Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies.

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.

Impact of cellulose properties on enzymatic degradation by bacterial GH48 enzymes: Structural and mechanistic insights from processive Bacillus licheniformis Cel48B cellulase.

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.

Low-resolution molecular shape, biochemical characterization and emulsification properties of a halotolerant esterase from Bacillus licheniformis.

Bacterial esterases are highly versatile enzymes, currently widely used in detergents, biosurfactants, bioemulsifiers and as biocatalysts in paper and food industries. Present work describes heterologous expression, purification, and biophysical and biochemical characterization of a halotolerant esterase from Bacillus licheniformis (BlEstA). BlEstA preferentially cleaves pNP-octanoate and both activity and stability of the enzyme increased in the presence of 2 M NaCl, and also with several organic solvents (ethanol, methanol and DMSO). Furthermore, BlEstA has considerable emulsifying properties, particularly with olive oil as substrate. Our studies also show that the enzyme is monomeric in solution and its small-angle X-ray scattering low-resolution molecular envelope fits well its high-resolution homology model.

Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Equilibrium partially folded states of B. licheniformis[Formula: see text]-lactamase.

[Formula: see text]-Lactamases (penicillinases) facilitate bacterial resistance to antibiotics and are excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A [Formula: see text]-lactamase with three tryptophan residues located one in each of its two domains and one in the interface between domains. The conformational landscape of three well-characterized ESP Trp[Formula: see text]Phe mutants was characterized in equilibrium unfolding experiments by measuring tryptophan fluorescence, far-UV CD, activity, hydrodynamic radius, and limited proteolysis. The Trp[Formula: see text]Phe substitutions had little impact on the native conformation, but changed the properties of the partially folded states populated at equilibrium. The results were interpreted in the framework of modern theories of protein folding.

Low-resolution structure, oligomerization and its role on the enzymatic activity of a sucrose-6-phosphate hydrolase from Bacillus licheniformis.

Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.

Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43.

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-ß-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys247 ) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-ß-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related ß-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the ß-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms.

Biochemical characterization and low-resolution SAXS structure of two-domain endoglucanase BlCel9 from Bacillus licheniformis.

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

Optimization of solid-state fermentation conditions of Bacillus licheniformis and its effects on Clostridium perfringens-induced necrotic enteritis in broilers

In the present study, we examined the growth parameters of Bacillus licheniformis in solid-state fermentation (SSF) and evaluated the effects of Bacillus licheniformis-fermented products on Clostridium perfringens-challenged broilers. During four and six days of SSF, the highest viable biomass was observed at 5% glucose, 10% soybean meal, 3% yeast, and 50% initial moisture content. The Bacillus licheniformis SSF products were heat- and acid-resistant. Furthermore, the fermented products were able to inhibit the growth of Clostridium perfringens and Staphylococcus aureus in vitro. In feeding experiments, in a similar manner to the antibiotic treatment group, dietary supplementation of Bacillus licheniformis-fermented products significantly improved intestinal morphology and necrotic lesions under Clostridium perfringens challenge, accompanied by increased IFN-γ mRNA expression in the spleen and bursa of Fabricius. These results together suggest that Bacillus licheniformis-fermented products have potential for development as feed additives and use as possible substitutes for antibiotics to treat Clostridium perfringens in the poultry industry.(AU)

Low-resolution envelope, biophysical analysis and biochemical characterization of a short-chain specific and halotolerant carboxylesterase from Bacillus licheniformis.

Esterases are widely applied in industrial processes due to their versatility, regio- and enantioselectivity, lack of cofactors and stability in organic solvents. Bacillus licheniformis, a microorganism frequently used in industrial and biotechnological applications such as dairy, baking, beverage, pulp and paper, detergent and cosmetics production, organic synthesis and waste management, is a promising source of esterases. Here we describe the biochemical and biophysical characterization of B. licheniformis carboxylesterase BlEst1 and its SAXS-derived molecular envelope. BlEst1 has optimal hydrolytic activity against p­nitrophenyl acetate at pH 7.0 and 40 °C. Furthermore, BlEst1 is stable in different organic solvents such as methanol, isopropanol and butanol. The BlEst1 homology model reveals a typical α/ß hydrolase core with an adjacent auxiliary domain, snuggly fitting the experimental low-resolution SAXS molecular envelope. Moreover, BlEst1 maintained considerable part of its activity in the presence of up to 5 M NaCl and its thermal stability was significantly enhanced by the presence of salt, revealing its halotolerant character. The ability to work under harsh conditions makes BlEst1 an interesting candidate for industrial applications.

Biotransformation of benzo[a]pyrene by the thermophilic bacterium Bacillus licheniformis M2-7.

