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

Abstract

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.

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

“…Specificity to LacNAc (␤-D-galactosyl-1,4-␤-D-N-acetylglucosamine) was also shown for a CBM32 of N-acetylhexosaminidase from C. perfringens (16). More recently, a mannan binding module belonging to the CBM32 family was found in a ␤-1,4-mannanase from Clostridium thermocellum (38). In this study thermal unfolding experiments suggested that DD1 in a chitosanase from Paenibacillus sp.…”

“…Binding Affinities of (GlcN) n -A recent study on a mannan binding module belonging to CBM32 revealed a greater contribution of the entropic penalty to interactions with ␤-1,4-mannno-oligosaccharides (ϪT⌬S r°ϭ about 5.0 kcal/mol) (38). This situation resulted in less favorable free energy changes in the interactions (⌬G r°ϭ Ϫ5.5 kcal/mol) than those obtained for (GlcN) n (n Ն 2) interactions with DD1 (⌬G r°ϭ Ϫ7.0 ϳ Ϫ8.0 kcal/mol).…”

Structure and functional investigation of ligand binding by a family 35 carbohydrate binding module (CtCBM35) of β-mannanase of family 26 glycoside hydrolase from Clostridium thermocellum

“…Mannotriose accesses the binding sites on CtCBM35 more easily than a galactose substituted branched polysaccharide like locust bean galactomannan. This was similarly reported earlier by Mizutani et al [25]. However, no significant binding of CtCBM35 was observed with carboxymethyl cellulose, rye arabinoxylan, birchwood xylan, oat spelt xylan, and glucuronoxylan, showing that CtCBM35 is mannan specific CBM ( Table 1).…”

“…This suggested that CtCBM35 bound less significantly with insoluble ivory nut mannan. The low affinity against insoluble mannan has been reported for the inability of "trans" form (as a separate fold and not appended to a hydrolytic enzyme) of CBM to disrupt the interchain interactions of the poly saccharide [25]. This is in contrast to some CBMs that have the ability to disintegrate the surface of crystalline insoluble polysaccharides displaying higher binding to soluble fractions of insoluble polysaccharides [27,28].…”

Structural and Biochemical Basis for Mannan Utilization by Caldanaerobius polysaccharolyticus Strain ATCC BAA-17

“…Unlike typical oligosaccharide-specific solute-binding proteins, CpMnBP1 engages mannan sugars through the nonreducing end. Although unexpected, such a binding mode is not without precedent as the family 32 carbohydrate-binding module from Clostridium thermocellum has been demonstrated to similarly engage manno-oligosaccharides through the non-reducing end (37). Likewise, structural studies of the family 6 carbohydrate-binding module reveal that ␤-1,3-glucan chains are targeted through the non-reducing end of the saccharide (38).…”