degradation of rice straw without chemical pretreatment. As main metabo- −1 −1 −1 lites, glucose (up to 165.18 ± 3.19 mg g ), xylose (64.21 ± 1.22 mg g ), and gluconic acid (5.17 ± 0.13 mg g ) could be identified during the synergistic conversion of this raw material with the fungal hydrolases (e.g., esterase, cellulase, and xylanase) and further optimization by using an RSM statistical approach. Keywords: Cellobiose dehydrogenase, Coprinellus aureogranulatus, Lignocellulose, Rice-straw degradation, Synergetic conversion for 90% of the worldwide amount [1]. Despite the wide Introduction potential of rice straw, the challenging goal in convert- As alternative feedstock for biotechnological and indus- ing this lignocellulosic biomass is to produce value-added trial applications, lignocellulosic materials like wood chemicals at high selectivity and commercial efficiency. and agricultural residues become more and more eco- uTh s, rice straw generally contains complex cell wall nomically important, not least against the background polymers such as cellulose (32–39 wt%), hemicellulose of intensified utilization of biomass in the sense of the (20–36 wt%), and lignin (14 wt%-22 wt%), along with sil- biorefinery concept and the idea of sustainable develop - ica and other minor components (10–17wt%) [2], which ment. Agricultural crops in Asia are primarily based on provide rigidity and mechanical stability and protecting it wet rice cultivation, annual production of which leaves from microbial and enzymatic attack. roughly 600 to 800 million tons of rice straw accounting Cellobiose dehydrogenase (CDH; EC 1.1.99.18; cel- lobiose: [acceptor] 1-oxidoreductase), an extracellular *Correspondence: nghi@inpc.vast.vn flavocytochrome, is a vital component of the extracel - Institute of Natural Products Chemistry, Vietnam Academy of Science lular lignocellulose-degrading enzyme system of both and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam Full list of author information is available at the end of the article basidiomycetous and ascomycetous fungi [3]. Based on © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Nghi et al. Appl Biol Chem (2021) 64:66 Page 2 of 11 its function to support polysaccharide depolymerization fungus Xylaria polymorpha (AE, specific activity of 1.3 U −1 by lytic polysaccharide monooxygenase (LPMO), CDH is mg ) as previously described [18]. classified into the auxiliary activity families (AA3_1) that Rice straw [45.7 wt% cellulose, 22.5 wt% hemicel- cover redox enzymes acting in conjunction with carbo- lulose, 19.6 wt% lignin, and 12.3 wt% ash content (dry hydrate active enzymes (CAZymes; www. cazy. org) [4]. basis)] was collected from Cu Chi District, Ho Chi Minh, The enzyme catalyzes the oxidation of soluble cellobiose, Vietnam. The paddy straw was thoroughly rinsed and cellodextrins, mannodextrins, and lactose to their cor- air-dried before ground into pieces of ~ 40 × 40  µm by a responding lactones in the presence of a suitable elec- planetary ball mill (Fritsch, Oberstein, Germany). tron acceptor such as 2,6-dichloro-indophenol (DCIP), cytochrome c (cyt c), or metal ions. Fungal isolation Although biological function of CDHs has not been The fungus (initially designated as MPG14) growing on comprehensively elucidated yet, the previous reports deciduous deadwood was collected in the Muong Phang implicate an important role of this oxidative enzyme for primeval forest (21°27’N, 103°09’E), Dien Bien Province, bioconversion of lignocellulosic biomass into sugars by Northwest Vietnam and isolated using 2% malt-agar catalyzing the redox-mediated cleavage of glycosidic plates supplemented with antibiotics (0.005% strepto- bonds in crystalline cellulose and hemicellulose [5, 6]. mycin, penicillin, chloramphenicol, benomyl and 0.004% It is assumed to play a role in reinforcement of manga- nystatin). The fungal strain used in this study was puri - nese peroxidase, lignin peroxidase, laccase and cellulase, fied by repeated sub-culturing on potato dextrose agar reducing aromatic radicals formed during lignin degrada- (PDA; Merck, Germany) and preserved at −  80  °C (25% tion, producing lignocellulose-modifying hydroxyl radi- glycerol); it has been deposited at the Vietnam Type Cul- cals and catalyzing quinone reduction as detoxification ture Collection (VTTC, Vietnam) under accession num- mechanism during fungal plant attack. Due to their bio- ber VTCC 930003. electrochemical properties, CDHs have a broad applica- tion potential in bioprocesses such as electrobiocatalysis, Molecular identification and phylogenetic analysis bioremediation, and in enzyme-based biofuel cells [4, 5, The fungal isolate was identified based on the sequence 7, 8]. Numerous CDHs have been isolated and character- of the internal transcribed spacer (ITS) region of the ized from fungal sources including white-rot fungi, such ribosomal gene repeat unit. The genomic DNA was iso - as Phanerochaete chrysosporium [9], Trametes hirsuta lated using Plant/Fungi DNA isolation Kit (Norgenbi- [10], Schizophyllum commune [11], Sporotrichum pul- otek, Canada) and purified using the GeneJET genomic verulentum [12], soft-rot fungi like Chaetomium cellulo- DNA purification kit (Thermo Scientific, USA), follow - lyticum [13], Sporotrichum thermophile [14], Humicola ing the manufacturer’s instructions. The genomic DNA insolens [15], and the brown-rot fungus Coniophora pute- amplification was achieved by PCR technique with the ana [16]. following primers ITS1: TCC GTA GGT GAA CCT GCG To get more insight into the fungal CDH system, we G and ITS4: TCC TCC GCT TAT TGA TAT GC [19]. The describe herein the production, purification and char - 25 μL reaction mixture contained 3 μL of genomic DNA acterization of a CDH from a newly isolated fungus (10–20  ng); 7  μL distilled H O; 12.5  µL of PCR Master Coprinellus aureogranulatus (designated as CauCDH). mix kit (2X); 1.25  μL of each primer (10  pmol/μL). The This agaricomycete was chosen as one of the most effi - following PCR thermal cycle parameters were used: 94 °C cient CDH producers from nearly fifty fungal strains in for 3  min, 35 cycles of 45  s at 94  °C, 45  s at 55  °C, and our previous screening and selection strategies [17]. Not 45  s at 72  °C and a final extension at 72  °C for 10  min. least, the enzymatic degradation of rice straw without The amplified products were purified by using Qiaquick chemical pretreatment was optimized by using the pure gel extraction kit (Qiagen, USA). The sequences obtained CauCDH catalysed synergistically with various hydro- were analyzed in the ABI Prism 3100 genetic analyzer lases (e.g., esterase, cellulase, and xylanase) to improve (Applied Biosystems) and compared with GenBank data- the efficiency of the conversion process. bases (nr) using BLAST tool. Phylogenetic trees were constructed by using maxi- mum likelihood (ML) and Bayesian inference (BI) [20, 21]. The BI summarized two independent runs of four Materials and methods Markov Chains for 10,000,000 generations. A tree was Materials sampled every 100 generations, and a consensus topol- Hydrolase preparation [cellulase and xylanase (Cell/Xyl) −1 ogy was calculated for 70,000 trees after discarding the activities, 1.0–1.6  U mg ] from Trichoderma reesei was first 30,001 trees (burn-in = 3,000,000). Parameter esti- obtained from AB Enzymes (Darmstadt, Germany). The mates and convergence was checked using Tracer v1.7.1 acetyl (xylan) esterase was purified from the wood-rot Nghi  et al. Appl Biol Chem (2021) 64:66 Page 3 of 11 [22]. Strength of nodal support in the ML tree was ana- using IEF precast gels (Novex IEF gel; Invitrogen) under lyzed using non-parametric bootstrapping (MLBS) with the conditions described previously [25]. The protein 1,000 replicates. Pairwise comparisons of uncorrected bands were visualized in the gels with a colloidal blue sequence divergences (p-distance) were calculated in staining kit (Invitrogen). Protein concentrations were Mega 7.0 [23]. determined by using a Roti-Nanoquant protein assay kit (Roth, Karlsruhe, Germany) with bovine serum albumin Enzyme activity assay as the standard. CDH activity was assayed by recording the decrease in absorbance of 2,6-dichlorophenol indophenol (DCIP) at Spectral studies 520 nm in a reaction mixture (200 μL) containing 0.3 mM Spectra of purified Cau CDH were recorded in the DCIP, 30 mM lactose and 4 mM NaF in 100 mM sodium range from 250 to 700  nm in both the oxidized and the acetate buffer (pH 5.0). The reaction was initiated by add - reduced states using a Spark microplate reader (Tecan, ing an appropriate amount of enzyme extract or purified Männedorf, Switzerland). The spectrum of the reduced CDH, and incubation of reaction mixtures occurred at enzyme was prepared from its oxidized form by adding 37 °C for 5 min [24]. 500 mM of cellobiose. The purity of the enzyme from C. aureogranulatus was also assessed using the ratio of the Production and purification of cellobiose dehydrogenase absorbances at 420 and 280  nm of CDH in its oxidized The production of the CDH of C. aureogranulatus (strain form [6]. MPG14) was performed in the sterile plastic bags, each containing about 500  g rice straw. After three weeks of Eec ff ts of temperature and pH on enzyme activity fungal growth, the proteins were extracted with the dis- and stability tilled water, then the mycelium and straw particles were The pH profile of purified Cau CDH was determined removed by centrifugation (10,000 × g for 10 min) and fil - using the assay with DCIP as described above at pH tration (filter GF6; Whatman, UK). The clear supernatant values ranging from pH 4.0 to 8.0. The pH stability of was repeatedly dialyzed and concentrated in a tangential CauCDH was evaluated by incubating the enzyme at flow ultrafiltration system at 11 °C (10 kDa cut-off; Sarto - 25  °C for different time periods in 100  mM sodium cit - rius, Germany). rate buffer, pH 4.0 as well as in sodium phosphate buffer, Subsequently, the crude enzyme preparation was puri- pH 7.0 and 8.0. fied by three steps of fast protein liquid chromatography The temperature optimum was determined by measur - (FPLC) using an ÄKTA Pure system (GE Healthcare, ing the enzyme activity of CauCDH at different tempera - Danderyd, SWE) with a detector operating at 280  nm. tures (30–70  °C) in 100  mM sodium acetate buffer (pH The first and final separation steps by ion-exchange chro - 5.5). The effect of temperature on enzyme stability