Appl Microbiol Biotechnol (2012) 93:203–214 DOI 10.1007/s00253-011-3419-8
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Recombinant expression, activity screening and functional characterization identifies three novel endo-1,4-β-glucanases that efficiently hydrolyse cellulosic substrates José Humberto Tambor & Huanan Ren & Sophia Ushinsky & Yun Zheng & Anja Riemens & Christopher St-Francois & Adrian Tsang & Justin Powlowski & Reginald Storms
Received: 1 February 2011 / Revised: 24 May 2011 / Accepted: 28 May 2011 / Published online: 28 June 2011 # Springer-Verlag 2011
Abstract The hydrolysis of cellulose into fermentable sugars is a costly and rate-limiting step in the production of biofuels from renewable feedstocks. Developing new cellulase systems capable of increased cellulose hydrolysis rates would reduce biofuel production costs. With this in mind, we screened 55 fungal endoglucanases for their abilities to be expressed at high levels by Aspergillus niger and to hydrolyze amorphous cellulose at rates significantly greater than that obtained with TrCel5A, one of the major endoglucanases in the Trichoderma reesei cellulase system. This screen identified three endoglucanases, Aureobasidium pullulans ApCel5A, Gloeophyllum trabeum GtCel12A and Sporotrichum thermophile StCel5A. We determined that A. niger expressed the three endoglucanases at relatively high levels (≥0.3 g/l) and that the hydrolysis rate of ApCel5A and StCel5A with carboxymethylcellulose 4M as substrate J. H. Tambor : H. Ren : S. Ushinsky : Y. Zheng : C. St-Francois : A. Tsang : J. Powlowski : R. Storms (*) Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montréal, Quebec H4B 1R6, Canada e-mail:
[email protected] J. Powlowski Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street West, Montréal, Quebec H4B 1R6, Canada A. Riemens Beijerinck Laboratory, DSM Biotechnology Center, PO Box 1, 2600 MA Delft, The Netherlands
was five and two times greater than the T. reesei Cel5A. The ApCel5A, GtCel12A and StCel5A enzymes also demonstrated significant synergy with Cel7A/CbhI, the major exoglucanase in the T. reesei cellulase system. The three endoglucanases characterized in this study are, therefore, promising candidate endoglucanases for developing new cellulase systems with increased rates of cellulose saccharification. Keywords Glycosyl hydrolase family 5 endoglucanase . Glycosyl hydrolase family 12 endoglucanase . Cellulase . Heterologous expression . Synergy . Fungus
Introduction Bacteria and fungi produce enzyme systems that hydrolyze cellulose to glucose. These enzyme systems usually include multiple members from three distinct classes of enzymes: the cellobiohydrolases (also known as exoglucanases or CBHs) that cleave cellulose polymers from their ends, the endoglucanases (EGLs) that cleave cellulose polymers internally and the β-glucosidases (BGLs) that convert cellobiose into glucose. The most thoroughly characterized cellulose degrading enzyme system is from Trichoderma reesei (teleomorph Hypocrea jecorina), a mesophilic soft rot fungus widely used as a source of commercial cellulases and hemicellulases. Not including the glycosyl hydrolase (GH) family 61 members which lack both cellulase activity and the conserved catalytic residues found in other GHs (Harris et al. 2010), the genes for 15 characterized cellulolytic
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GHs, two CBHs (Cel7A/CBH1 and Cel6A/CBH2), six EGLs (Cel7B/EG1, Cel5A/EG2, Cel12A/EG3, Cel45A/ EG5, Cel74A and Cel5B) and seven BGLs have been identified in the T. reesei genome sequence (Ouyang et al. 2006). The CBHs, Cel7A and Cel6A processively cleave cellulose polymers from the reducing and the non-reducing ends respectively (Divne et al. 1998; Jeoh et al. 2007). The EGLs cleave β-glycosidic bonds internal to the cellulose polymers (Karlsson et al. 2002). The combined action of the CBHs and EGLs on cellulose generates mainly the short soluble oligosaccharides cellobiose, cellotriose and cellotetraose, which are cleaved to glucose monomers by the BGLs, Cel3A, Cel1A, Cel3B, Cel3C, Cel1B, Cel3D and Cel3E (Takashima et al. 1999; Foreman et al. 2003). These 15 GHs work cooperatively to hydrolyze plant cellulose, which has a highly organized crystalline structure. Three types of synergy are demonstrated by these cellulases. EXO-EXO synergy is produced by the two types of CBHs, Cel7A and Cel6A (Medve et al. 1994). EXO-ENDO synergy occurs between EGLs and CBHs (Zhang and Lynd 2006). Synergy is also observed between EGLs and BGLs and between CBHs and BGLs (Gruno et al. 2004). Accessory proteins are also important. For example, although the glycosyl hydrolase family 61 members Cel61A and Cel61B lack activity on cellulose substrates alone or in combination with other cellulases on pure cellulosic substrates, the Cel61 members enhance the cellulose degrading activity of the “true cellulases” on pretreated biomass derived from woody and herbaceous plants (Harris et al. 2010). Cellulosic feedstocks derived from plant biomass have the potential to reduce fossil fuel dependence and greenhouse gas emissions while increasing energy security. Currently, fuels produced using cellulosic feedstocks are not cost competitive with fossil fuels due in part to the high cost associated with converting plant biomass polymers into fermentable sugars. One approach to address this issue is to use genome-based enzyme discovery to develop new enzyme systems capable of cost effectively converting cellulosic stocks into fermentable sugars. In this study. we screened 55 novel fungal endoglucanases and identified three, Aureobasidium pullulans ApCel5A, Gloeophyllum trabeum GtCel12A, and Sporotrichum thermophile StCel5A that were expressed as secreted proteins at relatively high levels and exhibited greater cellulose hydrolysis rates on a model amorphous substrate than is obtained with a commercial endoglucanase, T. reesei Cel5A. We also present the kinetic parameters, the pH and temperature optima and the cellulose hydrolyzing ability of these EGLs alone and in combination with the Cel7A cellobiohydrolase from T. reesei.
