A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium.

Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49, finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley ß-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo-ß-1,3-1,4-glucanase (EC 3.2.1.73) and exo-cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass.

A Novel Digestive GH16 ß-1,3(4)-Glucanase from the Fungus-Growing Termite Macrotermes barneyi.

ß-1,3-glucanases are the main digestive enzymes of plant and fungal cell wall. Transcriptomic analysis of the fungus-growing termite Macrotermes barneyi revealed a high expression of a predicted ß-1,3(4)-glucanase (Mbbgl) transcript in termite gut. Here, we described the cDNA cloning, heterologous expression, and enzyme characterization of Mbbgl. Sequence analysis and RT-PCR results showed that Mbbgl is a termite-origin GH16 ß-1,3(4)-glucanase. The recombinant enzyme showed the highest activity towards laminarin and was active optimally at 50 °C, pH 5.5. The enzyme displayed endo/exo ß-1,3(4)-glucanase activities. Moreover, Mbbgl had weak transglycosylation activity. The results indicate that Mbbgl is an endogenous digestive ß-1,3(4)-glucanase, which contributes to the decomposition of plant biomass and fungal hyphae. Additionally, the multiple activities, pH, and ion stabilities make Mbbgl a potential candidate for application in the food industry.

Microgravity Affects the Level of Matrix Polysaccharide 1,3:1,4-ß-Glucans in Cell Walls of Rice Shoots by Increasing the Expression Level of a Gene Involved in Their Breakdown.

The plant cell wall provides each cell with structural support and mechanical strength, and thus, it plays an important role in supporting the plant body against the gravitational force. We investigated the effects of microgravity on the composition of cell wall polysaccharides and on the expression levels of genes involved in cell wall metabolism using rice shoots cultivated under artificial 1 g and microgravity conditions on the International Space Station. The bulk amount of the cell wall obtained from microgravity-grown shoots was comparable with that from 1 g-grown shoots. However, the analysis of sugar constituents of matrix polysaccharides showed that microgravity specifically reduced the amount of glucose (Glc)-containing polysaccharides such as 1,3:1,4-ß-glucans, in shoot cell walls. The expression level of a gene for endo-1,3:1,4-ß-glucanase, which hydrolyzes 1,3:1,4-ß-glucans, largely increased under microgravity conditions. However, the expression levels of genes involved in the biosynthesis of 1,3:1,4-ß-glucans were almost the same under both gravity conditions. On the contrary, microgravity scarcely affected the level and the metabolism of arabinoxylans. These results suggest that a microgravity environment promotes the breakdown of 1,3:1,4-ß-glucans, which, in turn, causes the reduced level of these polysaccharides in growing rice shoots. Changes in 1,3:1,4-ß-glucan level may be involved in the modification of mechanical properties of cell walls under microgravity conditions in space.

Endo-ß-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

Characterisation of three novel ß-1,3 glucanases from the medically important house dust mite Dermatophagoides pteronyssinus (airmid).

The European house dust mite, Dermatophagoides pteronyssinus is a major source of airborne allergens worldwide and is found in half of European homes. Interactions between microbes and house dust mites (HDM) are considered important factors that allow them to persist in the home. Laboratory studies indicate the European HDM, D. pteronyssinus is a mycophagous mite, capable of utilising a variety of fungi for nutrients, however specific mycolytic digestive enzymes are unknown. Our previous work identified a number of putative glycosyl hydrolases present in the predicted proteome of D. pteronyssinus airmid and validated the expression of 42 of these. Of note, three GH16 proteins with predicted ß-1,3 glucanase activity were found to be consistently present in the mite body and excretome. Here, we performed an extensive bioinformatic, proteomic and biochemical study to characterize three-novel ß-1,3 glucanases from this medically important house dust mite. The genes encoding novel ß-1,3 glucanases designated Glu1, Glu2 and Glu3 were identified in D. pteronyssinus airmid, each exhibited more than 59% amino acid identity to one another. These enzymes are encoded by Glu genes present in a tri-gene cluster and protein homologs are found in other acari. The patchy phyletic distribution of Glu proteins means their evolutionary history remains elusive, however horizontal gene transfer cannot be completely excluded. Recombinant Glu1 and Glu2 exhibit hydrolytic activity toward laminarin, pachyman and barley glucan. Excreted ß-1,3 glucanase activity was increased in response to D. pteronyssinus airmid feeding on baker's yeast. Active ß-1,3 glucanases are expressed and excreted in the faeces of D. pteronyssinus airmid indicating they are digestive enzymes capable of breaking down ß-1,3 glucans of fungi present in house dust.