was matography were carried out on DEAE-Sepharose and studied at 25 °C, 50 °C, and 70 °C in sodium acetate buffer HiTrap Q FF columns, respectively, using sodium ace- (100 mM, pH 5.5). After incubation intervals of 2, 4, 6, 8, tate buffer (50 mM, pH 5.0) as the mobile phase. Elution and 12 h, aliquots of the samples were taken for measure- of the target protein was performed with the same buffer ment of the residual CDH activities. and an increasing sodium chloride gradient (0 to 1.5  M for DEAE-Sepharose and 0 to 0.5  M for the HiTrap Q Kinetic parameters and substrate specificity FF column respectively). The second purification step The Michaelis–Menten constant (K ), catalytic constant was performed by size-exclusion chromatography using (k ) of the purified enzyme from C. aureogranulatus cat a Superdex 75 column and sodium acetate buffer MPG14 were determined for various CDH substrates and (50  mM, pH 5.0) containing sodium chloride (100  mM) electron acceptors (Table  2). Kinetics of electron accep- as the eluent. The fractions with CDH activity were tors were measured at 520 nm with 20 mM cellobiose as pooled, dialyzed against 10  mM sodium acetate buffer the electron donor, while DCIP (0.3 mM) was used as the (pH 6.0), and stored at − 80 °C for further use. electron acceptor to determine the kinetics of substrate oxidations [28]. Enzyme characterization Molecular weight (M ) of the purified enzyme was Synergistic conversion of rice straw by an enzymatic determined by SDS-PAGE (NuPAGE Novex 10% Bis–Tris cocktail containing CauCDH gel; Invitrogen, Karlsruhe, Germany) using an omniPAGE The rice straw was ground to a fine powder (particle size Mini vertical electrophoresis system (Cleaver Scientific, 40 × 40  µm, determined microscopically). This mate - Warwickshire, UK). Analytical isoelectric focusing (IEF) rial (3%, wt/vol) was incubated with CauCDH (specific −1 was performed with the same electrophoresis system but activity ≥ 28.0 U mg ) and other selected enzymes Nghi et al. Appl Biol Chem (2021) 64:66 Page 4 of 11 (presented in supplementary material, Additional file  1: independent variables were expressed using the following Table  S1) in 100  mM sodium citrate buffer at pH 6.0, at second order polynomial Eq. (1). 37 °C under continuous shaking at 50 × g. To follow both k k k synergistic effects and the single effects of each enzyme, Y = b + b X + b X X + b X 0 j j u u j j (1) j j j enzymatic conversion was performed with each enzyme j=1 u,j=1 j=1 alone and in combinations on the one hand with all hydrolases [in-house AE from X. polymorpha and com- where Ŷ is the predicted response; b is the intercept mercial AB Enzymes preparation (Cell/Xyl)] and on the coefficient; b is the linear coefficient; b is the square j jj other hand, with all tested enzymes. Controls contain- coefficient; b is the interaction coefficient; X and X are uj u j ing heat-denatured enzymes (95  °C for 15  min) were the coded independent variables, terms of X X and X u j j used for comparison. After that, aliquots were taken are the intersection and quadratic terms, respectively. −1 and the amount of released carbohydrates was quanti- The three factors [Cell/Xyl, AE, and CauCDH (U g )] fied by using a high-performance liquid chromatogra - chosen for this study were designated as A, B, C, respec- phy (HPLC) system (1200 series, Agilent, Waldbronn, tively. The three variables were coded according to fol - Germany). The samples were equilibrated at 80 °C in the lowing equation: headspace oven, followed by injection onto a Rezex Z − Z ion-exclusion column [RPM-Monosaccharide Pb (8%), A, B, C = ; j = 1, 2, 3 (2) 7.8  mm × 300  mm, Phenomenex ] and monitoring the eluting substances with a refractive-index (RI) detector. max min max min Z +Z Z −Z j j j j where: Z = ;Z = Z was the For gluconic acid determination, the same HPLC system j j j 2 2 was used but equipped with a Shim-pack CLC-NH col- actual value of the variable [Cell/Xyl, AE, and CauCDH umn (150 mm × 6 mm, Shimadzu, CA, U.S.A) and a UV −1 0 (U g ); Additional file  1: Table  S1]; Z was the actual detector operating at 210 nm. value of the independent variable at the center point; and Z was the step change of the variable. A, B, C were Experimental design for optimization by response surface prescribed into three levels + 1, 0, − 1 for high, interme- methodology (RSM) diate, and low value, respectively. Enzymatic conversion of lignocellulose-rich material (3% rice straw, wt/vol) by pure CauCDH and further hydro- Statistical analysis lases was optimized to improve the efficiency of the con - Each experiment was performed in triplicate and the data version process. The ratio of enzyme to straw substrate are expressed as the mean ± SD. Statistically significant −1 (in Units per gram biomass; U g ) of each enzyme was differences were realized at p < 0.05 via Student’s t-test. varied according to the amplitude of the experiment. Statistical analysis was performed using the JMP Pro. The RSM approach, in conjunction with the Box- 13.2 software. Behnken design, was employed to estimate the effect of three main fators (enzyme concentrations of Cell/Xyl, Results AE, and CauCDH) on the product yields [Y] of glucose, Identification of the isolated fungal strain with high CDH xylose, and gluconic acid. Procedure for the construc- activity tion of the 15-experiment design matrix with the math- In order to identify the fungal isolate, total DNA was ematical-statistical treatments and the determination of extracted with suitable quality and amplified by PCR optimal conditions were carried out using Design-Expert with primers (ITS1/ITS4) at an annealing temperature 7.0.0 software (Stat-Ease, Minneapolis, MN, USA). of 55 °C. Both ends of the gene segment were sequenced Results of preliminary single factor experiments were and a sequence of 453 nucleotides in length was obtained used as inputs in an orthogonal design matrix to deter- and deposited in GenBank nucleotide database (http:// mine range and central points of variables (Additional www. ncbi. nlm. nih. gov/ genba nk/) under the acces- file  1: Tables S1 and S2). To be specific, in this investi - sion number MT427740. Nucleotide sequencing was gation, three parameters were varied individually with then compared to the reference sequences in Gen- other factors being kept in a fix (Additional file  1: Figure Bank database. The results revealed 100% identity with S2). Estimating results were tested with ANOVA analy- the sequences of species Coprinellus aureogranulatus sis to confirm model validity (Additional file  1: Table S3). GQ249274. Based on this genetic identification, and Then, optimal conditions were calculated from the final additional microscopic characteristics, the strain MPG14 model and verified by the actual experimental approach was classified as Coprinellus aureogranulatus (Psathyrel - (Fig.  5, Additional file  1: Tables S5 and S6). The depend - laceae, Agaricales, Agaricomycetes, Basidiomycota; Fig.  1 ent variables (the above product yields) as a function of and Additional file  1: Fig. S1), which is a new species for Nghi  et al. Appl Biol Chem (2021) 64:66 Page 5 of 11 Fig. 1 Phylogeny tree was constructed based on maximum likelihood ML method using the partial rDNA-ITS sequence of the MPG14 fungal strain. Psathyrella pygmaea MG734744 was used as an outgroup taxon in this tree Vietnam (www. gbif. org/ speci es/ 25345 97). Nevertheless, the few existing records indicate that it is a tropical to subtropical species [27]. Purification and physicochemical characterization of CDH from C. aureogranulatus For purification studies, a sufficient amount of CDH was produced under conditions of solid-state fermenta- tion with rice straw as growth substrate at a larger scale (0.5  kg bags) to obtain a crude extract with a total CDH activity of 1817 U (measured with DCIP). Subsequently, CDH was purified up to 41.9-fold with the yield of 22.7% Fig. 2 FPLC elution profile of the final purification step of Cau CDH starting from crude culture filtrate and resulting in a spe - performed on HiTrap Q a column: absorbance at 280 nm (solid −1 cific activity of 28.9 U mg (total 412 units) by a three- line), CDH activity (black circles), and NaCl gradient (dashed line). step purification procedure involving anion-exchange b SDS-PAGE (left) and native isoelectric focusing (right) of purified CauCDH (lane 2, 3); lanes 1 and 4, protein markers. c Spectra of the and size-exclusion chromatography. Thus, filtrated pro - oxidized (black line) and reduced (grey line) states of CauCDH tein aliquots of C. aureogranulatus’s culture extract was applied to the first chromatographic step on the weak anion exchanger DEAE-Sepharose and most of the dark polyphenolic pigments could be removed from the CDH- Figure  2c shows the UV–visible spectrum of purified exhibiting protein fraction. Thereby, the specific activity CauCDH with the typical characteristics of a flavocy - −1 of CDH increased 5.6-fold from 0.7 to 3.8 U mg . The tochrome-heme CDH. Thus, the Soret band at 422 nm of next purification step was performed on a Superdex 75 the oxidized enzyme indicates the heme b cofactor, while column and resulted in a significant increase of the spe - the broad shoulder between 450 and 500 nm is indicative cific activity of CauCDH (up to 30.9-fold), but also in a for a reduced Flavin (FAD) [3, 26]. The enzyme prepa - decrease of its total activity from 889 to 549 U. ration had a high purity with an A /A ratio of 0.6, 420 280 The purity of C. aureogranulatus CDH (Cau CDH), which is within the range of data reported previously for after the final step on HiTrap Q (Fig.  2a) columns, was other fungal CDHs [28, 29]. assessed by 12% SDS polyacrylamide gel electrophoresis The highest relative activities of Cau CDH were analysis, which revealed a distinct band with apparent observed at 50 °C and pH 5.5. The enzyme activity slightly molecular mass of 109 kDa. Purified Cau CDH exhibited decreased under suboptimal conditions, e.g., ≤ 15% activ- a weak acidic pI of 5.4 appearing as one homogeneous ity loss at 45  °C and 55  °C as well as at pH 5.0 and 6.0, protein band in the corresponding native IEF gel after respectively. A temperature of 70 °C caused a 50% activity staining with colloidal blue (Fig.  2b). The purification loss within 2 h, and at 40 °C, over 50% of the initial activ- results are summarized in Table 1. ity was still detectable after 12 h of incubation. CauCDH Nghi et