利用a凝集素在酿酒酵母表面展示解脂耶氏酵母脂肪酶Lip2

Expression, purification and crystallization of a truncated Saccharomyces cerevisiae diadenosion 5′,5′′′-P1,P4 -tetraphosphate phosphorylaseⅠ

摘要:

摘要：【目的】将解脂耶氏酵母胞外脂肪酶Lip2展示在酿酒酵母表面，构建全细胞催化剂。【方法】采用PCR方法扩增得到解脂耶氏酵母胞外脂肪酶Lip2成熟肽编码基因LIP2，将其连接到AGA2基因的下游构建表面展示载体pCTLIP2。分别以橄榄油、三丁酸甘油酯和对硝基苯酚棕榈酸酯（pNPP）为底物检测展示的脂肪酶酶活。在此基础上，对野生菌及工程菌的酶学性质进行比较。【结果】展示Lip2的酿酒酵母重组菌株在半乳糖的诱导下，表现出水解橄榄油、三丁酸甘油脂以及pNPP的活性，20℃诱导72h时酶活达到最高，为182 U/g干细胞。对展示的Lip2的酶学性质研究表明，其最适温度为40℃，最适pH为8.0，温度稳定性比自由酶有所提高，50℃温浴4 h后残余酶活为其最大酶活的23.2%。以不同碳链长度的对硝基苯酚酯为底物检测其底物特异性，结果显示其水解C8,C12,C16对硝基苯酚酯活性相近，均远高于对硝基苯酚丁酸酯（C4）的水解酶活。【结论】对于Lip2，a凝集素系统是一个有效的展示系统，利用该系统成功将Lip2展示在酿酒酵母表面，从而构建了酿酒酵母全细胞催化剂，该全细胞催化剂具有良好的潜在应用前景。

Abstract:

Abstract: [Objective] In order to display extracellular lipase Lip2 from Yarrowia lipolytica on the surface of yeast Saccharomyces cerevisiae for whole cell catalysts. [Methods] The mature Lip2 encoding fragment was amplified from Yarrowia lipolytica total DNA, and was inserted into the 3′terminal of AGA2 to give the plasmid pCTLIP2 for surface display of Lip2. Olive oil, tributyrin and p-nitrophenyl palmitate (pNPP) were used as substrates to measure lipase activity. Moreover, the characterization of displayed lipase and its free form was analyzed. [Results] The surface displayed lipase was confirmed to be active towards olive oil, tributyrin and p-nitrophenyl palmitate (pNPP), and reached its highest expression level at 182 U/g dry cell after induced by galactose for 72h. The optimum temperature of cell surface displayed Lip2 was 40℃ After incubated at 50℃ for 4h, the surface displayed lipase retained 23.2% of its full activity, improved a little compared to free Lip2. The surface displayed lipase showed a preference to medium-chain and long-chain fatty acids p-nitrophenyl esters (C8-C16). [Conclusion] The cell surface display system based on a-agglutinin is an effective system for displaying Lip2, and the whole cell EBY100-pCTLIP2 will be probably suited to a different range of applications.