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Materials and methods Plasmids, cDNA libraries, strains, strain propagation and transformation cDNA libraries were prepared using RNA isolated from 15 fungal species as described previously (Semova et al. 2006). The complete open reading frames (ORFs) of 55 putative EGL genes were PCR-amplified from full-length cDNAs identified by EST analysis of cDNA libraries. The forward and reverse primers used to clone each ORF, had at their 3′ ends about 20 nucleotides of identity to the Nterminal and C-terminal portions of the coding region of the targeted ORF, followed by Gateway BP reaction compatible recombination sites and finally two filler nucleotides (Table 1). The amplified ORFs were cloned into pDONR201, sequenced to verify their inserts and then cloned into pGBFIN-GTW, a Gateway compatible Aspergillus niger integrative expression vector, derived from pGBFIN (van den Brink et al. 1999). The resulting pGBFIN-GTW derivatives were verified by restriction enzyme analysis. Escherichia coli strain DB3.1 was used for the propagation of Gateway plasmids pDONR201 and pGBFIN-GTW containing the ccdB gene (encodes a cytotoxic protein which kills plasmid-free segregants) and TOP10 was used as the host for the propagation of recombinant pDONR201 and pGBFIN-GTW plasmids harbouring endoglucanase genes. A. niger strain CBS 513.88 (FGSC A1513) was used as the host for heterologous protein expression. The genes for 55 fungal endoglucanases were transformed into strain CBS 513.88. The preparation of A. niger protoplasts and protoplast transformation with the plasmid DNAs was performed as previously described (van den Berg et al. 2009). Fresh spores were used to inoculate the cultures used for endoglucanase expression. Chemicals, substrates and enzymes Avicel PH105 was kindly provided by FMC Biopolymer. Azo-CMC (partially depolymerised carboxymethyl-4Mcellulose dyed with Remazol Brilliant Blue R to a ratio of approximately one dye molecule per 20 sugar residues) and carboxymethylcellulose 4M (CMC) was purchased from Megazyme. Discs of Whatman No. 1 filter paper (7 mm in diameter) were used for filter paper assays, which were performed as described previously (Xiao et al. 2004). Cellobiose was purchased from Sigma-Aldrich. Cellotriose, cellotetraose, cellopentaose and cellohexaose were purchased from MJS BioLynx Inc. Silica gel thin-layer chromatography (TLC) plates were purchased from Fisher Scientific Canada. Phosphoric acid swollen cellulose (PASC) was prepared from Avicel as described previously
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Table 1 Oligonucleotides used to amplify the three endoglucanase ORFs 5′ to 3′ primer sequencesa ApCel5A F ApCel5A R GtCel12A F GtCel12A R StCel5A F StCel5A R
GGGGACAAGT TTGTACAAAA AAGCAGGCTA TGAAGTACTC AACTTTCG GGGGACCACT TTGTACAAGA AAGCTGGGTT TATTTGGAGT TGAAGAC GGGGACAAGT TTGTACAAAA AAGCAGGCTA TGTTCCGCTT CATCTCTGCT TTGC GGGGACCACT TTGTACAAGA AAGCTGGGTT CACCCGCTCA AGCTG GGGGACAAGT TTGTACAAAA AAGCAGGCTA TGAAGTCCTC CATCCTCG GGGGACCACT TTGTACAAGA AAGCTGGGTT TACGGCAAGT ACTTCTTCAA G
a
The underlined sequences in the forward primers (F) and reverse primers (R) represent sequences identical to the ORF nontemplate strand beginning at the ATG codon and to the ORF template strand beginning at the stop codon. The 5′ end of each oligo has two filler nucleotides followed by the Gateway BP recombination site
(Wood 1988) with the following modifications. The 600-ml suspension of 20 g of Avicel in phosphoric acid was kept on ice for 3 h with occasional grinding with a pestle and mortar. After centrifugation for 10 min at 14,000×g, the pelleted Avicel was suspended in 2 l of ice cold water and rinsed at 4°C for 15 min. The washes were repeated until the pH was between 5 and 7. Finally, the washed PASC was suspended in 10-mM citrate buffer of pH 5.0. Trypsin was purchased from Sigma-Aldrich. Purified T. reesei Cel7A (CBH1) and Cel5A (EG2) were obtained from Iogen Corporation (Ottawa, Ontario). Bioinformatic analysis Conserved domains and protein motifs were identified using the conserved Domain Database and Search Service v2.15 tool (Marchler-Bauer et al. 2007) and the EXPASY ScanProsite tool (Appel et al. 1994). Potential signal peptides were identified using the SignalP 3.0 server tools (Emanuelsson et al. 2007). Protein 3-D structures were generated using the SWISS-MODEL programme (Arnold et al. 2006) and template structures available at the Protein Data Bank (Berman et al. 2000). The T. reesei Cel12A structure, 1olqA, (Sandgren et al. 2003) was used to generate the model structure for GtCel12A and the Thermoascus aurantiacus Cel5A structures, 1h1nA and 1gzjB (Lo Leggio et al. 1997; Van Petegem et al. 2002), were used to model the GH5 domains of ApCel5A and StCel5A, respectively. All the models were subjected to quality analysis using the Qmean (Benkert et al. 2009) and PDBsum (Laskowski 2009) servers. Images of the modelled 3D structures were generated by the DeepView v4.0 programme (Guex and Peitsch 1997). Production and characterization of the secreted endoglucanases The 55 cloned EGL genes were expressed in A. niger strain CBS 513.88. The A. niger transformants and the empty
