Open AccessCloning and Expression of a Bacillus subtilis Endo-1,3-1,4- -D-Glucanase Gene in Escherichia coli K12
Author(s) -
Edward Hinchliffe
Publication year - 1984
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.019
H-Index - 179
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/00221287-130-5-1285
Subject(s) - bglii , periplasmic space , bacillus subtilis , ecori , biology , escherichia coli , glucanase , plasmid , molecular cloning , microbiology and biotechnology , bacillaceae , restriction enzyme , gene , hindiii , genetics , gene expression , bacteria
EcoRI fragments of DNA from Bacillus subtilis NCIB 8565, a high producer of an endo-1,3-1,4-beta-D-glucanase, were 'shot-gun' cloned in the plasmid vector pBR325. A 3.5 kb insert, carrying single restriction sites for AvaI, BglII, ClaI, PvuI and PvuII, was shown to direct the synthesis of beta-glucanase in Escherichia coli K12. Enzyme activity was demonstrated in extracellular fractions of E. coli harbouring the beta-glucanase gene; however, the largest proportion (greater than 50%) of total enzyme activity was periplasmic in location. beta-Glucanase activity and cellular location were independent of the orientation of the 3.5 kb fragment in pBR325.