A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis.
Incorporation of starch or casein into protoplast-regeneration medium facilitated shotgun cloning of alpha-amylase and neutral protease genes from an unidentified Bacillus sp. in Bacillus subtilis by polyethylene glycol-induced protoplast transformation. This modification and the use of the plasmid vector pPL603b enabled us to simultaneously select for promoter-bearing recombinant plasmids that expressed amylase or protease activity. The inserts were found to be 4 and 4.6 kb, respectively. Although protease activity directed by the cloned gene was only 2- to 4-fold higher than for the donor strain, that of alpha-amylase was 28-fold higher.
D001412:Bacillus subtilis / Q000235:genetics*; D002364:Caseins; D003001:Cloning, Molecular / Q000379:methods*; D005798:Genes, Bacterial; D010447:Peptide Hydrolases / Q000235:genetics*; D011523:Protoplasts / Q000201:enzymology; D013213:Starch; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000235:genetics*
D002364:Caseins; D013213:Starch; D000516:alpha-Amylases; D010447:Peptide Hydrolases