南京林业大学学报（自然科学版）Issue(6)：105-109,5.DOI:10.3969/j.issn.1000-2006.2014.06.020
痤疮丙酸杆菌亚油酸异构酶的克隆表达及酶学性质研究
Cloning, expression and characterization of a linoleic acid isomerase from Propionibacterium acnes
摘要
Abstract
In order to study on the feasibility for the enzymatic synthesis of conjugated linoleic acid ( CLA) , the linoleic acid ( LA) isomerase from Propionibacterium acnes was cloned and overexpressed in Escherichia coli, and characterized. The LA isomerase gene ( pai) from P. acnes was improved by using codon optimization as E. coli codon usage. The DNA sequence encoding modified LA isomerase was cloned into pET-20b, yielding pET-20b-pai, and transformed into E. coli BL21( DE3) . The recombinant LA isomerase had a molecular mass of 48 ku showing mainly in inclusion body on SDS-PAGE. The gene product was purified by Ni-NTA and the activity was 752.3μmol/( min·mg) after induction and purification. At pH 7�0 and 35 ℃, the activity of enzyme reached the maximum. The purified enzyme was stable from pH 6.5 to pH7�5, and retained approx. 90% of its activity after 2 h at 35 ℃. A low⁃level inhibition of LA isomerase (22.4%) and (15�8%) was observed with Mn2+ and Zn2+(1 mmol/L), respectively. The recombinant LA isomerase had a Km of 1.13 mmol/L and kcat of 4.67 s-1 for linoleic acid. The result showed that the P. acnes linoleate isomerase can effectively catalyze LA to trans-10, cis-12 conjugated linoleic acid ( t10, c12 CLA) .关键词
痤疮丙酸杆菌/亚油酸异构酶/共轭亚油酸/酶学性质Key words
Propionibacterium acnes/conjugated linoleic acid isomerase/conjugated linoleic acid/enzymatic properties分类
生物科学引用本文复制引用
李迅,顾华祥,王飞..痤疮丙酸杆菌亚油酸异构酶的克隆表达及酶学性质研究[J].南京林业大学学报（自然科学版）,2014,(6):105-109,5.基金项目
国家林业公益性行业科研重大项目（201004001）；国家自然科学基金面上项目（31270612，31170537）；江苏省高校自然科学研究重大项目（11KJA480001）；江苏高校优势学科建设工程资助项目 （）