vector control, CBS 513.88 transformed with pGBFINGTW, were grown in 100 ml of STIPT liquid medium as described previously (van den Brink et al. 1999) for 5 days at 34°C and 170 rpm. Culture supernatants were harvested by centrifugation at 5,000 rpm for 20 min followed by filtration through Whatman GF/A filters. Culture filtrates were screened for secreted protein expression by SDSPAGE analysis and for detectable endoglucanase activity on Congo red indicator plates seeded with carboxymethylcellulose (Wood 1988). SDS-PAGE analysis showed that six endoglucanases were expressed at relatively high levels (>0.1 mg per ml) and eight more were expressed at detectable levels but less than 0.1 mg/ml. Congo red indicator plates identified ten endoglucanases that could express detectable activity. The three endoglucanases, A. pullulans ApCel5A, S. thermophile StCel5A and G. trabeum GtCel12A, that produced the largest halos on the Congo red indicator plates and were also expressed highly (≥0.3 g/l) were selected for detailed characterization. G. trabeum strain Madison 617, ATCC 11539, A. pullulans strain NRRL Y-2311-1, ATCC 62921, and S. thermophile ATCC 42464 cDNA libraries, prepared as described previously (Semova et al. 2006), were the sources of these endoglucanase genes. Sugars were removed from the ApCel5A, StCel5A and GtCel12A culture filtrates using centrifugal filter devices (Ultrafree-0.5, Millipore) and the samples were buffer exchanged into sodium citrate buffer (10 mM, pH 5.0). Enzyme specific activities in culture filtrates from CBS 513.88 transformed with the three EGL genes and the empty vector control were determined using a 24-hour time course with samples taken at 3, 4, 7 and 24 h and the filter paper assay described previously (Xiao et al. 2004). The R2 values of the linear curves used to generate enzyme specific activities were 0.96, 0.97, 0.96 and 0.94 for ApCel5A, GtCel12A, StCel5A, and TrCel5A, respectively. The protein concentration of the EGLs was determined by running 5 μl of ApCel5A, GtCel12A and empty vector control and 1 μl of StCel5A on 14% SDS-PAGE gels and comparing
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band intensities to lanes loaded with 1, 0.75, 0.5 and 0.25 μg of a BSA standard using the SynGene GeneTools software. Maldi TOF mass spectrometry was performed essentially as described previously (Decelle et al. 2004). Biochemical characterization of the endoglucanases Temperature and pH profiles of the EGLs were determined using the filter paper assay (Xiao et al. 2004) with the following modifications. Filter paper discs were added to 40 μl of McIlvaine buffer prepared by combining 100-mM citrate buffer and 200 mM Na2HPO4 and then 20 μl of enzyme that had been suitably diluted in 10-mM citrate buffer of pH 4.5. Reaction mixtures were incubated at 37°C for 60 min and terminated by placing on ice. The extent of cellulose hydrolysis was measured using the bicinchoninic acid (BCA) reducing sugar assay (Doner and Irwin 1992). Temperature profiles were determined at the optimal pH of each enzyme and temperatures ranging from 30°C to 80°C. Enzyme thermostability was determined using AzoCMC as the substrate. Enzyme diluted in 10-mM citrate buffer of pH 4.5, was preincubated at temperatures ranging from 30°C to 100°C for 2 h with 15 μl McIlvaine buffer at the pH optimum of each EGL. After preincubation enzyme activity was quantitated at 40°C using 25 μl Azo-CMC (1%, w/v) as the substrate in 200 μl microplate wells according to the supplier’s instructions with the following modification. The Azo-CMC substrate was boiled and vortexed prior to use, the assays were done in triplicate and the absorbance was read at 590 nm. Enzyme kinetic parameters were determined using PASC (Wood 1988) or CMC as the substrate. Enzyme (40 μl) diluted in 10-mM citrate buffer of pH 5 was added to 60 μl of McIlvaine buffer at the pH optimum of each enzyme (pH 4.5 for ApCel5A and GtCel12A and 6.0 for StCel5A) and 100 μl of PASC suspension to yield final substrate concentrations of 1%, 0.5%, 0.25%, 0.125% and 0.05% or 