eIn 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 ␤-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 ␤-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.

A glucosyltransferase responsible for water-insoluble glucan synthesis was purified from the culture fluids of Streptococcus mutans 6715-15 strain by column chromatography on Toyopearl HW-60 and subsequently on hydroxyapatite. The enzyme preparation gave a single band on analysis by polyacrylamide gel electrophoresis. The pH dependency of the activity showed two optimal peaks at 5.8 and 7.3, and the Km values for sucrose were 1.4 and 3.3 mM at the respective optimal pHs. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 180,000. Although the enzyme scarcely synthesized waterinsoluble and water-soluble glucans from sucrose, water-insoluble glucan formed from sucrose in the presence of dextran T10 consisted of over 93% a-1,3glucosidic linkage. Analysis of the structure of water-insoluble glucan indicated that the enzyme catalyzed the formation of branch points in a-1 ,6-glucan (dextran) and transferred the glucosyl moiety of sucrose to the C-3 position of the branching glucose residue of dextran. Since this enzyme has not yet been registered, we named it mutansynthetase (EC 2.4.1.?).

a b s t r a c tThe 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.

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