caa a22 4500 605502552 CHVBK 20210128100606.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6923-4 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6923-4 Cloning a neutral protease of Clostridium histolyticum , determining its substrate specificity, and designing a specific substrate [Elektronische Daten] [Hiroshi Maeda, Kanako Nakagawa, Kazutaka Murayama, Masafumi Goto, Kimiko Watanabe, Michio Takeuchi, Youhei Yamagata] 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. Springer-Verlag Berlin Heidelberg, 2015 Clostridium histolyticum nationallicence Neutral protease nationallicence Purification nationallicence Substrate specificity nationallicence Islet separation nationallicence Maeda Hiroshi New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut Nakagawa Kanako New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut Murayama Kazutaka Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, 980-8575, Sendai, Miyagi, Japan aut Goto Masafumi New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut Watanabe Kimiko New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut Takeuchi Michio Graduate school of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, 183-8509, Fuchu, Tokyo, Japan aut Yamagata Youhei New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/24(2015-12-01), 10489-10499 0175-7598 99:24<10489 2015 99 253 https://doi.org/10.1007/s00253-015-6923-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6923-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Maeda Hiroshi New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut NATIONALLICENCE 700 1- Nakagawa Kanako New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut NATIONALLICENCE 700 1- Murayama Kazutaka Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, 980-8575, Sendai, Miyagi, Japan aut NATIONALLICENCE 700 1- Goto Masafumi New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut NATIONALLICENCE 700 1- Watanabe Kimiko New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut NATIONALLICENCE 700 1- Takeuchi Michio Graduate school of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, 183-8509, Fuchu, Tokyo, Japan aut NATIONALLICENCE 700 1- Yamagata Youhei New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki Aza Aoba, Aoba-ku, 980-0872, Sendai, Miyagi, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/24(2015-12-01), 10489-10499 0175-7598 99:24<10489 2015 99 253