Cloning of thermostable subtilisin-like protease gene from thermophilic bacillus species

Abstract

To clone a thermostable subtilisin-like protease gene, Chromosomal DNA was isolated from various thermophilic Bacillus species such as Bacillus coagulans NRRL B-5666, Bacillus licheniformis ATCC 27811, Bacillus stearothermophilus ATCC 12980 and Bacillvs Subtilis JCM 2889. The chromosomal DNA was digested with EcoR I and hybridized using aprA gene from Bacillus subtilis as a probe. The strong signal sized about 6.7kb appeared in B. coagulans. B. coagulans chromosomal DNA digested with EcoR I was electroeluted into 7 fractions and hybridized. F. II showed the positive signal and ligated with pUC9 and transformed into E. coli JM83. About 8000 transformants were screened to obtain recombianat with subtilisin-like protease gene by colony hybridization. Positive signal emerged in three transformants and these are rescreened by Southern hybridization.