Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2014 www.elsevier.com/locate/jbiosc

Identification of glycosyl hydrolases from a metagenomic library of microflora in sugarcane bagasse collection site and their cooperative action on cellulose degradation Pattanop Kanokratana,1, 2 Lily Eurwilaichitr,1 Kusol Pootanakit,2 and Verawat Champreda1, * Enzyme Technology Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phahonyothin Road, Klong Luang, PathumThani 12120, Thailand1 and Institute of Molecular Biosciences, Mahidol University, Salaya, Nakornpathom 73170, Thailand2 Received 19 May 2014; accepted 13 September 2014 Available online xxx

Lignocellulose decomposition is a natural process involving the cooperative action of various glycosyl hydrolases (GH) on plant cell wall components. In this study, a metagenomic library was constructed to capture the genetic diversity of microbes inhabiting an industrial bagasse collection site. A variety of putative genes encoding GH families 2, 3, 5, 9, 11, and 16 were identified using activity-based screening, which showed low to moderate homology to various cellulases and hemicellulases. The recombinant GH9 endoglucanase (Cel9) and GH11 endo-xylanase (Xyn11) were thermophilic with optimal activity between 75
C and 80
C and the maximal activity at slightly acidic to neutral pH range. The enzymes exhibited cooperative activity with Trichoderma reesei cellulase on the degradation of lignocellulosic substrates. Mixture design showed positive interactions among the enzyme components. The optimal combination was determined to be 41.4% Celluclast, 18.0% Cel9, and 40.6% Xyn11 with the predicted relative reducing sugar of 658% when compared to Celluclast alone on hydrolysis of alkaline-pretreated bagasse. The work demonstrates the potential of lignocellulolytic enzymes from a novel uncultured microbial resource for enhancing efficiency of biomass-degrading enzyme systems for bio-industries. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Glycosyl hydrolases; Lignocellulose; Metagenome; Mixture design; Sugarcane bagasse]

In the global biogeochemical organic carbon cycle, decomposition of lignocellulosic plant biomass by microbes is an essential process. The complex physical structure and chemical composition of lignocellulose confers high physicochemical stability; hence, lignocellulose decomposition involves cooperative actions of a diverse range of microbes producing various lignocellulolytic enzymes targeting cellulosic and non-cellulosic plant cell wall components (1). Lignocellulose has received increasing attention as a promising alternative renewable feedstock to depleting fossil resources, and it provides the basis of the biorefinery industry in which it is converted to fuels and value-added chemicals. Enzymatic hydrolysis of plant biomass to sugar is the key step in the sugar platform biorefinery. Exploration of microbial lignocellulose degradation mechanisms in nature and discovery of efficient lignocellulolytic enzyme systems are thus a key issue in current biotechnology research with potential for application in biomass industry. Synergistic or cooperative actions among lignocellulolytic enzymes have been reported for many cellulolytic and hemicellulolytic enzyme systems for both aerobic and anaerobic bacteria (2e5). The synergy of enzyme catalysis on hydrolysis of lignocelluloses is based on different mechanistic models of the enzymes on the substrates; for example, differences in their substrate

* Corresponding author. Tel.: þ66 2564 6700x3473; fax: þ66 2564 6707. E-mail address: [email protected] (V. Champreda).

specificities and their mode of actions among a variety of glycosyl hydrolases and auxiliary enzymes (6), proximity effects of enzyme components in cellulosomal systems (2), and physical alteration of the substrates by non-catalytic proteins, e.g., expansins (7). These enzyme cooperative interactions form the basis of efficient lignocellulose degradation in nature and provide a basis for development of efficient lignocellulolytic enzyme systems for biotechnological application. Culture-independent metagenomic technology has been used to explore novel genes and metabolic pathways from uncultured microbes, which can make up to 99% of total microbial diversity in environments. Different techniques, including functional-based and sequence-based gene identification and direct highthroughput pyrosequencing have been used to explore environmental metagenomes and reveal complex structures of microbial assemblages in various ecosystems related to plant biomass decomposition (8,9). These approaches have been used to identify a number of genes encoding various novel lignocellulose degrading enzymes from termite gut (10,11), cow rumen (12), compost (13), and symbiotic microbial consortia (14). A number of them show desirable properties for industrial applications such as alkaliphilic xylanases for pulp bio-bleaching (10). Industrial bagasse collection sites at sugar mills represent interesting habitats to explore lignocellulose decomposition because of their relatively high temperature, low nitrogen availability, and indigenous microbes. Recently, microbial communities occupying different regions of a bagasse pile have been explored for

