Kai Huang, Tao Zhang, Bo Jiang, Wanmeng Mu, Ming Miao
{"title":"pycnus Rummeliibacillus SK31.001耐热精氨酸酶的研究","authors":"Kai Huang, Tao Zhang, Bo Jiang, Wanmeng Mu, Ming Miao","doi":"10.1016/j.molcatb.2016.11.020","DOIUrl":null,"url":null,"abstract":"<div><p>L-arginase from <em>Rummeliibacillus pycnus</em> SK31.001 is newly discovered. A 906<!--> <!-->bp complete open reading frame, which encodes a 301 amino acid protein, was identified using degenerate PCR and inverse PCR techniques. The arginase was found to have a conserved active site with 6 amino acid residues binding to 2 manganese ions: D123, H125, D228, D230, H100 and D127. Bioinformatics analysis revealed that <em>R. pycnus</em> arginase is a hexamer with a subunit molecular mass of 33<!--> <!-->kDa and whole molecular mass of 195<!--> <!-->kDa. <em>R. pycnus</em> arginase is thermostable with an optimal temperature of 80<!--> <!-->°C and maintains 85% of its initial activity after 24<!--> <!-->h of incubation at 40 or 50<!--> <!-->°C. An arginase activity assay showed that <em>R. pycnus</em> arginase has an optimum pH of 9.5 and a preference for Mn<sup>2+</sup>. Using arginine as the substrate, the Michaelis-Menten constant (<em>K</em><sub>m</sub>) and catalytic efficiency (<em>k</em><sub>cat</sub>/<em>K</em><sub>m</sub>) were measured to be 0.212<!--> <!-->mM and 2970<!--> <!-->mM<sup>−1</sup>s<sup>−1</sup>, respectively. The biosynthesis yield of L-ornithine by the purified enzyme was 144.4<!--> <!-->g/L, and the molar yield was 95.2%.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"133 ","pages":"Pages S68-S75"},"PeriodicalIF":0.0000,"publicationDate":"2016-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.11.020","citationCount":"15","resultStr":"{\"title\":\"Characterization of a thermostable arginase from Rummeliibacillus pycnus SK31.001\",\"authors\":\"Kai Huang, Tao Zhang, Bo Jiang, Wanmeng Mu, Ming Miao\",\"doi\":\"10.1016/j.molcatb.2016.11.020\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>L-arginase from <em>Rummeliibacillus pycnus</em> SK31.001 is newly discovered. A 906<!--> <!-->bp complete open reading frame, which encodes a 301 amino acid protein, was identified using degenerate PCR and inverse PCR techniques. The arginase was found to have a conserved active site with 6 amino acid residues binding to 2 manganese ions: D123, H125, D228, D230, H100 and D127. Bioinformatics analysis revealed that <em>R. pycnus</em> arginase is a hexamer with a subunit molecular mass of 33<!--> <!-->kDa and whole molecular mass of 195<!--> <!-->kDa. <em>R. pycnus</em> arginase is thermostable with an optimal temperature of 80<!--> <!-->°C and maintains 85% of its initial activity after 24<!--> <!-->h of incubation at 40 or 50<!--> <!-->°C. An arginase activity assay showed that <em>R. pycnus</em> arginase has an optimum pH of 9.5 and a preference for Mn<sup>2+</sup>. Using arginine as the substrate, the Michaelis-Menten constant (<em>K</em><sub>m</sub>) and catalytic efficiency (<em>k</em><sub>cat</sub>/<em>K</em><sub>m</sub>) were measured to be 0.212<!--> <!-->mM and 2970<!--> <!-->mM<sup>−1</sup>s<sup>−1</sup>, respectively. The biosynthesis yield of L-ornithine by the purified enzyme was 144.4<!--> <!-->g/L, and the molar yield was 95.2%.</p></div>\",\"PeriodicalId\":16416,\"journal\":{\"name\":\"Journal of Molecular Catalysis B-enzymatic\",\"volume\":\"133 \",\"pages\":\"Pages S68-S75\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2016-11-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.11.020\",\"citationCount\":\"15\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Molecular Catalysis B-enzymatic\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1381117716302326\",\"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/S1381117716302326","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Chemical Engineering","Score":null,"Total":0}
Characterization of a thermostable arginase from Rummeliibacillus pycnus SK31.001
L-arginase from Rummeliibacillus pycnus SK31.001 is newly discovered. A 906 bp complete open reading frame, which encodes a 301 amino acid protein, was identified using degenerate PCR and inverse PCR techniques. The arginase was found to have a conserved active site with 6 amino acid residues binding to 2 manganese ions: D123, H125, D228, D230, H100 and D127. Bioinformatics analysis revealed that R. pycnus arginase is a hexamer with a subunit molecular mass of 33 kDa and whole molecular mass of 195 kDa. R. pycnus arginase is thermostable with an optimal temperature of 80 °C and maintains 85% of its initial activity after 24 h of incubation at 40 or 50 °C. An arginase activity assay showed that R. pycnus arginase has an optimum pH of 9.5 and a preference for Mn2+. Using arginine as the substrate, the Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) were measured to be 0.212 mM and 2970 mM−1s−1, respectively. The biosynthesis yield of L-ornithine by the purified enzyme was 144.4 g/L, and the molar yield was 95.2%.
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.
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