Source:Gene, Volume 642
Author(s): Anthony K.N. Chan, Alan K.L. Ng, Kate K.Y. Ng, W.K.R. Wong
Enzymatic degradation of cellulosic waste to generate renewable biofuels has offered an attractive solution to the energy problem. Synergistic hydrolysis of cellulose residues requires the participation of three different types of cellulases – endoglucanases, exoglucanases, and β-glucosidases (Bgl). Our group has been interested in using Bgl of Cellulomonas biazotea in studies designed to investigate cooperative action among different cellulases. We previously have cloned bgl genes encoding Cba and Cba3, which are C. biazotea Bgl isozymes representing two different Bgl families, respectively; specifically, Glycoside Hydrolase Family 3 (GH3) and Glycoside Hydrolase Family 1 (GH1). To gain an understanding of the complexity of Bgl in C. biazotea, we analyzed E. coli clones containing plasmids into which C. biazotea DNA had been inserted; these clones could hydrolyze 4-methylumbelliferyl β-d-glucopyranoside (MUG) supplemented in solid agar media, suggesting they might contain bgl genes. Through restriction analysis and DNA sequencing, two novel bgl genes, designated cba4 and cba5 and encoding Cba4 (484 amino acids) and Cba5 (758 amino acids) were identified. Cba4 and Cba5 appear to be members of GH1 and GH3, respectively. Both Cba4 and Cba5 were concluded to be genuine cellobiases as each was found to enable their E. coli hosts to survive on media in which cellobiose was the sole carbon source. Despite lacking a typical secretory signal sequence, Cba4 and Cba5 are secretory proteins. Although they are isoenzymes, Cba, Cba3, Cba4, and Cba5 were shown to possess distinct substrate specificities. These four Bgl members may play important roles in hydrolyzing a wide variety of β-glucosides including cellobiose and non-cellulosic substrates.
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