al. Appl Biol Chem (2021) 64:66 Page 6 of 11 Table 1 Purification table for the CDH extracted from solid-state cultures of Coprinellus aureogranulatus (Cau CDH) Purification steps Total activity, U Total protein, mg Specific activity, U Yield, % Purification, −1 mg fold Crude extract 1817 2636 0.7 100 1.0 Ultrafiltration 1687 2239 0.8 92.8 1.1 DEAE Sepharose 889 232 3.8 48.9 5.6 Superdex 75 549 26 21.3 30.2 30.9 HiTrap Q FF 412 14 28.9 22.7 41.9 Table 2 Substrate specificity of Cau CDH −1 Substrates* Specific K (µM) k (s ) k /K m cat cat m −1 −1 activity (U (M  s ) −1 mg ) Cellobiose 19.2 117.1 30.2 25.8 × 10 Cellotriose 16.9 205.5 26.6 12.9 × 10 Cellotetraose 17.1 232.0 26.8 11.6 × 10 Lactose 39.3 731.1 61.7 84.5 × 10 Glucose 5.7 49,200 8.9 180.3 Maltose 18.7 1,354,000 29.4 21.7 *Kinetics were measured using DCIP (0.3 mM) as the electron acceptor of/for the α-1,4-linked disaccharide maltose as well as for monosaccharides such as mannose and glucose was low. Michaelis–Menten constants (K = 117.1 up to 1,354,000  µM) and catalytic efficiencies (k /K values cat m 4 −1 −1 from 25.8 × 10 to 21.7  M  s for cellobiose and malt- ose, respectively) could be determined for all tested sub- strates. The highest affinity (K = 117.1 µM) and catalytic Fig. 3 Eec ff ts of temperature (at pH 5.5; a and pH (at 25 °C; b on 4 −1 −1 the activity and stability of CauCDH at different temperatures c and efficiency (k /K = 25.8 × 10  M  s ) was ascertained cat m pH-values (d); c 25 °C (circles), 40 °C (diamonds), 70 °C (triangles) at pH for cellobiose, which is also thought to be the physiologi- 5.5; d pH 8.0 (diamonds), pH 7.0 (triangles), pH 4.0 (circles) at 25 °C in cal substrate of CauCDH. Lactose used to assay CDH 100 mM citrate/phosphate buffers. All experiments were performed activity turned out be in fact a rather suitable substrate in triplicates, standard deviation (SD) < 5% for this purpose with a high specific activity (39.3 U −1 −1 mg ) and appreciable turnover number (k = 61.7  s ). cat Two-electron acceptors such as cyt c and DCIP showed nearly identical K values (1.3 and 1.6 µM, respectively), activity significantly decreased after 2  h incubation at whilst a much higher K was observed for another accep- 70  °C probably due to less efficient interdomain elec - tor, i.e., potassium ferricyanide (18.5 µM). Moreover, the tron transfer between heme and flavin of CDH [3]. The enzyme acted just poorly on one-electron acceptors such enzyme seems to be relatively stable at acidic and neu- as 2,6-dimethyl-1,4-benzoquinone and 2,6-dimethoxy- tral conditions (i.e., pH 4.0–7.0) but lost about 80% of its 1,4-benzoquinone with K -values around 300 µM. activity within 2 h at pH 8.0 (Fig. 3). Optimization of enzymatic conversion Kinetic parameters and substrate specificities Predicted model and statistical analysis As summarized in Tables  2 and 3, the broad substrate The fundamental levels and ranges of the reaction spectrum of purified Cau CDH becomes evident by its parameters were derived from the results of single factor ability to catalyze the oxidization of different β-1–4 investigation, of which the ratio of each enzyme activ- linked di- and oligosaccharides (cellobiose, cellotriose, ity to straw substrate (i.e., Cell/Xyl, AE, and CauCDH; U −1 cellotetraose, lactose), whereas conversion and affinity g ) was selected as independent variable. In general, the Nghi  et al. Appl Biol Chem (2021) 64:66 Page 7 of 11 Table 3 Kinetic constants of CauCDH towards various electron acceptors − −1 −1 −1 Electron acceptors* Stoichiometry** (# of e ) K (μM) k (s ) k /K (M  s ) m cat cat m cyt c 2 1.3 30.1 23.2 × 10 DCIP 2 1.6 30.0 18.4 × 10 •+ 5 ABTS 1 15.6 47.4 30.4 × 10 Potassium ferricyanide 2 18.5 37.7 20.4 × 10 1,4-Benzoquinone 1 37.1 25.2 67.9 × 10 2,6-Dimethyl-1,4-benzoquinone 1 292.2 19.3 66.1 × 10 2,6-Dimethoxy-1,4-benzoquinone 1 325.5 11.4 35.0 × 10 *Cellobiose (20 mM) was used as the electron donor **Moles of electron acceptor reduced per mole of cellobiose oxidized ABTS: 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) most significant increase of metabolites (monosaccha - y-axes of 3D responsive surfaces represent the interac- rides, carboxylic acid) could be observed at the enzyme tion of couple process parameters, i.e., the interaction −1 −1 −1 ratios up to 18 U g for Cell/Xyl, 35 U g for AE, and 50 of Cell/Xyl and AE (CauCDH, 50 U g ); Cell/Xyl and −1 −1 U g for CauCDH. As the higher relative enzyme ratios CauCDH (AE, 35 U g ); AE and CauCDH (Cell/Xyl, −1 led to only a slight increase of the released metabolites 18 U g ). The z-axis represents one of three evaluation (Additional file  1: Fig. S2). Accordingly, three ratios of indices (Y-glucose, Y -xylose, and Y -gluconic acid) 1 2 3 each enzyme were chosen as the survey area of the input and was reserved for the predicted response, resulting parameters to create an experimental design matrix, such in three separate plots. The 3D response surfaces were −1 −1 as 12, 18, 24 U g of Cell/Xyl, 25, 35, 45 U g of AE, constructed as shown in Fig.  4. It could be realized that and 40, 50, 60 of CauCDH, which corresponded to the the two-way interaction affected all objective functions low level (-1), the baseline (0), and the high level (+ 1), in descending order by AB > BC > AC. This is consist - respectively (Additional file  1: Table  S1). The depend - ent with the descending influence of single parameters ent variables, i.e., the yields (Y) of released glucose (mg in order of B > A > C (the coded variables of AE, Cell/ −1 −1 −1 g ), xylose (mg g ), and gluconic acid (mg g ) were Xyl, and CauCDH, respectively) as shown in the quad- determined experimentally as shown in the experi- ratic equations (Additional file  1: Table S4). As shown in mental design matrix (Additional file  1: Table  S2). The Fig. 4a, b, an improvement in the objective functions, i.e., model outcomes from analysis of variance (ANOVA) for Y and Y , could be reached following with the increased 1 2 the objective functions (product yields after enzymatic values of the single parameters A, B, C and vice versa. conversion) were evaluated using the F-value, p-value, Uniquely, the Y was improved following the increase and R -value (Additional file  1: Table  S3). The predicted of the single parameters A and B only, while it reduced responses of the three objective functions Y , Y , and Y with a raise of the parameter C (Fig. 4c). u Th s, there was 1 2 3 and the independent variables related by quadratic poly- a direct correlation between the objective functions (i.e., nomial equations were given (Additional file  1: Table S4). Y, Y , and Y ; Fig.  4) and the single parameters (A, B, 1 2 3 ANOVA of the quadratic regression model demonstrated and C) of the model equations as shown in the Additional that all three models corresponding to three objective file  1: Table S4. Of which, the influence of square param - 2 2 2 functions fitted well and were highly significant. The eters A, B, C was insignificant in comparison to A, B, F-values of the Y, Y and Y were 84.44, 110.35 and and C, respectively, due to their variety to be in the range 1 2 3 38.60, respectively, and the p-value was less than 0.05 of (-1, + 1). indicating all models to be statistically significant. The model coefficients of determination (R ) were 0.9835, Optimization and model verification 0.9850 and 0.9858, respectively, suggesting that most of The desired function was used to optimize the objective the yield variability can be explained by the experimental functions after the enzyme catalyzed process with the data. These data support the accuracy of the established expectation for the Y, Y , and Y can reach the maxi- 1 2 3 model as well as confirm a high agreement between the mum values. As the result, 10 experimental options were measured and theoretically calculated data. found, of which the best plan to maximize the predicted objective function (as shown in Fig.  5). Thus, the opti - Response surface analysis mal values of the objective functions were Y = 165,689, −1 The estimated quadratic model was analyzed and plot - Y = 65.56, Y = 5.26 (mg g ); and the values of the 2 3 ted by using Design Expert 7.0.0 software. The x- and encoded variables at the optimal conditions were A = 0.2, Nghi et al. Appl Biol Chem (2021) 64:66 Page 8 of 11 (a) (b) (c) Fig. 4 Surface plots illustrating interaction of parameters on product yields: a Y -glucose, b Y -xylose, and c Y -gluconic acid 1 2 3 Fig. 5 Optimal conditions by solution of ramps of the model, which exhibited a negligible difference B = 0.38 and C = 0.28, respectively. Then, the real varia - (Additional file  1: Table S6). This again confirms that the bles were determined according to formula (2) to be 19.2 −1 −1 construction model was compatible with the experimen- U g for Cell/Xyl, 38.8. U g for AE, and 52.8 U g-1 for tal conditions. CauCDH, respectively (Additional file  1: Table  S5). The After optimization of three dependent variables, the experimental values at optimal conditions were com- −1 optimal factors were determined to be 19.2 U g for pared with the predicted values to determine the validity Nghi  et al. Appl Biol Chem (2021) 64:66 Page 9 of 11 −1 −1 3 −1 −1 Cell/Xyl, 38.8 U g for AE, and 52.8 U g for CauCDH catalytic efficiency (84.5 × 10  M  s ) and moderate after incubating the rice straw for 48 h at 45 °C and pH affinity (K = 731  µM), cellobiose is without doubt the 5.0. Accordingly, the objective functions were deter- physiological (i.e., natural) substrate of the enzyme, with mined to be 165.18 ± 3.19; 64.21 ± 1.22, and 5.17 ± 0.13 the highest affinity and catalytic efficiency of all sugars −1 4 −1 −1 (mg g ) for glucose, xylose, and gluconic acid. tested (K = 117.1  µM, k /K = 25.8 × 10  M  s ) m cat m −1 and appreciable turnover number (30  s ) and specific −1 Discussion activity (28.9 U mg ). In this context, kinetic proper- Most efficient lignocellulolytic fungi are found among ties of CauCDH are consistent with those of the Class- the basidiomycetous fungi degrading all components II basidiomycetous CDHs, which exhibit higher affinities of lignocellulose and hence most of the characterized and turnover numbers for cellobiose than basidiomyce- CDHs have been isolated from this group of fungi. In the tous Class-I CDHs do [3]. The enzyme acted poorly on present study, a successful purification strategy involv - the electron acceptors 2,6-dimethyl-1,4-benzoquinone ing different steps of anion-exchange and size exclusion and 2,6-dimethoxy-1,4-benzoquinone with one to two chromatography was developed to obtain a homoge- orders of magnitude higher K -values compared to other neous CDH protein with high activity from solid-state acceptors (cyt c, DCIP, 1,4-benzoquinone) examined. cultures of the agaricomycete Coprinellus aureogranu- We assume that substitution of 1,4-benzoquinone with latus (CauCDH). The approx. 