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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.

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.

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&prime, CCRCCC-3&prime, 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.

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.

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.

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.

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.

The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli . Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited β-xylosidase activity toward a chromogenic substrate, p -nitrophenyl-β- d -xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, α- l -Ara f -(1→2)- d -Xyl p or α- l -Ara f -(1→3)- d -Xyl p and α- l -Ara f -(1→2)-[α- l -Ara f -(1→3)]-β- d -Xyl p . Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications. IMPORTANCE In this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) from P. curdlanolyticus B-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.

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.

Two different expression vectors were constructed to investigate two signal peptides on secretion of Geobacillus thermoleovorans IT-08 α-L-arabinofuranosidase (AbfA) in Saccharomyces cerevisiae . They were designed to direct the secretion of AbfA by the aid of one of the following signal peptides, the αF signal peptide in the plasmid YEpFLAG1-Af and HM-1 signal peptide in the plasmid pYHM1-Af. Although some successful results have been reported in proteins secretion with αF leader sequence, in this research no α-L-arabinofuranosidase activity could be observed in recombinants S. cerevisiae YEpFLAG1-Af. The HM-1 leader sequence, originated from Hansenula mrakii IFO 0895 killer toxin, showed the capability to AbfA secretion.

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.

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.

Plant biomass, or lignocellulosic biomass, is evaluated worldwide as a potential feedstock for the sustainable production of bioenergy in the near future due to its abundance, availability and renewability. Promising sources of plant biomass include agricultural residues and energy crops; however, the natural recalcitrance of this material is a major bottleneck for lignocellulose-derived ethanol production. The current process requires pre-treatment with severe conditions to disrupt the plant cell wall structures and remove hemicellulose and lignin components so that cellulose is more accessible to cellulases. However, the generation of enzyme inhibitors/deactivators and toxic substances during pre-treatment may subsequently affect enzymatic saccharification and fermentation processing. The pre-treatment and saccharification processability can be simplified if the plant biomass resistance to biochemical or enzymatic treatment is reduced. While there are many developed pre-treatment technologies and formulated enzyme cocktails that match pre-treated substrates, there has been attempt to design ideal energy crops via plant genetic manipulation. Cellulose engineering is aimed at reducing the crystallinity of cellulose structures. Expression of cellulose-disrupting proteins, including carbohydrate-binding modules, expansins, and swollenins, produces irregular forms of cellulose fibrils, which change from tightly packed fibrils to splayed ribbons with a high sugar release after enzymatic treatment. In addition, modifying genes and proteins involved in cellulose synthesis resulted in an unusual secondary cell wall deposition and composition and a lower crystallinity index. Reducing lignin content though engineering lignin biosynthesis pathways improves the saccharification process; however, abnormal growth and plant fitness remain problematic when improper genes are selected for manipulation. Lignin composition can be modified by introducing phenolic derivatives or peptide cross-links upon lignification, and these approaches might minimise the interference with plant growth and development. Hemicellulose biosynthesis is a complicated process. Currently, the reduction of hemicellulose content relies mostly on enzymes involved in xyloglucan/glucoarabinoxylan synthesis and the arrangements of those polymers in developing wood. Additionally, several glycosyltransferase and glycoside hydrolases are believed to be involved in hemicellulose modification in relation to loosened cell walls. Importantly, the expression of foreign glycoside hydrolases in plants may facilitate the reduction of enzyme loadings, thus making lignocellulosic ethanol production economically viable.

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-β-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.

Clostridial cellulosomes are cellulolytic complexes that are formed by highly specific interactions between one of the repeated cohesin modules present in the scaffolding protein and a dockerin module of the catalytic components. Although Clostridium thermocellum Xyn11A dockerin has a typical C. thermocellum dockerin sequence, in which two amino acid residues are species specifically conserved within the two segments of the dockerin modules, it can recognize Clostridium josui cohesin modules in a non-species-specific manner. The importance of these two conserved amino acids, which are part of the recognition site of the cohesin and dockerin interaction, was investigated by introducing mutations into the first and/or the second segments of the Xyn11A dockerin. Mutations in the first segment did not affect the interactions between dockerin and C. thermocellum and C. josui cohesins. However, mutations in the second segment prevented binding to cohesin proteins. A second round of mutations within the first segment re-established the affinity for both the C. thermocellum and the C. josui cohesins. However, this was not observed for a ‘conventional’ dockerin, Xyn10C. These results suggest that the combination of the first and second dockerin segments is important for the target recognition.