Benzo[a]pyrene (BaP) is recognized as a potentially carcinogenic and mutagenic hydrocarbon, and thus, its removal from the environment is a priority. The use of thermophilic bacteria capable of biodegrading or biotransforming this compound to less toxic forms has been explored in recent decades, since it provides advantages compared to mesophilic organisms. This study assessed the biotransformation of BaP by the thermophilic bacterium Bacillus licheniformis M2-7. Our analysis of the biotransformation process mediated by strain M2-7 on BaP shows that it begins during the first 3 h of culture. The gas chromatogram of the compound produced shows a peak with a retention time of 17.38 min, and the mass spectra shows an approximate molecular ion of m/z 167, which coincides with the molecular weight of the chemical formula C6H4(COOH)2, confirming a chemical structure corresponding to phthalic acid. Catechol 2,3-dioxygenase (C23O) enzyme activity was detected in minimal saline medium supplemented with BaP (0.33 U mg-1 of protein). This finding suggests that B. licheniformis M2-7 uses the meta pathway for biodegrading BaP using the enzyme C23O, thereby generating phthalic acid as an intermediate.

GH43 endo-arabinanase from Bacillus licheniformis: Structure, activity and unexpected synergistic effect on cellulose enzymatic hydrolysis.

The hydrolysis of the plant biomass provides many interesting opportunities for the generation of building blocks for the green chemistry industrial applications. An important progress has been made for the hydrolysis of the cellulosic component of the biomass while, for the hemicellulosic components, the advances are less straightforward. Here, we describe the cloning, expression and biochemical and structural characterization of BlAbn1, a GH43 arabinanase from Bacillus licheniformis. This enzyme is selective for linear arabinan and efficiently hydrolyzes this substrate, with a specific activity of 127 U/mg. The enzyme has optimal conditions for activity at pH 8.0 and 45 °C and its activity is only partially dependent of a bound calcium ion since 70% of the maximal activity is preserved even when 1 mM EDTA is added to the reaction medium. BlAbn1 crystal structure revealed a typical GH43 fold and narrow active site, which explains the selectivity for linear substrates. Unexpectedly, the enzyme showed a synergic effect with the commercial cocktail Accellerase 1500 on cellulose hydrolysis. Scanning Electron Microscopy, Solid-State NMR and relaxometry data indicate that the enzyme weakens the interaction between cellulose fibers in filter paper, thus providing an increased access to the cellulases of the cocktail.

Biocatalytic action of proteases in ionic liquids: Improvements on their enzymatic activity, thermal stability and kinetic parameters.

This study evaluated the effect of the addition of the following ionic liquids (IL): choline chloride (CC), tetramethylammonium bromide (TB) and 1­ethyl­3­methylimidazolium bromide (EM), on some biochemical properties including enzymatic activity and different kinetic parameters of commercial proteases. The enzyme-IL combinations that showed the highest increases in enzyme activities were as follows: CC (0.5mM) and Neutrase® 0.8L; CC (5mM) and Flavourzyme® 500L; TB (2000mM) and Alcalase® 2.4L, with relative increases of 20, 15 and 150% in protease activities, respectively, compared to the control assays. The combination TB and Alcalase® 2.4L showed a reduction of 50% of the activation energy (Ea), an increase of the relation Vmax/Km of 35% and a 16-fold rise in the values of t1/2, and D. Neutrase® 0.8L combined with CC showed an increase of 20% in the relation Vmax/Km. The combination Flavourzyme® 500L and CC presented a 20% higher value of the relation Vmax/Km and a 2-fold increase in the values of t1/2 and D compared to the control assay. In summary, the most positive effects observed in this study included proteases with improved activity and stability properties and a greater affinity for the substrate.

Biochemical characterization, low-resolution SAXS structure and an enzymatic cleavage pattern of BlCel48 from Bacillus licheniformis.

Economic sustainability of modern biochemical technologies for plant cell wall transformations in renewable fuels, green chemicals, and sustainable materials is considerably impacted by the elevated cost of enzymes. Therefore, there is a significant drive toward discovery and characterization of novel carbohydrate-active enzymes. Here, the BlCel48 cellulase from Bacillus licheniformis, a glycoside hydrolase family 48 member (GH48), was functionally and biochemically characterized. The enzyme is catalytically stable in a broad range of temperatures and pH conditions with its enzymatic activity at pH5.0 and 60°C. BlCel48 exhibits high hydrolytic activity against phosphoric acid swollen cellulose (PASC) and bacterial cellulose (BC) and significantly lower activity against carboxymethylcellulose (CMC). BlCel48 releases predominantly cellobiose, and also small amounts of cellotriose and cellotetraose as products from PASC hydrolysis. Small-angle X-ray scattering (SAXS) data analysis revealed a globular molecular shape and monomeric state of the enzyme in solution. Its molecular mass estimated based on SAXS data is ~77.2kDa. BlCel48 has an (αα)6-helix barrel-fold, characteristic of GH48 members. Comparative analyses of homologous sequences and structures reveal the existence of two distinct loops in BlCel48 that were not present in other structurally characterized GH48 enzymes which could have importance for the enzyme activity and specificity.

