【摘要】 【目的】本研究将推测的阿维链霉菌(Streptomyces avermitilis)脂肪酶基因lpsA2在大肠杆菌(Escherichia coli)中进行异源表达及系统的酶学性质分析。【方法】提取阿维链霉菌基因组,设计特异性引物,PCR扩增脂肪酶基因lpsA2,使其在大肠杆菌中异源表达,利用6个组氨酸标签纯化脂肪酶LpsA2,并进行酶学性质分析;对LpsA2进行序列比对和进化分析。【结果】氨基酸序列比对显示LpsA2具有脂肪酶典型的由Ser、His和Asp构成的活性部位,即Ser130-Asp221-His25,其中Ser位于保守的五肽结构(Gly128-His129-Ser130-Gln131-Gly132)中;分子系统学分析显示,LpsA2是脂肪酶第一家族亚家族成员(Subfamily I.7);实验测得纯化的重组脂肪酶LpsA2的最适反应pH为8.0,最适反应温度为50℃;最适底物为对硝基苯酚豆蔻酸酯;在10℃-50℃范围内该酶的激活自由能为6.3 kcal/mol;1 mmol/L Co2+、Hg2+、Zn2+可使酶活性提高至250%以上;15%的二甲基甲酰胺和二甲基亚砜使酶活分别提高至110.7%和138%;0.1%和1%的Span-20可使酶活性分别提高至352.7%和189.7%。【结论】本研究对推测的来源于S.avermitilis的脂肪酶基因lpsA2进行了异源表达和酶学功能鉴定,不仅为脂肪酶的研究积累了更多数据,也为具有优良性能的脂肪酶生物工程菌的筛选奠定了基础,更为其在食品加工、药物合成等工业生产中的应用提供了依据。更多还原

【Abstract】 [Objective]We expressed the lipase gene lpsA2 from Streptomyces avermitilis in Escherichia coli and characterized the enzymatic properties of LpsA2.[ Methods ] We extracted the genomic DNA of S.avermitilis and amplified lpsA2 gene by PCR with specific primers.We then expressed lpsA2 gene in E.coli and determined the enzymatic properties of LpsA2.We also analyzed the evolutionary relationship between LpsA2 and lipase family by using phylogenetic tree based on the alignment of amino acid sequences.[ Results ] According to the alignment of amino acid sequences,we found that LpsA2 had the active site(Ser130-Asp221-His253) which was consistent with the typical characteristics of lipases that their active centers always consisted of Ser,His and Asp,and Ser always located in the conserved five peptide structure(Gly128-His129-Ser130-Gln131-Gly132).We constructed the evolutionary tree and found that LpsA2 belonged to Subfamily I.7 of lipase Family I.Furthermore,the optimal pH and temperature of LpsA2 were pH 8.0 and 50oC,respectively.The best substrate of LpsA2 was p-nitrophenol myristate.The activation energy(Ea) of LpsA2 between 10oC and 50oC was 6.3 kcal/mol.The enzyme activity of LpsA2 was increased to 250% or above by 1 mmol/L Co2+,Hg2+ and Zn2+,respectively.The activity was also elevated to 110.7% and 138.0% by 15% dimethylformamide and dimethyl sulfoxide,and 352.7% and 189.7% by 0.1% and 1% Span-20,respectively.[ Conclusion ] The enzymatic properties of LpsA2 from S.avermitilis were characterized that may provide the fundament to the screening of bio-engineered bacteria and the industrial applications in food processing and drug synthesis.更多还原