A cellulase gene cluster of Clostridium josui was sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josui cellulosome. Among these genes, the man5A gene encoding β-mannanase was expressed in Escherichia coli and the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source.

In previous work, we reported on the identification and purification of a multi-enzyme complex (MEC) from Bacillus licheniformis SVD1. The predominant activity within the MEC was xylanase activity and this study examined the effect of various environmental parameters such as pH, temperature, substrate concentration and compounds such as Mg 2+ , Mn 2+ , Fe 2+ , Zn 2+ , Ca 2+ , EDTA, SDS, xylose, xylobiose and ethanol on complexed xylanase activity. The pH optimum was found to be between pH 6.0 and 7.0 and the temperature optimum at 55 °C. High levels of residual activity were present over a broad range of pH values. Enhancement of complexed xylanase activity was found in the presence of Mg 2+ at 2 mM and 10 mM, while Ca 2+ displayed a slight activation at 2 mM but inhibition at 10 mM. Mn 2+ , Fe 2+ , Zn 2+ , EDTA and SDS all displayed an inhibitory effect on complexed xylanase activity, with the greatest inhibition found in the presence of Mn 2+ . Xylose and xylobiose were found to enhance complexed xylanase activity up to 50%, which has not been reported in literature previously. Ethanol was found to inhibit complexed xylanase activity in a competitive manner, but 58% residual activity was still present at concentrations of 50 g/l ethanol. Complexed xylanases from B. licheniformis SVD1, being uninhibited by products of degradation and only mildly inhibited by ethanol, would be suitable for use in biotechnological applications such as bioethanol production.

Micro-organisms that degrade plant biomass require a range of enzymes to effectively degrade such substrates into sugar monomers. Some organisms secrete a host of free, extracellular enzymes while others produce a multi-enzyme complex (MEC) consisting of a variety of these enzymes. This study examined the cellulolytic and hemi-cellulolytic system of Bacillus licheniformis SVD1 with respect to the presence of key enzymes within the MEC found in this organism. When cultured on birchwood xylan, the MEC in B. licheniformis SVD1 was found to contain two endoglucanases (at 25–27 kDa, 30–32 kDa), seven xylanases (at 20 kDa, 38 kDa, 42 kDa, 45 kDa, 48 kDa, 54 kDa and 70 kDa), two mannanases (25 kDa, 40–42 kDa) and one pectinase (a pectate lyase at 70–72 kDa). Identification of enzymes was based on zymogram analysis. The MEC contained 17 different protein species based on SDS-PAGE analysis and 9 of these proteins could be identified through zymograms. Further work will be conducted to confirm the identity of other proteins in the MEC. The crude extract contained a further endoglucanase of 55 kDa, a mannanase of 53 kDa and a pectin methyl esterase of 38 kDa, indicating that not all enzymes are incorporated into the MEC. Understanding the composition of a hemi-cellulolytic system when cultured on a substrate may assist in utilising such a system for the synergistic degradation of complex lignocellulose substrates.

