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J. Biochem, 1988, Vol. 103, No. 6 1060-1065
© 1988 Japanese Biochemical Society

research-article

Cloning and Expression of Subtilisin Amylosacchariticus Gene1

Tadashi Yoshimoto, Hiroshi Oyama, Takashi Honda, Hiroshi Tone, Tomoaki Takeshita, Tomomitsu Kamiyama and Daisuke Tsuru

School of Pharmaceutical Sciences, Nagasaki University Nagasaki, Nagasaki 852

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosac-chariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.

1This study was supported in part by Research Funds from Nagasaki Super Technology Development Association and Applied Enzymology.


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