Abstract
The gene for an alkaline serine protease from alkalophilic Bacillus sp. NKS-21 (subtilisin ALP I) was cloned, and its nucleotide sequence was determined. The gene (aprQ) contained an open reading frame of 1125 bp, encoding a primary product of 374 amino acids. The mature protease, composed of 272 amino acids, was preceded by a putative signal sequence of 37 amino acids and a pro-sequence of 65 amino acids. The mature protease conserved the catalytic triad, Asp, His, and Ser, as subtilisin BPN′ or other subtilisins, and the subtilisin ALP I might belong to the subtilisin super family. The primary structure of subtilisin ALP I was compared and discussed with those of 13 subtilisins, 5 subtilisins from alkalophilic Bacillus, and 8 from neutrophiles. Low homology was shown between subtilisin ALP I and subtilisins from alkalophiles or subtilisins from neutrophiles. Forty-five amino acid residues of the mature protein of subtilisin ALP I were entirely independent of other subtilisins. According to the homology of ALP I with other subtilisins, subtilisin ALP I might be in the middle point between alkaline subtilisins and neutral ones.
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Literature Cited
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current protocols in molecular biology, vol 1. New York, Chichester, Brisbane, Toronto, Singapore: Greene Associates and Wiley-Interscience
Betzel C, Klupsch S, Papendorf G, Hastrup S, Branner S, Wilson KS (1992) Crystal structure of the alkaline proteinase Savinase™ from Bacillus lentus at 1.4 Å resolution. J Mol Biol 223:427–445
Heijne GV (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690
Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ (1981) A gas-liquid solid phase peptide and protein sequenator. J Biol Chem 256:7990–7997
Jacobs M, Eliasson M, Uhlen M, Flock J-I (1985) Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res 13:8913–8926
Jang JS, Kang DO, Chun MJ, Byun SM (1992) Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis. Biochem Biophys Res Commun 184:277–282
Kaneko R, Koyama N, Tsai Y-C, Juang R-Y, Yoda K, Yamazaki M (1989) Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by alkalophilic Bacillus strain. J Bacteriol 171:5232–5236
von der Laan JC, Gerritse G, Mulleners LJSM, von der Hoek RAC, Quax WJ (1991) Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene. Appl Environ Microbiol 57:901–909
Moran Jr CP, Lang N, LeGrice SFJ, Lee G, Stephens M, Sonenshein AL, Pero J, Losick R (1982) Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet 186:339–346
Nakamura T, Yamagata Y, Ichishima E (1992) Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci Biotechnol Biochem 56:1869–1871
Nedkov P, Obserthuer W, Braunitzer G (1985) Determination of the complete amino acid sequence of subtilisin DY and its comparison with the primary structure of the subtilisin BPN′, Carlsberg and amylosacchariticus. Biol Chem Hoppe-Seyler 366:421–430
Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629
Shinde U, Li Y, Chatterjee S, Inouye M (1993) Folding pathway mediated by an intramolecular chaperone. Proc Natl Acad Sci USA 90:6924–6928
Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16 S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346
Silen JL, Agard DA (1989) The α-lytic protease pro-region does not require a physical linkage to activate the proteae domain in vivo. Nature 341:462–464
Stahl ML, Ferrari E (1984) Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro derived deletion mutation. J Bacteriol 158:411–418
Svendsen I, Genov N, Idakieva K (1986) Complete amino acid sequence of alkaline mecentericopeptidase. FEBS Lett 196:228–232
Takami H, Kobayashi T, Kobayashi M, Yamamoto M, Nakamura S, Aono R, Horikoshi K (1992) Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp. 221. Biosci Biotechnol Biochem 56:1455–1460
Tsuchida O, Yamagata Y, Ishizuka T, Arai T, Yamada J, Takeuchi M, Ichishima E (1986) An alkaline proteinase of an alkalophilic Bacillus sp. Curr Microbiol 14:7–12
Vieira J, Messing J (1987) Production of single-stranded plasmid DNA. In: Wu R, Grossman L (ed) Methods Enzymol 153:3–11
Wells JA, Ferrari E, Henner DJ, Estell DA, Chen EY (1983) Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res 11:7911–7925
Yamagata Y, Ichishima E (1989) A new alkaline proteinase with pI 2.8 from alkalophilic Bacillus sp. Curr Microbiol 19:259–264
Yamagata Y, Isshiki K, Ichishima E (1993) Subtilisin Sendai from alkalophilic Bacillus sp.: Molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS. Enz Microbial Technol (in press)
Yamaguchi M, Hanzawa S, Hirano K, Yamagata Y, Ichishima E (1993) Specificity and molecular properties of penicillolysin, a metalloproteinase from Penicillium citrinum. Phytochemistry 33:1317–1321
Yang JT, Wu C-SC, Martinez HM (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol 130:208–269
Yoshimoto T, Oyama H, Honda T, Tone H, Takeshita T, Kamiyama T, Tsuru D (1988) Cloning and expression of subtilisin amylosacchariticus gene. J Biochem 103:1060–1065
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Yamagata, Y., Sato, T., Hanzawa, S. et al. The structure of subtilisin ALP I from alkalophilic Bacillus sp. NKS-21. Current Microbiology 30, 201–209 (1995). https://doi.org/10.1007/BF00293634
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DOI: https://doi.org/10.1007/BF00293634