The cellulolytic and hemi-cellulolytic system of Bacillus licheniformis SVD1 was isolated and characterised in birchwood xylan cultures. The predominant activity in the crude culture was xylanase activity, but the crude culture also displayed Avicelase, carboxymethylcellulase (CMCase), mannanase, and pectinase activity. Most of the xylanase activity was found in the culture supernatant, but some activity was cell-associated. Using Sepharose 4B size exclusion chromatography, a 2000 kDa multi-enzyme complex (MEC) was purified. The MEC contained predominantly xylanase activity, as well as significant levels of mannanase and CMCase activity, but no Avicelase activity. SDS-PAGE revealed up to eight visible bands in the MEC while zymograms of the MEC displayed two xylanase active bands at 21 kDa and 45 kDa, and two CMCase active bands at 25 kDa and 30 kDa. More active bands were visible in the crude supernatant with an additional xylanase active band at 40 kDa and an additional CMCase active band at 55 kDa. Using thin layer chromatography (TLC), it was established that the crude fraction could release xylose from insoluble birchwood xylan, while the MEC was only able to produce xylobiose from this substrate. The MEC was further able to bind to insoluble xylan, but was unable to bind to crystalline cellulose. This MEC lacks many of the characteristic features of a cellulosome and is most likely a different type of complex. The presence of both high xylanase and mannanase activity makes this MEC unusual.

We identified 16S rRNA gene sequences in sediment samples from Ago Bay in Japan, forming a new branch of the anammox group or closely related to anaerobic ammonium oxidizing (anammox) bacterial sequences. Anammox activity in the sediment samples was detected by (15)N tracer assays. These results, along with the results of fluorescence in situ hybridization (FISH) analysis, suggest the presence of anammox bacteria in the marine sediments.

The effect of polymeric substances such as alpha-cellulose, birchwood xylan, corn hull, and sugarcane bagasse, and of soluble sugars such as L-arabinose, D-galactose, D-glucose, D-xylose, and cellobiose, on the induction of multienzyme complexes in a facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, was investigated under aerobic conditions. Cells and culture supernatants of strain B-6 grown on different carbon sources were analyzed. Cells grown on each carbon source adhered to cellulose. Hence strain B-6 cells from all carbon sources must have an essential component responsible for anchoring the cells to the substrate surfaces. Native-polyacrylamide gel electrophoresis (native-PAGE), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), zymogram analysis, and enzymatic assays indicated that many proteins having xylanolytic and cellulolytic activities from P. curdlanolyticus B-6 grown on each carbon source were produced as two multienzyme complexes in the culture supernatants. These results indicate that P. curdlanolyticus B-6 produced multienzyme complexes when grown on both polymeric and soluble sugars. The multienzyme complexes of P. curdlanolyticus B-6 consisted of the main enzymes and non-enzymatic subunits and the production of some different subunits, depending on the carbon source.

To examine the possibility of module interaction in the thermal unfolding of different modular architectures, four truncated proteins were constructed from Clostridium stercorarium Xyn10B: a family 10 catalytic module (CM10), a polypeptide compound of one family 22 carbohydrate-binding module (CBM22-2) and the catalytic module (CBM22-CM10), two family 22 CBMs and the catalytic module (2CBM22-CM10), and only two family 22 CBMs (2CBM22). Thermal unfolding of four proteins were observed by differential scanning calorimetry. CM10 was unfolded reversibly and denatured as one component. The unfolding of protein CBM22-CM10 comprising CBM22-2 connected with CM10 was irreversible, and can be assumed to be one-component denaturation. Protein 2CBM22, with two CBM22s in tandem, unfolded as two independent modules. However, 2CBM22-CM10, with two CBM22s, unfolded as two and not the expected three separate components. These findings constitute the first reported case in which differences in thermal unfolding units and mechanisms were derived from differences in the modular architectures of proteins.

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with β-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a β-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to β-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to β-1,3–1,4-glucans while substantially decreasing the affinity for decorated β-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified β-1,3–1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant.

Clostridium paraputrificum M-21 beta-N-acetylglucosaminidase 3A (Nag3A) is an enzyme classified in family 3 of the glycoside hydrolases. To identify catalytic residues of this enzyme, mutations were introduced into highly conserved Glu and Asp residues. Replacement of Asp175 with Ala abolished the catalytic activity without change in the circular dichroism spectrum, strongly suggesting that this residue is a catalytic residue, a nucleophile/base or a proton donor. Since the K(m) values of mutant enzymes D119N, D229N, D229A and D274N increased 17 to 41 times as compared with that of wild-type enzyme, Asp119, Asp229, and Asp274 appear to be involved in substrate recognition and binding. Taking previous studies into consideration, we presume that Asp303 is the catalytic nucleophile and Asp175 is the proton donor of C. paraputrificum Nag3A.