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.09.010

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their metabolic potential using high-throughput pyrosequencing approaches, which revealed complex microbial communities with different niches varying according to microenvironmental conditions (15). In particular, substantial numbers of lignocellulolytic enzymes are found in the inner pile based regions suggesting that this unique environment provides a rich potential resource of biomass degrading microbes and enzymes. In this study, a fosmid metagenomic library was constructed to represent the diversity of microbes residing in a bagasse pile. Activity-based screening was used to identify genes encoding various glycosyl hydrolases attacking lignocellulosic components. A recombinant thermophilic endo-glucanase and a xylanase from the metagenome were characterized and their cooperative action on enhancing efficiency of Trichoderma reesei cellulase was demonstrated using a mixture design approach. The work shows potential of enzymes from unexplored microbial resources and provides the basis for development of efficient biomass-degrading enzyme systems for biotechnological application. MATERIALS AND METHODS Sample collection Sugarcane bagasse samples were collected from an industrial bagasse collection site at Phu Khieo Bio-Energy, Chaiyapoom province,
0 00 Thailand (N 16 28 54 , W 102
070 0500 ). Bagasse was collected from a large open-air pile, approximately 10 m in height covering the area of several acres. The bagasse had been left in the field for approximately 6 months at the time of collection. A bagasse sample was taken at a depth of 1 m from the base of bagasse pile in June 2009. The temperatures of the samples inside the pile at the collection time ranged from 49 to 52
C. The sample was rapidly frozen in liquid nitrogen and kept at 80
C for subsequent experiments. Lignocellulosic biomass preparation Intact bagasse sample collected from the same site was physically processed using a SM2000 cutting mill (Retsch, Haan, Germany) and sieved to 80% activity was retained after incubation at 60
C for 3 h (Fig. 2c). However, no activity was detected against other cellulosic substrates [Avicel, carboxymethylcellulose (CMC), and filter paper]. Recombinant Xyn11 expression and characterization The mature GH11 xylanase gene was amplified and cloned into pET-32a for expression of the enzyme with an N-terminal 109-aa Trx$Tag thioredoxin tag and a C-terminal His6-tag. Recombinant Xyn11 of the expected Mw of 45 kDa was detected on SDS-PAGE. The induced protein was mostly insoluble when expressed at 37
C. Reduction of induction temperature (30
C) and IPTG concentration (0.2 mM) led to a higher fraction of soluble Xyn11 protein. The recombinant Xyn11 was purified on Ni2þ matrix for subsequent characterization. The enzyme activity profiles of Xyn11 were characterized against 1% beechwood xylan as the substrate. The recombinant xylanase worked optimally at 80
C and at pH 6 (Fig. 2a, b). Under these conditions, the enzyme showed a specific activity of 435 U/mg. The enzyme also retained >80% activity after incubation at 60
C for 3 h (Fig. 2d). The mature gene sequences of cel5 and bgl3A and the full length glu16 were also cloned into protein expression vectors. Recombinant Cel5 was obtained when expressed as a Trx/His6 fusion protein with the apparent Mw of 55 kDa. However, this recombinant protein showed no activity against AZCL-b-glucan or other cellulosic substrates including avicel, CMC and filter paper. Glu16 was expressed as an N-terminal His6 fusion. This protein, however, was expressed mainly in the insoluble form and no activity against bglucan was detected. No expression of recombinant Bgl3A was observed under the experimental conditions used in this study. Optimization of ternary enzyme system The optimal composition of the ternary enzyme mixture comprising Celluclast, Cel9, and Xyn11 was studied using the mixture design method (22). Pretreated bagasse was used as the model substrate. As the enzyme activities were determined based on their corresponding substrates, the model was thus designed based on the amount of each component as a percentage in the mixture. Celluclast was diluted from a commercial stock to obtain final concentration of 0.061 FPU/ml while the concentration of purified Cel9 and Xyn11 were 180 Unit/ml and 280 Unit/ml respectively. All experimental points generated by the software are located inside the triangular graph, which means that the sum of Celluclast, Cel9, and Xyn11 loading for each experimental point is always 100% (Fig. 3a). The responses of the relative reducing sugar yield obtained from hydrolysis of pre-treated bagasse are graphically illustrated as a

TABLE 1. Annotation of genes encoding glycosyl hydrolases from the positive fosmid library clones. Gene

Length (bp)