Enzymatic properties of a multi-specific ß-(1,3)-glucanase from Corallococcus sp. EGB and its potential antifungal applications.

The lamC gene encoding a novel ß-(1,3)-glucanase was cloned from Corallococcus sp. EGB and successfully expressed in the industrial yeast Pichia pastoris. The mature protein without the initial 26 residues of signal peptide, designated LamC27, was found to be composed of fascin-like module and laminarinase-like catalytic module. The purified recombinant enzyme (rLamC27) with a calculated molecular mass of 45.3 kDa displays activities toward a broad range of ß-linked polysaccharides, including laminarin, curdlan, pachyman, lichenan, and CMC. Enzymological characterization showed that rLamC27 performes its optimal activity under the condition of 45 °C and pH 7.0, respectively, and preferentially catalyzes the hydrolysis of glucans with a ß-1,3-linkage, which is similar to the LamC previously expressed in E. coli. TherLamC27 enzyme was activated by Mn2+ and Ba2+, while it was inhibited by Cu2+, Zn2+, and Co2+. Moreover, rLamC27 was strongly inhibited by 10 mM EDTA with 7.5% of its original activity remiaining, and weakly by SDS and Triton X-100. In antifungal assay, rLamC27 was conformed to possess lytic and antifungal activity against rice blast fungus. Specifically, a significant decrease germ tube and appressorium formation ratios from 94% to 59% and 97%-51%, respectively, were observed following exposure to rLamC27. H2DCFDA and CFW staining further demonstrated that the fungistasis capability of rLamC27 could be contributed by its ability to hydrolyze components of the cell wall. All these favorable properties indicate a promising potential for using rLamC27 as a biological antifungal agent in areas such as plant protection and food preservation.

Expression in Lactococcus lactis of a ß-1,3-1,4-glucanase gene from Bacillus sp. SJ-10 isolated from fermented fish.

Bacterial ß-1,3-1,4-glucanase (BG) is an endoglucanase that hydrolyzes linear ß-glucans containing ß-1,3 and ß-1,4 linkages, such as barley ß-glucans. In this study, a BG gene was transformed into the food-grade plasmid pNZ8149 and successfully expressed in Lactococcus lactis NZ3900 using the nisin-controlled gene expression system. To facilitate extracellular secretion, the signal peptide Usp45 was added during vector construction. A histidine tag was also added for affinity purification. BG was extracellularly secreted and was also present in the cells in soluble form. N-terminal amino acid residue analysis of secreted BG revealed that the Usp45 peptide was removed. The optimum temperature and pH for both intracellular and extracellular BG were 40 °C and 6, respectively. The enzyme kinetic parameters, Vmax, Km, kcat, and kcat/Km, of extracellular BG were 1317.51 µmol min-1, 1.97 mg ml-1, 588.54 s-1, and 298.26 ml s-1∙mg-1, respectively. There was no significant difference in the enzyme kinetic parameters of intracellular and extracellular BG. The growth pattern of transformed L. lactis NZ3900 in ß-glucan-containing liquid medium confirmed ß-glucan degradation by BG. The transformed strain degraded ß-glucans, produced gluco-oligosaccharide, and produced lactic acid. The strain and expression system constructed in this study could be applied to industrial fields requiring BG produced in food-grade lactococcal secretory expression system.

Overexpression of the Key Virulence Factor 1,3-1,4-ß-d-Glucanase in the Endophytic Bacterium Bacillus halotolerans Y6 To Improve Verticillium Resistance in Cotton.