A gene encoding an exo-β-1,3-galactanase from Clostridium thermocellum , Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-β-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli , and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50°C and catalyzes hydrolysis only of β-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from β-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a β-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-β-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including β-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a β-1,3-galactobiose, β-1,4-galactosyl glucose, or β-1,4-galactosyl N -acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward β-1,3-galactan and α- l -arabinofuranosidase-treated AGP.

Clostridium stercorarium F-9 pectate lyase Pel9A is a modular enzyme composed of two hypothetical family-9 catalytic modules of the polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. In this study, we constructed and characterized CM9-1 and CM9-2 polypeptides as rCM9-1 and rCM9-2 respectively. Both of them, like the full-length Pel9A, required the Ca2+ ion for their enzyme activities and showed high activity toward polygalacturonic acid but lower activity toward pectin. The specific activity of rCM9-2 was three times higher than that of rCM9-1 and rCM9-2 by itself efficiently catalyzed the depolymerization reaction of polygalacturonic acid into monosaccharide as the major product. It was found that rCM9-1 and rCM9-2 adsorbed to polygalacturonic acid and pectin on native affinity PAGE analysis, suggesting that they contain an independent carbohydrate-binding module separable from a catalytic module or consist of a catalytic module with a binding affinity for pectic substrates.

Five distinct genomic DNA fragments (EcPT1, EcPT2, EcPT3, EcPT4 and EcPT5) encoding phosphate transporters were isolated from Eucalyptus camaldulensis. EcPT2 and EcPT3 were exclusively expressed in the root, but were not enhanced by phosphate deprivation. The transcript level of EcPT2 was much higher than that of EcPT3. The EcPT2 is present as a single copy gene in Eucalyptus genome, and phylogenetic analysis and deduced amino acid sequence revealed that EcPT2 can be classified into the Pht1 family, a high-affinity phosphate transporter. These results suggest that EcPT2 functions in phosphate transport in root tissues not only under low-phosphate conditions as with typical Pht1 family members, but also under high-phosphate conditions.

The PHT1 promoter::GUS fusion gene was constructed and introduced into Arabidopsis and rice by Agrobacterium-mediated transformation. Strong beta-glucuronidase (GUS) activity was detected in roots and showed phosphate starvation induction both in Arabidopsis and rice. In contrast, GUS activity in aerial tissues such as those of the leaf and stem was low. In situ GUS staining of root tissue indicated that PHT1 was expressed in root hairs and the outer layer of the main roots, but not in root tips. The PHT1 promoter has a desirable character for biotechnological transgene expression in monocot rice plants.

Clostridium stercorarium Xyn10B having hydrolytic activities on xylan and beta-1,3-1,4-glucan is a modular enzyme composed of two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of the glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. We investigated the function of family-9 and family-22 CBMs in a modular enzyme by comparing the enzymatic properties of a truncated enzyme composed of two family-22 CBMs and the catalytic module (rCBM22-CM), an enzyme composed of the catalytic module and family-9 CBM (rCM-CBM9), an enzyme composed of two family-22 CBMs, the catalytic module, and family-9 CBM (rCBM22-CM-CBM9), and the catalytic module polypeptide (rCM). Although the addition of family-9 CBM to rCM and rCBM22-CM did not significantly change catalytic activity toward xylan and beta-1,3-1,4-glucan, the addition of family-22 CBM to rCM and rCM-CBM9 drastically enhanced catalytic activity toward xylan and especially beta-1,3-1,4-glucan. Furthermore, the addition of family-22 CBM to rCM and rCM-CBM9 shifted the optimum temperature from 65 degrees C to 75 degrees C, but that of family-9 CBM to rCM and rCBM22-CM did not affect the optimum temperature. These facts suggest that the enzyme properties of Xyn10B were mainly dependent on the presence of the family-22 CBMs but not family-9 CBM.