42-fold purified CauCDH two methyl or methoxy groups caused a drastic activity recovered after the final step on a HiTrap Q column reduction due to steric hindrance that was also observed was comparatively high with a yield of 22.7%. Thus, the for a CDH from the corticoid plant pathogen Sclerotium recovery of purified Cau CDH is comparable to percent- (Athelia) rolfsii [35]. ages reported for the CDHs from the polypores Cer- The ability of purified Cau CDH to catalyze the con- rena unicolor [28] and Trametes hirsuta [10] and the version of the lignocellulosic material as an auxiliary agaric Termitomyces clypeatus [30], but higher than that enzyme in cooperation with polysaccharide hydrolases, reported for the CDH of another polypore, Pycnoporus i.e., cellulase/xylanase (Cell/Xyl) and acetyl esterase (AE), −1 sanguineus (14.7%) [31]. The specific activity (28.9  mg was tested with rice straw as target, without any chemi- at pH 5.0 and 37 °C) of purified Cau CDH for DCIP was cal pre-treatment. As main metabolites, glucose, xylose, found to be noticeably higher than the respective activi- and gluconic acid were detected, which are all biotech- ties of other CDHs from basidiomycetous fungi [10, 29, nological interest (as renewable resources for subsequent 30]. In general, however, the physical and catalytic prop- biological or chemical conversions). Thus, supplementa - erties of CauCDH, i.e., M (109 kDa), pI (5.4), optimum tion of purified CDH to the optimized rice-straw reac - pH and temperature (pH 5.5 and 50  °C, respectively), tion setup, led to a moderate but significant increase of have been expectable and within the range of reported all product yields. Cellobiose accumulation during the values for other basidiomycetous CDHs, such as of P. hydrolytic process may have caused a feedback inhibition chrysosporium [32], Schizophyllum commune [11], T. hir- of cellobiohydrolases and endoglucanases. It is reason- suta [10], and Trametes versicolor [33]. able to assume that the role of fungal CDHs is to facilitate Among the various carbohydrates tested, di- and oligo- lignocellulose degradation for the concomitant attack, saccharides with β-(1,4) glycosidic bonds were the best depolymerizing enzymes. Similar effects were reported substrates, for which CauCDH exhibited high specific for the synergistic acting CDHs from P. chrysosporium activities, whereas substrates with α-(1,4)-interlinkages [36] or V. volvacea [5] and cellulases from T. reesei. Here, as well as monosaccharides were just converted very carbohydrate yields were increased by adding CDH, slowly and with low catalytic efficiencies. Thus, CDHs resulting in an increased formation of gluconic acid due preferably catalyse the oxidation of soluble β-(1,4)-linked to the hydrolysis of cellobionic acid by β-glucosidase that saccharides over that of monosaccharides, which seems was also present in the T. reesei enzymatic cocktail [37]. to be an inherent property of this fungal enzyme type In addition, the desired enzymatic activity/function was [34]. Increasing the number of glucosyl units in the cello- applied in this study to optimize the objective functions oligosaccharides tested resulted in decreasing substrate (i.e., the yields of carbohydrates and gluconic acid) dur- affinity (higher K values) and lower catalytic efficien - ing the enzyme-catalyzed process with the expectation of cies (k /K ). This phenomenon has also been observed their max values will reach. Accordingly, the conversion cat m for other CDHs such as those from Volvariella volvacea of rice straw meal by hydrolyzing enzymes, i.e., cellulases, [5] and T. clypeatus [30]. Although lactose is structurally xylanases (Cell/Xyl), and/or acetyl esterase (AE), in the similar to cellobiose and CauCDH exhibited the highest presence of CauCDH could be optimized with increased specific activity and turnover number for this substrate amounts of released C-5 and C-6 sugars (glucose, xylose) −1 −1 (39.3 U mg and 61.7  s , respectively) along with high as well as of gluconic acid. Nghi et al. Appl Biol Chem (2021) 64:66 Page 10 of 11 2. Goodman BA (2020) Utilization of waste straw and husks from rice pro- Future approaches in the field of lignocellulose con - duction: a review. J Biores Biopro 5:143–162 version by CDH-containing enzymatic cocktails should 3. Harreither W, Sygmund C, Augustin M, Narciso M, Rabinovich ML, Gorton include LPMOs, the most probable physiological part- L, Haltrich D, Ludwig R (2011) Catalytic properties and classification of cellobiose dehydrogenases from ascomycetes. Appl Envir Microbiol ners of CDHs. 77:1804–1815 4. Scheiblbrandner S, Ludwig R (2020) Cellobiose dehydrogenase: bioelec- Supplementary Information trochemical insights and applications. Bioelectrochem. https:// doi. org/ 10. 1016/j. bioel echem. 2019. 107345 The online version contains supplementary material available at https:// doi. 5. Chen K, Liu X, Long L, Ding S (2017) Cellobiose dehydrogenase from Vol- org/ 10. 1186/ s13765- 021- 00637-y. variella volvacea and its effect on the saccharification of cellulose. Proces Biochem 60:52–58 Additional file 1: Fig. S1. Internal Transcribed Spacer (ITS) region of nuclear 6. Rai R, Basotra N, Kaur B, Falco MD, Tsang A, Chadha BS (2020) Exopro- DNA (rDNA) was used to identify fungal taxonomy of MPG14. Fig. S2. teome profile reveals thermophilic fungus Crassicarpon thermophilum Single factor investigations of the effect of enzyme ratio (units per gram (strain 6GKB; syn. Corynascus thermophilus) as a rich source of cellobiose substrate; U g-1) on the yields of carbohydrates [glucose (diamond; mg dehydrogenase for enhanced saccharification of bagasse. Biomas Bioen- g-1), xylose (triangle; mg g-1)] and gluconic acid (circle; mg g-1). A The erg. https:// doi. org/ 10. 1016/j. biomb ioe. 2019. 105438 effect of various Cell/Xyl ratio and fixed AE (25 U g-1) and CaCDH (40 7. Baldrian P, Valášková V (2008) Degradation of cellulose by basidiomycet- U g-1); B The effect of various AE ratio and fixed Cell/Xyl (18 U g-1) and ous fungi. FEMS Microbiol Rev 32:501–521 CaCDH (40 U g-1); C The effect of various CaCDH ratio and fixed Cell/Xyl 8. Cameron MD, Aust SD (2001) Cellobiose dehydrogenase-an extracellular (18 U g-1) and CaCDH (40 U g-1). Table S1. Ranges of parameters deter- fungal flavocytochrome. Enzym Microb Technol 28:129–138 mined by experimental design. Table S2. Empirical data compared with 9. Bao W, Usha S, Renganathan V (1993) Purification and characterization actual and predicted response values. Table S3. Regression coefficients of cellobiose dehydrogenase, a novel extracellular hemoflavoenzyme of the predicted second-order polynomial models for the amount of from the white-rot fungus Phanerochaete chrysosporium. Arch Biochem released glucose (Y1), xylose (Y2) and gluconic acid (Y3). Table S4. Empiri- Biophys 300:705–713 cal second-order polynomial models of the glucose (Y1), xylose (Y2) and 10. Nakagame S, Furujyo A, Sugiura J (2006) Purification and characterization gluconic acid content (Y3). Table S5. Values of the independent variable of cellobiose dehydrogenase from white-rot basidiomycete Trametes and real variables. Table S6. Predicted response values and experimental hirsuta. Biosci Biotechnol Biochem 70:1629–1635 response values obtained under optimum conditions. 11. Fang J, Liu W, Gao P (1998) Cellobiose dehydrogenase from Schizophyl- lum commune: Purification and study of some catalytic, inactivation, and cellulose-binding properties. Arch Biochem Biophys 353:37–46 Acknowledgements 12. Ayers AR, Ayers SB, Eriksson KE (1978) Cellobiose oxidase, purification and This research is partly funded by Vietnam National Foundation for Science and partial characterization of a hemoprotein from Sporotrichum pulverulen- Technology Development (NAFOSTED) under grant number FWO.104.2017.03, tum. Eur J Biochem 90:171–181 and Ministry of Science and Technology (NĐT.45.GER/18), as well as the 13. Fähnrich P, Irrgang K (1982) Conversion of cellulose to sugars and cellobi- Bundesministerium für Bildung und Forschung (BMBF) VnmDiv 031B0627 and onic acid by the extracellular enzyme system of Chaetomium cellulolyti- CEFOX 031B0831B. cum. Biotechnol Lett 4:775–780 14. Coudray MR, Canevascini G, Meier H (1982) Characterization of a cello- Authors’ contributions biose dehydrogenase in the cellulolytic fungus Sporotrichum (Chrys- DHN, MH, HK project design and funding acquisition. DHN, HK, TTNH, DTQ, CL osporium) thermophile. Biochem J 203:277–284 experimental performance, data analysis, and manuscript preparation. LMH, 15. Schou C, Christensen HM, Schulein M (1998) Characterization of a cel- AV, LD, CL, MH, participated in data analysis and manuscript revision. LXD lobiose dehydrogenase from Humicola insolens. Biochem J 330:565–571 joined in experimental design for optimization. All authors read and approved 16. Schmidhalter DR, Canevascini G (1993) solation and characterization of the final manuscript. the cellobiose dehydrogenase from the brown-rot fungus Coniophora puteana (Schum ex Fr.) Karst. Arch Biochem Biophys 300:559–563 17. Giap VD, Hiep TTM, Nghi DH (2020) Cellulose-dehydrogenase produc- Declarations tion by some fungal species isolated in rain forests of northern Vietnam. Vietnam J Biotechnol 18:135–145 Competing interests 18. Nghi D, Ullrich R, Moritz F, Huong L, Giap V, Chi D, Hofrichter M, Liers C The authors declare no competing interests. (2015) The ascomycete Xylaria polymorpha produces an acetyl esterase that solubilises beech wood material to release water-soluble lignin frag- Author details ments. Appl Biol Chem 58:415–421 Institute of Natural Products Chemistry, Vietnam Academy of Science 19. White T, Bruns T, Lee S, Taylor F, White TJ, Lee SH, Taylor L, Taylor JS (1990) and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam. Graduate Universit y Amplification and direct sequencing of fungal ribosomal RNA genes for of Science and Technology, Vietnam Academy of Science and Technology, 18 phylogenetics. Academic Press, New York Hoang Quoc Viet, Hanoi, Vietnam. 