100 μl of CMC suspension to yield final substrate concentrations of 1%, 0.75%, 0.5%, 0.25%, 0.125%, 0.0625% and 0.03125% . The 200 μl reaction mixtures were incubated in a 96 well flat bottom microplate at 37°C with shaking at 250 rpm. Aliquots of 10 μl were removed at intervals of 5 min over a time course of 60 min and the BCA assay was performed. Reducing sugar end production was determined using the BCA assay and a D-glucose 5 to 25 μM standard curve. Each assay was done in triplicate; the values with the standard deviation are presented. The Km and Vmax parameters of each enzyme were determined by Michaelis–Menten fitting using the GraFit v6.0 software programme (Erithacus Software Limited). A comparison of the activity on both PASC and CMC substrates was also done to determine the relative amounts of each endoglucanase required to obtain equivalent levels
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of hydrolysis. Reaction mixtures (200 μl) containing each enzyme at various concentrations (each concentration repeated in triplicate) were incubated in 96 well flat bottom microplates at 37°C with shaking at 250 rpm. Each reaction included 40 μl of enzyme appropriately diluted in 10-mM citrate buffer of pH 5, 60 μl of McIlvaine buffer at the pH optimum of each enzyme (pH 4.5 for ApCel5A and GtCel12A and 6.0 for StCel5A) and 100 μl of substrate (1%). Reactions were incubated for 30 min for CMC or 20 h for PASC. Aliquots of 10 μl were removed for reducing sugar end determination by the BCA assay. The results, the amount of enzyme versus reducing sugar ends produced, were plotted and used to derive the picomoles (pmol) of enzyme necessary to hydrolyse 2% of substrate. The R2 values of the linear curves for CMC hydrolysis were 0.99, 0.99, 0.90 and 0.91 for ApCel5A, GtCel12A, StCel5A, and TrCel5A, respectively. The R2 values of the linear curves for PASC hydrolysis were 0.97, 0.85, 0.89 and 0.97 for ApCel5A, GtCel12A, StCel5A and TrCel5A, respectively. Analysis of glucooligosaccharide degradation products using thin-layer chromatography Products for TLC analysis were generated in 100 μl reaction mixtures (200-mM citrate buffer) at each enzyme’s pH optimum with cellobiose (0.5%, w/v), cellotriose (0.5%, w/v), cellotetraose (0.5%, w/v), cellopentaose (0.125%, w/v) or cellohexaose (0.1%, w/v). The reaction mixtures were incubated at 37°C for 16 h. Following incubation, 10 μl of the cellobiose, cellotriose and, cellotetraose reactions, 40 μl of the cellopentaose reaction, and 50 μl of the cellohexaose reaction were resolved by TLC using chloroform/acetic acid/H2O (6:7:1) as the solvent. The resolved reaction products were visualized by spraying the TLC plate with a 1:20 solution of concentrated sulphuric acid and ethanol solution (v/v) followed by baking at 110°C for 15 min (Blanco et al. 1999). Cellulase synergy Synergy assays using filter paper as the substrate were done in a final volume of 120 μl prepared by adding 20 μl of each EGL (0.9 pmol) and/or 20 μl of CBH (30.6 pmol), 80 μl of McIlvaine buffer (pH 4.5 for ApCel5A, GtCel12A, TrCel5A and pH 6.0 for StCel5A) and a 0.7-cm disc of Whatman No. 1 filter paper. Reactions were incubated for 2 h at 37°C and 250 rpm. Enzyme reactions were terminated by placing on ice and the production of reducing sugar ends quantified using the BCA assay. The total reaction volume (120 μl of buffer) including the filter paper was subjected to the colorimetric reaction for 30 min in a water bath at 80°C and an aliquot of 80 μl was used to
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measure the reducing sugar ends. For synergy assays using Avicel as the substrate, 30 μl of McIlvaine buffer at each enzyme’s pH optimum was added to the wells of a flat bottom 96 well reading plate along with 20 μl of the following enzyme combinations (1.8 pmol of EGL+0 pmol CBH, 1.35 pmol EGL+10 pmol CBH, 0.9 pmol EGL+ 20 pmol CBH, 0.45 pmol EGL+30 pmol CBH or 0 pmol EGL+40 pmol CBH) and 50 μl of 1% Avicel in 10-mM citrate buffer of pH 5.0. The plate was sealed and incubated for 2 h at 37°C with shaking at 250 rpm. Enzyme reactions were terminated by placing on ice and the production of reducing sugar ends determined as described for the filter paper assay except that before transferring the reaction to a reading plate the reaction was centrifuged for 3 min at 3,000 rpm to remove the Avicel. Each assay was done in triplicate; the values with the standard deviation are presented. Nucleotide sequence accession numbers The sequences have been deposited in GenBank; the accession numbers are HQ163778 for GtCel12A, HQ163777 for ApCel5A and HQ163779 for StCel5A.
