Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks.     

Carbon nanotubes tuned foam structures as novel nanostructured biocarriers for lignocellulose hydrolysis

The use of immobilized enzymes during saccharification of lignocelluloses enables the continuous process of enzymatic hydrolysis and repeatable use of enzyme, resulting in reduced operational cost. Novel nano-biocarriers were developed by layer-by-layer deposition of carbon nanotube (CNT) on the foam structures, and their efficiency for enzyme immobilization was demonstrated with cellulase and β-glucosidase. A three-fold enhancement was achieved in the activity of cellulase immobilized on CNT coated polyurethane foam. In addition, both cellulase and β-glucosidase immobilized on the CNT-foam showed much better storage stability and operational stability than the ones immobilized on the commercial biocarrier (Celite), which is critical for a continuous operation. CNT coated monolith was also developed as a biocarrier, offering high surface area and geometric stability. These nano-biocarriers are promising candidates for the efficient saccharification of biomass and to reduce carbon footprint and cost of the equipment.     

β-glucosidases from a new Aspergillus species can substitute commercial β-glucosidases for saccharification of lignocellulosic biomass

β-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for β-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus ) showed 10 times greater β-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial β-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis-Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3?U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4?°C for Novozym 188. The significant thermostability of strain AP β-glucosidases was further confirmed when compared with Cellic CTec. The β-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for β-glucosidase activity.     

Enhanced production of cellulase from a novel strain Trichoderma gamsii M501 through response surface methodology and its application in biomass saccharification

Cellulolytic enzymes produced by Trichoderma sp. have attracted interest in converting the biomass to simple sugars in the production of cellulosic ethanol. In this work, a novel cellulolytic strain M501 was isolated and identified as T. gamsii by sequencing the ITS rDNA region. The production of cellulase (CMCase) by T. gamsii M501 was enhanced by employing statistical methods. The strain grown in the optimized production medium composed of mineral salts, microcrystalline cellulose (13.7 g/l), tryptone (4.8 g/l) and trace elements (2 mL/l) at pH 5.5 and 28 °C for 72 h produced a maximum CMCase of 61.3 U/mL. The optimized production medium also showed the other enzyme activity of FPU (2.6 U/mL), β-glucosidase (2.1 U/mL), xylanase (681 U/mL) and β- xylosidase (0.6 U/mL). The crude cellulase cocktail produced by T. gamsii M501 efficiently hydrolyzed alkali pretreated sugarcane bagasse with glucose and xylose yield of 78 % and 74 % respectively at 10 % solid loading. This study is the first of its kind research on biomass saccharification using T. gamsii cellulase cocktail. Therefore, the novel strain T. gamsii M501 would be useful for further development of an enzyme cocktail for cellulosic ethanol production.     

Salivary β-glucosidase as a direct factor influencing the occurrence of halitosis

β-Glucosidases are enzymes present in all living organisms, playing a pivotal role in diverse biological processes. These enzymes cleave β-glycosidic bonds between carbohydrates, or between a carbohydrate and a non-carbohydrate moiety, which may result in the liberation of volatile aglycones. Released compounds execute diverse physiological roles, while the industry takes advantage of exogenously added β-glucosidases for aroma enrichment during food and beverage production. β-Glucosidase enzymatic activity has been reported in human saliva and given the fact that these enzymes are involved in aroma release, we investigated here the correlation between β-glucosidase activity in human saliva and the occurrence of halitosis. Measurement of salivary enzyme activity of 48 volunteers was performed using p-nitrophenyl-β-d-glucopyranoside as substrate. Each volunteer was clinically evaluated by a dental surgeon and clinical and laboratorial data were statistically analyzed. Gas-chromatography of saliva headspace allowed the analysis of the direct role of exogenous β-glucosidase on aromatic /volatile profile of saliva samples. The data demonstrated a positive correlation between halitosis and enzymatic activity, suggesting that the enzyme exerts a direct role in the occurrence of bad breath. Gas-chromatography analysis demonstrated that exogenously added enzyme led to the alteration of volatile organic content, confirming a direct contribution of β-glucosidase activity on saliva volatile compounds release. Although halitosis is a multifactorial condition, the complete understanding of all governing factors may allow the development of more effective treatment strategies. Such studies may pave the way to the use of β-glucosidase inhibitors for halitosis clinical management.     

