Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2.
A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C.
Faculty of Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Sun-Young
SY
An
Min
M
Ok
Ji-Youn
JY
Kim
Moon-Sun
MS
Jang
Young-Su
YS
Cho
Yong-Lark
YL
Choi
Cheorl-Ho
CH
Kim
Young-Choon
YC
Lee
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001671:Biochemistry / Q000379:methods; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D057057:Serine Proteases / Q000096:biosynthesis* / Q000235:genetics*; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; D057057:Serine Proteases