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Molecul Biolog Evolut 33:1870–1874 24. Harreither W, Sygmund C, Dunhofen E, Vicuña R, Haltrich D, Ludwig R (2009) Cellobiose dehydrogenase from the ligninolytic basidiomycete References Ceriporiopsis subvermispora. Appl Environ Microbiol 75:2750–2757 1. Abraham A, Mathew AK, Sindhu R, Pandey A, Binod P (2016) Potential of rice straw for bio-refining: an overview. Bioresour Technol 215:29–36 Nghi  et al. Appl Biol Chem (2021) 64:66 Page 11 of 11 25. Nghi DH, Bittner B, Kellner H, Jehmlich N, Ullrich R, Pecyna MJ, Nousiainen 32. Lehner D, Zipper P, Henriksson G, Pettersson G (1996) Small-angle X-ray P, Sipilä J, Huong LM, Hofrichter M, Liers C (2012) The wood-rot ascomy- scattering studies on cellobiose dehydrogenase from Phanerochaete cete Xylaria polymorpha produces a novel GH78 glycoside hydrolase that chrysosporium. Biochem Biophys Acta 1293:161–169 exhibits α-L-rhamnosidase and feruloyl esterase activity and releases 33. 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Saha T, Ghosh D, Mukherjee S, Bose S, Mukherjee M (2008) Cellobiose Publisher’s Note dehydrogenase production by the mycelial culture of the mushroom Springer Nature remains neutral with regard to jurisdictional claims in pub- Termitomyces clypeatus. Proces Biochem 43:634–641 lished maps and institutional affiliations. 31. Sulej J, Janusz G, Osińska-Jaroszuk M, Małek P, Mazur A, Komaniecka I, Rogalski J (2013) Characterization of cellobiose dehydrogenase and its FAD-domain from the ligninolytic basidiomycete Pycnoporus sanguineus. Enzym Microb Technol 53:427–437 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Biological Chemistry Springer Journals

Cellobiose dehydrogenase from the agaricomycete Coprinellus aureogranulatus and its application for the synergistic conversion of rice straw

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Abstract

From the biotechnological viewpoint, the enzymatic disintegration of plant lignocellulosic biomass is a promising goal since it would deliver fermentable sugars for the chemical sector. Cellobiose dehydrogenase (CDH) is a vital com- ponent of the extracellular lignocellulose-degrading enzyme system of fungi and has a great potential to improve catalyst efficiency for biomass processing. In the present study, a CDH from a newly isolated strain of the agaricomy- −1 cete Coprinellus aureogranulatus (CauCDH) was successfully purified with a specific activity of 28.9 U mg . This pure enzyme (MW = 109 kDa, pI = 5.4) displayed the high oxidative activity towards β-1–4-linked oligosaccharides. Not least, CauCDH was used for the enzymatic degradation of rice straw without chemical pretreatment. As main metabo- −1 −1 −1 lites, glucose (up to 165.18 ± 3.19 mg g ), xylose (64.21 ± 1.22 mg g ), and gluconic acid (5.17 ± 0.13 mg g ) could be identified during the synergistic conversion of this raw material with the fungal hydrolases (e.g., esterase, cellulase, and xylanase) and further optimization by using an RSM statistical approach. Keywords: Cellobiose dehydrogenase, Coprinellus aureogranulatus, Lignocellulose, Rice-straw degradation, Synergetic conversion for 90% of the worldwide amount [1]. Despite the wide Introduction potential of rice straw, the challenging goal in convert- As alternative feedstock for biotechnological and indus- ing this lignocellulosic biomass is to produce value-added trial applications, lignocellulosic materials like wood chemicals at high selectivity and commercial efficiency. and agricultural residues become more and more eco- uTh s, rice straw generally contains complex cell wall nomically important, not least against the background polymers such as cellulose (32–39 wt%), hemicellulose of intensified utilization of biomass in the sense of the (20–36 wt%), and lignin (14 wt%-22 wt%), along with sil- biorefinery concept and the idea of sustainable develop - ica and other minor components (10–17wt%) [2], which ment. Agricultural crops in Asia are primarily based on provide rigidity and mechanical stability and protecting it wet rice cultivation, annual production of which leaves from microbial and enzymatic attack. roughly 600 to 800 million tons of rice straw accounting Cellobiose dehydrogenase (CDH; EC 1.1.99.18; cel- lobiose: [acceptor] 1-oxidoreductase), an extracellular *Correspondence: nghi@inpc.vast.vn flavocytochrome, is a vital component of the extracel - Institute of Natural Products Chemistry, Vietnam Academy of Science lular lignocellulose-degrading enzyme system of both and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam Full list of author information is available at the end of the article basidiomycetous and ascomycetous fungi [3]. Based on © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Nghi et al. Appl Biol Chem (2021) 64:66 Page 2 of 11 its function to support polysaccharide depolymerization fungus Xylaria polymorpha (AE, specific activity of 1.3 U −1 by lytic polysaccharide monooxygenase (LPMO), CDH is mg ) as previously described [18]. classified into the auxiliary activity families (AA3_1) that Rice straw [45.7 wt% cellulose, 22.5 wt% hemicel- cover redox enzymes acting in conjunction with carbo- lulose, 19.6 wt% lignin, and 12.3 wt% ash content (dry hydrate active enzymes (CAZymes; www. cazy. org) [4]. basis)] was collected from Cu Chi District, Ho Chi Minh, The enzyme catalyzes the oxidation of soluble cellobiose, Vietnam. The paddy straw was thoroughly rinsed and cellodextrins, mannodextrins, and lactose to their cor- air-dried before ground into pieces of ~ 40 × 40  µm by a responding lactones in the presence of a suitable elec- planetary ball mill (Fritsch, Oberstein, Germany). tron acceptor such as 2,6-dichloro-indophenol (DCIP), cytochrome c (cyt c), or metal ions. Fungal isolation Although biological function of CDHs has not been The fungus (initially designated as MPG14) growing on comprehensively elucidated yet, the previous reports deciduous deadwood was collected in the Muong Phang implicate an important role of this oxidative enzyme for primeval forest (21°27’N, 103°09’E), Dien Bien Province, bioconversion of lignocellulosic biomass into sugars by Northwest Vietnam and isolated using 2% malt-agar catalyzing the redox-mediated cleavage of glycosidic plates supplemented with antibiotics (0.005% strepto- bonds in crystalline cellulose and hemicellulose [5, 6]. mycin, penicillin, chloramphenicol, benomyl and 0.004% It is assumed to play a role in reinforcement of manga- nystatin). The fungal strain used in this study was puri - nese peroxidase, lignin peroxidase, laccase and cellulase, fied by repeated sub-culturing on potato dextrose agar reducing aromatic radicals formed during lignin degrada- (PDA; Merck, Germany) and preserved at −  80  °C (25% tion, producing lignocellulose-modifying hydroxyl radi- glycerol); it has been deposited at the Vietnam Type Cul- cals and catalyzing quinone reduction as detoxification ture Collection (VTTC, Vietnam) under accession num- mechanism during fungal plant attack. Due to their bio- ber VTCC 930003. electrochemical properties, CDHs have a broad applica- tion potential in bioprocesses such as electrobiocatalysis, Molecular identification and phylogenetic analysis bioremediation, and in enzyme-based biofuel cells [4, 5, The fungal isolate was identified based on the sequence 7, 8]. Numerous CDHs have been isolated and character- of the internal transcribed spacer (ITS) region of the ized from fungal sources including white-rot fungi, such ribosomal gene repeat unit. The genomic DNA was iso - as Phanerochaete chrysosporium [9], Trametes hirsuta lated using Plant/Fungi DNA isolation Kit (Norgenbi- [10], Schizophyllum commune [11], Sporotrichum pul- otek, Canada) and purified using the GeneJET genomic verulentum [12], soft-rot fungi like Chaetomium cellulo- DNA purification kit (Thermo Scientific, USA), follow - lyticum [13], Sporotrichum thermophile [14], Humicola ing the manufacturer’s instructions. The genomic DNA insolens [15], and the brown-rot fungus Coniophora pute- amplification was achieved by PCR technique with the ana [16]. following primers ITS1: TCC GTA GGT GAA CCT GCG To get more insight into the fungal CDH system, we G and ITS4: TCC TCC GCT TAT TGA TAT GC [19]. The describe herein the production, purification and char - 25 μL reaction mixture contained 3 μL of genomic DNA acterization of a CDH from a newly isolated fungus (10–20  ng); 7  μL distilled H O; 12.5  µL of PCR Master Coprinellus aureogranulatus (designated as CauCDH). mix kit (2X); 1.25  μL of each primer (10  pmol/μL). The This agaricomycete was chosen as one of the most effi - following PCR thermal cycle parameters were used: 94 °C cient CDH producers from nearly fifty fungal strains in for 3  min, 35 cycles of 45  s at 94  °C, 45  s at 55  °C, and our previous screening and selection strategies [17]. Not 45  s at 72  °C and a final extension at 72  °C for 10  min. least, the enzymatic degradation of rice straw without The amplified products were purified by using Qiaquick chemical pretreatment was optimized by using the pure gel extraction kit (Qiagen, USA). The sequences obtained CauCDH catalysed synergistically with various hydro- were analyzed in the ABI Prism 3100 genetic analyzer lases (e.g., esterase, cellulase, and xylanase) to improve (Applied Biosystems) and compared with GenBank data- the efficiency of the conversion process. bases (nr) using BLAST tool. Phylogenetic trees were constructed by using maxi- mum likelihood (ML) and Bayesian inference (BI) [20, 21]. The BI summarized two independent runs of four Materials and methods Markov Chains for 10,000,000 generations. A tree was Materials sampled every 100 generations, and a consensus topol- Hydrolase preparation [cellulase and xylanase (Cell/Xyl) −1 ogy was calculated for 70,000 trees after discarding the activities, 1.0–1.6  U mg ] from Trichoderma reesei was first 30,001 trees (burn-in = 3,000,000). Parameter esti- obtained from AB Enzymes (Darmstadt, Germany). The mates and convergence was checked using Tracer v1.7.1 acetyl (xylan) esterase was purified from the wood-rot Nghi  et al. Appl Biol Chem (2021) 64:66 Page 3 of 11 [22]. Strength of nodal support in the ML tree was ana- using IEF precast gels (Novex IEF gel; Invitrogen) under lyzed using non-parametric bootstrapping (MLBS) with the conditions described previously [25]. The protein 1,000 replicates. Pairwise comparisons of uncorrected bands were visualized in the gels with a colloidal blue sequence divergences (p-distance) were calculated in staining kit (Invitrogen). Protein concentrations were Mega 7.0 [23]. determined by using a Roti-Nanoquant protein assay kit (Roth, Karlsruhe, Germany) with bovine serum albumin Enzyme activity assay as the standard. CDH activity was assayed by recording the decrease in absorbance of 2,6-dichlorophenol indophenol (DCIP) at Spectral studies 520 nm in a reaction mixture (200 μL) containing 0.3 mM Spectra of purified Cau CDH were recorded in the DCIP, 30 mM lactose and 4 mM NaF in 100 mM sodium range from 250 to 700  nm in both the oxidized and the acetate buffer (pH 5.0). The reaction was initiated by add - reduced states using a Spark microplate reader (Tecan, ing an appropriate amount of enzyme extract or purified Männedorf, Switzerland). The spectrum of the reduced CDH, and incubation of reaction mixtures occurred at enzyme was prepared from its oxidized form by adding 37 °C for 5 min [24]. 