GH family

NCBI accession number

Closest relative protein

Organism

% Identity

glu2 glu16 cel5 bgl3A bgl3B cel9 xyn11

1374 921 1221 3147 2478 2424 1077

GH2 GH16 GH5 GH3 GH3 GH9 GH11

AGN70395 AGN70392 AGN70391 AGN70393 AGN70394 AGN70390 AGN70389

b-Galactosidase

Myxococcus xanthus Halothermothrix orenii Ignavibacterium album Marinilabilia salmonicolor Ignavibacterium album Micromonospora aurantiaca Uncultured bacterium

26 57 57 43 50 66 74

Laminarinase Endoglucanase b-Glucosidase b-Glucosidase Endoglucanase Xylanase

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KANOKRATANA ET AL.

J. BIOSCI. BIOENG., Carboxypeptidase regulatory-like domain GH2 domain

Glu2

457

GH16 domain

Glu16

306

GH5 domain

Cel5

406

Por secretion system C-terminal sorting domain GH3 N-terminal domain Signal sequence

Discoidin domain

GH3 C-terminal domain

Cell adhesion related domain 1048

Bgl3A Fibronectin type III-like domain GH3 N-terminal domain Signal sequence

GH3 C-terminal domain

PA 14 domain

Bgl3B

825

Fibronectin type III-like domain GH9 domain Signal sequence

CBM 3

Cel9

CBM 2 807

Signal sequence GH11 domain

Xyn11

352

FIG. 1. Putative conserved domains found in each protein. Numbers indicates the number of amino acid in polypeptide chain.

ternary contour plot (Fig. 3b). The area of the graph that highlighted the greatest relative sugar yield was in the middle of the Celluclast and Xyn11 axes and near the bottom of the Cel9 vertex. This indicates that a high level of reducing sugars can be obtained when the concentration of Celluclast and Xyn11 are nearly equal whereas a lower amount of Cel9 is needed. The relative reducing sugar yield of each experimental point after the hydrolysis of the biomass for 24 h is demonstrated in Table 2. The binary combination between Celluclast and Xyn11 (No. 6 and 7) demonstrated a cooperative effect between both enzymes as a significantly increased reducing sugar yield was observed when compared to the reactions containing only one enzyme (No. 1 and 3). Cel9 also showed slight enhancement on hydrolytic activity of Xyn11 (No. 8 and 9) and Celluclast (No. 4 and 5) in the binary complex relative to the reactions using the single enzyme. Moreover, cooperative interactions among Celluclast, Cel9, and Xyn11 were found in the ternary combinations (No. 10, 11, 12, and 13). These results suggest that the recombinant enzymes obtained from environmental metagenome can enhance the biomass hydrolysis efficiency of the T. reesei cellulase. The response data for the relative sugar yield was further analyzed using multiple regression analysis. The full cubic model was found to be the best fitted model for the response data (R2 ¼ 92.82%).

ANOVA analysis data of the full cubic model regression analysis are provided in Table 3 in order to give an insight into the importance of individual components and their interaction on enzyme cooperation (23). For a single factor, Xyn11 showed the highest positive relation to the relative sugar yield, while the Cel9 coefficient was the lowest, indicating that Cel9 alone had a relatively low contribution to the reducing sugar yield compared with the other two components. For pairwise interactions, a marked cooperation between Celluclast and Xyn11 is shown (1244.79). Significant interactions between Cel9 and Xyn11 (957.48) and between Celluclast and Cel9 (55.36) were also observed. The highest coefficient was detected for the mixture of all three components, indicating significant interactions among these three enzymes. In addition, the reduced model equations for the relative reducing sugar yield based on the component amount after removal of non-significant factors (p  0.05) are: Relative sugar (%) ¼ 0.99459Celluclast þ 0.12338Cel9 þ 5.00545Xyn11 þ 0.12448CelluclastXyn11 þ 0.09575Cel9Xyn11 þ 0.00471 CelluclastCel9xyn11 þ 0.00139CelluclastXyn11() þ 0.00095Cel9Xyn11(). The optimal composition of the three enzyme components was predicted using Minitab 16.0 for maximizing the relative reducing sugar yield. The optimal combination for pretreated bagasse

