Enzymatic and molecular characterization of an endoglucanase E from Clostridium cellulovorans 743B

Abstract

Endoglucanase E (EngE) is a cellulosomal enzyme of the glycoside hydrolase family 5 generated by the cellulosome-producing bacterium Clostridium cellulovorans 743B. Although its basic activities and properties have been characterized, its substrate specificity, product range, and steady-state kinetics remain unclear. The current study prepared recombinant EngE (rEngE) and analyzed its substrate specificity and product range using thin layer chromatography. When carboxymethyl cellulose (CMC) or phosphoric acid swollen cellulose was used as a substrate, disaccharides and trisaccharides were the main products. However, no product was detected with microcrystalline cellulose as the substrate. This indicated that rEngE is a cellulase that hydrolyzes low-crystallinity cellulose. Furthermore, products were detected when glucomannan, lichenan, or β-glucan was used, but no product was obtained with xylan. These results suggested that rEngE hydrolyzes the β-1,4 glycosidic bond between glucose residues of the substrate. In the kinetic analysis, at CMC concentrations of ≥3mg/mL, the reaction rate decreased. Application of the above data to three substrate inhibition models generated a better fit to a model that generates products not only from the enzyme-substrate complex but also from enzyme-substrate-substrate (ESS) complexes, in which two substrates are bound to the enzymes. In addition, it was found that a carbohydrate-binding module (CBM) contained in EngE binds to cellulose. Therefore, substrate inhibition likely occurred because the binding site of CBM may correspond to one of the substrate-binding sites in the ESS complex.