Unique behavioral patterns of wandering colonies of Brevibacillus thermoruber on agar plates.

Brevibacillus thermoruber strain Nabari cells grow as widely spreading dendritic colonies on reasoner's 2A-agar (1.5%) plates at around 55°C but as small motile colonies at 37°C. Motile colonies can be divided into colonies that move in straight or curved lines over long distances (wandering colonies), and colonies that rotate at a fixed location (rotating colonies). The addition of surfactant to the agar medium greatly increased the frequency of wandering colonies and facilitated the study of such colonies. The morphology of the wandering colonies varied: circular at the tip and pointed at the back, lemon-shaped with pointed ends, crescent-shaped, bullet-shaped, fish-like, and so on. A single colony may split into multiple colonies as it moves, or multiple colonies may merge into a single colony. The most surprising aspect of the movement of wandering colonies was that when a moving colony collides with another colony, it sometimes does not make a U-turn, but instead retreats straight back, as if bouncing back. The migration mechanisms of wandering colonies are discussed based on optical microscopic observations of swimming patterns of single cells in water and scanning electron microscopy of the arrangement of bacterial cells in wandering colonies.

Swarming behavior of a novel strain of Brevibacillus thermoruber.

Brevibacillus thermoruber strain Nabari was isolated from compost and identified based on 16 S rRNA gene sequencing and DNA-DNA hybridization using B. thermoruber DSM 7064 T as the standard, despite some differences in their physiological and structural characteristics. When B. thermoruber Nabari was cultivated on various solid media containing 1.5% agar at 60°C, it rapidly propagated over the entire plate. In particular, on R2A-agar medium, it formed fine dendritic colonies. Macroscopic and microscopic observations of peripheral regions of the colonies indicated that the dendritic patterns were formed by bacterial swarming of some of the cells; large flows of bacterial cell populations were observed in the peripheral regions of the dendritic colonies. The cells were highly flagellated, but no extreme elongation of cells was observed. When B. thermoruber Nabari cells were cultivated at 37°C on R2A-agar plates, most colonies were nonmotile, but some colonies were motile. For example, a wandering colony moved on the plate and split into two, and then they collided to become one again. Additionally, a simple incubation system was devised to record the movement of colonies at high temperatures in this study while protecting the cameras from thermal damage.

A novel AA10 from Paenibacillus curdlanolyticus and its synergistic action on crystalline and complex polysaccharides.

Lytic polysaccharide monooxygenases (LPMOs) play an important role in the degradation of complex polysaccharides in lignocellulosic biomass. In the present study, we characterized a modular LPMO (PcAA10A), consisting of a family 10 auxiliary activity of LPMO (AA10) catalytic domain, and non-catalytic domains including a family 5 carbohydrate-binding module, two fibronectin type-3 domains, and a family 3 carbohydrate-binding module from Paenibacillus curdlanolyticus B-6, which was expressed in a recombinant Escherichia coli. Comparison of activities between full-length PcAA10A and the catalytic domain polypeptide (PcAA10A_CD) indicates that the non-catalytic domains are important for the deconstruction of crystalline cellulose and complex polysaccharides contained in untreated lignocellulosic biomass. Interestingly, PcAA10A_CD acted not only on cellulose and chitin, but also on xylan, mannan, and xylan and cellulose contained in lignocellulosic biomass, which has not been reported for the AA10 family. Mutation of the key residues, Trp51 located at subsite - 2 and Phe171 located at subsite +2, in the substrate-binding site of PcAA10A_CD revealed that these residues are substantially involved in broad substrate specificity toward cellulose, xylan, and mannan, albeit with a low effect toward chitin. Furthermore, PcAA10A had a boosting effect on untreated corn hull degradation by P. curdlanolyticus B-6 endo-xylanase Xyn10D and Clostridium thermocellum endo-glucanase Cel9A. These results suggest that PcAA10A is a unique LPMO capable of cleaving and enhancing lignocellulosic biomass degradation, making it a good candidate for biotechnological applications. KEY POINTS: • PcAA10A is a novel modular LPMO family 10 from Paenibacillus curdlanolyticus. • PcAA10A showed broad substrate specificity on ß-1,4 glycosidic linkage substrates. • Non-catalytic domains are important for degrading complex polysaccharides. • PcAA10A is a unique LPMO capable of enhancing lignocellulosic biomass degradation.