The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover

Recycling of enzymes during biomass conversion is one potential strategy to reduce the cost of the hydrolysis step of cellulosic ethanol production. Devising an efficient enzyme recycling strategy requires a good understanding of how the enzymes adsorb, distribute, and interact with the substrate during hydrolysis. We investigated the interaction of individual Trichoderma reesei enzymes present in a commercial cellulase mixture during the hydrolysis of steam-pretreated corn stover (SPCS). The enzyme profiles were followed using zymograms, gel electrophoresis, enzyme activity assays and mass spectrometry. The adsorption and activity profiles of 6 specific enzymes Cel7A (CBH I), Cel7B (EG I), Cel5A (EG II), Xyn 10 (endo-1,4-β-xylanase III), Xyn 11 (endo-xylanase II), and β-glucosidase were characterized. Initially, each of the enzymes rapidly adsorbed onto the SPCS. However, this was followed by partial desorption to an adsorption equilibrium where the Cel7A, Cel7B, Xyn 10, and β-glucosidase were partially adsorbed to the SPCS and also found free in solution throughout the course of hydrolysis. In contrast, the Cel5A and Xyn 11 components remained primarily free in the supernatant. The Cel7A component also exhibited a partial desorption when the rate of hydrolysis leveled off as evidenced by MUC zymogram and SDS-PAGE. Those cellulase components that did not bind to the substrate were generally less stable and lost their activities within the first 24h when compared to enzymes that were distributed in both the liquid and solid phases. Therefore, to ensure maximum enzyme activity recovery, enzyme recycling seems to be most effective when short-term rounds of hydrolysis are combined with the recovery of enzymes from both the liquid and the solid phases and potentially enzyme supplementation to replenish lost activity.     

The US Department of Energy Great Lakes Bioenergy Research Center: Midwestern Biomass as a Resource for Renewable Fuels

The Great Lakes Bioenergy Research Center is one of three Bioenergy Research Centers establish by the US Department of Energy and the only one based at an academic institution. The Center’s mission is to perform basic and applied science to enable economically and environmentally sustainable production of liquid fuels derived from biomass. The research is focused on converting plant biomass into soluble sugars and the sugars into fuels. A large group focused on sustainability informs and guides the applied research to ensure that new technology will provide the required environmental benefits.     

Direct ethanol production from hemicellulosic materials of rice straw by use of an engineered yeast strain codisplaying three types of hemicellulolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells

The cost of the lignocellulose-hydrolyzing enzymes used in the saccharification process of ethanol production from biomass accounts for a relatively high proportion of total processing costs. Cell surface engineering technology has facilitated a reduction in these costs by integrating saccharification and fermentation processes into a recombinant microbe strain expressing heterologous enzymes on the cell surface. We constructed a recombinant Saccharomyces cerevisiae that not only hydrolyzed hemicelluloses by codisplaying endoxylanase from Trichoderma reesei, β-xylosidase from Aspergillus oryzae, and β-glucosidase from Aspergillus aculeatus but that also assimilated xylose through the expression of xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. The recombinant strain successfully produced ethanol from rice straw hydrolysate consisting of hemicellulosic material containing xylan, xylooligosaccharides, and cellooligosaccharides without requiring the addition of sugar-hydrolyzing enzymes or detoxication. The ethanol titer of the strain was 8.2g/l after 72h fermentation, which was approximately 2.5-fold higher than that of the control strain. The yield (grams of ethanol per gram of total sugars in rice straw hydrolysate consumed) was 0.41g/g, which corresponded to 82% of the theoretical yield. The cell surface-engineered strain was thus highly effective for consolidating the process of ethanol production from hemicellulosic materials.     

Hydrolysis of lignocellulosic materials for ethanol production: a review

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.     

基于重组策略的一体化生物加工过程最新进展

木质纤维素材料具有储量丰富、原料成本低及可再生等优点,人们期望其能替代石油作为原料来生产多种燃料和化学品,如生物柴油、生物氢、生物乙醇等,而木质纤维素解聚过程的高成本成为实现这一过程的主要障碍。一体化生物加工过程 (Consolidated bioprocessing,CBP) 是指在不添加任何外源水解酶的情况下,直接将木质纤维素原料一步转化为生物化学品的生物加工过程。通过基因工程,将水解酶的生成、木质纤维素的降解和生物产品的生产等功能集成到一个生物体上。对于CBP,人们通常有两种策略可供选择,即本地策略和重组策略。文中重点介绍了基于重组策略的CBP的原理、两种不同的应对方式、合成生物学及代谢工程对其的贡献以及未来所面临的挑战与展望。     

Economic analysis of fuel ethanol production from winter hulled barley by the EDGE (Enhanced Dry Grind Enzymatic) process

A process and cost model was developed for fuel ethanol production from winter barley based on the EDGE (Enhanced Dry Grind Enzymatic) process. In this process, in addition to β-glucanases, which are added to reduce the viscosity of the mash, β-glucosidase is also added to completely hydrolyze the oligomers obtained during the hydrolysis of β-glucans to glucose. The model allows determination of capital costs, operating costs, and ethanol production cost for a plant producing 40 million gallons of denatured fuel ethanol annually. A sensitivity study was also performed to examine the effects of β-glucosidase and barley costs on the final ethanol production cost. The results of this study clearly demonstrate the economic benefit of adding β-glucosidase. Lower ethanol production cost was obtained compared to that obtained without β-glucosidase addition in all cases except one where highest β-glucosidase cost allowance and lowest barley cost were used.     

