黑曲霉脂肪酶Lip A的异源表达与酶学性质分析  

Heterologous expression and enzymatic characterization of Aspergillus niger lipase Lip A

作　　者：王雅琪 凡林林 李文瑶 李弘轩 杜丽平[1,2] 王洪 马立娟[1,2] WANG Yaqi;FAN Linlin;LI Wenyao;LI Hongxuan;DU Liping;WANG Hong;MA Lijuan(College of Biotechnology,Tianjin University of Science&Technology,Tianjin,300457,China;Key Laboratory of Industrial Fermentation Microbiology Ministry of Education,Tianjin,300457,China;Key Laboratory of Wuliangye-Flavor Liquor Solid-state Fermentation,China National Light Industrial,Yibin 644000,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]工业发酵微生物教育部重点实验室,天津300457 [3]中国轻工业浓香型白酒固态发酵重点实验室,四川宜宾644000

出　　处：《食品与发酵工业》2023年第5期25-31,共7页Food and Fermentation Industries

基　　金：中国轻工业浓香型白酒固态发酵重点实验室开放基金项目(2021zd012)。

摘　　要：脂肪酶在油脂分解和脂质合成方面具有广泛应用,是一种很有前途的生物催化剂。该研究对来源于黑曲霉的脂肪酶Lip A进行了异源表达及催化特性研究。结果表明,该重组脂肪酶的最适温度为50℃,最适pH为8.0,并且在40~60℃和pH 5.0~8.0有较好的稳定性。该酶对有机试剂有较好的耐受性,金属离子Ca^(2+)和Mg^(2+)对脂肪酶水解活性有促进作用,Fe^(3+)、Zn^(2+)和Cu^(2+)严重抑制该酶活性。反应动力学参数表明,对硝基苯酚乙酸酯是脂肪酶Lip A的最适底物,其反应Km为(0.228±0.05)mmol/L,催化常数Kcat为(0.023±0.001)s-1。生物信息学分析表明,脂肪酶Lip A是abH23同源家族Rhizomucor mihei脂肪酶中的一员。黑曲霉脂肪酶Lip A具有催化乳酸和乙醇生成乳酸乙酯特性,为其在酿造领域的应用奠定了基础。Lipase has been widely used in the decomposition and synthesis of lipid, which is a promising biocatalyst. In this study, a putative lipase(Lip A) from Aspergillus niger was heterologous expressed in E. coli Rosetta(DE3) with success. The optimal reaction temperature and pH of lipase Lip A was at 50℃and pH 8.0, which means this lipase was a thermo-alkaline lipase. Lipase Lip A showed good stability in a range of temperature of 40-60℃ and pH of 5.0-8.0, and it had good tolerance to organic reagent. Lipase Lip A activity was enhanced by Ca^(2+)and inhibited seriously by Fe^(3+)and Cu^(2+). The kinetic parameters ofshowed that pNP-C2 was its optimal substrate, and the Kmand Kcattowards pNP-C2 were(0.228±0.05) mmol/L and(0.023±0.001) s-1, respectively. Phylogenetic analysis showed that lipase Lip A was a member of the abH23 homologous family Rhizomucor mihei lipase. The lipase Lip A could catalyze the synthesis of the flavor ester ethyl lactate under aqueous phase, which showed a bright prospect for its application in the field of brewing.

关 键 词：黑曲霉 脂肪酶 酶学性质 酯化特性 

分 类 号：Q78[生物学—分子生物学] TS201.25[轻工技术与工程—食品科学]