Significance of a family-6 carbohydrate-binding module in a modular feruloyl esterase for removing ferulic acid from insoluble wheat arabinoxylan.

Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α-l-arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6's preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation.

Light Regulation of Two New Manganese Peroxidase-Encoding Genes in Trametes polyzona KU-RNW027.

To better understand the light regulation of ligninolytic systems in Trametes polyzona KU-RNW027, ligninolytic enzymes-encoding genes were identified and analyzed to determine their transcriptional regulatory elements. Elements of light regulation were investigated in submerged culture. Three ligninolytic enzyme-encoding genes, mnp1, mnp2, and lac1, were found. Cloning of the genes encoding MnP1 and MnP2 revealed distinct deduced amino acid sequences with 90% and 86% similarity to MnPs in Lenzites gibbosa, respectively. These were classified as new members of short-type hybrid MnPs in subfamily A.2 class II fungal secretion heme peroxidase. A light responsive element (LRE), composed of a 5'-CCRCCC-3' motif in both mnp promoters, is reported. Light enhanced MnP activity 1.5 times but not laccase activity. The mnp gene expressions under light condition increased 6.5- and 3.8-fold, respectively. Regulation of laccase gene expression by light was inconsistent with the absence of LREs in their promoter. Blue light did not affect gene expressions but impacted their stability. Reductions of MnP and laccase production under blue light were observed. The details of the molecular mechanisms underlying enzyme production in this white-rot fungus provide useful knowledge for wood degradation relative to illumination condition. These novel observations demonstrate the potential of enhancing ligninolytic enzyme production by this fungus for applications with an eco-friendly approach to bioremediation.

Two Manganese Peroxidases and a Laccase of Trametes polyzona KU-RNW027 with Novel Properties for Dye and Pharmaceutical Product Degradation in Redox Mediator-Free System.

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10-30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.

The modular arabinanolytic enzyme Abf43A-Abf43B-Abf43C from Ruminiclostridium josui consists of three GH43 modules classified in different subfamilies.

The abnA gene from Ruminiclostridium josui encodes the large modular arabinanolytic enzyme, Abf43A-Abf43B-Abf43C, consisting of an N-terminal signal peptide, a Laminin_G_3 module, a GH43_22 module, a Laminin_G_3 module, a Big_4 module, a GH43_26 module, a GH43_34 module and a dockerin module in order with a calculated molecular weight of 204,108. Three truncated enzymes were recombinantly produced in Escherichia coli and biochemically characterized, RjAbf43A consisting of the first Laminin_G_3 module and GH43_22 module, RjAbf43B consisting of the second Laminin_G_3 module, Big_4 module and GH43_26 module, and RjAbf43C consisting of the GH43_34 module. RjAbf43A showed a strong α-l-arabinofuranosidase activity toward sugar beet arabinan, highly branched arabinan but not linear arabinan, thus it acted in the removal of arabinose side chains from sugar beet arabinan. By contrast, RjAbf43B showed a strong exo-α-1,5-l-arabinofuranosidase activity toward linear arabinan and arabinooligosaccharides whereas RjAbf43C showed low activity toward these substrates. Although RjAbf43B was activated by the presence of some metal ions such as Zn2+, Mg2+ and Ni2+, RjAbf43A was inhibited by these ions. RjAbf43A and RjAbf43B attacked sugar beet arabinan in a synergistic manner. By comparison, RjAbf43A-Abf43B containing both GH43_22 and GH43_26 modules showed lower hydrolytic activity toward sugar beet arabinan but higher activity toward sugar beet fiber than the sum of the individual activities of RjAbf43A and RjAbf43B, suggesting that the coexistence of two distinct GH43 modules in a single polypeptide is important for the efficient hydrolysis of an insoluble and natural polysaccharide but not a soluble substrate.

