A Gene Encoding an Exo-β-Glucosidase from Cellvibrio mixtus

Abstract.

Cellvibrio mixtus produces an array of endohydrolytic enzymes involved in the initial phases of β-glycan polysaccharide degradation in the soil. These enzymes convert complex, high-molecular-weight, insoluble polysaccharides into low-molecular-weight, soluble oligosaccharides which must be further degraded for cellular uptake and catabolism. Little is known about the enzymes involved in this latter process in C. mixtus. In this paper we report the cloning of the lam2 gene, which encodes an exohydrolase of low-molecular-weight, soluble β-glucans and which may be involved in the latter stages of β-glucan degradation in C. mixtus. The Lam2 enzyme has a broad substrate specificity including β-1,3-, β-1,4-, β-1,6-, and β-1,3-1,4-linked glucans. Analysis of the end-products of laminarin hydrolysis showed that the Lam2 enzyme is an exohydrolase that removes terminal glucose residues from its substrate. The substrate specificity and action pattern of this enzyme indicate that it belongs to EC3.2.1.21 (β-glucosidase). The 2.5-kb lam2 insert encodes a protein of approximately 91 kDa as determined by in vivo transcription/translation and deletion analyses. Southern analysis showed that a single copy of the gene exists on the C. mixtus chromosome.