Verticillium wilt, caused by Verticillium dahliae, results in a dramatic loss of cotton yields in China. There is great potential for biocontrol to manage this destructive crop disease. In this study, we obtained the endophytic bacterium Bacillus halotolerans Y6 from Verticillium wilt-resistant cotton Gossypium barbadense Xinhai15; this bacterium possesses strong antagonistic abilities that inhibit V. dahliae spore germination and mycelial growth. The results of the enzyme activity assay, heterologous expression, and gene knockdown showed that the key virulence factor of Y6 for antagonizing V. dahliae was ß -glucanase Bgy6. To facilitate field tests of biological control, we constructed the homologous Bgy6-overexpression strain OY6. Compared with the wild-type Y6 strain, the ß-glucanase activity of OY6 was increased by 91.79%, and the inhibition rate of OY6 against V. dahliae V991 exceeded 96.7%. Moreover, the spores of V. dahliae V991 treated with OY6 showed more mucus and larger holes on the surface, as observed by scanning electron microscopy. Potting test results illustrated that both OY6 and Y6 could improve the resistance of upland cotton to Verticillium wilt. With the inoculation of V. dahliae V991 for 45 days, the disease index of G. hirsutum TM-1 treated with OY6 was only 8.33, which was significantly lower than that in plants treated with the wild-type strain Y6 (17.86) or the controls without bacteria (35.94). Our research provides a new idea for the control of Verticillium wilt in upland cotton via transforming endophytic bacteria of Verticillium wilt-resistant cotton and proposes a new solution to prevent and control Verticillium wilt.

Expression of a wheat ß-1,3-glucanase in Pichia pastoris and its inhibitory effect on fungi commonly associated with wheat kernel.

ß-1,3-glucanases, the plant PR-2 family of pathogenesis-related (PR) proteins, can be constitutively expressed and induced in wheat crop to enhance its anti-fungal pathogen defense. This study aimed to investigate the inhibitory effect of wheat ß-1,3-glucanase on fungi most commonly associated with wheat kernel. A ß-1,3-glucanase from wheat was successfully expressed in Pichia pastoris X-33 and its biochemical and antifungal properties were characterized herein. The molecular weight of recombinant ß-1,3-glucanase is approximately 33 kDa. ß-1,3-glucanase displays optimal activity at pH 6.5, remaining relatively high at pH 5.5-8.0. The optimal reaction temperature of ß-1,3-glucanase is 50 °C, retaining approximately 84.0% residual activity after heat-treated at 50 °C for 1 h. The steady-state kinetic parameters of ß-1,3-glucanase against laminarin was determined and the Km and Vmax were 1.32 ±â€¯0.20 mg/ml and 96.4 ±â€¯4.4 U mg-1 protein, respectively. The inhibitory effect of purified ß-1,3-glucanase against the seven fungi commonly associated with wheat kernel was assessed in vitro. ß-1,3-glucanase exerted differential inhibitory effects on hyphal growth of Fusarium graminearum, Alternaria sp., A. glaucus, A. flavus, A. niger, and Penicillium sp. Spore formation and mycelial morphology of Alternaria sp., A. flavus, and A. niger were significantly affected by ß-1,3-glucanase (1U). The present results would help elucidate the mechanism underlying the inhibition of wheat ß-1,3-glucanases on pathogens.

Endo-ß-1,3-glucanase digestion combined with the HPAEC-PAD-MS/MS analysis reveals the structural differences between two laminarins with different bioactivities.

To resolve the structure of laminarin, the recombinant endo-ß-1,3-glucanase from Coprinopsis cinerea, which has specific activity on ß-1,3 glycosidic bond and could hydrolyze the laminarin with complex structure, was used to hydrolyze laminarin. Then, the structures of enzyme-resistant oligosaccharides were quantitatively and qualitatively analysed by high-performance anion exchange chromatography coupled with mass spectrometry. The laminarin from Laminaria digitata contains 9.51% ß-1,6 glycosidic bonds only in the branches (branch degree 7.68%). The laminarin from Eisenia bicyclis contains more ß-1,6 glycosidic bonds: 19.42% ß-1,6 glycosidic bonds in backbone and more and longer ß-1,6 branches (branch degree 25.99%). The differences in the ratio of glycosidic bonds and branch degree influence their bioactivity: the antioxidant activity and the antimicrobial activity against Gram positive bacteria of the laminarin from E. bicyclis is stronger than the laminarin from L. digitata, but the antimicrobial activity on Gram negative bacteria of the laminarin from E. bicyclis is weaker.

Autolytic enzymes are responsible for increased melanization of carbon stressed Aspergillus nidulans cultures.

