- Original Article
- Open Access
Characterization of acidic endoglucanase Cel12A from Gloeophyllum trabeum and its synergistic effects on hydrogen peroxide–acetic acid (HPAC)-pretreated lignocellulose
Journal of Wood Science volume 65, Article number: 24 (2019)
Gloeophyllum trabeum is a potent filamentous fungus that rapidly decomposes lignocellulose. In the present study, we cloned the G. trabeum cel12a gene and expressed it in Pichia pastoris strain GS115. The purified recombinant GtCel12A exhibited high pH stability and very high specific enzymic activity against β-glucan (6546 U mg−1) and carboxymethyl cellulose (1129 U mg−1) compared to GtCel5B, endoglucanases from Trichoderma reesei, and other glycoside hydrolase family 12 (GH12) enzymes. GtCel12A exhibited high enzymic activity with regard to hydrogen peroxide–acetic acid (HPAC)-pretreated lignocellulose biomass, and produced cellobiose as a major product with a small quantity of glucose. In combination with commercial cellulase, this enzyme also showed synergistic effects of 14.5, 16.1, 29.0, and 13.4% on filter paper, HPAC-pretreated pine, corn stover, and rice straw, respectively. The acidic endoglucanase GtCel12A from G. trabeum is a promising tool that can be used in combination with cellulase against HPAC-pretreated lignocellulose.
Approximately 200 billion tons of lignocellulose are produced every year. These renewable polymers can be used to produce biofuel or biochemicals without competing with the first-generation feedstocks . Enzymatic hydrolysis, which converts lignocellulose into its monomeric sugars, requires a hydrolytic enzyme complex . A synergistic cooperation of cellulases, comprising exo-1,4-β-glucanases (EC 22.214.171.124 and EC 126.96.36.199) and endoglucanase (EC188.8.131.52), is important for the complete hydrolysis of lignocellulose. Endoglucanases, which hydrolyze the internal glucosidic bonds, provide exo-1,4-β-glucanases with new reducing and nonreducing ends. Enzymes have been widely used in the pulp and paper, textile, bioethanol, wine and brewery, food processing, animal feed, agricultural, biomolecular chemical products, and pharmaceutical industries [1, 3, 4]. Cellulases comprise approximately 20% of the global enzyme market . Currently, the main source for commercial and industrial cellulases is Trichoderma reesei, a well-studied and -established fungus used to produce cellulase at a scale of more than 100 g L−1 [6, 7]. However, the production cost of cellulase accounts for approximately 40% of the total cost of bioethanol production from lignocellulose biomass . Therefore, the development of effective enzymes or an enzyme cocktail is essential to enhance enzyme efficiency.
Among bacteria and fungi, brown rot basidiomycetes have been studied extensively for their ability to rapidly decompose lignocellulosic biomass, despite lacking the processive cellobiohydrolases . Cellulase-encoding gene sequences have been reported in Gloeophyllum trabeum . This fungus is also one of the most potent and a well-studied microorganism that degrades lignocelluloses [8, 10,11,12]. G. trabeum is able to depolymerize lignocellulose biomass using a unique system combining nonenzymatic lignocellulose degradation, which is Fenton-type reactions mediated by reducing Fe3+ and O2 with two hydroquinones [11, 12], with enzymatic lignocellulose degradation [8, 10].
Endoglucanases are grouped into 14 glycoside hydrolase (GH) families within the Carbohydrate-Active enZYmes database (CAZy; http://www.cazy.org/Glycoside-Hydrolases.html). Among these, the GH 12 family has a β-jelly roll structure with relatively low molecular mass and several catalytic activities, including endoglucanase (EC 184.108.40.206), xyloglucan hydrolase (EC 220.127.116.11), β-1,3-1,4-glucanase (EC 18.104.22.168), and xyloglucan endotransglucosylase (EC 22.214.171.124). Most of GH12 have two conserved cystines region that form a disulfide bridge, which offers enzyme structures local stabilization. This family of endoglucanases plays a critical role during the hydrolysis of β-1,3-1,4 and β-1,4-glucan with high activities .