Function of a laminin_G_3 module as a carbohydrate-binding module in an arabinofuranosidase from Ruminiclostridium josui.

Laminin_G_3 modules can exist together with family-43 catalytic modules of glycoside hydrolase (GH43), but their functions are unknown. Here, a laminin_G_3 module and a GH43 module derived from a Ruminiclostridium josui modular arabinofuranosidase Abf43A-Abf43B-Abf43C were produced individually as RjLG3 and RjGH43_22, respectively, or combined as RjGH43-1 to gain insights into their activities. Isothermal calorimetry analysis showed that RjLG3 has high affinity toward 32 -α-l-arabinofuranosyl-(1,5)-α-l-arabinotriose but not for α-1,5-linked arabinooligosaccharides, which suggests that RjLG3 interacts specifically with a branched arabinofuranosyl residue of an arabinooligosaccharide but not an arabinofuranosyl residue at the end of α-1,5-linked arabinooligosaccharides. RjGH43-1 (with CBM) shows higher activity toward sugar beet arabinan than RjGH43_22 (without CBM), which suggests that the LG3 module in RjGH43-1 plays an important role in substrate hydrolysis as a carbohydrate-binding module.

Ruminiclostridium josui Abf62A-Axe6A: A tri-functional xylanolytic enzyme exhibiting α-l-arabinofuranosidase, endoxylanase, and acetylxylan esterase activities.

Ruminiclostridium josui Abf62A-Axe6A is a modular enzyme comprising (in order from the N-terminus): an N-terminal signal peptide, a glycoside hydrolase family 62 (GH62) catalytic module, a family 6 carbohydrate binding module (CBM6), a dockerin module and an additional carbohydrate esterase family 6 catalytic module (CE6). In this study, three Abf62A-Axe6A derivatives were constructed, overexpressed in Escherichia coli, purified, and biochemically characterized: RjAbf62A-Axe6A, containing all four modules but lacking the signal peptide; RjAbf62A-CBM6, containing the GH62 and CBM6 modules; and RjAxe6A, containing only CE6. RjAbf62A-Axe6A was highly active toward arabinoxylan and moderately active toward sugar beet arabinan, and released mainly arabinose. Analysis of the arabinoxylooligosaccharide hydrolysis products revealed that RjAbf62A-Axe6A released α-1,2- and α-1,3-linked arabinofuranose from both singly and doubly substituted xylosyl residues. Furthermore, RjAbf62A-Axe6A exhibited a weak activity toward linear 1,5-α-l arabinan and arabinooligosaccharides, indicating that it is capable of cleaving α-1,5-linkage. Surprisingly, RjAbf62A-Axe6A also demonstrated an endoxylanase activity toward birchwood and beechwood xylans and xylooligosaccharides. Although RjAbf62A-CBM6 exhibited a similar substrate specificity to RjAbf62A-Axe6A, RjAbf62A-CBM6 showed lower activities toward soluble arabinoxylans, birchwood and beechwood xylans and arabinoxylooligosaccharides but not toward insoluble arabinoxylan. RjAbf62A-Axe6A is the first reported GH62 enzyme with α-l-arabinofuranosidase and endoxylanase activities. Although both RjAbf62A-Axe6A and RjAxe6A had acetylxylan esterase activities, RjAbf62A-Axe6 exhibited a higher activity toward insoluble wheat arabinoxylan compared with RjAxe6.

Development of an efficient host-vector system of Ruminiclostridium josui.