Efficient screening of potential cellulases and hemicellulases produced by <Emphasis Type="Italic">Bosea</Emphasis> sp. FBZP-16 using the combination of enzyme assays and genome analysis

Identification of bacteria that produce carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. Twenty lignocellulose-degrading bacterial isolates from Algerian compost and different soils were screened for their potential to produce different enzymes involved in biomass deconstruction. Based on 16S rRNA gene sequencing, the isolates belonged to Proteobacteria and Actinobacteria. Differences among species were reflected both as the presence/absence of enzymes or at the level of enzyme activity. Among the most active species, Bosea sp. FBZP-16 demonstrated cellulolytic activity on both amorphous cellulose (CMC) and complex lignocellulose (wheat straw) and was selected for whole-genomic sequencing. The genome sequencing revealed the presence of a complex enzymatic machinery required for organic matter decomposition. Analysis of the enzyme-encoding genes indicated that multiple genes for endoglucanase, xylanase, β-glucosidase and β-mannosidase are present in the genome with enzyme activities displayed by the bacterium, while other enzymes, such as certain cellobiohydrolases, were not detected at the genomic level. This indicates that a combination of functional screening of bacterial cultures with the use of genome-derived information is important for the prediction of potential enzyme production. These results provide insight into their possible exploitation for the production of fuels and chemicals derived from plant biomass.     

Ecoenzymatic Stoichiometry in Relation to Productivity for Freshwater Biofilm and Plankton Communities

The degradation of detrital organic matter and assimilation of carbon (C), nitrogen (N), and phosphorus (P) by heterotrophic microbial communities is mediated by enzymes released into the environment (ecoenzymes). For the attached microbial communities of soils and freshwater sediments, the activities of β-glucosidase, β-N-acetylglucosaminidase, leucine aminopeptidase, and phosphatase show consistent stoichiometric patterns. To determine whether similar constraints apply to planktonic communities, we assembled data from nine studies that include measurements of these enzyme activities along with microbial productivity. By normalizing enzyme activity to productivity, we directly compared the ecoenzymatic stoichiometry of aquatic biofilm and bacterioplankton communities. The relationships between β-glucosidase and α-glucosidase and β-glucosidase and β-N-acetylglucosaminidase were statistically indistinguishable for the two community types, while the relationships between β-glucosidase and phosphatase and β-glucosidase and leucine aminopeptidase significantly differed. For β-glucosidase vs. phosphatase, the differences in slope (biofilm 0.65, plankton 1.05) corresponded with differences in the mean elemental C:P ratio of microbial biomass (60 and 106, respectively). For β-glucosidase vs. leucine aminopeptidase, differences in slope (0.80 and 1.02) did not correspond to differences in the mean elemental C:N of biomass (8.6 and 6.6). β-N-Acetylglucosaminidase activity in biofilms was significantly greater than that of plankton, suggesting that aminosaccharides were a relatively more important N source for biofilms, perhaps because fungi are more abundant. The slopes of β-glucosidase vs. (β-N-acetylglucosaminidase + leucine aminopeptidase) regressions (biofilm 1.07, plankton 0.94) corresponded more closely to the estimated difference in mean biomass C:N. Despite major differences in physical structure and trophic organization, biofilm and plankton communities have similar ecoenzymatic stoichiometry in relation to productivity and biomass composition. These relationships can be integrated into the stoichiometric and metabolic theories of ecology and used to analyze community metabolism in relation to resource constraints.     

Three amino acid changes contribute markedly to the thermostability of β-glucosidase BglC from Thermobifida fusca

Thermostability of β-glucosidase was enhanced by family shuffling, site saturation mutagenesis, and site-directed mutagenesis. Family shuffling was carried out based on β-glucosidase BglC from Thermobifida fusca and β-glucosidase BglB from Paebibacillus polymxyxa with the help of synthetic primers. High-throughput screening revealed mutants with higher thermostability than both parental enzymes. The most thermostable mutant VM2 containing three key amino acid changes in L444Y/G447S/A433V had a 144-fold increase in half-life of inactivation as compared to the parental enzyme BglC. The mutant VM2 showed 28% and 94% increase in k(cat) towards p-nitrophenyl-β-D-glucopyranoside (pNPG) and cellobiose, respectively. The mutant with enhanced stability would facilitate the recycle of β-glucosidase in the bioconversion of cellulosic biomass.     