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DISCUSSION

FIG. 2. Effects of pH and temperature on activity and stability on the recombinant enzymes. The enzyme activities were analyzed using 0.5% b-glucan and 1% beechwood xylan as the substrates for Cel9 and Xyn11, respectively. (A) pH: Reactions contained 50 mM Mcllvaine’s buffer and incubated for 10 min 75
C or 80
C for Cel9 and Xyn11, respectively. (B) Temperature: Reactions contained 50 mM Mcllvaine’s buffer pH 6.0 and incubated at varying temperatures for 10 min. The reactions were incubated in 50 mM phosphate buffer, pH 6.0 for 3 h at the corresponding temperature to determine the thermostability of Cel9 (C) and Xyn11 (D).

hydrolysis was determined to be 41.4% Celluclast, 18.0% Cel9, and 40.6% Xyn11 with the predicted relative reducing sugar of 658%. The experimentally-determined relative reducing sugar yields were 642% equivalent to the reducing sugar yield of 104 mg/g substrate with the Celluclast dosage of 0.01 FPU/g substrate. The results thus validate the response model equations (5% confidence interval).

Exploration of lignocellulose degrading enzymes from uncultured microbe metagenomes is a promising approach for increasing our understanding on lignocellulose decomposition in nature. This knowledge could be applied to enhance bioconversion efficiency on biomass degradation in bio-industry. In this study, the genetic diversity of microbes inhabiting a bagasse pile was explored for lignocellulolytic enzymes using activity-based screening. Microbial diversity in this habitat, as shown by clonal library analysis (24) and tagged 16S rRNA pyrosequencing (15) revealed novel and complex microbial communities with high metabolic potential on lignocellulose degradation. Activity screening based on target enzyme function has been demonstrated to be useful for identifying genes and gene clusters encoding various glycosyl hydrolases and auxiliary enzymes involved in biomass degradation from various environments, e.g., soil (25), rumen (26), and termite gut (10). On the one hand, activity-based screening is limited by differences among species in codon usage, transcription and translation signals, protein folding elements, and post-translational modification, which affect heterologous expression in E. coli host. On the other hand, this technique allows discovery of completely novel genes in new families which would not be found by conventional screening based on gene sequence homology to known genes. The lack of success in expressing Cel5, Glu16, and Bgl3A as soluble, active enzymes in E. coli in this study highlighted the limitation of activitybased screening of metagenomic libraries. One solution to this problem is to perform heterologous expression in alternative hosts, e.g., Sinorhizobium meliloti and Rhizobium leguminosarum (27,28). A range of genes encoding glycosyl hydrolases in different GH families have been found from screening a metagenomic fosmid library in this study. Many of them are related to major classes of enzymes involved in degradation of plant biomass. The Cel9 enzyme belongs to the GH9 family and is of a similar structural organization to a previously identified endoglucanase from Cellulomonas flavigena and Thermobifida fusca containing a GH9 catalytic domain linked to a family 3 carbohydrate binding module (CBM), two fibronectin III-like domains, and a family 2 CBM (29,30). According to the CAZy database (http://www.cazy.org) (31), all members of GH9 are cellulases including endoglucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91) or b-glucosidase (EC 3.2.1.21). Although Cel9 is closely related to endoglucanase and was expected to hydrolyze cellulosic substrates (CMC, Avicel and filter paper), it only exhibited high activity against the b-glucan, while its activity against other cellulosic substrate was not detected. Compared to most GH9 endoglucanases reported to date which showed broader substrate specificity, its narrow substrate specificity is distinct. A gene cluster consisting of five contiguous genes (glu2, glu16, cel5, bgl3A and bgl3B) encoding proteins related to different GH families was found from the metagenomic library. The structural arrangement of these genes was consistent with their arrangement as an operon. Operons containing sets of genes encoding cellulases and hemicellulases have been found in anaerobic bacteria such as Clostridium, Bacteroidetes, and Acitovibrio. These operons mostly encode catalytic units and scaffoldin protein, which collectively constitute cellulosome systems. These enzyme complexes have been characterized in several anaerobic bacteria and show high activity against crystalline cellulose owing to the composite enzyme synergy based on their catalytic specificities and proximity effects (4). However, the operon identified in this study lacks dockerin regions characteristic of cellulosomal components, suggesting that they do not encode cellulosomal enzymes. Arrangement of non-cellulosomal genes in an operon has been found in some bacteria, e.g., Cellvibrio mixtus (32). A xylanase gene cluster

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J. BIOSCI. BIOENG.,

FIG. 3. The ternary plots of the experimental design optimization of the ternary enzyme complex. (A) The design points for the {3,3}-augmented simplex lattice design and (B) the contour plot of the relative reducing sugar yield from hydrolysis of pretreated bagasse.