500 mM of cellobiose. The purity of the enzyme from C. aureogranulatus was also assessed using the ratio of the Production and purification of cellobiose dehydrogenase absorbances at 420 and 280  nm of CDH in its oxidized The production of the CDH of C. aureogranulatus (strain form [6]. MPG14) was performed in the sterile plastic bags, each containing about 500  g rice straw. After three weeks of Eec ff ts of temperature and pH on enzyme activity fungal growth, the proteins were extracted with the dis- and stability tilled water, then the mycelium and straw particles were The pH profile of purified Cau CDH was determined removed by centrifugation (10,000 × g for 10 min) and fil - using the assay with DCIP as described above at pH tration (filter GF6; Whatman, UK). The clear supernatant values ranging from pH 4.0 to 8.0. The pH stability of was repeatedly dialyzed and concentrated in a tangential CauCDH was evaluated by incubating the enzyme at flow ultrafiltration system at 11 °C (10 kDa cut-off; Sarto - 25  °C for different time periods in 100  mM sodium cit - rius, Germany). rate buffer, pH 4.0 as well as in sodium phosphate buffer, Subsequently, the crude enzyme preparation was puri- pH 7.0 and 8.0. fied by three steps of fast protein liquid chromatography The temperature optimum was determined by measur - (FPLC) using an ÄKTA Pure system (GE Healthcare, ing the enzyme activity of CauCDH at different tempera - Danderyd, SWE) with a detector operating at 280  nm. tures (30–70  °C) in 100  mM sodium acetate buffer (pH The first and final separation steps by ion-exchange chro - 5.5). The effect of temperature on enzyme stability was matography were carried out on DEAE-Sepharose and studied at 25 °C, 50 °C, and 70 °C in sodium acetate buffer HiTrap Q FF columns, respectively, using sodium ace- (100 mM, pH 5.5). After incubation intervals of 2, 4, 6, 8, tate buffer (50 mM, pH 5.0) as the mobile phase. Elution and 12 h, aliquots of the samples were taken for measure- of the target protein was performed with the same buffer ment of the residual CDH activities. and an increasing sodium chloride gradient (0 to 1.5  M for DEAE-Sepharose and 0 to 0.5  M for the HiTrap Q Kinetic parameters and substrate specificity FF column respectively). The second purification step The Michaelis–Menten constant (K ), catalytic constant was performed by size-exclusion chromatography using (k ) of the purified enzyme from C. aureogranulatus cat a Superdex 75 column and sodium acetate buffer MPG14 were determined for various CDH substrates and (50  mM, pH 5.0) containing sodium chloride (100  mM) electron acceptors (Table  2). Kinetics of electron accep- as the eluent. The fractions with CDH activity were tors were measured at 520 nm with 20 mM cellobiose as pooled, dialyzed against 10  mM sodium acetate buffer the electron donor, while DCIP (0.3 mM) was used as the (pH 6.0), and stored at − 80 °C for further use. electron acceptor to determine the kinetics of substrate oxidations [28]. Enzyme characterization Molecular weight (M ) of the purified enzyme was Synergistic conversion of rice straw by an enzymatic determined by SDS-PAGE (NuPAGE Novex 10% Bis–Tris cocktail containing CauCDH gel; Invitrogen, Karlsruhe, Germany) using an omniPAGE The rice straw was ground to a fine powder (particle size Mini vertical electrophoresis system (Cleaver Scientific, 40 × 40  µm, determined microscopically). This mate - Warwickshire, UK). Analytical isoelectric focusing (IEF) rial (3%, wt/vol) was incubated with CauCDH (specific −1 was performed with the same electrophoresis system but activity ≥ 28.0 U mg ) and other selected enzymes Nghi et al. Appl Biol Chem (2021) 64:66 Page 4 of 11 (presented in supplementary material, Additional file  1: independent variables were expressed using the following Table  S1) in 100  mM sodium citrate buffer at pH 6.0, at second order polynomial Eq. (1). 37 °C under continuous shaking at 50 × g. To follow both k k k synergistic effects and the single effects of each enzyme, Y = b + b X + b X X + b X 0 j j u u j j (1) j j j enzymatic conversion was performed with each enzyme j=1 u,j=1 j=1 alone and in combinations on the one hand with all hydrolases [in-house AE from X. polymorpha and com- where Ŷ is the predicted response; b is the intercept mercial AB Enzymes preparation (Cell/Xyl)] and on the coefficient; b is the linear coefficient; b is the square j jj other hand, with all tested enzymes. Controls contain- coefficient; b is the interaction coefficient; X and X are uj u j ing heat-denatured enzymes (95  °C for 15  min) were the coded independent variables, terms of X X and X u j j used for comparison. After that, aliquots were taken are the intersection and quadratic terms, respectively. −1 and the amount of released carbohydrates was quanti- The three factors [Cell/Xyl, AE, and CauCDH (U g )] fied by using a high-performance liquid chromatogra - chosen for this study were designated as A, B, C, respec- phy (HPLC) system (1200 series, Agilent, Waldbronn, tively. The three variables were coded according to fol - Germany). The samples were equilibrated at 80 °C in the lowing equation: headspace oven, followed by injection onto a Rezex Z − Z ion-exclusion column [RPM-Monosaccharide Pb (8%), A, B, C = ; j = 1, 2, 3 (2) 7.8  mm × 300  mm, Phenomenex ] and monitoring the eluting substances with a refractive-index (RI) detector. max min max min Z +Z Z −Z j j j j where: Z = ;Z = Z was the For gluconic acid determination, the same HPLC system j j j 2 2 was used but equipped with a Shim-pack CLC-NH col- actual value of the variable [Cell/Xyl, AE, and CauCDH umn (150 mm × 6 mm, Shimadzu, CA, U.S.A) and a UV −1 0 (U g ); Additional file  1: Table  S1]; Z was the actual detector operating at 210 nm. value of the independent variable at the center point; and Z was the step change of the variable. A, B, C were Experimental design for optimization by response surface prescribed into three levels + 1, 0, − 1 for high, interme- methodology (RSM) diate, and low value, respectively. Enzymatic conversion of lignocellulose-rich material (3% rice straw, wt/vol) by pure CauCDH and further hydro- Statistical analysis lases was optimized to improve the efficiency of the con - Each experiment was performed in triplicate and the data version process. The ratio of enzyme to straw substrate are expressed as the mean ± SD. Statistically significant −1 (in Units per gram biomass; U g ) of each enzyme was differences were realized at p < 0.05 via Student’s t-test. varied according to the amplitude of the experiment. Statistical analysis was performed using the JMP Pro. The RSM approach, in conjunction with the Box- 13.2 software. Behnken design, was employed to estimate the effect of three main fators (enzyme concentrations of Cell/Xyl, Results AE, and CauCDH) on the product yields [Y] of glucose, Identification of the isolated fungal strain with high CDH xylose, and gluconic acid. Procedure for the construc- activity tion of the 15-experiment design matrix with the math- In order to identify the fungal isolate, total DNA was ematical-statistical treatments and the determination of extracted with suitable quality and amplified by PCR optimal conditions were carried out using Design-Expert with primers (ITS1/ITS4) at an annealing temperature 7.0.0 software (Stat-Ease, Minneapolis, MN, USA). of 55 °C. Both ends of the gene segment were sequenced Results of preliminary single factor experiments were and a sequence of 453 nucleotides in length was obtained used as inputs in an orthogonal design matrix to deter- and deposited in GenBank nucleotide database (http:// mine range and central points of variables (Additional www. ncbi. nlm. nih. gov/ genba nk/) under the acces- file  1: Tables S1 and S2). To be specific, in this investi - sion number MT427740. Nucleotide sequencing was gation, three parameters were varied individually with then compared to the reference sequences in Gen- other factors being kept in a fix (Additional file  1: Figure Bank database. The results revealed 100% identity with S2). Estimating results were tested with ANOVA analy- the sequences of species Coprinellus aureogranulatus sis to confirm model validity (Additional file  1: Table S3). GQ249274. Based on this genetic identification, and Then, optimal conditions were calculated from the final additional microscopic characteristics, the strain MPG14 model and verified by the actual experimental approach was classified as Coprinellus aureogranulatus (Psathyrel - (Fig.  5, Additional file  1: Tables S5 and S6). The depend - laceae, Agaricales, Agaricomycetes, Basidiomycota; Fig.  1 ent variables (the above product yields) as a function of and Additional file  1: Fig. S1), which is a new species for Nghi  et al. Appl Biol Chem (2021) 64:66 Page 5 of 11 Fig. 1 Phylogeny tree was constructed based on maximum likelihood ML method using the partial rDNA-ITS sequence of the MPG14 fungal strain. Psathyrella pygmaea MG734744 was used as an outgroup taxon in this tree Vietnam (www. gbif. org/ speci es/ 25345 97). Nevertheless, the few existing records indicate that it is a tropical to subtropical species [27]. Purification and physicochemical characterization of CDH from C. aureogranulatus For purification studies, a sufficient amount of CDH was produced under conditions of solid-state fermenta- tion with rice straw as growth substrate at a larger scale (0.5  kg bags) to obtain a crude extract with a total CDH