Enzymatic hydrolysis of cellulose to glucose lung immobilized β-glucosidase

The production of sugars by enzymatic hydrolysis of cellulose is a multistep process which includes conversion of the intermediate cellobiose to glucose by β-glucosidase. Aside from its role as an intermediate, cellobiose inhibits the endoglucanase components of typical cellulase enzyme systems. Because these enzyme systems often contain insufficient concentrations of β-glucosidase to prevent accumulation of inhibitory cellobiose, this research investigated the use of supplemental immobilized β-glucosidase to increase yield of glucose. Immobilized β-glucosidase from Aspergillus phoenicis was produced by sorption at controlled-pore alumina with about 90% activity retention. The product lost only about 10% of the original activity during an on-stream reaction period of 500 hr with cellobiose as substrate; maximum activity occurred near pH 3.5 and the apparent activation energy was about 11 kcal/mol. The immobilized β-glucosidase was used together with Trichoderma reesei cellulase to hydrolyze cellulosic materials, such as Solka Floc, corn stove and exploded wood. Increased yields of glucose and greater conversions of cellobiose of glucose were observed when the reaction systems contained supplemental immobilized β-glucosidase.     

不同施肥模式对设施菜田土壤酶活性的影响

利用天津日光温室蔬菜不同施肥模式定位试验,研究了6种施肥模式对设施菜田土壤酶活性的影响.结果表明: 番茄生育期间不同施肥模式土壤α-葡萄苷酶、β-木糖苷酶、β-葡萄苷酶、β-纤维二糖苷酶、几丁质酶和磷酸酶的活性总体上均呈先增后降的趋势,土壤脲酶活性呈先增高后趋于平缓的趋势.与全部施用化肥氮相比,5种有机无机肥料配施模式土壤酶活性均有所提升,且随猪粪施用量的增加,尤其是配施秸秆条件下,土壤酶活性显著增加.番茄各生育期土壤酶活性与土壤微生物生物量碳、氮和可溶性有机碳、氮之间总体上呈显著或极显著正相关关系.同等养分投入量下,有机无机肥配施,特别是配施一定的秸秆可有效提高设施菜田土壤酶活性,维持较高的菜田土壤肥力,有利于设施蔬菜的可持续生产.     

Direct ethanol production from cellulosic materials by Zymobacter palmae carrying Cellulomonas endoglucanase and Ruminococcus β-glucosidase genes

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we conferred the ability to ferment cellulosic materials directly on Zymobacter palmae by co-expressing foreign endoglucanase and β-glucosidase genes. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, the six genes encoding the cellulolytic enzymes (CenA, CenB, CenD, CbhA, CbhB, and Cex) from Cellulomonas fimi were introduced and expressed in Z. palmae. Of these cellulolytic enzyme genes cloned, CenA degraded carboxymethylcellulose and phosphoric acid-swollen cellulose (PASC) efficiently. The extracellular CenA catalyzed the hydrolysis of barley β-glucan and PASC to liberate soluble cello-oligosaccharides, indicating that CenA is the most suitable enzyme for cellulose degradation among those cellulolytic enzymes expressed in Z. palmae. Furthermore, the cenA gene and β-glucosidase gene (bgl) from Ruminococcus albus were co-expressed in Z. palmae. Of the total endoglucanase and β-glucosidase activities, 57.1 and 18.1 % were localized in the culture medium of the strain. The genetically engineered strain completely saccharified and fermented 20 g/l barley β-glucan to ethanol within 84 h, producing 79.5 % of the theoretical yield. Thus, the production and secretion of CenA and BGL enabled Z. palmae to efficiently ferment a water-soluble cellulosic polysaccharide to ethanol.     

Induction of β-Glucosidase in Botrytis cinerea by Cell Wall Fractions of the Host Plant

The pathogenicity of Botrytis cinerea has been found to correlate positively with the β-glucosidase activity. In this report, the relationship between the induction of β-glucosidase and the components of host plant tissues was studied by the use of tissue fractions and cellulose-related compounds.

The most active enzyme induced by the crude fiber fraction and Avicel was β-glucosidase, among the cell wall degrading enzymes tested. The β-glucosidase was very inducible in the strains with strong pathogenicity, and intensively degraded the fiber fraction made from apple fruit tissues. The same degradation of the cell wall fraction was demonstrated with the purified enzyme.     

Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation

The structures of rice BGlu1 β-glucosidase, a plant β-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 Å and 1.55 Å resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) β-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long β-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa β-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-β-glucanase/β-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/β-II β-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.     

Biomass hydrolyzing enzymes from plant pathogen Xanthomonas axonopodis pv. punicae: optimizing production and characterization

Xanthomonas axonopodis pv. punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28 °C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55 °C while xylanase had highest activity at 45 °C. Heat treatment of enzyme extract at 75 °C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.