Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (CtCBM11) of cellulosomal bifunctional cellulase from Clostridium thermocellum

Abstract

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyperexpressed in E. coli and functionally characterized. Affi nity fi electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affi nity electrophoresis and binding adsorption fi isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affi nity with polysaccharides. fi These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum

“…3) It has been reported that CjCBM3 is not able to bind oat-spelt xylan, whereas CtCBM11 and CsCBM6 do. [11][12][13][14] The affinity of CtCBM11 and CsCBM6 to oat-spelt xylan is 1.7 × 10 1 M −1 and 2.3 × 10 6 M −1 , respectively. [12][13][14] This suggests that the quite low affinity of CtCBM11, which should cause low binding of CtCel5E-CtCBM11 to oat-spelt xylan, confers a small increase in CtCel5E activity (Fig.…”

Advances in molecular engineering of carbohydrate-binding modules

“…The replacement of any of these residues by alanine resulted in decreased binding of carboxymethylcellulose (CMC), β‐glucan, glucomannan, and xylan. The highest effect was observed with the W65A mutant, whereas lesser impacts are observed with the Y129A and Y22A mutants …”

Structural and biochemical properties of lichenase from Clostridium thermocellum

“…Native polyacrylamide gels were prepared consisting of 7.5% (w/v) acrylamide in 25 mM Tris/250 mM glycine buffer [5]. The substrates (1.0%) were added to the polyacrylamide mixture before polymerization.…”

“…A bifunctional cellulase from Clostridium thermocellum was shown to contain an N-terminal family 26 glycoside hydrolase called lichenase (Lic26A) [2] and an internal family 5 glycoside hydrolase (GH5) [3] catalytic domains and a C-terminal family 11 carbohydrate-binding module (CBM11) [4,5]. It was demonstrated that the carbohydratebinding module (CBM11) of bifunctional cellulase binds to ligands that are substrates for both Lic26A and GH5 catalytic modules [4].…”