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.

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.

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.

Preparation, characterisation and use for antioxidant oligosaccharides of a cellulase from abalone (Haliotis discus hannai) viscera.

BACKGROUND: In China, abalone (Haliotis discus hannai) production is growing annually. During industrial processing, the viscera, which are abundant of cellulase, are usually discarded or processed into low-value feedstuff. Thus, it is of interest to obtain cellulase from abalone viscera and investigate its application for preparation of functional oligosaccharides. RESULTS: A cellulase was purified from the hepatopancreas of abalone by ammonium sulfate precipitation and two-steps column chromatography. The molecular weight of the cellulase was 45 kDa on SDS-PAGE. Peptide mass fingerprinting analysis yielded 103 amino acid residues, which were identical to cellulases from other species of abalone. Substrate specificity analysis indicated that the cellulase is an endo-1,4-ß-glucanase. Hydrolysis of seaweed Porphyra haitanensis polysaccharides by the enzyme produced oligosaccharides with degree of polymerisation of two to four, whose monosaccharide composition was 58% galactose, 4% glucose and 38% xylose. The oligosaccharides revealed 2,2'-diphenyl-1-picrylhydrazyl free radical as well as hydrogen peroxide scavenging activity. CONCLUSION: It is feasible and meaningful to utilise cellulase from the viscera of abalone for preparation of functional oligosaccharides. © 2015 Society of Chemical Industry.

Enhanced xyloglucan-specific endo-ß-1,4-glucanase efficiency in an engineered CBM44-XegA chimera.

Xyloglucan-specific endo-ß-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for ß-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.

ZmGns, a maize class I ß-1,3-glucanase, is induced by biotic stresses and possesses strong antimicrobial activity.

Plant ß-1,3-glucanases are members of the pathogenesis-related protein 2 (PR-2) family, which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses. One of the differentially expressed proteins (spot 842) identified in a recent proteomic comparison between five pairs of closely related maize (Zea mays L.) lines differing in aflatoxin resistance was further investigated in the present study. Here, the corresponding cDNA was cloned from maize and designated as ZmGns. ZmGns encodes a protein of 338 amino acids containing a potential signal peptide. The expression of ZmGns was detectible in all tissues studied with the highest level in silks. ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus. ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid. In vivo, ZmGns showed a significant inhibitory activity against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis. Its high level of expression in the silk tissue and its induced expression by phytohormone treatment, as well as by bacterial and fungal infections, suggest it plays a complex role in maize growth, development, and defense.

Recombinant expression and characterization of an acid-, alkali- and salt-tolerant ß-1,3-1,4-glucanase from Paenibacillus sp. S09.

A new ß-1,3-1,4-glucanase gene (PlicA) was cloned from Paenibacillus sp. S09. The ORF contained 717 bp coding for a 238 amino acid protein. PlicA, expressed in Escherichia coli and purified by Ni(2+)-affinity chromatography, had optimum activity at 55 °C and pH 6.2. The specific activity toward barley ß-glucan reached 7,055 U/mg. K m and V max values with barley ß-glucan were 3.7 mg/ml and 3.3 × 10(3) µmol/min mg, respectively. The enzyme exhibited acid- and alkali-tolerance with more than 80 % activity remaining after incubation for 4 h at pH 3.5-12. PlicA was salt-tolerant (>90 % activity retained in 4 M NaCl at 25 °C for 24 h) and salt-activated: activity rising 1.5-fold in 0.5 M NaCl. The thermostability was improved by NaCl and CaCl2. This is the first report of an acid-, alkali- and salt-tolerant bacterial ß-1,3-1,4-glucanase with high catalytic efficiency.

Truncation of a novel C-terminal domain of a ß-glucanase improves its thermal stability and specific activity.

Enzymes that degrade ß-glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo-ß-1,3(4)-glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum, was cloned and expressed in Pichia pastoris. Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C-terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR-mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A-ΔC, exhibited significantly enhanced thermal stability at 50°C, with D-values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A-ΔC demonstrated a 20%-25% increase in halotolerance at 1.25 and 1.5 M NaCl, respectively, compared to the full-length enzymes. Notably, specific activity against cereal ß-glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress-tolerant fungus C. sphaerospermum and the first report of a halotolerant and engineered endo-ß-1,3(4)-glucanase. Additionally, it sheds light on a group of endo-ß-1,3(4)-glucanases from Antarctic rock-inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function.

Biochemical properties and application of a multi-domain ß-1,3-1,4-glucanase from Fibrobacter sp.