Clostridium thermocellum xylanase Xyn10C (formerly XynC) is a modular enzyme, comprising a family-22 carbohydrate-binding module (CBM), a family-10 catalytic module of the glycoside hydrolases, and a dockerin module responsible for cellulosome assembly consecutively from the N-terminus. To study the functions of the CBM, truncated derivatives of Xyn10C were constructed: a recombinant catalytic module polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBM and CM (rCBM-CM). The recombinant proteins were characterized by enzyme and binding assays. Although the catalytic activity of rCBM-CM toward insoluble xylan was four times higher than that of rCM toward the same substrate, removal of the CBM did not severely affect catalytic activity toward soluble xylan or beta-1,3-1,4-glucan. rCBM showed an affinity for amorphous celluloses and insoluble and soluble xylan in qualitative binding assays. The optimum temperature of rCBM-CM was 80 degrees C and that of rCM was 60 degrees C. These results indicate that the family-22 CBM of C. thermocellum Xyn10C not only was responsible for the binding of the enzyme to the substrates, but also contributes to the stability of the CM in the presence of the substrate at high temperatures.

Clostridium josui xylanase Xyn10A is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module (CM), a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. To study the functions of the family-22 CBMs, truncated derivatives of Xyn10A were constructed: a recombinant CM polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBMs and CM (rCBM-CM). Recombinant proteins were characterized by enzyme and binding assays. rCBM-CM showed the highest activity toward xylan and weak activity toward some polysaccharides such as barley beta-glucan and carboxymethyl-cellulose. Although rCBM showed an affinity for insoluble and soluble xylan as well as barley beta-glucan and Avicel in qualitative binding assays, removal of the CBMs negligibly affected the catalytic activity and thermostability of the CM.

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the KD values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.

A β- N -acetylglucosaminidase gene ( nag 84A) was cloned from Clostridium paraputrificum M-21 in Escherichia coli . The nag84A gene consists of an open reading frame of 4647 by encoding I549 amino acids, with a deduced molecular weight of 174,311, which have a catalytic domain belonging to family 84 of the glycoside hydrolases. Nag84A was purified from a recombinant E. coli and characterized. Although Nag84A exhibited high homology to the hyaluronidase from Clostridium perfringens , it did not degrade hyluronic acid. The enzyme hydrolyzed chitooligomers such as di-, tri-, tetra-, penta- and hexa- N -cetylchitobexaose, and synthetic substrates such as 4-methylumbelliferyl N -acetyl β- d -glucosaminide [4-MU-(G1cNAc)], but did not hydrolyze 4-MU-β- d -glucoside, 4-MU-α- d -glycoside, 4-MU-α- d -GlcNAc, 4-MU-α- d -galactoside, 4-MU-β- d -xyloside, PNP-β- d -galactoside, and PNP-α- d -xyloside. The enzyme was optimally active at 50°C and pH 6.5, and the apparent K m and V max values for 4-MU-(G1cNAc) were 8.5 μM and 1.39 μmol/min/mg of protein, respectively. SDS-PAGE, zymogram, and immunological analyses suggested that Nag84A was inducible by ball-milled chitin. Since Nag84A has a high molecular weight with a family 84 catalytic domain with high homology to hyaluronidases but no hyaluronidase activity, the enzyme is a novel β- N -acetylglucosaminidase different from others reported having low molecular weights and belonging to family 3 and family I8.

