地衣芽孢杆菌碱性果胶酶PelA和PelC的异源表达优化及其在不同表达宿主中的酶学性质

Optimization of heterologous expression of alkaline pectinases PelA and PelC from Bacillus licheniformis and their enzymatic properties in different expression hosts

为提高碱性果胶酶的酶活并挖掘其耐碱、耐热属性,选用Bacillus licheniformis X-01的碱性果胶酶基因pelA和pelC为研究对象,对质粒载体、表达元件和宿主进行优化,提高其异源表达的水平,并将相关基因在不同宿主内进行表达,分析碱性果胶酶在不同宿主中的酶学差异。将含有强启动子P43并共表达pelA和pelC的重组质粒pHY-P43-pelA-pelC转化至B.subtilis 168,得到的重组菌的碱性果胶酶酶活比仅表达pelA的重组菌的碱性果胶酶酶活提高了24.8%。碱性果胶酶编码基因pelA和pelC在不同宿主表达时,碱性果胶酶酶学性质有较大差异:以E.coli BL21为宿主,碱性果胶酶PelA的最适温度为75℃,Ca^(2+)对EcPelA酶活没有明显的促进效果;以B.subtilis 168为宿主时,碱性果胶酶BsPelA的最适温度为60℃,Ca^(2+)对BsPelA和BsPelC酶活均有明显促进作用,相对酶活分别达116%和141%。研究结果表明:碱性果胶酶BsPelC的最适反应温度为60℃、最适pH值为10;在温度为35~55℃时保温1 h,BsPelC的热稳定性良好,相对酶活均在90%以上;Mg 2+、Ca^(2+)对BsPelC有激活作用,其中Ca^(2+)激活效果最显著,相对酶活达到141%;Cu 2+、Zn 2+对BsPelC有明显的抑制作用,相对酶活分别下降到30%和47%。

In order to improve the enzymatic activity of alkaline pectinases and explore their alkali-resistant and heat-resistant properties,alkaline pectinase coding genes pelA and pelC of Bacillus licheniformis X-01 were selected.Plasmid vectors,expression elements and hosts were optimized to improve the level of heterologous expression of genes,and both genes were expressed in different hosts to investigate differences of enzymatic properties.The results show that when recombinant plasmid pHY-P43-pelA-pelC containing a strong promoter P43 and co-expressing pelA and pelC is transformed into B acillus.subtilis 168,alkaline pectinase activity of the recombinant bacterial is 24.8%higher than that of the recombinant bacterial only with pelA expression.When pelA and pelC were expressed in different hosts respectively,enzymatic properties of alkaline pectinases were quite different.Using E scherichia.coli BL21 as the host,the optimum temperature of alkaline pectinase EcPelA was 75℃and Ca^(2+)had no obvious promoting effect on the enzyme activity.Whereas using B.subtilis 168 as the host,the optimum temperature of alkaline pectinase BsPelA was 60℃and Ca^(2+)significantly promoted the enzymatic activities of BsPelA and BsPelC,reached 116%and 141%,respectively.The results show that:the optimum temperature and optimum pH of the alkaline pectinase BsPelC are 60℃and 10,respectively.When the temperature is kept at 35-55℃for 1 h,the relative enzyme activities are all above 90%,indicating that BsPelC has good thermal stability.Both Mg 2+and Ca^(2+)can activate BsPelC,and Ca^(2+)promoted the relative enzyme activity to reach 141%.Cu 2+and Zn 2+has obvious inhibitory effects on the relative enzyme activities,which decrease to 30%and 47%,respectively.