Subtilisin Sendai from alkalophilic Bacillus sp.: molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS.
We purified a new extracellular serine proteinase (designated subtilisin Sendai) from the culture broth of alkalophilic Bacillus sp. G-825-6, and its properties were characterized. Its optimum pH was at 10.0, when succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-4-methylcoumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) was used as a substrate. The substrate specificity of subtilisin Sendai was determined with oxidized insulin B-chain and fluorogenic peptidyl-MCA substrates. The isoelectric point of subtilisin Sendai was over 11.0. The molecular mass of the enzyme was estimated as 28,000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The circular dichroism spectrum of the enzyme was measured, and we discuss the relationship between the secondary structure of the enzyme and alkaline stability at pH 12 in comparison with that of subtilisin NAT. The structural gene (aprS) was cloned and sequenced. The deduced amino acid sequence for the mature protein (269 amino acids) was preceded by a putative signal sequence of 27 residues and a putative pro-sequence of 86 amino acids. The homology of the primary structure for 13 subtilisins was compared. The catalytic triad (Asp32, His64, and Ser221 with the numbering of subtilisin BPN') and the amino acid sequences near these amino acid residues were well conserved. As a special feature, it was observed that there was an extensive number of negatively charged amino acids in the pro-region of subtilisin Sendai and alkaline subtilisins. This was different from those of subtilisin from neutrophiles.
Yamagata
Y
Y
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Isshiki
K
K
Ichishima
E
E
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D007328:Insulin; D011994:Recombinant Proteins; C094600:AprS protein, Bacillus; D013381:Subtilisins