lacking a dockerin region was also recently reported in termite gut metagenome (10). The Bgl3A and Bgl3B enzymes contain GH3 catalytic modules and other domains with various putative functions. Bgl3B shows an unusual protein arrangement in that a PA14 domain interrupts the catalytic core domain. A similar structure has been found in the GH3 enzyme from Kluyveromyces marxianus, and a role for PA14 in carbohydrate binding and substrate specificity was suggested (33). A type III-like fibronectin domain present in both Bgl3A and Bgl3B is commonly found among bacterial glycosyl hydrolase enzymes (34). This domain aids the hydrolysis of cellulose by modifying its surface (34), and removal of this domain reduces cellulase activity on bacterial microcrystalline cellulose (30). Moreover, a Por secretion system C-terminal sorting domain is located at the C-terminal end of Bgl3A. This domain has also been shown to be involved in translocation of some proteins to the bacterial cell surface (35); therefore, Bgl3A may function as an extracellular enzyme. The protein sequence of all five gene products in the gene cluster showed low to moderate similarity to related genes from phylum Bacteroidetes, suggesting the origin of this gene cluster. Bacteroidetes is a major bacterial group in many lignocellulose degrading environments, such as the crop of an invasive snail (36), and in the bagasse collection site, as shown by direct shotgun pyrosequencing

TABLE 2. {3,3} simplex lattice design for the optimization of the Celluclast, Cel9 and Xyn11 ternary enzyme complex for pretreated bagasse hydrolysis and the associated response data. No.

1 2 3 4 5 6 7 8 9 10 11 12 13

Variable (%)

Relative reducing sugar (%)

Celluclast

Cel9

Xyn11

Average

SD

100.0 0.0 0.0 66.7 33.3 66.7 33.3 0.0 0.0 33.3 66.7 16.7 16.7

0.0 100.0 0.0 33.3 66.7 0.0 0.0 66.7 33.3 33.3 16.7 66.7 16.7

0.0 0.0 100.0 0.0 0.0 33.3 66.7 33.3 66.7 33.3 16.7 16.7 66.7

100.0 12.6 497.6 70.5 50.5 594.1 562.7 442.3 501.5 620.9 511.3 418.7 525.7

0.0 3.7 71.2 20.0 26.6 84.2 106.9 57.9 81.7 141.1 76.1 42.2 66.6

Fit

SE fit

99.459 12.338 500.454 79.966 56.435 613.100 540.146 458.587 479.842 629.493 485.538 397.728 555.281

40.693 40.693 40.693 37.258 37.258 37.258 37.258 37.258 37.258 31.362 27.982 27.982 27.982

The fit value refers to point estimates of the mean response for the given values of the predictors, factor levels, or components calculated by the Minitab 16.0 software. SE of fit refers to the standard error of the fit value calculated by the Minitab 16.0 software. Relative reducing sugar of 100% was equivalent to the released sugars of 23.6 mg/g biomass.

(15). A recent study of genes encoding proteins related to cellulose utilization among 5123 sequenced bacterial genomes revealed that Bacteroidetes are highly represented as potent cellulose degraders in various environments (37). In this study, two genes, cel9 and xyn11, were successfully expressed in E. coli in active forms and were characterized biochemically. Both enzymes are thermophilic, working optimally at 75e80
C under acidic conditions reflecting the high-temperature conditions in the bagasse pile. Many enzymes screened from metagenomic libraries also exhibit this habitat-related property, for example, a genes encoding thermophilic cellulases working optimally at 60e65
C were identified from the metagenome of microbial community in a thermophilic anaerobic digester (38,39). The results thus indicate the potential of metagenomic approaches for discovering novel genes with promising properties for bio-industry and the importance of selecting environments under related physical conditions as the sources for metagenomes. In order to develop an enzyme mixture for efficient biomass degradation, the recombinants Cel9 and Xyn11 targeting different lignocellulosic substrates were evaluated for their cooperative activity with a commercial T. reesei cellulase (Celluclast). T. reesei cellulase has been shown as a highly efficient enzyme system on lignocelluloses degradation (40,41). It comprises a broad range of cellulolytic enzyme activities, most notably cellobiohydrolase (CBH) and endoglucanase. It has been utilized as a core enzyme on lignocellulose hydrolysis in various biotechnological applications including biomass saccharification in biorefinery. In this study, the mixture design method (42) was employed to determine optimal enzyme mixture between Celluclast, Cel9, and Xyn11 for pretreated bagasse hydrolysis. This experimental design approach has TABLE 3. The regression model analysis of the {3,3} full cubic model developed for the relative reducing sugar yield (component proportion) from pretreated bagasse hydrolysis. Factor Celluclast Cel9 Xyn11 Celluclast*Cel9 Celluclast*Xyn11 Cel9*Xyn11 Celluclast*Cel9*Xyn11 Celluclast*Cel9*() Celluclast*Xyn11*() Cel9*Xyn11*() S ¼ 70.6310 R2 ¼ 92.82%