Although Ruminiclostridium josui (formerly Clostridium josui), a strictly anaerobic mesophilic cellulolytic bacterium, is a promising candidate for biomass utilization via consolidated bioprocessing, its host-vector system has not yet been established. The existence of a restriction and modification system is a significant barrier to the transformation of R. josui. Here, we partially purified restriction endonuclease RjoI from R. josui cell extract using column chromatography. Further characterization showed that RjoI is an isoschizomer of DpnI, recognizing the sequence 5'-Gmet ATC-3', where the A nucleotide is Dam-methylated. RjoI cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. We then successfully introduced plasmids prepared from Escherichia coli C2925 (dam- /dcm- ) into R. josui by electroporation. The highest transformation efficiency of 6.6 × 103 transformants/µg of DNA was obtained using a square-wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin-encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui, a truncated form of RjCel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of fundamental technology for molecular breeding of R. josui.

Characterization of Ruminiclostridium josui arabinoxylan arabinofuranohydrolase, RjAxh43B, and RjAxh43B-containing xylanolytic complex.

A novel gene (axh43B) from Ruminiclostridium josui encoding a cellulosomal enzyme consisting of a catalytic module of subfamily GH43_10, a family-6 carbohydrate-binding module, and a dockerin module, was expressed using Escherichia coli. RjAxh43B released only arabinose from arabinoxylan and 23,33-di-α-l-arabinofuranosyl xylotriose, but not 32-α-l-arabinofuranosyl xylobiose or 23-α-l-arabinofuranosyl xylotriose, strongly suggesting that RjAxh43B is an arabinoxylan α-l-1,3-arabinofuranohydrolase capable of cleaving α-1,3-linked arabinose residues of doubly arabinosylated xylan. When Axh43B was mixed with the recombinant scaffolding protein RjCipA of R. josui at a molar ratio of 6:1, the activity of the RjAxh43B-RjCipA complex (6:1) toward insoluble wheat arabinoxylan was similar to that of RjAxh43B alone, suggesting that RjAxh43B does not show a proximity effect, which is defined as an activity enhancement effect caused by the presence of plural catalytic subunits adjoining each other. When RjAxh43A was mixed with xylanase RjXyn10C, they acted synergistically toward insoluble wheat arabinoxylan and rice straw powder in the absence of RjCipA. Furthermore, the RjAxh43B-RjXyn10C-RjCipA (3:3:3) complex had higher activity toward insoluble wheat arabinoxylan than a mixture of RjAxh43B and RjXyn10C without RjCipA, suggesting that incorporation of a xylanase and an α-l-arabinofuranosidase into a cellulosome is beneficial for more efficiently degrading arabinoxylan.

Recombinant cellulolytic or xylanolytic complex comprising the full-length scaffolding protein RjCipA and cellulase RjCel5B or xylanase RjXyn10C of Ruminiclostridium josui.

Three cellulosomal subunits of Ruminiclostridium josui, the full-length scaffolding protein CipA (RjCipA), a cellulase Cel5B (RjCel5B) and a xylanase Xyn10C (RjXyn10C), were successfully produced by Escherichia coli recombinant clones. RjCel5B and RjXyn10C were characterized as an endoglucanase and an endoxylanase, respectively. RjCipA, RjCel5B and Xyn10C adsorbed to microcrystalline cellulose (Funacel) and rice straw powder. Interaction between RjCel5B and RjCipA, and RjXyn10C and RjCipA were confirmed by qualitative assays. When a fixed amount of RjCel5B was mixed with different amounts of RjCipA, i.e., at the molar ratio of 6:1 or 6:6, the 6:6 complex showed 6.6-fold higher activity toward Funacel and 11.5-fold higher activity toward rice straw powder than RjCel5B, whereas the 6:1 complex showed only 2.8- and 3.9-folds higher activities toward Funacel and rice straw powder, respectively, than RjCel5B. These results suggest that the family-3 carbohydrate binding module (CBM3) of RjCipA in the RjCel5B-RjCipA complex plays an important role for hydrolysis of cellulose and the substrate-targeting effect of the CBM is more significant than the proximity effect caused by the presence of plural catalytic subunits adjoining each other. In contrast, the 6:1 complex of RjXyn10C and RjCipA showed 45% and 28% of the activities of RjXyn10C toward insoluble wheat arabinoxylan and rice straw powder, respectively. These results suggest that both a negative proximity effect and substrate-isolating effect, but not substrate-targeting effect, are caused by the CBM3 with inappropriate polysaccharide specificity. Substrate-targeting, proximity and substrate-isolating effects are discussed.

