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Applied and Environmental Microbiology, January 2003, p. 162-169, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.162-169.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Naoki Tsuruoka,1 Toru Nakayama,1 Masako Ashida,1 Hisashi Hemmi,1 Masahiro Nakao,2 Hiroyuki Minakata,3 Hiroshi Oyama,4 Kohei Oda,4 and Tokuzo Nishino1*

Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579,1 Suntory Research Center,2 Suntory Institute for Bioorganic Research (SUNBOR), Suntory Ltd., Mishima-gun, Osaka 618-8503,3 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan4

Received 27 June 2002/ Accepted 7 October 2002

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (kcat, 5.41 s-1; Km, 32 µM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (kcat, 351 s-1; Km, 214 µM), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.

* Corresponding author. Mailing address: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 07 Aoba-yama, Sendai 980-8579, Japan. Phone and fax: 81-22-217-7270. E-mail: nishino{at}mail.cc.tohuku.ac.jp.

Applied and Environmental Microbiology, January 2003, p. 162-169, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.162-169.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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