A variety of recombinant endoglucanases and xyloglucan-specific endoglucanases in the GH12 family have been cloned and studied using various hetero-expression hosts [14,15,16,17,18]. A G. trabeum endoglucanase expressed in a host, Aspergillus niger, has been characterized in terms of molecular weight, thermal stability, and optimal activity conditions [16, 19]. Tambor et al.  have also reported a synergistic relationship between endoglucanases and T. reesei Cel7A with regard to the digestion of filter paper. However, there is insufficient information about enzyme activity with regard to the digestion of lignocellulose substrates and synergistic relationship with cellulase. Pichia pastoris provides a well-studied eukaryotic heterologous expression system with numerous advantages, including protein folding, protein processing, posttranslational modification, and high expression [20,21,22]. In the present study, we describe the expression, purification, and biochemical characterization of acidic GtCel12A from P. pastoris. We also studied the synergistic effects of GtCel12A with commercial cellulase on hydrogen peroxide–acetic acid (HPAC)-pretreated lignocellulose from pine, corn stover, and rice straw.
Materials and methods
Pine wood and chips were obtained from the arboretum of Chonnam National University, South Korea. Rice straw and corn stover were purchased online (http://www.gmarket.co.kr/). Lignocellulose biomass were pretreated with HPAC, according to a procedure reported previously . Following pretreatment, residues after the pretreatment were washed with hot water until attaining neutral pH and then freeze-dried.
Microbial strains, media, and cultivation conditions
The filamentous fungus G. trabeum strain ATCC11539 was used as a gene source for cel12a. The fungus was grown on potato dextrose agar (PDA) medium at 30 °C for 5 days and then transferred into 100 mL Avicel medium (10 g L−1 Avicel, 5 g L−1 (NH4)2SO4, 2 g L−1 KH2PO4, 0.3 g L−1 CaCl2, 0.3 g L−1 MgSO4·7H2O, 1 g L−1 protease peptone, 0.2% (v/v) Tween 80, and 0.2% (v/v) trace elements solution) trace elements solution; 5 g L−1 FeSO4·7H2O, 1.6 g L−1 MnSO4·H2O, 1.4 g L−1 ZnSO4·7H2O, and 2 g L−1 CoCl2) contained in a 500-mL baffle flask at 30 °C for 5 days in a 180 rpm shaking incubator. The TOP10 E. coli strain was transformed and grown on Luria–Bertani (LB) medium at 37 °C for 18 h. Yeast strain Pichia pastoris GS115 was grown step-by-step on YPD media (10 g L−1 yeast extract, 20 g L−1 protease peptone, and 10 g L−1 dextrose), YPG (10 g L−1 yeast extract, 20 g L−1 protease peptone, and 1% (v/v) glycerol) at 30 °C for 24 h, respectively. After cultivation on the YPG medium, the yeast cell was harvested and transferred onto YP medium (10 g L−1 yeast extract, 20 g L−1 protease peptone) with 0.8% (v/v) methyl alcohol added every 24 h, for heterologous protein expression at 30 °C for 4 days.
Gtcel12a gene cloning
The G. trabeum was harvested for total RNA extraction by centrifuging at 13,000 rpm (Avanti J-E, Beckman, Fullerton, CA, USA) for 30 min after cultivation in PDB medium with 1% (w/v) Avicel. To extract total RNA from G. trabeum, the TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) was used. The SMART cDNA library construction kit (TaKaRa, Mountain View, CA, USA) was used for cDNA synthesis with isolated RNA. Gene sequences of GtCel12a were amplified by Polymerase chain reaction (PCR) using designed primers (GtCel12A-F 5′-GAA TTC GCG ACC GTG CTC ACT GGT CAA TAC-3′ and GtCel12A-R 5′-CTG CAG CCC GCT CAA GCT GAC GCT GAA-3′) without the signal peptide. PCR was performed in a volume of 20 μL, containing 3 μL first strand cDNA, 2 μL 10× Ex Taq buffer, 2 μL dNTP, 1 μL each of forward and reverse primers, and 0.1 μL Ex Taq DNA polymerase (5 U μL−1) under the following conditions: 1 cycle of denaturation at 95 °C for 30 s, and 35 cycles of annealing at 60 °C for 30 s, extension at 72 °C for 60 s. The resulting PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) with EcoRI and PstI restriction enzymes and then sequenced and transformed into TOP10F E. coli strain. The transformants were selected on LB medium with ampicillin.