Results Screening for functionally expressed endoglucanases We screened 55 heterologous fungal endoglucanases for their ability to be functionally expressed as secreted proteins. This screen identified three endoglucanases that were functionally expressed at relatively high levels as assessed by Congo red indicator plates (data not shown). SDS-PAGE analysis determined that the three endoglucanases were expressed as secreted proteins at greater than 0.3 g L −1 . These three endoglucanases, ApCel5A, GtCel12A and StCel5A, were subject to basic bioinformatic and biochemical characterization and their ability to hydrolyze model cellulosic substrates was determined to assess their suitability as enzymes for the hydrolysis of cellulosic substrates for biofuels production.
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16 amino acid residues, respectively. The predicted presequences, catalytic domains and catalytic residues for ApCel5A, StCel5A and GtCel12A as well as the fCBDs for ApCel5A and StCel5A are indicated in Fig. 1. ApCel5A is most similar to the T. reesei Cel5A showing 54% identity over the entire protein whereas StCel5A is most similar to thermophilic T. aurantiacus Cel5A showing 65% identity overall. GtCel12A shares 34% identity with TrCel12A enzyme overall. The T. aurantiacus Cel5A 1h1nA and 1gzjB structures were used as templates to model the GH5 domains of ApCel5A and StCel5A, respectively (Fig. 2a, c). The T. reesei Cel12A 1olqA structure was used in SWISSMODEL “Alignment mode” to generate GtCel12A structure (Fig. 2b). The predicted structures were evaluated using a number of tools including, QMEAN, QMEAN Zscore (Benkert et al. 2009) and Procheck (Laskowski 2009). This revealed that the models exhibited few unfavourable packaging environments, disallowed bond angles or bond lengths and that the modelled structures compared very favourably to a set of protein structures solved by X-ray crystallography (Table 2). Other quality control programmes from SWISS-MODEL Workstation (data not shown) were also used and corroborate the quality assessment results summarized in Table 2. The model structures of ApCel5A and StCel5A were typical of GH5s with a central (β/α)8-barrel structure with β-sheet in the inner region surrounded by an outer shell of α-helices (Fig. 2a, c). The GtCel12A model, with 15 β-ribbons and a single α-helix, was a β-jelly roll structure typical of Cel12A structures (Fig. 2b). The predicted pairs of catalytic glutamates in ApCel5A and StCel5A are located on opposite sides of the respective substrate binding clefts. The positions, orientations and distances between the two catalytic glutamates (4.22 and 4.32Å for ApCel5A and StCel5A, respectively) correlate well with the X-ray crystal structures of other GH5 family members (Fig. 2a, c). In the GtCel12A structure, the 6.27 Å distance between the two catalytic residues (Fig. 2b) was similar to the distance between the two catalytic residues in the 1olqA template. Endoglucanase characterization
Sequence analysis Searching the NCBI Conserved Domain Database (CDD) (Marchler-Bauer et al. 2007) determined that ApCel5A and StCel5A had GH5 domains, GtCel12A had a GH12 domain. The CDD search also revealed and that ApCel5A and StCel5A harboured type 1 fungal carbohydrate binding domains (fCBD) (Gilkes et al. 1991). The SignalP algorithm (Bendtsen et al. 2004) predicted that ApCel5A, GtCel12A and StCel5A had signal peptides of 23, 20 and
The secreted peptides were verified by MALDI-TOF mass spectrometry peptide mass mapping (Table 3). SDS-PAGE revealed that ApCel5A, GtCel12A and StCel5A had relative molecular masses of 47, 29 and 46 kDa, and that they were expressed at levels of 0.32, 0.46 and 0.92 mg/ml, respectively. The filter paper hydrolyzing ability of culture filtrates from A. niger transformed with pGBFIN-GTW harbouring the heterologous EGLs was compared with A. niger harbouring pGBFIN-GTW without an ORF. The
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Fig. 1 ClustalW sequence alignments. a Alignment of ApCel5A and StCel5A with T. aurantiacus TaCel5A (NCBI accession number AAL88714) and T. reesei TrCel5A (NCBI accession P07982). b Alignment of GtCel12A with T. reesei TrCel12A wild type (NCBI accession BAA20140) and the mutated TrCel12A (NCBI accession
1OLQ_A). Regions identified with a single thin line correspond to secretory N-terminal pre-sequences, a dotted line correspond to the fCBDs, conserved glycosyl hydrolase domains are identified by a thin double line and predicted catalytic residues are highlighted in grey
specific activity of the endoglucanases ApCel5A, GtCel12A, StCel5A and TrCel5A were 4.8, 0.87, 1.1, and 1.4 pmol of glucose equivalents released per picomoles of enzyme per minute, respectively. A. niger transformed with the empty vector did not express detectable activity (data not shown).