The concentration of estrogenic substances in water and sediment was surveyed in Ago Bay, which is a typical enclosed sea area. Eight water samples and seven sediment samples were collected from the mouth of the Maekawa River at the entrance of the Ago Bay. To examine estrogenic substances, a yeast screen assay developed by Prof. J. P. Sumpter was used. The highest concentrations of estrogenic substances in water and sediment were noted in the Maekawa River (16.6ng·l-1 and 24.3ng·g-1). The concentrations gradually decreased towards the entrance of Ago Bay from the mouth of the Maekawa River. It was suggested that this decrease may be the result of dilution, adsorption to the seabed and biodegradation of the estrogenic substances. The water and sediment assay samples were fractionated by high pressure liquid chromatography and assayed to determine the chemical species of estrogenic substances (high pressure liquid chromatography-bioassay detection method). The estrogenic substances detected corresponded to E1 and/or E2, and were noted in all samples. These are typical natural female hormones, and their origin is presumably domestic sewage and other wastewater containing feces and urine.

Two distinct domains of the xynA gene from Clostridium thermocellum encoding a xylanase catalytic domain (XynAl) and a xylanase catalytic domain with a cellulose binding domain (XynA2) under the control of the cauliflower mosaic virus 35S promoter were electroporated into cultured tobacco BY-2 cells. Transgenic BY -2 calli expressing xylan-hydrolyzing activity were obtained at high frequency for both genes. Western blot analysis using an anti-XynA antibody indicated that XynAl and XynA2 were produced in these calli.

The alpha-galactosidase gene aga36A of Clostridium stercorarium F-9 was cloned, sequenced, and expressed in Escherichia coli. The aga36A gene consists of 2,208 nucleotides encoding a protein of 736 amino acids with a predicted molecular weight of 84,786. Aga36A is an enzyme classified in family 36 of the glycoside hydrolases and showed sequence similarity with some enzymes of family 36 such as Geobacillus (formerly Bacillus) stearothermophilus GalA (57%) and AgaN (52%). The enzyme purified from a recombinant E. coli is optimally active at 70 degrees C and pH 6.0. The enzyme hydrolyzed raffinose and guar gum with specific activities of 3.0 U/mg and 0.46 U/mg for the respective substrates.

The kinetic parameters of Cex, a family 10 xylanase from Cellulomonas fimi , were determined at various pH levels using soluble birchwood xylan (BWX) as a natural polymeric substrate along with three other synthetic aryl-β- d -xylobioside substrates. Using BWX, a high level of substrate inhibition was observed which increased with decreasing pH. In contrast, typical Michaelis-Menten-type profiles were obtained using the three aryl-β- d -xylobiosides as substrates. The k cat values determined using o -nitrophenyl-β- d -xylobioside did not change as the pH increased, whereas the k cat values obtained with BWX, phenyl-β- d -xylobioside and p -nitrophenyl-β- d -xylobioside decreased, suggesting that the presence of an ortho nitro group affects the activity displayed by Cex. These differences were not observed with XynB from Clostridium stercorarium F9, a member of the same family of xylanases as Cex. These results indicate that a careful evaluation is required when employing substituted aryl-β- d -xylobiosides in the characterization of xylanases.

Cellulases catalyze the hydrolysis of cellulose, the major constituent of plant biomass and the most abundant organic polymer on earth. Cellulases are modular enzymes containing catalytic domains connected,vialinker sequences, to noncatalytic carbohydrate-binding modules (CBMs). A putative modular endo-β-1,4-glucanase (BhCel5B) is encoded at locus BH0603 in the genome ofBacillus halodurans. It is composed of an N-terminal glycoside hydrolase family 5 catalytic module (GH5) followed by an immunoglobulin-like module and a C-terminal family 46 CBM (BhCBM46). Here, the crystallization and preliminary X-ray diffraction analysis of the trimodularBhCel5B are reported. The crystals ofBhCel5B belonged to the orthorhombic space groupP21212 and data were processed to a resolution of 1.64 Å. A molecular-replacement solution has been found.

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.