Biochemical characterization and low-resolution SAXS structure of an exo-polygalacturonase from Bacillus licheniformis.

Among the structural polymers present in the plant cell wall, pectin is the main component of the middle lamella. This heterogeneous polysaccharide has an α-1,4 galacturonic acid backbone, which can be broken by the enzymatic action of pectinases, such as exo-polygalacturonases, that sequentially cleave pectin from the non-reducing ends, releasing mono or di-galacturonic acid residues. Constant demand for pectinases that better suit industrial requirements has motivated identification and characterization of novel enzymes from diverse sources. Bacillus licheniformis has been used as an important source for bioprospection of several industrial biomolecules, such as surfactants and enzymes, including pectate lyases. Here we cloned, expressed, purified, and biochemically and structurally characterized an exo-polygalacturonase from B. licheniformis (BlExoPG). Its low-resolution molecular envelope was derived from experimental small-angle scattering data (SAXS). Our experimental data revealed that BlExoPG is a monomeric enzyme with optimum pH at 6.5 and optimal temperature of approximately 60°C, at which it has considerable stability over the broad pH range from 5 to 10. After incubation of the enzyme for 30min at pH ranging from 5 to 10, no significant loss of the original enzyme activity was observed. Furthermore, the enzyme maintained residual activity of greater than 80% at 50°C after 15h of incubation. BlExoPG is more active against polygalacturonic acid as compared to methylated pectin, liberating mono galacturonic acid as a unique product. Its enzymatic parameters are Vmax=4.18µM.s-1,Km=3.25mgmL-1 and kcat=2.58s-1.

Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost.

During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C) and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1). For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1) for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization.

Combined strategies for improving production of a thermo-alkali stable laccase in Pichia pastoris

Background: Laccases are copper-containing enzymes which have been used as green biocatalysts for many industrial processes. Although bacterial laccases have high stabilities which facilitate their application under harsh conditions, their activities and production yields are usually very low. In this work, we attempt to use a combinatorial strategy, including site-directed mutagenesis, codon and cultivation optimization, for improving the productivity of a thermo-alkali stable bacterial laccase in Pichia pastoris. Results: A D500G mutant of Bacillus licheniformis LS04 laccase, which was constructed by site-directed mutagenesis, demonstrated 2.1-fold higher activity when expressed in P. pastoris. The D500G variant retained similar catalytic characteristics to the wild-type laccase, and could efficiently decolorize synthetic dyes at alkaline conditions. Various cultivation factors such as medium components, pH and temperature were investigated for their effects on laccase expression. After cultivation optimization, a laccase activity of 347 ± 7 U/L was finally achieved for D500G after 3 d of induction, which was about 9.3 times higher than that of wild-type enzyme. The protein yield under the optimized conditions was about 59 mg/L for D500G. Conclusions: The productivity of the thermo-alkali stable laccase from B. licheniformis expressed in P. pastoris was significantly improved through the combination of site-directed mutagenesis and optimization of the cultivation process. The mutant enzyme retains good stability under high temperature and alkaline conditions, and is a good candidate for industrial application in dye decolorization.

Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost

ABSTRACT During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C) and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1). For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1) for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization.

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism.

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

GH53 Endo-Beta-1,4-Galactanase from a Newly Isolated Bacillus licheniformis CBMAI 1609 as an Enzymatic Cocktail Supplement for Biomass Saccharification.

Galactanases (endo-ß-1,4-galactanases-EC 3.2.1.89) catalyze the hydrolysis of ß-1,4 galactosidic bonds in arabinogalactan and galactan side chains found in type I rhamnogalacturan. The aim of this work was to understand the catalytic function, biophysical properties, and use of a recombinant GH53 endo-beta-1,4-galactanase for commercial cocktail supplementation. The nucleotide sequence of the endo-ß-1,4-galactanase from Bacillus licheniformis CBMAI 1609 (Bl1609Gal) was cloned and expressed in Escherichia coli, and the biochemical and biophysical properties of the enzyme were characterized. The optimum pH range and temperature of Bl1609Gal activity were 6.5-8 and 40 °C, respectively. Furthermore, Bl1609Gal showed remarkable pH stability, retaining more than 75 % activity even after 24 h of incubation at pH 4-10. The enzyme was thermostable, retaining nearly 100 % activity after 1-h incubation at pH 7.0 at 25-45 °C. The enzymatic efficiency (K cat /K m ) against potato galactan under optimum conditions was 241.2 s(-1) mg(-1) mL. Capillary zone electrophoresis demonstrated that the pattern of galactan hydrolysis by Bl1609Gal was consistent with that of endogalactanases. Supplementation of the commercial cocktail ACCELLERASE(®)1500 with recombinant Bl1609Gal increased hydrolysis of pretreated sugarcane bagasse by 25 %.