Makiko Sakka

Essential role of the family‐22 carbohydrate‐binding modules for β‐1,3‐1,4‐glucanase activity of Clostridium stercorarium Xyn10B

Clostridium stercorarium Xyn10B is a modular enzyme comprising two family‐22 carbohydrate‐binding modules (CBMs), a family‐10 catalytic module of glycoside hydrolases, a family‐9 CBM, and two S‐layer homologous modules consecutively from the N‐terminus. To investigate the role of the family‐22 CBMs, truncated proteins were constructed: a recombinant catalytic module polypeptide (rCD), a CBM polypeptide composed of two family‐22 CBMs (rCBM) and a polypeptide composed of the family‐22 CBMs and the catalytic module (rCBM‐CD). We found that rCBM‐CD was highly active toward β‐1,3‐1,4‐glucan; however, rCD was negligibly active toward the same substrate. The V max/K m value of rCBM‐CD for β‐1,3‐1,4‐glucan was 7.8 times larger than that for oat‐spelt xylan, indicating that rCBM‐CD should be specified as a β‐1,3‐1,4‐glucanase rather than a xylanase despite the fact that family‐10 catalytic modules are well‐known xylanase modules. These results indicate that the family‐22 CBMs in rCBM‐CD are essential for hydrolysis of β‐1,3‐1,4‐glucan.

Binding of S-layer homology modules from Clostridium thermocellum SdbA to peptidoglycans.

S-layer homology (SLH) module polypeptides were derived from Clostridium josui xylanase Xyn10A, Clostridium stercorarium xylanase Xyn10B, and Clostridium thermocellum scafoldin dockerin binding protein SdbA as rXyn10A-SLH, rXyn10B-SLH, and rSdbA-SLH, respectively. Their binding specificities were investigated using various cell wall preparations. rXyn10A-SLH and rXyn10B-SLH bound to native peptidoglycan-containing sacculi consisting of peptidoglycan and secondary cell wall polymers (SCWP) prepared from these bacteria but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWP, suggesting that SCWP are responsible for binding with SLH modules. In contrast, rSdbA-SLH interacted with HF-EPCS, suggesting that this polypeptide had an affinity for peptidoglycans but not for SCWP. The affinity of rSdbA-SLH for peptidoglycans was confirmed by a binding assay using a peptidoglycan fraction prepared from Escherichia coli cells. The SLH modules of SdbA must be useful for cell surface engineering in bacteria that do not contain SCWP.

Influence of a Mannan Binding Family 32 Carbohydrate Binding Module on the Activity of the Appended Mannanase

ABSTRACT In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.

Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a Xylanase That Contains a Family 3 Carbohydrate-Binding Module

ABSTRACT Paenibacillus curdlanolyticus B-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass.

Analysis of a Clostridium josui Cellulase Gene Cluster Containing the man5A Gene and Characterization of Recombinant Man5A

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.

Stable Production of Thermotolerant Xylanase B of Clostridium stercorarium in Transgenic Tobacco and Rice

The xylanase B gene encoding a thermostable family 10 xylanase of Clostridium stercorarium was expressed in plants under the control of a constitutive promoter. Two forms of the xylanase B gene, the xynB gene encoding the full length of the xylanase B gene including the bacterial signal sequence and the xynBM gene without the signal sequence region, were introduced into tobacco BY-2 cells and tobacco plants respectively under the control of the cauliflower mosaic virus 35S promoter. Transgenic BY-2 cells and tobacco plants showed xylanase activity and normal growth. The recombinant enzyme produced in transgenic BY-2 cells harboring the xynB gene was secreted into the culture supernatant, and the recombinant enzyme produced in transgenic BY-2 cells harboring the xynBM gene was localized in the cells. In contrast to tobacco plants, expression of the xynB gene under the control of the rice actin promoter in rice plants was toxic to host cells. However, the recombinant XynBM accumulated in leaf cells, and no phenotypic effect of expression of the xynBM gene was observed. Enzyme activity was maintained in cell-free extracts of transgenic rice leaves at 60 °C for 72 h, and the recombinant XynBM degraded hemicellulosic polymers in cell-free extracts of transgenic rice leaves.

High-Level Heterologous Expression of Bacillus halodurans Putative Xylanase Xyn11A (BH0899) in Kluyveromyces lactis

The putative xyn11A structural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus halodurans strain C-125 was heterologously expressed in the yeast Kluyveromyces lactis CBS 1065 and secreted to a level of 156 μg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactis CBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or β-xylosidase activity.

Characterization of Paenibacillus curdlanolyticus Intracellular Xylanase Xyn10B Encoded by the xyn10B Gene

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.

Characterization of a Dihydrolipoyl Dehydrogenase Having Diaphorase Activity of Clostridium kluyveri

The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD+ as a specific electron acceptor, and the apparent K m values for DLA and NAD+ were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K m values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 °C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 °C.

Functions of family-22 carbohydrate-binding module in Clostridium thermocellum Xyn10C.

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 β-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 °C and that of rCM was 60 °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.