activity of 1817 U (measured with DCIP). Subsequently, CDH was purified up to 41.9-fold with the yield of 22.7% Fig. 2 FPLC elution profile of the final purification step of Cau CDH starting from crude culture filtrate and resulting in a spe - performed on HiTrap Q a column: absorbance at 280 nm (solid −1 cific activity of 28.9 U mg (total 412 units) by a three- line), CDH activity (black circles), and NaCl gradient (dashed line). step purification procedure involving anion-exchange b SDS-PAGE (left) and native isoelectric focusing (right) of purified CauCDH (lane 2, 3); lanes 1 and 4, protein markers. c Spectra of the and size-exclusion chromatography. Thus, filtrated pro - oxidized (black line) and reduced (grey line) states of CauCDH tein aliquots of C. aureogranulatus’s culture extract was applied to the first chromatographic step on the weak anion exchanger DEAE-Sepharose and most of the dark polyphenolic pigments could be removed from the CDH- Figure  2c shows the UV–visible spectrum of purified exhibiting protein fraction. Thereby, the specific activity CauCDH with the typical characteristics of a flavocy - −1 of CDH increased 5.6-fold from 0.7 to 3.8 U mg . The tochrome-heme CDH. Thus, the Soret band at 422 nm of next purification step was performed on a Superdex 75 the oxidized enzyme indicates the heme b cofactor, while column and resulted in a significant increase of the spe - the broad shoulder between 450 and 500 nm is indicative cific activity of CauCDH (up to 30.9-fold), but also in a for a reduced Flavin (FAD) [3, 26]. The enzyme prepa - decrease of its total activity from 889 to 549 U. ration had a high purity with an A /A ratio of 0.6, 420 280 The purity of C. aureogranulatus CDH (Cau CDH), which is within the range of data reported previously for after the final step on HiTrap Q (Fig.  2a) columns, was other fungal CDHs [28, 29]. assessed by 12% SDS polyacrylamide gel electrophoresis The highest relative activities of Cau CDH were analysis, which revealed a distinct band with apparent observed at 50 °C and pH 5.5. The enzyme activity slightly molecular mass of 109 kDa. Purified Cau CDH exhibited decreased under suboptimal conditions, e.g., ≤ 15% activ- a weak acidic pI of 5.4 appearing as one homogeneous ity loss at 45  °C and 55  °C as well as at pH 5.0 and 6.0, protein band in the corresponding native IEF gel after respectively. A temperature of 70 °C caused a 50% activity staining with colloidal blue (Fig.  2b). The purification loss within 2 h, and at 40 °C, over 50% of the initial activ- results are summarized in Table 1. ity was still detectable after 12 h of incubation. CauCDH Nghi et al. Appl Biol Chem (2021) 64:66 Page 6 of 11 Table 1 Purification table for the CDH extracted from solid-state cultures of Coprinellus aureogranulatus (Cau CDH) Purification steps Total activity, U Total protein, mg Specific activity, U Yield, % Purification, −1 mg fold Crude extract 1817 2636 0.7 100 1.0 Ultrafiltration 1687 2239 0.8 92.8 1.1 DEAE Sepharose 889 232 3.8 48.9 5.6 Superdex 75 549 26 21.3 30.2 30.9 HiTrap Q FF 412 14 28.9 22.7 41.9 Table 2 Substrate specificity of Cau CDH −1 Substrates* Specific K (µM) k (s ) k /K m cat cat m −1 −1 activity (U (M  s ) −1 mg ) Cellobiose 19.2 117.1 30.2 25.8 × 10 Cellotriose 16.9 205.5 26.6 12.9 × 10 Cellotetraose 17.1 232.0 26.8 11.6 × 10 Lactose 39.3 731.1 61.7 84.5 × 10 Glucose 5.7 49,200 8.9 180.3 Maltose 18.7 1,354,000 29.4 21.7 *Kinetics were measured using DCIP (0.3 mM) as the electron acceptor of/for the α-1,4-linked disaccharide maltose as well as for monosaccharides such as mannose and glucose was low. Michaelis–Menten constants (K = 117.1 up to 1,354,000  µM) and catalytic efficiencies (k /K values cat m 4 −1 −1 from 25.8 × 10 to 21.7  M  s for cellobiose and malt- ose, respectively) could be determined for all tested sub- strates. The highest affinity (K = 117.1 µM) and catalytic Fig. 3 Eec ff ts of temperature (at pH 5.5; a and pH (at 25 °C; b on 4 −1 −1 the activity and stability of CauCDH at different temperatures c and efficiency (k /K = 25.8 × 10  M  s ) was ascertained cat m pH-values (d); c 25 °C (circles), 40 °C (diamonds), 70 °C (triangles) at pH for cellobiose, which is also thought to be the physiologi- 5.5; d pH 8.0 (diamonds), pH 7.0 (triangles), pH 4.0 (circles) at 25 °C in cal substrate of CauCDH. Lactose used to assay CDH 100 mM citrate/phosphate buffers. All experiments were performed activity turned out be in fact a rather suitable substrate in triplicates, standard deviation (SD) < 5% for this purpose with a high specific activity (39.3 U −1 −1 mg ) and appreciable turnover number (k = 61.7  s ). cat Two-electron acceptors such as cyt c and DCIP showed nearly identical K values (1.3 and 1.6 µM, respectively), activity significantly decreased after 2  h incubation at whilst a much higher K was observed for another accep- 70  °C probably due to less efficient interdomain elec - tor, i.e., potassium ferricyanide (18.5 µM). Moreover, the tron transfer between heme and flavin of CDH [3]. The enzyme acted just poorly on one-electron acceptors such enzyme seems to be relatively stable at acidic and neu- as 2,6-dimethyl-1,4-benzoquinone and 2,6-dimethoxy- tral conditions (i.e., pH 4.0–7.0) but lost about 80% of its 1,4-benzoquinone with K -values around 300 µM. activity within 2 h at pH 8.0 (Fig. 3). Optimization of enzymatic conversion Kinetic parameters and substrate specificities Predicted model and statistical analysis As summarized in Tables  2 and 3, the broad substrate The fundamental levels and ranges of the reaction spectrum of purified Cau CDH becomes evident by its parameters were derived from the results of single factor ability to catalyze the oxidization of different β-1–4 investigation, of which the ratio of each enzyme activ- linked di- and oligosaccharides (cellobiose, cellotriose, ity to straw substrate (i.e., Cell/Xyl, AE, and CauCDH; U −1 cellotetraose, lactose), whereas conversion and affinity g ) was selected as independent variable. In general, the Nghi  et al. Appl Biol Chem (2021) 64:66 Page 7 of 11 Table 3 Kinetic constants of CauCDH towards various electron acceptors − −1 −1 −1 Electron acceptors* Stoichiometry** (# of e ) K (μM) k (s ) k /K (M  s ) m cat cat m cyt c 2 1.3 30.1 23.2 × 10 DCIP 2 1.6 30.0 18.4 × 10 •+ 5 ABTS 1 15.6 47.4 30.4 × 10 Potassium ferricyanide 2 18.5 37.7 20.4 × 10 1,4-Benzoquinone 1 37.1 25.2 67.9 × 10 2,6-Dimethyl-1,4-benzoquinone 1 292.2 19.3 66.1 × 10 2,6-Dimethoxy-1,4-benzoquinone 1 325.5 11.4 35.0 × 10 *Cellobiose (20 mM) was used as the electron donor **Moles of electron acceptor reduced per mole of cellobiose oxidized ABTS: 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) most significant increase of metabolites (monosaccha - y-axes of 3D responsive surfaces represent the interac- rides, carboxylic acid) could be observed at the enzyme tion of couple process parameters, i.e., the interaction −1 −1 −1 ratios up to 18 U g for Cell/Xyl, 35 U g for AE, and 50 of Cell/Xyl and AE (CauCDH, 50 U g ); Cell/Xyl and −1 −1 U g for CauCDH. As the higher relative enzyme ratios CauCDH (AE, 35 U g ); AE and CauCDH (Cell/Xyl, −1 led to only a slight increase of the released metabolites 18 U g ). The z-axis represents one of three evaluation (Additional file  1: Fig. S2). Accordingly, three ratios of indices (Y-glucose, Y -xylose, and Y -gluconic acid) 1 2 3 each enzyme were chosen as the survey area of the input and was reserved for the predicted response, resulting parameters to create an experimental design matrix, such in three separate plots. The 3D response surfaces were −1 −1 as 12, 18, 24 U g of Cell/Xyl, 25, 35, 45 U g of AE, constructed as shown in Fig.  4. It could be realized that and 40, 50, 60 of CauCDH, which corresponded to the the two-way interaction affected all objective functions low level (-1), the baseline (0), and the high level (+ 1), in descending order by AB > BC > AC. This is consist - respectively (Additional file  1: Table  S1). The depend - ent with the descending influence of single parameters ent variables, i.e., the yields (Y) of released glucose (mg in order of B > A > C (the coded variables of AE, Cell/ −1 −1 −1 g ), xylose (mg g ), and gluconic acid (mg g ) were Xyl, and CauCDH, respectively) as shown in the quad- determined experimentally as shown in the experi- ratic equations (Additional file  1: Table S4). As shown in mental design matrix (Additional file  1: Table  S2). The Fig. 4a, b, an improvement in the objective functions, i.e., model outcomes from analysis of variance (ANOVA) for Y and Y , could be reached following with the increased 1 2 the objective functions (product yields after enzymatic values of the single parameters A, B, C and vice versa. conversion) were evaluated using the F-value, p-value, Uniquely, the Y was improved following the increase and R -value (Additional file  1: Table  S3). The predicted of the single parameters A and B only, while it reduced responses of the three objective functions Y , Y , and Y with a raise of the parameter C (Fig. 4c). u Th s, there was 1 2 3 and the independent variables related by quadratic poly- a direct correlation between the objective functions (i.e., nomial equations were given (Additional file  1: Table S4). Y, Y , and Y ; Fig.  4) and the single parameters (A, B, 1 2 3 ANOVA of the quadratic regression model demonstrated and C) of the model equations as shown in the Additional that all three models corresponding to three objective file  1: Table S4. Of which, the influence of square param - 2 2 2 functions fitted well and were highly significant. The eters A, B, C was insignificant in comparison to A, B, F-values of the Y, Y and Y were 84.44, 110.35 and and C, respectively, due to their variety to be in the range 1 2 3 38.60, respectively, and the p-value was less than 0.05 of (-1, + 1). indicating all models to be statistically significant. The model coefficients of determination (R ) were 0.9835, Optimization and model verification 0.9850 and 0.9858, respectively, suggesting that most of The desired function was used to optimize the objective the yield variability can be explained by the experimental functions after the enzyme catalyzed process with the data. These data support the accuracy of the established expectation for the Y, Y , and Y can reach the maxi- 1 2 3 model as well as confirm a high agreement between the mum values. As the result, 10 experimental options were measured and theoretically