Melanization of carbon stressed Aspergillus nidulans cultures were studied. Melanin production showed strong positive correlation with the activity of the secreted chitinase and ß-1,3-glucanase. Deletion of either chiB encoding an autolytic endochitinase or engA encoding an autolytic ß-1,3-endoglucanase, or both, almost completely prevented melanization of carbon stressed cultures. In contrast, addition of Trichoderma lyticase to cultures induced melanin production. Synthetic melanin could efficiently inhibit the purified ChiB chitinase activity. It could also efficiently decrease the intensity of hyphal fragmentation and pellet disorganization in Trichoderma lyticase treated cultures. Glyphosate, an inhibitor of L-3,4-dihydroxyphenylalanine-type melanin synthesis, could prevent melanization of carbon-starved cultures and enhanced pellet disorganization, while pyroquilon, a 1,8-dihydroxynaphthalene-type melanin synthesis inhibitor, enhanced melanization, and prevented pellet disorganization. We concluded that cell wall stress induced by autolytic cell wall hydrolases was responsible for melanization of carbon-starved cultures. The produced melanin can shield the living cells but may not inhibit the degradation and reutilization of cell wall materials of dead hyphae. Controlling the activity of autolytic hydrolase production can be an efficient approach to prevent unwanted melanization in the fermentation industry, while applying melanin synthesis inhibitors can decrease the resistance of pathogenic fungi against the chitinases produced by the host organism.

A Salivary Endo-ß-1,4-Glucanase Acts as an Effector That Enables the Brown Planthopper to Feed on Rice.

The brown planthopper (BPH) Nilaparvata lugens is one of the most destructive insect pests on rice (Oryza sativa) in Asia. After landing on plants, BPH rapidly accesses plant phloem and sucks the phloem sap through unknown mechanisms. We discovered a salivary endo-ß-1,4-glucanase (NlEG1) that has endoglucanase activity with a maximal activity at pH 6 at 37°C and is secreted into rice plants by BPH NlEG1 is highly expressed in the salivary glands and midgut. Silencing NlEG1 decreases the capacity of BPH to reach the phloem and reduces its food intake, mass, survival, and fecundity on rice plants. By contrast, NlEG1 silencing had only a small effect on the survival rate of BPH raised on artificial diet. Moreover, NlEG1 secreted by BPH did not elicit the production of the defense-related signal molecules salicylic acid, jasmonic acid, and jasmonoyl-isoleucine in rice, although wounding plus the application of the recombination protein NlEG1 did slightly enhance the levels of jasmonic acid and jasmonoyl-isoleucine in plants compared with the corresponding controls. These data suggest that NlEG1 enables the BPH's stylet to reach the phloem by degrading celluloses in plant cell walls, thereby functioning as an effector that overcomes the plant cell wall defense in rice.

Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-ß-1,3(4)-glucanase.

A gene coding endo-ß-1,3(4)-glucanase (ENG16A) was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris. ENG16A only acts on substrates containing ß-1,3 glycosidic bonds but not on substrates containing only ß-1,4- or ß-1,6-glycosidic bonds. Interestingly, compared to the activity of this enzyme towards carboxymethyl (CM)-pachyman containing only ß-1,3-glycosidic bonds, its activity towards barley ß-glucan containing both ß-1,3-glycosidic and ß-1,4-glycosidic bonds was increased by 64.72 %,, its activity towards laminarin containing both ß-1,3-glycosidic and ß-1,6-glycosidic bonds was decreased by 50.83 %. In addition, ENG16A has a higher Km value and Vmax for barley ß-glucan than laminarin, which may be related to the fact that barley ß-glucan contains mainly ß-1,4-glycosidic bonds mixed with a few ß-1,3-glycosidic bonds, whereas laminarin mainly contains ß-1,3-glycosidic bonds with a few ß-1,6-branched glucose residues. The adjacent ß-1,4-glycosidic bond promotes ENG16A to hydrolyse ß-1,3-glycosidic bonds, leading to an increased Vmax; the nearby ß-1,6-glycosidic bonds inhibited its hydrolysis of ß-1,3-glycosidic bonds, resulting in a decreased Vmax. This property is suggested to be related to the mechanism that C. cinerea uses to degrade and utilize hemicellulose in straw medium and to protect its cell wall during the mycelium growth stage.