Heterologous enzyme expression, purification, western blot, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis
The Gtcel12a gene was subcloned into the pPICZαA vector (Invitrogen) with restriction enzymes, EcoRI and PstI, and then it was transformed into host strain, P. pastoris GS115, for heterologous expression. The transformants were selected on YPD medium with the antibiotics zeocin. Cells were harvested by centrifuging at 13,000 rpm for 30 min. The supernatant was then filtered through a GF/C glass microfiber filter (1.2 μm) to remove any remaining yeast cells, and then purified using a column, containing 1 mL Ni–NTA matrix (Qiagen, Hilden, Germany). GtCel12A expression was confirmed through a western blot assay with mouse anti-His-tagged antibody (Abfrontier, Seoul, South Korea). The purified enzymes were dialyzed in 20 mM tris-hydrochloride buffer (pH 8.0). The protein concentration was determined by a Bradford assay at 595 nm with bovine serum albumin (BSA) as a standard. SDS-PAGE analysis was conducted to determine the molecular mass of a heterologous expressed enzyme using a 12% (w/v) polyacrylamide gel stained with Coomassie brilliant blue R-250.
The optimal pH was determined by measuring the relative activity of enzymes in 50 mM buffers ranging from pH 2.0 to 9.0, glycine-hydrochloric acid (pH 2.0–3.0), sodium acetate (pH 4.0–5.0), sodium phosphate (pH 6.0–7.0), and tris-hydrochloride (pH 8.0–9.0) in 1% (w/v) solution of carboxymethyl cellulose (CMC) at 50 °C, following a dinitrosalicylic acid (DNS) assay by using a spectrophotometer (Multiskan EX, Thermo Fisher Scientific, Vantaa, Finland) at 550 nm. The optimal temperature was determined by monitoring the relative activity at temperature ranging from 20 to 70 °C in 50 mM glycine-hydrochloric acid buffer (pH 2.5) with CMC. pH stability was assessed according to the residual enzyme activity after preincubation in various buffers (ranging from pH 2.0 to 9.0) at 4 °C for 18 h. Thermal stability was likewise determined by monitoring residual enzyme activity after preincubation at 30–60 °C for 0–120 min.
Nine materials were used as enzyme assay substrates: Avicel PH-101 (microcrystalline cellulose, ca. 50 μm), xyloglucan from tamarind, β-glucan from barley (Megazyme), filter paper (Whatman number 1), locust bean gum from Ceratonia siliqua seeds, xylan from beech wood, carrageenan, CMC, and soluble starch (all purchased from Sigma). These substrates were prepared as 1% stock solutions. Each enzymatic assay was performed in triplicate in a final volume of 100 μL, containing 50 μL substrate stocks, 50 mM glycine-hydrochloric acid buffer (pH 2.5), and diluted enzyme. The kinetic parameters, Km and Vmax, were evaluated with 0.25–2.0% CMC as a substrate for 5 min under optimal conditions (pH 2.5, 50 °C) and estimated using Lineweaver–Burk plot. To determine the effects of chemical and ions, 50 mM of EDTA, NaCl, LiCl, KCl, CoCl2, ZnCl2, CaCl2, MnCl2, HgCl2 AlCl3, and FeCl3 were tested. Reducing sugars were measured using dinitrosalicylic acid reagent with a glucose standard curve . One unit (U) was defined as the amount of enzyme required to produce 1 μmol of reducing sugars per min. Products degraded from oligosaccharides were prepared by purified enzymes in a 100 μL final volume with 50 mM glycine-hydrochloric acid buffer (pH 2.5), diluted enzyme, and each substrate, containing 1% (w/v) cellobiose, cellotriose, cellotetraose, or cellopentaose. These mixtures were incubated at 50 °C for 1 to 20 min, followed by loading 1 μL of released products on thin-layer chromatography (TLC) silica gel 60 plates and separated with n-BuOH/CH3O2H/H2O (2:1:1 v/v). Sugars were detected using a 5% (v/v) H2SO4 in methanol staining solution .
Enzymatic hydrolysis and synergistic effect with commercial cellulase
Hydrolysis was performed under their optimal conditions of GtCel12A and GtCel5B . Synergistic effects were examined using a 3% (w/v) solution of HPAC-pretreated lignocellulose and filter paper, 50 mM citrate buffer (pH 4.8), 10 FPU g−1 commercial cellulase (Celluclast 1.5L, Novozymes, Denmark), and 30 U g−1 in pine, 50 U g−1 GtCel12A in filter paper, corn stover, and rice straw at 48 °C for 24 h. The reducing sugars were determined by a DNS assay and high-performance liquid chromatography (HPLC) (Waters, Milford, MA, USA) with a Rezex RPM column (300 × 4.6 mm; Phenomenes, Torrance, CA, USA). The analysis conditions were 85 and 40 °C in the column and refractive index detector, respectively, with deionized water at a flow rate of 0.6 mL min−1.
