TY - JOUR
T1 - Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate
AU - Maeda, Hiroshi
AU - Nakagawa, Kanako
AU - Murayama, Kazutaka
AU - Goto, Masafumi
AU - Watanabe, Kimiko
AU - Takeuchi, Michio
AU - Yamagata, Youhei
N1 - Funding Information:
This study was partly supported by the grant of “Coordination, Support and Training Program for Translational Research” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors of this publication received a partial financial support by Meiji Seika Pharma Co., Ltd. according to a collaborative research contract.
Publisher Copyright:
© 2015, Springer-Verlag Berlin Heidelberg.
PY - 2015/12/1
Y1 - 2015/12/1
N2 - 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.
AB - 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.
KW - Clostridium histolyticum
KW - Islet separation
KW - Neutral protease
KW - Purification
KW - Substrate specificity
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U2 - 10.1007/s00253-015-6923-4
DO - 10.1007/s00253-015-6923-4
M3 - Article
C2 - 26307443
AN - SCOPUS:84948386069
VL - 99
SP - 10489
EP - 10499
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 24
ER -