Overexpression and characterization of a novel endo-ß-1,3(4)-glucanase from thermophilic fungus Humicola insolens Y1.

A novel endo-ß-1,3(4)-glucanase gene, cel16A, was cloned from the fungus Humicola insolens Y1. The 988-bp full-length gene encoded a 286-residue polypeptide consisting of a putative signal peptide of 20 residues and a catalytic domain belonging to glycosyl hydrolase family 16. It was successfully overexpressed in Pichia pastoris GS115. The purified recombinant Cel16A exhibited highest specific activity toward barley ß-glucan, followed by lichenan and laminarin, but not toward CMC-Na, birchwood xylan, Avicel and filter paper, indicating that Cel16A is an endo-ß-1,3(4)-glucanases. Recombinant Cel16A had a pH optimum at 5.5 and a temperature optimum at 55 °C with a specific activity of 693 U/mg toward barley ß-glucan. It exhibited good stability over pH 5.0-9.0 and at temperatures up to 50 °C, retaining over 80% maximum activity. The Km and Vmax values of Cel16A for barley ß-glucan were 0.91 mg ml-1 and 1530 µmol min-1·mg-1, respectively. All these favorable enzymatic properties of Cel16A make it a good candidate for applications in various industries.

Improvement of thermostability and halostability of ß-1,3-1,4-glucanase by substituting hydrophobic residue for Lys48.

The aim of this study was to improve the stability of ß-1,3-1,4-glucanase by substituting hydrophobic residue for specific amino acid. The results indicated that the catalytic efficiency, thermostability and halostability were enhanced simultaneously by replacement of Lys48 with Ala (K48A) or Leu (K48L). Comparison of kinetic parameters revealed that catalytic efficiency of mutants is enhanced as a result of the increase in substrate affinity. A great improvement in thermostability and halostability was observed. The half-lives of mutants significantly increased (up to ∼7-fold) at 60-70°C. Moreover, relative enzymatic activities of mutants were observed more than 80% even in the presence of 30% NaCl, and half-lives were increased to 3-fold that of wild-type. Based on above results, when applied to ionic liquid, mutants were more active and stable compared to wild-type. These were the results from improvement of protein functions by the substitution of hydrophobic single residue in adjacent with forming carbohydrate binding cavity. Therefore, this report could be helpful for improvement of the enzyme property and for biotechnological application as well.

Antagonistic activity of endo-ß-1,3-glucanase from a novel isolate, Streptomyces sp. 9X166, against black rot in orchids.

A total of 123 actinomycetes was isolated from 12 varieties of wild orchids and screened for potential antagonistic activity against Phytophthora, which causes black rot disease in orchids. In vitro and in vivo experimental results revealed that Streptomyces sp. strain 9X166 showed the highest antagonistic activity; its ß-1,3-glucanase production ability was a key mechanism for growth inhibition of the pathogen. PCR amplification and DNA sequencing of the 16S ribosomal RNA gene allowed the identification of this strain, with high similarity (99.93%) to the novel species Streptomyces similaensis. The glucanase enzyme, purified to homogeneity by anion exchange and gel filtration chromatography, showed a specific activity of 58 U mg(-1) (a 3.9-fold increase) and yield of 6.4%. The molecular weight, as determined by SDS-PAGE and gel filtration, was approximately 99 and 80 kDa, respectively, suggesting that the enzyme was a monomer. The purified enzyme showed the highest substrate specificity to laminarin, indicating that it was ß-1,3-glucanase. The hydrolyzed products of cello-oligosaccharides suggested that this enzyme was endo-type ß-1,3-glucanase. Streptomyces sp. 9X166 culture filtrate, possessing ß-1,3-glucanase activity, could degrade both freeze-dried and living mycelium. This is the first report on a ß-1,3-glucanase-producing Streptomyces sp. that could be an effective biocontrol agent for black rot disease in orchids.

Characterization of a thermostable endo-1,3(4)-ß-glucanase from Caldicellulosiruptor sp. strain F32 and its application for yeast lysis.