Results and discussion
Gene cloning and sequence analysis
We obtained the cel12a gene (National Center for Biotechnology Information (NCBI) accession number HQ163778) from G. trabeum ATCC 11539 and confirmed it by sequence analysis. This gene includes 741 nucleotides, encoding 246 amino acids with a calculated molecular weight of 26.17 kDa and a theoretical isoelectric point of 4.10. The SignalP 4.1 server (http://www.cbs.dtu.dk/server/SignalP) predicted that GtCel12A has signal peptides from amino acids 1 to 21. The amino acid sequence of the GtCel12A was aligned by BLAST (http://www.uniprot.org/blast/) with 12 other fungal family endoglucanases and visualized using the Vector NTI software. Multiple alignment analysis showed that the GtCel12A had high identities with other brown-rot fugal GH12 endoglucanases, Neolentinus lepideus (87.3% identity), Laetiporus sulphureus (65.2% identity), Postia placenta (67.4% identity), and Daedalea quercina (63.8% identity) (Additional file 1: Fig. S1). Therefore, these results indicate that GtCel12A belongs to the typical glycolyl hydrolase family 12, which is a specific endoglucanase of brown-rot fungi.
Expression and purification of recombinant GtCel12A in the P. pastoris
GtCel12A has been reported in its native form, and in heterologous protein expression from Aspergillus niger [10, 16, 19]. The Gtcel12a gene was expressed in the host P. pastoris GS115 using a pPICZαA vector without signal peptides, owing to an unfavorable N-terminal secretion signal in the yeast . The expressed enzyme was purified using a Ni–NTA agarose affinity column and dialyzed with 20 mM tris-hydrochloride buffer (pH 8.0). The expressed GtCel12A protein was confirmed by western blot assay (Additional file 1: Fig. S2a). SDS-PAGE showed that the molecular weight of the purified protein from P. pastoris was approximately 31 kDa, which was larger than the calculated molecular mass (Additional file 1: Fig. S2b). Recombinant GtCel12A expressed from A. niger strain CBS513.88 and py11 had different protein molecular masses, of 29 and 26.1 kDa, respectively [16, 19]. Recombinant EGIIIs (Cel12A) of Trichoderma reesei was expressed in heterologous hosts E. coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, which likewise showed different molecular masses (25, 28, and 29 kDa, respectively) due to N-glycosylation by the different hosts. The molecular mass was decreased to 25 kDa by endoglycosidase H and α-mannosidase treatment . These increases in molecular weight may be at least partly related to glycosylation by a heterologous host in the recombinant GtCel12A protein.
Characterization of the GtCel12A
To determine the biochemical characterization of GtCel12A, the effect of pH and temperatures was conducted on the CMC substrate. The GtCel12A expressed from P. pastoris exhibited enzyme activity over a range of extremely acidic conditions (pH 2.0–4.0) with an optimal pH 2.5, showed residual relative enzyme activity exceeding 80% across the range of pH conditions tested, and was highly stable at base conditions of pH 8.0–9.0 (Fig. 1a). In contrast, the GtCel12A expressed by A. niger has an optimal pH of 4.5, indicating N-glycosylation of the protein molecule [16, 19]. Similarly, other GH12 endoglucanases from fungi have been reported to have an optimal (pH values pH 5.0–6.0) [17, 27]. Optimal enzyme activity was found to occur at 50 °C within a tested range of 20–80 °C (Fig. 1b). GtCel12A was stable below 40 °C but lost approximately half of its activity at 50 °C after 120 min (Fig. 1c). This result consistent with the observed stability of GtCel12A expressed from A. niger .
