title: Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate.
abstract: Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.
authors: New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, Sendai, Miyagi, 980-0872, Japan.
authors: Hiroshi
authors: H
authors: Maeda
authors: New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, Sendai, Miyagi, 980-0872, Japan.|School of Bioscience and Biotechnology, Tokyo University of Technology, 1401-1 Katakura-machi, Hachioji, Tokyo, 192-0982, Japan.
authors: Kanako
authors: K
authors: Nakagawa
authors: Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
authors: Kazutaka
authors: K
authors: Murayama
authors: New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, Sendai, Miyagi, 980-0872, Japan.|Division of Advanced Surgical Science and Technology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Miyagi, 980-0872, Japan.
authors: Masafumi
authors: M
authors: Goto
authors: New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, Sendai, Miyagi, 980-0872, Japan.
authors: Kimiko
authors: K
authors: Watanabe
authors: Graduate school of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan.
authors: Michio
authors: M
authors: Takeuchi
authors: New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, Sendai, Miyagi, 980-0872, Japan. y-yama@cc.tuat.ac.jp.|Graduate school of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan. y-yama@cc.tuat.ac.jp.
authors: Youhei
authors: Y
authors: Yamagata
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D047009:Hathewaya histolytica / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D010447:Peptide Hydrolases
keywords: Clostridium histolyticum; Islet separation; Neutral protease; Purification; Substrate specificity