Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis.
The structural gene for a subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was determined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start site (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprised of 275 residues. The productivity of subtilisin in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence of the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69% identity with subtilisin Carlsberg, 89% with subtilisin BPN' and 70% with subtilisin DY. Some properties of the subtilisin J that had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. It retains about 50% of its activity even after treatment at 60 degrees C for 30 min in the presence of 2 mM calcium chloride.
Jang
J S
JS
Department of Life Science, Korea Advanced Institute of Science and Technology, Taejon.
Kang
D O
DO
Chun
M J
MJ
Byun
S M
SM
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D013381:Subtilisins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D009842:Oligopeptides; D011994:Recombinant Proteins; D013381:Subtilisins