The specific activity of the purified recombinant enzyme was assayed using various substrates (Additional file 1: Table S1). The enzyme exhibited significantly higher specific activity toward β-glucan (6546 U mg−1) and CMC (1129 U mg−1), which represent the β-1,3-1,4- and β-1,4-linkages, respectively, than toward xyloglucan (10.1 U mg−1) and filter paper (0.21 U mg−1). It did not degrade Avicel, xylan, soluble starch, locust bean gum, or carrageenan. The results of substrate preference were similar to that of GtCel12A expressed by A. niger . The GtCel12A had detectable enzyme activities against both β-glucan and xyloglucan. Thus, GtCel12A can be classified into SF 12-1 of the GH12 family . The kinetic parameters of GtCel12A were as follows: Vmax = 3452 μmol min−1 mg−1, Km = 13.83 mg mL−1, and kcat = 1726 s−1 as determined by a Lineweaver–Burk plot. Table 1 shows the characteristics of GtCel12A in comparison with other endoglucanases from various fungal genera including Trichoderma, Aspergillus, and Penicillium. Such fungi have been widely studied because they produce large quantities of cellulases and accepted commercial enzymes [29, 30]. The endoglucanases TrEGIII, TrEGII, PoXEG12A, and AtGH12 had low or zero enzyme activities with respect to β-glucan and CMC, while GtCel5B in previous study showed high specific activities. However, GtCel12A exhibited notable specific activities on both substrates compared to them. [15, 17, 18, 25, 31, 32].
The influences of metal ions and EDTA were tested. The relative activity of GtCel12A was 126.9% when EDTA was added, and relative activities in the presence of these metal ions (Ca2+, Co2+, Cu2+, Mn2+, Zn2+, K+, Li+, or Na+) were 90–100% of the relative activities observed in the absence of these cofactors. However, relative activity was entirely lost following exposure to Al3+, Fe3+, and Hg2+. GtCel12A did not employ metal ions as a cofactor. Naika and Tiku  have shown that enzyme activity of GH12 endoglucanase was enhanced in the presence of EDTA, which can affect molecule to form an intermediate state that is well disposed for protein–substrate interaction.
The hydrolysis pattern of oligosaccharides, including cellobiose, cellotriose, cellotetraose, and cellopentaose, was assessed by TLC analysis (Fig. 2). GtCel12A did not hydrolyze cellobiose into glucose; however, it did exhibit hydrolytic activity toward cellotriose, converting it into glucose and cellobiose. Cellotetraose was mainly degraded into cellobiose and minimally into glucose and cellotriose. Cellopentaose was likewise hydrolyzed by attacking its internal bonds, releasing cellobiose and cellotriose. In a previous study, TrCel12A did not degrade cellotriose into cellobiose and glucose, unlike GtCel12A . However, the GtCel5B exhibited similar cello-oligomers hydrolysis patterns to those of GtCel12A, but with weaker hydrolysis activity for cellotriose . For these reasons, recombinant acidic GtCel12A could be a candidate for use in numerous commercial industrial applications, as a superior enzyme with high catalytic efficiency and stability across a wide range of pH conditions.
Enzymatic hydrolysis and synergistic effects of GtCel12A on the HPAC-pretreated lignocellulose
Enzymatic hydrolysis of lignocellulose is impaired by several factors such as lignin, cellulose crystallinity, enzyme accessibility, and degree of polymerization. Lignin is the most important limiting factor for enzymatic hydrolysis because it limits the accessibility of cellulases to their substrates and directly inhibits cellulases [35, 36]. As a result, the enzymatic hydrolysis efficiency of lignocellulose is reduced by lignin. Therefore, an effective pretreatment method for lignocellulose is crucial to ensure a high conversion rate during enzymatic hydrolysis. A pretreatment method for lignocellulose biomass has been reported to effectively eliminate (97.2%) of lignin from biomass. . In the present study, we used the HPAC-pretreated lignocellulose as a substrate to assess enzyme activity and synergistic effects. Purified GtCel12A exhibited enzyme activities on HPAC-pretreated pine, rice straw, and corn stover and its characteristics were compared to those of GtCel5B endoglucanases because the GtCel5B showed more higher enzyme activities than other family 12 of glycoside hydrolase (GH12s) against β-glucan and CMC  (Fig. 3a). The pine substrate released 0.219 and 0.185 mg mL−1 reducing sugars via GtCel5B and GtCel12A, respectively. As a result, similar reducing sugar yield were achieved by both endoglucanases with respect to the pine substrates. However, GtCel12A achieved reducing sugar yields of 0.270 and 0.292 mg mL−1 from rice straw and corn stover, respectively, which were higher than the 0.144 mg mL−1 of reducing sugars that GtCel5B produced from each substrate. HPLC showed that GtCel12A produced cellobiose as a major product, and glucose as a minor product, from HPAC-pretreated lignocellulose (Fig. 3b). This result may correspond with the hydrolysis patterns of cello-oligomers determined by TLC analysis. Cellulose comprises a combination of crystalline and amorphous forms that can be easier to degrade than the crystalline regions . The relative ratio of crystalline and amorphous cellulose regions varies according to the biomass source, which results in different degrees of degradability by enzymes. Certain grass-based biomasses contain significant quantities of β-glucan component in their cell walls. These conditions enabled GtCel12A to exhibit a high specific activity on β-glucan and CMC (Table 1). Miotto et al.  also reported that GtCel12A expressed by A. niger had high enzyme activity with respect to β-glucan, and exhibited hydrolytic effects with respect to oat spelt. These results suggest that GtCel12A hydrolyzed grass-based biomass more efficiently than pine wood compared to GtCel5B. Furthermore, GtCel12A exhibited a higher specific activity on CMC, which is amorphous cellulose, compared to GtCel5B. However, GtCel5B had higher specific activity on filter paper (Table 1). These results suggest that GtCel12A hydrolyzes amorphous cellulose more efficiently than GtCel5B, perhaps due to the cellulose crystallinity of the cellulose in HPAC-pretreated lignocellulose.