ß-1,3-Glucans, important structural components of cell wall or nutritional components of the endosperm, are extensively found in bacteria, fungi, yeast, algae, and plants. The structural complexity of ß-1,3-glucans implies that the enzymatic depolymerization of polysaccharides needs combined activities of distinct enzymes. In this study, Lam16A-GH, the catalytic module of a putative glycoside hydrolase (GH) family 16 laminarinase/lichenase from thermophilic bacterium Caldicellulosiruptor sp. F32, was purified and characterized through heterologous expression in Escherichia coli. Lam16A-GH can hydrolyze both ß-1,3-glucan (laminarin) and ß-1,3-1,4-glucan (barley ß-glucan) revealed by analysis of the products of polysaccharide degradation using thin-layer chromatography (TLC). The time required for the loss of 50 % of its activity is 45 h under the optimal condition of 75 °C and pH 6.5. Oligosaccharides degradation assay indicated that Lam16A-GH can catalyze endo-hydrolysis of the ß-1,4 glycosidic linkage adjacent to a 3-O-substituted glucosyl residue in the mixed linked ß-glucans, as well as the ß-1,3 linkage. The survival rate of Saccharomyces cerevisiae cells depends on the addition of Lam16A-GH, and the cytoplasm protein was released from the apparently deconstructed yeast cells. These results indicate that the bi-functional thermostable Lam16A-GH exhibits unique enzymatic properties and potential for yeast lysis.

ß-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi.

Bacillus velezensis is a known antifungal bacteria. To understand the role of ß-1, 3-1, 4-glucanase played on B. velezensis about the mechanism which exerts effect on fungi, we isolated and cloned the ß-1, 3-1, 4-glucanase gene (Bglu1) from B. velezensis ZJ20. The Bglu1 open reading frame was 732 bp that encoded a protein with 243 amino acids and a calculated molecular weight of 27.3 kDa. The same gene without the signal peptide, termed Bglu2, was also cloned and expressed in E. coli BL21. Among the two variants, only Bglu2 protein was expressed. Purified Bglu2 could be eluted with imidazole solution at concentrations ranging from 100 to 500 mM although the highest expression was observed at 150 and 200 mM and the purest was at 500 mM. In addition, activity of the crude enzyme was 1527 U ml(-1) and the highest activity of the purified enzyme was 1706 U ml(-1). The purified ß-1, 3-1, 4-glucanase had activity on a wide range of pH and temperatures and displayed optimal activity at pH 5.0 and 35 °C. More importantly, the mycelial morphology of three pathogenic fungi was destroyed by the purified ß-1, 3-1, 4-glucanase. In conclusion, ß-1, 3-1, 4-glucanase from B. velezensis ZJ20 can be highly expressed in E. coli BL21 and the recombinant protein is pathogenic to fungi.

Structure-Function Analysis of a Mixed-linkage ß-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B(1)4.

The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixed-linkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixed-linkage ß-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.

Engineering of a thermostable ß-1,3-1,4-glucanase from Bacillus altitudinis YC-9 to improve its catalytic efficiency.

BACKGROUND: Error-prone polymerase chain reaction (PCR) is frequently used in directed evolution of enzymes to modify their quality. In this study, error-prone PCR was used to improve the catalytic efficiency of ß-1,3-1,4-glucanase from Bacillus altitudinis YC-9. RESULTS: By screening, the mutant Glu-3060 with higher activity was selected among 5000 transformants. After induction with isopropyl ß-D-1-thiogalactopyranoside (IPTG), the activity of the mutant Glu-3060 reached 474.6 U mL(-1), resulting in a 48.6% increment of the parent enzyme activity. Research on the characterization of the mutated enzyme showed the optimal pH of the mutated enzyme to be 5.0, which is lower than the parent enzyme, but thermal stability was almost the same between them. Sequence analysis of the mutated enzyme revealed that three amino acids were changed compared with the parent enzyme, including K142N, Q203L and N214D. CONCLUSION: The three-dimensional structure predicted by SWISS-MODEL of the mutated enzyme Glu-3060 showed that the substitution of three amino acids had an effect on the catalytic activity, stability and optimal pH of the enzyme, through changing the charge properties or electron density, forming secondary keys, the acidity of the amino acids and the side chain group. The sum effects of all the factors were increased activity of the mutated enzyme and decreased optimal pH, while the same thermostability was maintained, thereby increasing the suitability of the enzyme for industrial use.