Basic biochemical characterization was performed on each EGL. The pH optima of ApCel5A and GtCel12A were 4.5 whereas StCel5A showed optimal activity at pH 6.0 (Fig. 3a). The temperature optima were 40°C, 50°C and 70°C for ApCel5A, GtCel12A and StCel5A, respectively (Fig. 3b). In measuring thermal stability, ApCel5A lost approximately
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Fig. 2 Predicted ribbon structures of the three EGLs. Predicted ribbon structures for ApCel5A (a), GtCel12A (b) and StCel5A (c). The T. aurantiacus Cel5A 1h1nA and 1gzjB structures were used to generate the GH5 domains of ApCel5A and StCel5A, respectively. TaCel5A has 30% and 65% primary sequence identity with ApCel5A and
StCel5A. The T. reesei Cel12A structure 1olqA, which has 34% primary sequence identity with GtCel12A, was used to generate the GH12 domain. Unstructured regions in the proteins are indicated by the thin lines. Distances between the catalytic amino acids in the active site are also indicated
half its activity following 2 h at 45°C and GtCel12A lost at least half its activity following 2 h at 50°C. StCel5A from the thermophilic fungus S. thermophile was the most stable and retained greater than 50% of its activity following 2 h of incubation at 55°C (Fig. 3c). The kinetic parameters of the three enzymes were determined at 37°C and their pH optimum using PASC and CMC as substrates. PASC was used because it is a model substrate that can be reproducibly produced yet approximates the cellulose in pretreated cellulosic biomass and is commonly used (Zhang and Lynd 2004). When PASC was the substrate, the Vmax values for ApCel5A, GtCel12A and StCel5A were 40.7±6.8, 15.7±3.1 and 8.5± 0.6 μmol mg−1 min−1, respectively. The respective Km values were 10.3±2.8, 12.9±3.9 and 3.3±0.6 mg/ml. For comparison, TrCel5A was also assayed at 37°C, pH 4.5 with PASC as a substrate and had a Vmax value of 5.7± 0.6 μmol mg−1 min−1 and a Km value of 2.6±0.7 mg/ml. When CMC was the substrate, the Vmax values were 264± 40, 101±7 and 194±86 μmol mg−1 min−1 for ApCel5A, GtCel12A and StCel5A, respectively. The respective Km values were 7.4±1.4, 2.6±0.3 and 11±5 mg/ml. For comparison, TrCel5A was also assayed at 37°C, pH 4.5 with CMC as a substrate and had a Vmax of 135±5 μmol mg−1 min−1 and a Km value of 3.2±0.3 μmol mg−1 min−1. The catalytic
efficiencies (Kcat/Km) of ApCel5A, GtCel12A, StCel5A and TrCel5A with both PASC and CMC as substrates were also determined. With PASC and CMC as substrates the catalytic efficiencies were: 2.8 and 12 ml s−1 mg−1 for ApCel5A, 0.46 and 7.7 ml s−1 mg−1 for GtCel12A, 1.8 and 6.1 ml s−1 mg−1 for StCel5A and 1.5 and 15 ml s−1 mg−1 for TrCel5A. In addition, a comparison of the activity on both PASC and CMC substrates was done to obtain the amount of each endoglucanase (in pmol) required to obtain equivalent levels of hydrolysis. The pmol of ApCel5A, GtCel12A, StCel5A and TrCel5A necessary to obtain 2% PASC hydrolysis in 20 h were 3.68, 4.72, 2.05 and 1.43 and 0.033, 0.335, 0.079 and 0.182 for 2% CMC hydrolysis in 30 min, respectively. Analysis of hydrolysis products by thin-layer chromatography We compared the ability of ApCel5A, GtCel12A and StCel5A to hydrolyse various oligosaccharides using TLC. Culture filtrates prepared from the empty vector control did not hydrolyze any of the oligosaccharides at either pH 4.5 or 6.0 even after 24-h incubations (data not shown). The three EGLs efficiently hydrolysed G5 and G6; however, GtCel12A and ApCel5A produced glucose as the major product whereas StCel5A produced cellobiose as the
Table 2 Quality scores for the three models by Qmean and PDBsum servers Ramachandran-within limits
ApCel5A GtCel12A StCel5A a
Qmeana
Z-score
Bond length (in %)
Bond angle (in %)
Planar group (in %)
Core (in %)
0.678 0.57 0.731
−0.87 −1.508 −0.03
99.1 97.5 99.6
95.0 95.1 99.2
78.1 81.2 84.6
82.6 77.7 88.2
The result of a combination of six structural descriptors whose values range from 0 to 1, where model quality correlates with higher values (Benkert et al. 2009)