The synergistic cooperation of different types of cellulases is important for a high lignocellulose conversion rate. Endoglucanase, which acts on the internal glycosidic bonds in cellulose, creates new reducing and nonreducing ends for the exo-1,4-β-glucanases. When acting alone, GtCel12A achieved a reducing sugar yield of only 0.185 mg mL−1 from HPAC-pretreated pine. However, we detected a synergistic effect when we compared the amount of reducing sugars released from HPAC-pretreated pine lignocellulose by commercial cellulase alone with that released by commercial cellulase together with purified GtCel12A with enzymic activity of 10 filter paper units per gram (FPU g−1). An external supplement of purified GtCel12A increased reducing sugar production by 16.1, 29.0, 13.4, and 14.5% on the HPAC-pretreated pine, corn stover, rice straw, and filter paper, respectively, compared to commercial cellulase alone (Fig. 4). In a previous study, synergistic effects of endoglucanases were reported to enhance hydrolysis efficiency: the GH12 family endoglucanase from A. terreus increased hydrolysis efficiency by 38.7% compared to crude cellulase alone on delignified rice straw . Endoglucanase from Armillaria gemina KJS114 additionally showed enhanced reducing sugar production when combined with commercial cellulase mixtures on the pretreated poplar . Increased enzymatic hydrolysis may be related to a synergistic action between the purified endoglucanase and commercial cellulase, and could also be enhanced by added endoglucanase, which causes an increase in the total amount of enzyme in the mixture . Therefore, acidic GtCel12A could be a superior candidate for enzymatic hydrolysis of HPAC-pretreated lignocellulose.
The recombinant G. trabeum acidic endoglucanase Cel12A expressed by P. pastoris GS115 had an approximate molecular weight of 31 kDa, which differs from that of the enzyme expressed by A. niger. The purified GtCel12A expressed by P. pastoris had a more acidic optimal pH than that expressed by A. niger and exhibited superior pH stability across a range of pH values (2.0–9.0). Furthermore, GtCel12A exhibited a notable specific activity with regard to CMC and β-glucan compared to GtCel5B, TrCel12A, and other GH12 endoglucanases. GtCel12A exhibited better enzymatic hydrolysis on HPAC-pretreated rice straw and corn stover than GtCel5B. Moreover, it showed synergistic effects of 16.1, 29.0, 13.4, and 14.5% on HPAC-pretreated pine, corn stover, rice straw, and filter paper, respectively, when GtCel12A was added as a supplement to cellulase. The GtCel12A with superior pH stability and enzyme activity could be a potential candidate as an additive enzyme for biotechnological application.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and Additional file 1.