Coefficient

SE

99.46 40.69 12.34 40.69 500.55 40.69 55.36 181.91 1244.79 181.91 957.48 181.91 4712.33 1185.99 37.18 348.46 1394.88 348.46 954.99 348.46 PRESS ¼ 250907 R2 (pred) ¼ 87.55%

T

p-value

e e e 0.30 6.84 5.26 3.97 0.11 4.00 2.74

e e e 0.763 0.000 0.000 0.000 0.916 0.000 0.010

R2 (adj) ¼ 90.59%

Asterisks indicate interaction between factors.

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been previously used for examining the synergistic interactions among six purified fungal cellulases (43) and between a crude fungal enzyme, bacterial expansin, and Celluclast (44). Based on our results, all three enzymes displayed cooperative interactions for the pretreated biomass hydrolysis, and optimal enzyme mixtures with maximal activity toward degradation of the target cellulosic substrates were identified. High degree of enzyme’s cooperativity (642%) was shown at a low cellulase dosage (0.01 FPU/g substrate). Lower degree of co-operativity was observed at a higher cellulase dosage, which is similar to that found with other mixture systems comprising a core cellulase and accessory enzymes or proteins (7). Our result shows cooperation of enzymes from different microbial origins and represents the first example on cooperative actions between enzymes from metagenomes and a fungal core cellulase. Synergistic and cooperative actions of lignocellulolytic enzymes have been reported for many cellulolytic and hemicellulolytic enzyme systems for both aerobic and anaerobic bacteria (2e5). The basis on cooperation of lignocelluloses degrading enzymes generally involves (i) different substrate specificities of endo-acting enzymes and downstream enzymes acting on smaller substrates and (ii) cooperative action of cellulolytic enzymes and enzymes attacking non-cellulosic polysaccharides in the plant cell wall, e.g., hemicellulases, glucanases, and pectinases, which creates new sites of attack for the other enzymes (6). In addition to the synergistic interaction of the enzymes, expansins, a class of non-catalytic protein, has been recently reported to increase the hydrolysis of lignocelluloses by a physical alteration process (7,44e46). The synergistic and cooperative enzyme action forms the basis of efficient lignocellulose degradation in nature and could be applicable for the development of efficient polysaccharide degrading enzyme systems for biotechnological application. The optimal mixture in this study contained a relatively low dosage of Celluclast for biomass saccharification in biorefinery process as it resulted in relatively low sugar yield (104 mg/g pretreated biomass). However, this level of polysaccharide degradation is considered practical for many other biotechnological processes, for examples, in textile or pulp processing where only partial hydrolysis or modification of the cellulose/hemicellulose in the substrates is required (47). Chemical pretreatment by acids and alkalis are widely used as a basic pretreatment method for improving digestibility of lignocellulosic biomass (48). Alkaline-catalyzed hydrothermal treatment results in delignification of the biomass and swelling of the cellulose fibers while most of the hemicellulose fraction is intact. In contrast, diluted acid pretreatment leads to hemicellulose removal as the major effect. The differences in chemical and physical characteristic of the biomass pretreated by different methods thus lead to variation in optimal enzyme composition for hydrolysis. The strategy demonstrated in our work could be efficiently applied to optimize enzyme mixture for various cellulosic substrates with difference in intrinsic properties. In conclusion, a metagenomic approach has been used to explore the genetic diversity of microbial community in a bagasse collection site. Several genes and a gene cluster were discovered encoding various biomass-degrading enzymes with different reaction specificities from uncultured bacteria. Cooperation between the enzymes from metagenomic approach and the core cellulase from T. reesei was demonstrated. The mixture design approach provides a promising strategy for improving enzyme performance used in bio-industry. ACKNOWLEDGMENTS This project was supported by a research grant from the National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency (P-10-10848). Kanokratana P. was granted by the Royal Golden Jubilee Scholarship

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Please cite this article in press as: Kanokratana, P., et al., Identification of glycosyl hydrolases from a metagenomic library of microflora in sugarcane bagasse collection site and their cooperative action..., J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.09.010