Plant cell-wall polysaccharides offer an abundant energy source utilized by many microorganisms, thus playing a central role in carbon recycling. Aerobic microorganisms secrete carbohydrate-active enzymes (CAZymes) that catabolize this composite structure, comprising cellulose, hemicellulose and lignin, into simple compounds such as glucose. Carbohydrate-binding modules (CBMs) enhance the efficacy of associated CAZYmes. They are organized into families based on primary-sequence homology. CBM family 46 contains more than 40 different members, but has yet to be fully characterized. Here, a recombinant derivative of the C-terminal family 46 CBM module (BhCBM46) ofBacillus haloduransendo-β-1,4-glucanase B (CelB) was overexpressed inEscherichia coliand purified by immobilized metal-ion affinity chromatography. Preliminary structural characterization was carried out onBhCBM46 crystallized in different conditions. The crystals ofBhCBM46 belonged to the tetragonal space groupI4122. Data were collected for the native form and a selenomethionine derivative to 2.46 and 2.3 Å resolution, respectively. TheBhCBM46 structure was determined by a single-wavelength anomalous dispersion experiment usingAutoSolfrom thePHENIXsuite.

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.

The cells of Clostridium stercorarium F-9 grown on cellobiose bound to insoluble cellulose allomorphs such as phosphoric acid-swollen cellulose (ASC). Treatment of the cells with 3 M guanidine hydrochloride extracted surface-layer proteins from the cells and abolished the affinity of the cells for ASC. SDS-polyacrylamide gel electrophoresis, zymogram, and immunological analyses indicated that one of the major surface layer proteins was Xyn10B, which is a modular xylanase comprising two family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, and two S-layer homologous (SLH) domains. The C. stercorarium F-9 cells treated with guanidine hydrochloride coprecipitated with ASC upon the addition of a derivative of Xyn10B containing both a CBM and SLH domain in addition to a catalytic domain, but not a derivative without Xyn10B-SLH domains, suggesting that Xyn10B functioned as a cellulose-binding protein in C. stercorarium F-9.

The man26B gene of Clostridium thermocellum strain F1 was found in pKS305, which had been selected as a recombinant plasmid conferring endoglucanase activity on Escherichia coli. The open reading frame of man26B consists of 1,773 nucleotides encoding a protein of 591 amino acids with a predicted molecular weight of 67,047. Man26B is a modular enzyme composed of an N-terminal signal peptide and three domains in the following order: a mannan-binding domain, a family 26 mannanase domain, and a dockerin domain responsible for cellulosome assembly. We found that this gene was a homologue of the man26A gene of C. thermocellum strain YS but that there were insertion or deletion mutations that caused a frame-shift mutation affecting a stretch of 26 amino acids in the catalytic domain. Man26B devoid of the dockerin domain was constructed and purified from a recombinant E. coli, and its enzyme properties were examined. Immunological analysis indicated that Man26B was a catalytic component of the C. thermocellum F1 cellulosome.

The chitinolytic bacterium Clostridium paraputrificum strain M-21 produced 2.2 and 1.5 mol hydrogen gas from 1 mol N-acetyl-d-glucosamine (GlcNAc) and ball-milled chitin equivalent to 1 mol of GlcNAc, respectively, at pH 6.0. In addition, strain M-21 efficiently degraded and fermented ball-milled raw shrimp and lobster shells to produce hydrogen gas: 11.4 mmol H2 from 2.6 g of the former and 7.8 mmol H2 from 1.5 g of the latter. Hydrogen evolution from these shell wastes were enhanced two fold by employing acid and alkali pretreatment. Waste from the starch industry was also converted to hydrogen. When C. paraputrificum M-21 was cultivated on ball-milled chitin and ball-milled shrimp shells for 14 and 12 h, respectively, chitinases ChiA and/or ChiB were detected as the major chitinase species in the supernatant of the cultures, suggesting that the play a critical role in the degradation of chitinous materials.

The Paenibacillus curdlanolyticus xyn10B gene encoding a family-10 xylanase was cloned and expressed in Escherichia coli, and the recombinant enzyme rXyn10B was characterized. Immunological analysis suggested that Xyn10B is an intracellular enzyme. rXyn10B hydrolyzed birch-wood xylan and xylooligosaccharides to produce mainly xylobiose, suggesting that it is an endoxylanase. Its properties were significantly different from those of some homologous enzymes.