bovine serum albumin
Carbohydrate-Active enZYmes database
hydrogen peroxide–acetic acid
high-performance liquid chromatography
Polymerase chain reaction
potato dextrose agar
sodium dodecyl sulfate polyacrylamide gel electrophoresis
yeast extract peptone dextrose
yeast extract peptone glycerol
Behera SS, Ray RC (2016) Solid state fermentation for production of microbial cellulases: recent advances and improvement strategies. Int J Biol Macromol 86:656–669
Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG (2012) Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 5:45
Hasunuma T, Okazaki F, Okai N, Hara KY, Ishii J, Kondo A (2013) A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology. Bioresour Technol 135:513–522
Kuhad RC, Gupta R, Singh A (2011) Microbial cellulases and their industrial applications. Enzyme Res 2011:280696
Srivastava N, Rawat R, Singh Oberoi H, Ramteke PW (2015) A review on fuel ethanol production from lignocellulosic biomass. Int J Green Energy 12(9):949–960
Cherry JR, Fidantsef AL (2003) Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol 14(4):438–443
Schuster A, Schmoll M (2010) Biology and biotechnology of Trichoderma. Appl Microbiol Biotechnol 87(3):787–799
Baldrian P, Valaskova V (2008) Degradation of cellulose by basidiomycetous fungi. FEMS Microbiol Rev 32(3):501–521
Floudas D, Binder M, Riley R, Barry K, Blanchette RA, Henrissat B, Martinez AT, Otillar R, Spatafora JW, Yadav JS, Aerts A, Benoit I, Boyd A, Carlson A, Copeland A, Coutinho PM, de Vries RP, Ferreira P, Findley K, Foster B, Gaskell J, Glotzer D, Gorecki P, Heitman J, Hesse C, Hori C, Igarashi K, Jurgens JA, Kallen N, Kersten P, Kohler A, Kues U, Kumar TK, Kuo A, LaButti K, Larrondo LF, Lindquist E, Ling A, Lombard V, Lucas S, Lundell T, Martin R, McLaughlin DJ, Morgenstern I, Morin E, Murat C, Nagy LG, Nolan M, Ohm RA, Patyshakuliyeva A, Rokas A, Ruiz-Duenas FJ, Sabat G, Salamov A, Samejima M, Schmutz J, Slot JC, St John F, Stenlid J, Sun H, Sun S, Syed K, Tsang A, Wiebenga A, Young D, Pisabarro A, Eastwood DC, Martin F, Cullen D, Grigoriev IV, Hibbett DS (2012) The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336(6089):1715–1719
Cohen R, Suzuki MR, Hammel KE (2005) Processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum. Appl Environ Microbiol 71(5):2412–2417
Kerem Z, Jensen KA, Hammel KE (1999) Biodegradative mechanism of the brown rot basidiomycete Gloeophyllum trabeum: evidence for an extracellular hydroquinone-driven fenton reaction. FEBS Lett 446(1):49–54
Suzuki MR, Hunt CG, Houtman CJ, Dalebroux ZD, Hammel KE (2006) Fungal hydroquinones contribute to brown rot of wood. Environ Microbiol 8(12):2214–2223
Takeda T, Takahashi M, Nakanishi-Masuno T, Nakano Y, Saitoh H, Hirabuchi A, Fujisawa S, Terauchi R (2010) Characterization of endo-1,3–1,4-β-glucanases in GH family 12 from Magnaporthe oryzae. Appl Microbiol Biotechonol 88:1113–1123
Linger JG, Adney WS, Darzins A (2010) Heterologous expression and extracellular secretion of cellulolytic enzymes by Zymomonas mobilis. Appl Environ Microbiol 76(19):6360–6369
Nakazawa H, Okada K, Kobayashi R, Kubota T, Onodera T, Ochiai N, Omata N, Ogasawara W, Okada H, Morikawa Y (2008) Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli. Appl Microbiol Biotechnol 81(4):681–689
Miotto LS, de Rezende CA, Bernardes A, Serpa VI, Tsang A, Polikarpov I (2014) The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on beta-glucan. PLoS ONE 9(9):e108393
Segato F, Dias B, Berto GL, de Oliveira DM, De Souza FHM, Citadini AP, Murakami MT, Damasio ARL, Squina FM (1865) Polikarpov I (2017) Cloning, heterologous expression and biochemical characterization of a non-specific endoglucanase family 12 from Aspergillus terreus NIH2624. Biochim Biophys Acta Proteins Proteom 4:395–403
Xian L, Wang F, Yin X, Feng JX (2016) Identification and characterization of an acidic and acid-stable endoxyloglucanase from Penicillium oxalicum. Int J Biol Macromol 86:512–518
Tambor JH, Ren H, Ushinsky S, Zheng Y, Riemens A, St-Francois C, Tsang A, Powlowski J, Storms R (2012) Recombinant expression, activity screening and functional characterization identifies three novel endo-1,4-beta-glucanases that efficiently hydrolyse cellulosic substrates. Appl Microbiol Biotechnol 93(1):203–214
Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98(12):5301–5317
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24(1):45–66
Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T (2009) Expression in the yeast Pichia pastoris. Methods Enzymol 463:169–189
Wi SG, Cho EJ, Lee DS, Lee SJ, Lee YJ, Bae HJ (2015) Lignocellulose conversion for biofuel: a new pretreatment greatly improves downstream biocatalytic hydrolysis of various lignocellulosic materials. Biotechnol Biofuels 8:228
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Kim HM, Lee YG, Patel DH, Lee KH, Lee DS, Bae HJ (2012) Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum. J Ind Microbiol Biotechnol 39(7):1081–1089
Okada H, Tada K, Sekiya T, Yokoyama K, Takahashi A, Tohda H, Kumagai H, Morikawa Y (1998) Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414. Appl Environ Microbiol 64(2):555–563
Master ER, Zheng Y, Storms R, Tsang A, Powlowski J (2008) A xyloglucan-specific family 12 glycosyl hydrolase from Aspergillus niger: recombinant expression, purification and characterization. Biochem J 411(1):161–170
Calzado F, Prates ET, Goncalves TA, Rubio MV, Zubieta MP, Squina FM, Skaf MS, Damasio AR (2016) Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases. Biotechnol Bioeng 113(12):2577–2586
Imran M, Anwar Z, Irshad M, Asad MJ, Ashfaq H (2016) Cellulase production from species of fungi and bacteria from agricultural wastes and its utilization in industry: a review. Adv Enzyme Res 4:44–55
Vaishnav N, Singh A, Adsul M, Dixit P, Sandhu SK, Mathur A, Puri SK, Singhania RR (2018) Penicillium: the next emerging champion for cellulase production. Bioresour Technol Rep 2:131–140
Grishutin SG, Gusakov AV, Dzedzyulya EI, Sinitsyn AP (2006) A lichenase-like family 12 endo-(1→4)-beta-glucanase from Aspergillus japonicus: study of the substrate specificity and mode of action on beta-glucans in comparison with other glycoside hydrolases. Carbohydr Res 341(2):218–229
Samanta S, Basu A, Halder UC, Sen SK (2012) Characterization of Trichoderma reesei endoglucanase II expressed heterologously in Pichia pastoris for better biofinishing and biostoning. J Microbiol 50(3):518–525
Naika GS, Tiku PK (2011) Influence of ethylenediaminetetraacetic acid (EDTA) on the structural stability of endoglucanase from Aspergillus aculeatus. J Agric Food Chem 59(13):7341–7345
Karlsson J, Siika-aho M, Tenkanen M, Tjerneld F (2002) Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei. J Biotechnol 99(1):63–78
Nakagame S, Chandra RP, Saddler JN (2010) The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates, on enzymatic hydrolysis. Biotechnol Bioeng 105(5):871–879
Várnai A, Siika-aho M, Viikari L (2010) Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose. Enzyme Microb Technol 46(3–4):185–193
Karimi K, Taherzadeh MJ (2016) A critical review of analytical methods in pretreatment of lignocelluloses: composition, imaging, and crystallinity. Bioresour Technol 200:1008–1018
Narra M, Dixit G, Divecha J, Kumar K, Madamwar D, Shah AR (2014) Production, purification and characterization of a novel GH 12 family endoglucanase from Aspergillus terreus and its application in enzymatic degradation of delignified rice straw. Int Biodeterior Biodegrad 88:150–161
Jagtap SS, Dhiman SS, Kim T-S, Kim I-W, Lee J-K (2014) Characterization of a novel endo-β-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis. Appl Microbiol Biotechnol 98(2):661–669
This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MSIP) (No. 2018R1A2A2A05018238).
This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MSIP) (No. 2018R1A2A2A05018238).
Consent for publication
Informed consent was obtained from all individual participants in the study.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Oh, C.H., Park, C.S., Lee, Y.G. et al. Characterization of acidic endoglucanase Cel12A from Gloeophyllum trabeum and its synergistic effects on hydrogen peroxide–acetic acid (HPAC)-pretreated lignocellulose. J Wood Sci 65, 24 (2019). https://doi.org/10.1186/s10086-019-1803-7
- Synergistic effects
- Pichia pastoris