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Table 3 MALDI-TOF mass spectrometry
Mass (Daltons) Position ApCel5A 72– 86* 148–155*b 156–179 180–204 205–230 343–348 GtCel12A 81–91 151–160
StCel5A 89–110 133–139 140–155 170–180 181–193 194–201 305–313
Peptide sequencea 25.8% covered in 39.6% possible VVTSAKASTTQAPSK DDKLNAFR LPVGWQYLVNSQLGGTLD STFFAK YDQQMTYCLNSGAALCIL DLHNYAR WNGQIVGTSGGPTNAQFA SVWSQLAK LATWLR
Predicted
Observed
1,475.81 978.50 2,641.37
1,475.76 978.53 2,641.84
2,876.32
2,876.07
2,704.35
2,704.90
759.45
759.44
1,181.62 955.56
1,181.58 955.53
2,355.07
2,355.22
897.41 1,837.94 1,304.64 1,547.75 980.52 1,029.58
897.46 1,838.04 1,304.73 1,547.85 980.58 1,029.66
SDS-PAGEc EGL
pGBFINGTW
9.3% covered in 9.3% possible TYANVLSNTAK GGIQPVGSLK
23.1% covered in 35.4% possible WLGSNESGAEFGEGNYPG LWGK IDFSMER LVPNQLTSSFDQGYLR YAVLDPHNYGR YYGNIITDTNAFR TFWTNLAK VVGATQWLR
a
Protein fragments detected by MALDI-TOF analysis of trypsin-digested SDS-PAGE resolved ApCel5A, GtCel12A and StCel5A. The detection range was 500–3,000 Da
b
Fragments harbouring one missed cleavage
c
SDS-PAGE of culture filtrates produced by A. niger transformed with pGBFIN-GTW harbouring the ApCel5A, GtCel12A and StCel5A ORFs and pGBFIN-GTW alone
*
Indicates the fragments with missed cleavages
major product (Fig. 4). Contrasting the ability of all three endoglucanases to hydrolyze G5 and G6, only GtCel12A efficiently hydrolyzed G2, G3 and G4 (Fig. 4). GtCel12A and StCel5A also showed transglycosylation activity when using G2 and G3, and G3, respectively, as substrates. Synergy Next, we wanted to compare the ability of ApCel5A, GtCel12A, StCel5A and TrCel5A to hydrolyze filter paper and crystalline Avicel both individually and in combination with the well-characterized exoglucanase TrCel7A. All four endoglucanases exhibited significant synergy with
TrCel7A when either filter paper (Fig. 5) or Avicel (Fig. 6) was used as the substrate. ApCel5A exhibited the highest activity on filter paper showing 10-fold, 4.3-fold, 1.9-fold and 60-fold higher activity per mole of enzyme than GtCel12A, StCel5A, TrCel5A and TrCel7A (Fig. 5). When combined with the TrCel7A exoglucanase, the ApCel5A and TrCel5A combinations exhibited the highest activities on filter paper. The degree of synergy (dividing the experimental value by the expected value) exhibited with TrCel7A was 1.8, 2.3, 2.5 and 2.5 for ApCel5A, GtCel12A, StCel5A and TrCel5A, respectively. The lower degree of synergy obtained with the ApCel5A versus TrCel5A apparently occurred because when used individ-
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211
Fig. 3 Biochemical characterization of the EGLs: a effect of pH on activity, b effect of temperature on activity and c effect of preincubation for 2 h at various temperatures on activity. Curves for ApCel5A (circles), GtCel12A (diamonds) and StCel5A (squares)
ually in equal molar amounts ApCel5A exhibited significantly higher activity than TrCel5A. ApCel5A, StCel5A and TrCel5A, when assayed using Avicel as the substrate, exhibited very similar activity with 1.8 pmol of enzyme producing 0.21, 0.22 and 0.17 nanomoles (nmol) of reducing sugar ends during the reaction, respectively (Fig. 6). The GH12 EGL was less active and produced about 0.11 nmol of reducing sugar ends (Fig. 6). Under the assay conditions tested, the maximum degree of synergy with TrCel7A was 1.7-fold for ApCel5A, GtCel12A and StCel5A and 3.3-fold for TrCel5A; however, the ApCel5A/TrCel7A combination produced comparable amounts of reducing sugar ends to the TrCel5A/TrCel7A combination (Fig. 6).
scale industrial protein production, it should be relatively easy to achieve commercially viable production levels for these three endoglucanases. The pH 4.5 optima of ApCel5A and GtCel12A fall within the pH 4 to 4.5 range, which is optimal for growth of ethanol producing Saccharomyces cerevisiae strains. Expressing ApCel5A and/or GtCel12A in combination with other cellulase system enzymes with similar pH optima could be used to engineer yeast strains capable of cellulose saccharification and glucose fermentation to ethanol.
