Xiao lan Liu, Narasimha kumar Kopparapu, Hong chen Zheng, Priti Katrolia, Yong ping Deng, Xi qun Zheng
{"title":"食用级真菌嗜sitophila神经孢子菌纤维蛋白溶解酶的纯化及特性研究","authors":"Xiao lan Liu, Narasimha kumar Kopparapu, Hong chen Zheng, Priti Katrolia, Yong ping Deng, Xi qun Zheng","doi":"10.1016/j.molcatb.2016.10.006","DOIUrl":null,"url":null,"abstract":"<div><p>A fibrinolytic protease was purified from the culture supernatant of a GRAS fungus, <em>Neurospora sitophila</em>. The enzyme displayed a molecular mass of 34<!--> <!-->kDa, as estimated by SDS-PAGE and gel filtration chromatography. The isoelectric point (pI) of the enzyme was 9.3<!--> <!-->±<!--> <!-->0.2 as determined by iso-electric focusing (IEF). It was maximally active at pH 7.6 and 41<!--> <!-->°C and displayed remarkable stability in a wide pH range (4–11) and up to 52<!--> <!-->°C. The enzyme activity was inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), indicating that it is a metal-dependent serine protease. It was found to be a direct acting plasmin like protein which efficiently cleaved the α-chain of fibrin(ogen), followed by β-chain and γ-chain. Three internal peptide sequences LASTANSGVLSGLLAGTVGGK; AYTSKSSVPSSVGLAR; LLDTGLNTAHSDFNR were determined by Q-TOF2. These results indicate no sequence similarities with other fibrinolytic enzymes suggesting it to be a novel enzyme. This fibrinolytic enzyme may be developed as a safe potential candidate for oral administration as a functional food additive or as a drug for prevention and/or treatment of thrombolytic diseases.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 98-104"},"PeriodicalIF":0.0000,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.006","citationCount":"16","resultStr":"{\"title\":\"Purification and characterization of a fibrinolytic enzyme from the food-grade fungus, Neurospora sitophila\",\"authors\":\"Xiao lan Liu, Narasimha kumar Kopparapu, Hong chen Zheng, Priti Katrolia, Yong ping Deng, Xi qun Zheng\",\"doi\":\"10.1016/j.molcatb.2016.10.006\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>A fibrinolytic protease was purified from the culture supernatant of a GRAS fungus, <em>Neurospora sitophila</em>. The enzyme displayed a molecular mass of 34<!--> <!-->kDa, as estimated by SDS-PAGE and gel filtration chromatography. The isoelectric point (pI) of the enzyme was 9.3<!--> <!-->±<!--> <!-->0.2 as determined by iso-electric focusing (IEF). It was maximally active at pH 7.6 and 41<!--> <!-->°C and displayed remarkable stability in a wide pH range (4–11) and up to 52<!--> <!-->°C. The enzyme activity was inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), indicating that it is a metal-dependent serine protease. It was found to be a direct acting plasmin like protein which efficiently cleaved the α-chain of fibrin(ogen), followed by β-chain and γ-chain. Three internal peptide sequences LASTANSGVLSGLLAGTVGGK; AYTSKSSVPSSVGLAR; LLDTGLNTAHSDFNR were determined by Q-TOF2. These results indicate no sequence similarities with other fibrinolytic enzymes suggesting it to be a novel enzyme. This fibrinolytic enzyme may be developed as a safe potential candidate for oral administration as a functional food additive or as a drug for prevention and/or treatment of thrombolytic diseases.</p></div>\",\"PeriodicalId\":16416,\"journal\":{\"name\":\"Journal of Molecular Catalysis B-enzymatic\",\"volume\":\"134 \",\"pages\":\"Pages 98-104\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2016-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.006\",\"citationCount\":\"16\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Molecular Catalysis B-enzymatic\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1381117716301990\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"Chemical Engineering\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Molecular Catalysis B-enzymatic","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1381117716301990","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Chemical Engineering","Score":null,"Total":0}
Purification and characterization of a fibrinolytic enzyme from the food-grade fungus, Neurospora sitophila
A fibrinolytic protease was purified from the culture supernatant of a GRAS fungus, Neurospora sitophila. The enzyme displayed a molecular mass of 34 kDa, as estimated by SDS-PAGE and gel filtration chromatography. The isoelectric point (pI) of the enzyme was 9.3 ± 0.2 as determined by iso-electric focusing (IEF). It was maximally active at pH 7.6 and 41 °C and displayed remarkable stability in a wide pH range (4–11) and up to 52 °C. The enzyme activity was inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), indicating that it is a metal-dependent serine protease. It was found to be a direct acting plasmin like protein which efficiently cleaved the α-chain of fibrin(ogen), followed by β-chain and γ-chain. Three internal peptide sequences LASTANSGVLSGLLAGTVGGK; AYTSKSSVPSSVGLAR; LLDTGLNTAHSDFNR were determined by Q-TOF2. These results indicate no sequence similarities with other fibrinolytic enzymes suggesting it to be a novel enzyme. This fibrinolytic enzyme may be developed as a safe potential candidate for oral administration as a functional food additive or as a drug for prevention and/or treatment of thrombolytic diseases.
期刊介绍:
Journal of Molecular Catalysis B: Enzymatic is an international forum for researchers and product developers in the applications of whole-cell and cell-free enzymes as catalysts in organic synthesis. Emphasis is on mechanistic and synthetic aspects of the biocatalytic transformation.
Papers should report novel and significant advances in one or more of the following topics;
Applied and fundamental studies of enzymes used for biocatalysis;
Industrial applications of enzymatic processes, e.g. in fine chemical synthesis;
Chemo-, regio- and enantioselective transformations;
Screening for biocatalysts;
Integration of biocatalytic and chemical steps in organic syntheses;
Novel biocatalysts, e.g. enzymes from extremophiles and catalytic antibodies;
Enzyme immobilization and stabilization, particularly in non-conventional media;
Bioprocess engineering aspects, e.g. membrane bioreactors;
Improvement of catalytic performance of enzymes, e.g. by protein engineering or chemical modification;
Structural studies, including computer simulation, relating to substrate specificity and reaction selectivity;
Biomimetic studies related to enzymatic transformations.