Characterization of endoglucanase from Paenibacillus sp. M33, a novel isolate from a freshwater swamp forest.

The newly isolated Paenibacillus sp. M33 from freshwater swamp forest soil in Thailand demonstrated its potential as a cellulose degrader. One of its endoglucanase genes from Paenibacillus sp., celP, was cloned to study the molecular characteristics of its gene product. The celP gene was recognized firstly by degenerate primer designed from Paenibacillus endoglucanase gene, and subsequently identified flanking region by inverse PCR technique. The celP gene consists of an open reading frame of 1707 bp encoding for 569 amino acids including 33-amino acids signal sequence. CelP is a member of glycoside hydrolase family 5 appended with a family 46 carbohydrate-binding module. CelP from recombinant Escherichia coli was purified by affinity chromatography. SDS-PAGE analysis of purified CelP showed a protein band at about 60 kDa. The purified enzyme gave a specific CMCase activity of 0.03 µmol min-1 mg-1 . It had higher activities on lichenan (0.19 µmol min-1 mg-1 ) and barley ß-glucan (0.14 µmol min-1 mg-1 ). Maximum activity on lichenan was obtained at 50 °C, pH 5.0. CelP was stable over a pH range of 3.0-10.0 and retained 80% activity when incubated at 50 °C for 1 h. The properties of its CelP endoglucanase, especially substrate specificity, will make it useful in various biotechnological applications including biomass hydrolysis.

The proteolytic profile of human cancer procoagulant suggests that it promotes cancer metastasis at the level of activation rather than degradation.

Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

Exopolysaccharide assay in Escherichia coli microcolonies using a cleavable fusion protein of GFP-labeled carbohydrate-binding module.

A fused protein composed of a carbohydrate-binding module and green fluorescence protein (GFP) was developed to measure the exopolysaccharides (EPShs) present in Escherichia coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative evaluation of the EPShs.

Paenibacillus curdlanolyticus B-6 xylanase Xyn10C capable of producing a doubly arabinose-substituted xylose, α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-D-Xylp, from rye arabinoxylan.

Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis α-L-arabinofuranosidase Axh43A indicated that U1 was α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-D-Xylp and U2 was α-L-Araf-(1→2)-D-Xylp, suggesting that rXyn10C had strong activity toward a xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an α-1,2- and α-1,3-linked arabinose-substituted xylose unit in both the -1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite -1. This is the first report of a xylanase capable of producing α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-D-Xylp from highly arabinosylated xylan.

Probing of exopolysaccharides with green fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced by bcsB overexpression.

Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a carbohydrate-binding module 3, from Paenibacillus curdlanolyticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing.

Essential role of a family-32 carbohydrate-binding module in substrate recognition by Clostridium thermocellum mannanase CtMan5A.

The family-5 glycoside hydrolase domain (GH5) and the family-32 carbohydrate-binding module (CBM32) of Clostridium thermocellum mannanase CtMan5A, along with their genetically inactivated derivatives, were collectively or separately expressed. Their catalytic and substrate-binding abilities were measured to investigate importance of CBM32 in substrate recognition by CtMan5A. Characterization of the truncated derivatives of CtMan5A and isothermal calorimetry analysis of the interaction between the inactivated proteins and mannooligosaccharides suggested that GH5 and CBM32 collectively formed a substrate-binding site capable of accommodating a mannotetraose unit in CtMan5A. This suggested that CBM32 directly participated in the substrate recognition required for catalytic action.

ß-D-xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme.

The gene encoding a thermostable ß-D-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6-8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s(-1) mM(-1) on p-nitrophenyl-ß-D-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel ß-strands) and catalytic module (residues 157 to 604 forming five-bladed ß-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.

Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis.

The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-ß-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-D-glucurono-D-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-L-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.