Discussion The three cellulases, ApCel5A, GtCel12A and StCel5A were expressed as a secreted protein at high levels by A. niger. This is an attractive feature, since it is often difficult to efficiently express proteins in a heterologous host. Given that A. niger has a long history of use as a host for large
Fig. 4 Thin-layer chromatography of glucooligosaccharide hydrolysis products. TLC analysis of reaction products released during a 16h incubation of EGLs GtCel12A, ApCel5A and StCel5A with cellobiose (G2), cellotriose (G3), cellotetraose (G4), cellopentaose (G5) and cellohexaose (G6). The size standard lane L is a mixture of glucose and G2 to G6
Fig. 5 Reducing sugar equivalents produced from filter paper by individual cellulases and binary combinations. Activities obtained for the individual EGLs (EGL) using 0.9 pmol ApCel5A (black bar), GtCel12A (medium-grey bar), StCel5A (light-grey bar), TrCel5A (white bar) and for CBHI (CBH) using 30.6 pmol TrCel7A (lined bar) are shown. Binary combinations were performed using 0.9 pmol of the individual EGLs and 30.6 pmol of the TrCel7A (Experimental). Also presented are the sum of the activities obtained with 0.9 pmol of the individual EGLs and 30.6 pmol of the TrCel7A (Expected). Bars designating (Expected) and (Experimental) are indicated according to the designation used for the EGL included in the mixture
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Fig. 6 Reducing sugar equivalents produced from Avicel by individual cellulases and binary combinations. The upper x-axis is the pmol amount used for each EGL (ApCel5A in (a), GtCel12A in (b), StCel5A in (c) and TrCel5A in (d)) and lower x-axis is the pmol amount used in each reaction of commercially obtained CBH
(TrCel7A). The y-axis is the amount of reducing sugar ends produced in nmol. The activities with an EGL alone (squares) or the cellobiohydrolase alone (diamonds) are shown: the predicted combined rate (triangles) may be compared with the experimentally determined combined rate (circles) to estimate synergy
It was interesting that GtCel12A hydrolysed cellotriose, since a previous study found that T. reesei Cel12A was unable to hydrolyze G3 (Karlsson et al. 2002). This demonstrates that GH12 EGLs exhibit differences in substrate specificity. The substrate specificity of StCel5A also differed remarkably from that of the other EGLs. A study investigating the hydrolytic action of a Bacillus agaradhaerens Cel5A found that G5 was cleaved to yield G3 and G2. The authors postulated that G2 might inhibit the B. agaradhaerens enzyme (Schagerlöf et al. 2009). Our results for StCel5A are similar to those observed with B. agaradhaerens Cel5A in that very little glucose was produced. ApCel5A, on the other hand, generated significant amounts of glucose, particularly when G5 and G6 were the substrates, suggesting that compared with other Cel5A enzymes ApCel5A has different specificity and efficiency for glucooligosaccharide hydrolysis. ApCel5A, GtCel12A, StCel5A and TrCel5A all displayed significantly higher activity on filter paper than on Avicel with this being most significant for ApCel5A, which had 10-fold
higher activity on the less structured substrate. That cellulases are more active on a less structured substrate was noted previously in a comprehensive survey of cellulases from different Trichoderma species (Zhang and Lynd 2004). Each of the novel EGLs characterized here exhibited potentially attractive characteristics for various biotechnological applications. ApCel5A is an acid tolerant enzyme, and its Vmax on both PASC and CMC was significantly greater than that of TrCel5A. ApCel5A was also able to generate glucose from glucooligosaccharides, had the highest activity alone on filter paper, and exhibited a high level of synergism when combined with TrCel7A. StCel5A was thermostable, had a Vmax comparable to TrCel5A with both substrates and showed synergism in combination with TrCel7A. Lastly, GtCel12A exhibited significant synergism with TrCel7A, had an acidic pH optimum, an almost 3-fold higher Vmax than TrCel5A with the PASC substrate and produced glucose from glucooligosaccharides. The pH optima and acid tolerance of ApCel5A and GtCel12A make them potentially attractive candidates for
Appl Microbiol Biotechnol (2012) 93:203–214
biomass conversion, because the substrate will not have to be neutralized before cellulase treatment. These attributes would also be advantageous for engineering consolidated bioprocessing competent S. cerevisiae, since this efficient ethanol producer prefers growth under low pH conditions. With the increased importance of biofuels derived from renewable cellulosic feedstocks, it is imperative that cost effective cellulase formulations be developed. An attractive approach is to identify novel cellulases with hydrolysis capabilities superior to that exhibited by the cellulases in presently available commercial cellulase formulations. The catalytic efficiencies (Kcat/Km) of ApCel5A, and StCel5A using both CMC and the model pretreated cellulosic substrate PASC as substrate, were comparable to TrCel5A suggesting they may be attractive candidates for developing next generation cellulase systems. When using either PASC or CMC as a substrate the Vmax of both Cel5A enzymes was higher than T. reesei endoglucanase, Cel5A/EG2. This was most dramatic for ApCel5A, which had a Vmax about seven and two times greater than T. reesei Cel5A/EG2 using PASC and CMC, respectively. The same level of CMC hydrolysis was obtained using significantly less ApCel5A and StCel5A than TrCel5A the wellcharacterized endoglucanase found in many commercial cellulase formulations. ApCel5A and StCel5A might therefore, be good candidate enzymes for developing improved cellulase formulations. Synergy assays with the model cellulosic feedstock PASC as substrate also suggested that ApCel5A was an attractive candidate for cellulosic biorefinery applications. Acknowledgement This work was supported by a Strategic Project Grant STPGP336896-06 and a collaborative Research and Development Grant CRDPJ385439-09 from the Natural Sciences and Engineering Research Council of Canada to RS and JP.
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