A novel glycoside hydrolase (GH) family 16 multi-domain ß-1,3-1,4-glucanase (FsGlc16A) from Fibrobacter sp. UWP2 was identified, heterogeneously expressed, and its enzymatic properties, protein structure and application potential were characterized. Enzymological characterization showed that FsGlc16A performed the optimal catalytic activity at pH 4.5 and 50 °C with a specific activity of 3263 U/mg. FsGlc16A exhibited the substrate specificity towards oat ß-glucan, barley ß-glucan and lichenan, and in addition, it hydrolyzed oat ß-glucan and lichenan into different ß-glucooligosaccharides with polymerization degrees of 3-4, which further illustrated that it belonged to the endo-type ß-1,3-1,4-glucanase. FsGlc16A was classified in subfamily25 of GH16. A 'PXSSSS' repeats domain was identified at the C-terminus of FsGlc16A, which was distinct from the typical GH family 16 ß-1,3-1,4-glucanases. Removing the 'PXSSSS' repeats domain affected the binding of the substrate to FsGlc16A and reduced the enzyme activity. FsGlc16A displayed good potential for the applications, which hydrolyzed oat bran into ß-glucooligosaccharides, and reduced filtration time (18.89 %) and viscosity (3.64 %) in the saccharification process. This study investigated the enzymatic properties and domain function of FsGlc16A, providing new ideas and insights into the study of ß-1,3-1,4-glucanase.

Catalytic efficiency diversification of duplicate ß-1,3-1,4-glucanases from Neocallimastix patriciarum J11.

Four types of ß-1,3-1,4 glucanase (ß-glucanase, EC 3.2.1.73) genes, designated bglA13, bglA16, bglA51, and bglM2, were found in the cDNA library of Neocallimastix patriciarum J11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias to Streptococcus equinus. The presence of expansion and several predicted secondary structures in the 3' untranslated regions (3'UTRs) of bglA16 and bglM2 suggest that these two genes were duplicated recently, whereas bglA13 and bglA16, which contain very short 3'UTRs, were replicated earlier. These findings indicate that the ß-glucanase genes from N. patriciarum J11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. ß-Glucanase genes of Streptococcus equinus, Ruminococcus albus 7, and N. patriciarum J11 were cloned and expressed by Escherichia coli. The recombinant ß-glucanases cloned from S. equinus, R. albus 7, and N. patriciarum J11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial ß-glucanases were also significantly lower than those of the fungal ß-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal ß-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived ß-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated ß-glucanases, showed much higher k(cat) values than others. These results support the notion that duplicated ß-glucanase genes, namely, bglA16 and bglM2, increase the reaction efficiency of ß-glucanases and suggest that the catalytic efficiency of ß-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms in N. patriciarum J11.

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-ß-D-glucanase.

1,3-1,4-ß-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-ß-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.

Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima.

Cellulases have been used in many applications to treat various carbohydrate-containing materials. Thermotoga maritima cellulase 12A (TmCel12A) belongs to the GH12 family of glycoside hydrolases. It is a ß-1,4-endoglucanase that degrades cellulose molecules into smaller fragments, facilitating further utilization of the carbohydrate. Because of its hyperthermophilic nature, the enzyme is especially suitable for industrial applications. Here the crystal structure of TmCel12A was determined by using an active-site mutant E134C and its mercury-containing derivatives. It adopts a ß-jellyroll protein fold typical of the GH12-family enzymes, with two curved ß-sheets A and B and a central active-site cleft. Structural comparison with other GH12 enzymes shows significant differences, as found in two longer and highly twisted ß-strands B8 and B9 and several loops. A unique Loop A3-B3 that contains Arg60 and Tyr61 stabilizes the substrate by hydrogen bonding and stacking, as observed in the complex crystals with cellotetraose and cellobiose. The high-resolution structures allow clear elucidation of the network of interactions between the enzyme and its substrate. The sugar residues bound to the enzyme appear to be more ordered in the -2 and -1 subsites than in the +1, +2 and -3 subsites. In the E134C crystals the bound -1 sugar at the cleavage site consistently show the α-anomeric configuration, implicating an intermediate-like structure.

Construction and characterization of a fusion ß-1,3-1,4-glucanase to improve hydrolytic activity and thermostability.

A new fusion gene (Bgl-licMB), encoding ß-1,3-1,4-glucanase both from Bacillus amyloliquefaciens (Bgl) and Clostridium thermocellum (licMB), was constructed via end-to-end fusion and expressed in Escherichia coli to improve hydrolytic activity and thermostability of ß-1,3-1,4-glucanase. The results of enzymatic properties showed that the catalytic efficiency (K(cat)/K(m)) of the fusion enzyme for oat ß-glucan was 2.7 and 20-fold higher than that of the parental Bgl and licMB, respectively, and that the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bgl and licMB were both less than 30%. These properties make this particular ß-1,3-1,4-glucanase a good candidate for application in brewing and animal-feed industries.

A novel family 9 beta-1,3(4)-glucanase from thermoacidophilic Alicyclobacillus sp. A4 with potential applications in the brewing industry.