calculated data. found, of which the best plan to maximize the predicted objective function (as shown in Fig.  5). Thus, the opti - Response surface analysis mal values of the objective functions were Y = 165,689, −1 The estimated quadratic model was analyzed and plot - Y = 65.56, Y = 5.26 (mg g ); and the values of the 2 3 ted by using Design Expert 7.0.0 software. The x- and encoded variables at the optimal conditions were A = 0.2, Nghi et al. Appl Biol Chem (2021) 64:66 Page 8 of 11 (a) (b) (c) Fig. 4 Surface plots illustrating interaction of parameters on product yields: a Y -glucose, b Y -xylose, and c Y -gluconic acid 1 2 3 Fig. 5 Optimal conditions by solution of ramps of the model, which exhibited a negligible difference B = 0.38 and C = 0.28, respectively. Then, the real varia - (Additional file  1: Table S6). This again confirms that the bles were determined according to formula (2) to be 19.2 −1 −1 construction model was compatible with the experimen- U g for Cell/Xyl, 38.8. U g for AE, and 52.8 U g-1 for tal conditions. CauCDH, respectively (Additional file  1: Table  S5). The After optimization of three dependent variables, the experimental values at optimal conditions were com- −1 optimal factors were determined to be 19.2 U g for pared with the predicted values to determine the validity Nghi  et al. Appl Biol Chem (2021) 64:66 Page 9 of 11 −1 −1 3 −1 −1 Cell/Xyl, 38.8 U g for AE, and 52.8 U g for CauCDH catalytic efficiency (84.5 × 10  M  s ) and moderate after incubating the rice straw for 48 h at 45 °C and pH affinity (K = 731  µM), cellobiose is without doubt the 5.0. Accordingly, the objective functions were deter- physiological (i.e., natural) substrate of the enzyme, with mined to be 165.18 ± 3.19; 64.21 ± 1.22, and 5.17 ± 0.13 the highest affinity and catalytic efficiency of all sugars −1 4 −1 −1 (mg g ) for glucose, xylose, and gluconic acid. tested (K = 117.1  µM, k /K = 25.8 × 10  M  s ) m cat m −1 and appreciable turnover number (30  s ) and specific −1 Discussion activity (28.9 U mg ). In this context, kinetic proper- Most efficient lignocellulolytic fungi are found among ties of CauCDH are consistent with those of the Class- the basidiomycetous fungi degrading all components II basidiomycetous CDHs, which exhibit higher affinities of lignocellulose and hence most of the characterized and turnover numbers for cellobiose than basidiomyce- CDHs have been isolated from this group of fungi. In the tous Class-I CDHs do [3]. The enzyme acted poorly on present study, a successful purification strategy involv - the electron acceptors 2,6-dimethyl-1,4-benzoquinone ing different steps of anion-exchange and size exclusion and 2,6-dimethoxy-1,4-benzoquinone with one to two chromatography was developed to obtain a homoge- orders of magnitude higher K -values compared to other neous CDH protein with high activity from solid-state acceptors (cyt c, DCIP, 1,4-benzoquinone) examined. cultures of the agaricomycete Coprinellus aureogranu- We assume that substitution of 1,4-benzoquinone with latus (CauCDH). The approx. 42-fold purified CauCDH two methyl or methoxy groups caused a drastic activity recovered after the final step on a HiTrap Q column reduction due to steric hindrance that was also observed was comparatively high with a yield of 22.7%. Thus, the for a CDH from the corticoid plant pathogen Sclerotium recovery of purified Cau CDH is comparable to percent- (Athelia) rolfsii [35]. ages reported for the CDHs from the polypores Cer- The ability of purified Cau CDH to catalyze the con- rena unicolor [28] and Trametes hirsuta [10] and the version of the lignocellulosic material as an auxiliary agaric Termitomyces clypeatus [30], but higher than that enzyme in cooperation with polysaccharide hydrolases, reported for the CDH of another polypore, Pycnoporus i.e., cellulase/xylanase (Cell/Xyl) and acetyl esterase (AE), −1 sanguineus (14.7%) [31]. The specific activity (28.9  mg was tested with rice straw as target, without any chemi- at pH 5.0 and 37 °C) of purified Cau CDH for DCIP was cal pre-treatment. As main metabolites, glucose, xylose, found to be noticeably higher than the respective activi- and gluconic acid were detected, which are all biotech- ties of other CDHs from basidiomycetous fungi [10, 29, nological interest (as renewable resources for subsequent 30]. In general, however, the physical and catalytic prop- biological or chemical conversions). Thus, supplementa - erties of CauCDH, i.e., M (109 kDa), pI (5.4), optimum tion of purified CDH to the optimized rice-straw reac - pH and temperature (pH 5.5 and 50  °C, respectively), tion setup, led to a moderate but significant increase of have been expectable and within the range of reported all product yields. Cellobiose accumulation during the values for other basidiomycetous CDHs, such as of P. hydrolytic process may have caused a feedback inhibition chrysosporium [32], Schizophyllum commune [11], T. hir- of cellobiohydrolases and endoglucanases. It is reason- suta [10], and Trametes versicolor [33]. able to assume that the role of fungal CDHs is to facilitate Among the various carbohydrates tested, di- and oligo- lignocellulose degradation for the concomitant attack, saccharides with β-(1,4) glycosidic bonds were the best depolymerizing enzymes. Similar effects were reported substrates, for which CauCDH exhibited high specific for the synergistic acting CDHs from P. chrysosporium activities, whereas substrates with α-(1,4)-interlinkages [36] or V. volvacea [5] and cellulases from T. reesei. Here, as well as monosaccharides were just converted very carbohydrate yields were increased by adding CDH, slowly and with low catalytic efficiencies. Thus, CDHs resulting in an increased formation of gluconic acid due preferably catalyse the oxidation of soluble β-(1,4)-linked to the hydrolysis of cellobionic acid by β-glucosidase that saccharides over that of monosaccharides, which seems was also present in the T. reesei enzymatic cocktail [37]. to be an inherent property of this fungal enzyme type In addition, the desired enzymatic activity/function was [34]. Increasing the number of glucosyl units in the cello- applied in this study to optimize the objective functions oligosaccharides tested resulted in decreasing substrate (i.e., the yields of carbohydrates and gluconic acid) dur- affinity (higher K values) and lower catalytic efficien - ing the enzyme-catalyzed process with the expectation of cies (k /K ). This phenomenon has also been observed their max values will reach. Accordingly, the conversion cat m for other CDHs such as those from Volvariella volvacea of rice straw meal by hydrolyzing enzymes, i.e., cellulases, [5] and T. clypeatus [30]. Although lactose is structurally xylanases (Cell/Xyl), and/or acetyl esterase (AE), in the similar to cellobiose and CauCDH exhibited the highest presence of CauCDH could be optimized with increased specific activity and turnover number for this substrate amounts of released C-5 and C-6 sugars (glucose, xylose) −1 −1 (39.3 U mg and 61.7  s , respectively) along with high as well as of gluconic acid. Nghi et al. Appl Biol Chem (2021) 64:66 Page 10 of 11 2. Goodman BA (2020) Utilization of waste straw and husks from rice pro- Future approaches in the field of lignocellulose con - duction: a review. J Biores Biopro 5:143–162 version by CDH-containing enzymatic cocktails should 3. Harreither W, Sygmund C, Augustin M, Narciso M, Rabinovich ML, Gorton include LPMOs, the most probable physiological part- L, Haltrich D, Ludwig R (2011) Catalytic properties and classification of cellobiose dehydrogenases from ascomycetes. Appl Envir Microbiol ners of CDHs. 77:1804–1815 4. Scheiblbrandner S, Ludwig R (2020) Cellobiose dehydrogenase: bioelec- Supplementary Information trochemical insights and applications. Bioelectrochem. https:// doi. org/ 10. 1016/j. bioel echem. 2019. 107345 The online version contains supplementary material available at https:// doi. 5. Chen K, Liu X, Long L, Ding S (2017) Cellobiose dehydrogenase from Vol- org/ 10. 1186/ s13765- 021- 00637-y. variella volvacea and its effect on the saccharification of cellulose. Proces Biochem 60:52–58 Additional file 1: Fig. S1. Internal Transcribed Spacer (ITS) region of nuclear 6. Rai R, Basotra N, Kaur B, Falco MD, Tsang A, Chadha BS (2020) Exopro- DNA (rDNA) was used to identify fungal taxonomy of MPG14. Fig. S2. teome profile reveals thermophilic fungus Crassicarpon thermophilum Single factor investigations of the effect of enzyme ratio (units per gram (strain 6GKB; syn. Corynascus thermophilus) as a rich source of cellobiose substrate; U g-1) on the yields of carbohydrates [glucose (diamond; mg dehydrogenase for enhanced saccharification of bagasse. Biomas Bioen- g-1), xylose (triangle; mg g-1)] and gluconic acid (circle; mg g-1). A The erg. https:// doi. org/ 10. 1016/j. biomb ioe. 2019. 105438 effect of various Cell/Xyl ratio and fixed AE (25 U g-1) and CaCDH (40 7. Baldrian P, Valášková V (2008) Degradation of cellulose by basidiomycet- U g-1); B The effect of various AE ratio and fixed Cell/Xyl (18 U g-1) and ous fungi. FEMS Microbiol Rev 32:501–521 CaCDH (40 U g-1); C The effect of various CaCDH ratio and fixed Cell/Xyl 8. Cameron MD, Aust SD (2001) Cellobiose dehydrogenase-an extracellular (18 U g-1) and CaCDH (40 U g-1). Table S1. Ranges of parameters deter- fungal flavocytochrome. Enzym Microb Technol 28:129–138 mined by experimental design. Table S2. Empirical data compared with 9. Bao W, Usha S, Renganathan V (1993) Purification and characterization actual and predicted response values. Table S3. Regression coefficients of cellobiose dehydrogenase, a novel extracellular hemoflavoenzyme of the predicted second-order polynomial models for the amount of from the white-rot fungus Phanerochaete chrysosporium. Arch Biochem released glucose (Y1), xylose (Y2) and gluconic acid (Y3). Table S4. Empiri- Biophys 300:705–713 cal second-order polynomial models of the glucose (Y1), xylose (Y2) and 10. Nakagame S, Furujyo A, Sugiura J (2006) Purification and characterization gluconic acid content (Y3). Table S5. Values of the independent variable of cellobiose dehydrogenase from white-rot basidiomycete Trametes and real variables. Table S6. Predicted response values and experimental hirsuta. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Cellobiose dehydrogenase; Coprinellus aureogranulatus; Lignocellulose; Rice-straw degradation; Synergetic conversion

References