Cloning and expression of a Bacteroides succinogenes mixed-linkage beta-glucanase (13-14-beta-D-glucan 4-glucanohydrolase) gene in Escherichia coli.

摘要

A pseudorandom genomic library of Bacteroides succinogenes DNA, cloned into pUC8 in Escherichia coli, was screened for beta-glucanase activity on 0.1% lichenan plates. Six high-activity clones, containing identical 5.2-kilobase inserts of B. succinogenes DNA, were obtained. The clones exhibited activity solely on beta-glucan substrates containing beta-(1----3)(1----4) linkages, thus manifesting a specific fibrolytic enzyme previously unrecognized in B. succinogenes. A subclone (pJI10) of the original insert (1.35 kilobases in size) expressed full beta-glucanase activity under control of its own promoter. The expression of beta-glucanase in pJI10 appeared subject to catabolite regulation by glucose. Detailed analysis of enzyme activity in the parental and deleted derivatives, subcloned into pUC18 and pUC19, suggested that the apparent glucose repression was an artifact arising as a consequence of interactions with the lac transcriptional unit in the plasmid vector.