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 subtilis in Sendai) from the culture broth of alkalophilic Bacillus sp. G-825-6 , and its properties were characterized. Its optimum pH was at 10.0, w hen -leucyl-L-valyl-L-tyrosyl-4-methylcoumaryl-7-amide (Suc-Leu-Leu-Va l-Tyr-MCA) was used as a substrate. The substrate specificity of subti lisin Sendai was determined with oxidized insulin B-chain and fluoroge nic peptidyl-MCA substrates. The isoelectric point of subtilisin Senda i was over 11.0. The molecular mass of the enzyme was estimated as 28, 000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T he circular dichroism spectrum of the enzyme was measured, and we disc uss 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 ami no acid sequence for the mature protein (269 amino acids) was preceded by a putative signal sequence of 27 residues and a putative pro-seque nce of 86 amino acids. The homology of the primary structure for 13 su btilisins was compared. The catalytic triad (Asp32, His64, and Ser221 with the numbering of subtilisin BPN') and the amino acid sequences ne ar these amino acid residues were well conserved. As a special feature , it was observed that there was an extensive number of negatively cha rged amino acids in the pro-region of subtilisin Sendai and alkaline s ubtilisins. This was different from those of subtilisin from neutrophi les.