An endo-beta-1,3(4)-glucanase gene, Agl9A, was cloned from Alicyclobacillus sp. A4 and expressed in Pichia pastoris. Its deduced amino acid sequence shared the highest identity (48%) with an endo-beta-1,4-glucansae from Alicyclobacillus acidocaldarius that belongs to family 9 of the glycoside hydrolases. The purified recombinant Agl9A exhibited relatively wide substrate specificity, including lichenan (109%), barley beta-glucan (100%), CMC-Na (15.02%), and laminarin (6.19%). The optimal conditions for Agl9A activity were pH 5.8 and 55 degrees C. The enzyme was stable over a broad pH range (>60% activity retained after 1-h incubation at pH 3.8-11.2) and at 60 degrees C (>70% activity retained after 1-h incubation). Agl9A was highly resistant to various neutral proteases (e.g., trypsin, alpha-chymotrypsin, and collagenase) and Neutrase 0.8L (Novozymes), a protease widely added to the mash. Under simulated mashing conditions, addition of Agl9A (20 U/ml) or a commercial xylanase (200 U/ml) reduced the filtration rate (26.71% and 20.21%, respectively) and viscosity (6.12% and 4.78%, respectively); furthermore, combined use of Agl9A (10 U/ml) and the xylanase (100 U/ml) even more effectively reduced the filtration rate (31.73%) and viscosity (8.79%). These characteristics indicate that Agl9A is a good candidate to improve glucan degradation in the malting and brewing industry.

Purification and characterization of a novel endo-ß-1,4-glucanases , AfEG22, from the giant snail, Achatina fulica frussac.

In this study, we confirmed that at least three endo-ß-1,4-glucanases existed in the digestive juice of the giant snail, Achatina fulica ferussac, by Congo red staining assay. One of these enzymes, a novel endo-ß-1,4-glucanase (AfEG22), was purified 29.5-fold by gel filtration, anion exchange, and hydrophobic interaction chromatography. The carboxymethyl cellulose (CMC) hydrolytic activity of the purified enzyme was 12.3 U/mg protein. The molecular mass of AfEG22 was 22081 Da determined by MALDI-TOF. N-terminal amino acid sequencing revealed a sequence of EQRCTNQGGILKYYNT, which did not have significant homology with any proteins in BLAST database. The optimal pH and temperature for hydrolytic activity toward CMC were pH 4.0 and 50°C, respectively. AfEG22 was stable between pH 3.0 and pH 12.0 when incubated at 4°C for 3 h or at 37°C for 1 h. The enzyme remained more than 80% activity between pH 4.5 and pH 7.0 after incubation at 50°C for 1 h. AfEG22 possessed excellent thermostability as more than 70% activity was remained after incubation at 60°C for 3 h. Substrate specific analysis revealed that AfEG22 was a typical endo-ß-1,4-glucanase. This is the first time to report a novel endo-ß-1,4-glucanase with high stability from the digestive juice of A. fulica.

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8.

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.

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.

Crystallization and preliminary crystallographic analysis of laminarinase from Rhodothermus marinus: a case of pseudomerohedral twinning.

Thermophilic endo-1,3(4)-beta glucanase (laminarinase) from Rhodothermus marinus was crystallized by the hanging-drop vapor diffusion method. The needle-like crystals belong to space group P2(1) and contain two protein molecules in the asymmetric unit with a solvent content of 51.75 %. Diffraction data were collected to a resolution of 1.95A and resulted in a dataset with an overall R(merge) of 10.4% and a completeness of 97.8%. Analysis of the structure factors revealed pseudomerohedral twinning of the crystals with a twin fraction of approximately 42%.

Catalytic properties and mode of action of endo-(1-->3)-beta-D-glucanase and beta-D-glucosidase from the marine mollusk Littorina kurila.

A complex of the enzymes from the liver of the marine mollusk Littorina kurila that hydrolyzes laminaran was investigated. Two (1-->3)-beta-d-glucanases (G-I and G-II) were isolated. The molecular mass of G-I as estimated by gel-permeation chromatography and SDS-PAGE analysis was 32 and 40kDa, respectively. The G-II molecular mass according to SDS-PAGE analysis was about 200kDa. The pH optimum for both G-I and G-II was pH 5.4. The G-I had narrow substrate specificity and hydrolyzed only the (1-->3)-beta-d-glucosidic bonds in the mixed (1-->3),(1-->6)- and (1-->3),(1-->4)-beta-d-glucans down to glucose and glucooligosaccharides. This enzyme acted with retention of the anomeric configuration and catalyzed a transglycosylation reaction. G-I was classified as the glucan endo-(1-->3)-beta-d-glucosidase (EC 3.2.1.39). G-II exhibited both exo-glucanase and beta-d-glucoside activities. This enzyme released from the laminaran glucose as a single product, but retained the anomeric center configuration and possessed transglycosylation activity. The hydrolysis rate of glucooligosaccharides by G-I decreased with an increase of the substrate's degree of polymerization. In addition to (1-->3)-beta-d-glucanase activity, the enzyme had the ability to hydrolyze p-nitrophenyl beta-d-glucoside and beta-d-glucobioses: laminaribiose, gentiobiose, and cellobiose, with the rate ratio of 50:12:1. G-II may correspond to beta-d-glucoside glucohydrolase (EC 3.2.1.21).