Cloning and expression in Escherichia coli of dextranase genes from Bacteroides thetaiotaomicron

Abstract

A genomic bank was constructed in Escherichia coli HB101, consisting of DNA fragments from Bacteroides thetaiotaomicron strain 489 inserted within the vector pBR322. By screening on complex medium containing blue dextran, 10 stable dextranase-positive (Dex+) clones were isolated. Seven groups of Dex+ inserts were identified on the basis of their restriction maps and hybridization responses. Dextanase activity of the recombinant clones was weak, and was revealed on the selection medium after 15 days. Subcloning of a Sau3AI partially digested 3.2-kb insert in the expression vector pDR720 greatly enhanced dextranse activity on blue dextran plates in one clone, but the delay remained unaltered. This suggested that the enzyme was released by cell lysis. Expression of this 0.7-kb subcloned insert was dependent on the promoter region of tryptophan operon carried by pDR720.

Molecular cloning and expression in Escherichia coli of genes encoding pectate lyase and pectin methylesterase activities from Bacteroides thetaiotaomicron

“…THETAIOTAOMICRON 593 (Whitehead and Hespell 1990), N-acetyl-neuraminidase pectins by B. thetaiotaomicron, and (ii) to compare some (Russo et al 1990), dextranase (Joncquiert et al 1991) biochemical properties of the cloned genes products with activities and gum guar hydrolases (Valentine et al 1992).…”