为了研究使用生物酶法制备共轭亚油酸的可行性,笔者对痤疮丙酸杆菌亚油酸异构酶进行了异源表达和理化性质研究。根据大肠杆菌偏好密码子,优化了来源于痤疮丙酸杆菌亚油酸异构酶(PAI)的基因(pai),将克隆的基因在BL21(DE3)中进行表达,纯化目的蛋白,获得重组PAI蛋白。SDS-PAGE结果显示,重组PAI分子质量为48 ku,重组蛋白主要以包涵体沉淀的形式存在。经测定重组PAI在35 ℃、pH 7.0时酶活最高,比酶活为752.3 μmol/(min·mg)。经35 ℃保温2 h,酶活保持90%以上,pH 为6.5～7.0时,PAI具有较好的稳定性,Mn2+和Zn2+对酶活力分别有22.4%和15.8%的抑制作用,以亚油酸为底物时该酶的Km值为1.13 mmol/L, kcat为4.67 s-1。相关分析表明,痤疮丙酸杆菌亚油酸异构酶性质优良,适合以亚油酸为底物生物催化制备共轭亚油酸。
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 752.3 μmol/(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).
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基金
收稿日期:2014-03-17 修回日期:2014-08-28
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(PAPD)
第一作者:李迅,副教授,博士。*通信作者:王飞,教授。E-mail: hgwf@njfu.edu.cn。
引文格式: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,2014,38(6):105-109.
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