碱性果胶酯裂解酶的异源表达与性质研究

Heterologous Expression and Characteristics of Alkaline Polygalacturonate Lyase that Specifically Depolymerize Substrate with High Methyl Esterification Degree

目的：异源表达、纯化特异性降解高甲酯化果胶的碱性果胶酯裂解酶,研究其酶学性质。方法：采用PCR方法从枯草芽孢杆菌7-3-3中扩增到编码碱性果胶酯裂解酶的结构基因pelB,将去信号肽基因序列连接甲醇诱导型质粒pPIC9K,得到重组质粒p PIC9K-pelB,并转入毕赤酵母GS115进行表达,对该表达产物进行酶学性质分析。结果：纯化得到PelB及其糖基化蛋白,糖基化不影响PelB的酶活力。该酶可降解甲酯化程度为85%的果胶,比酶活达2 746 U/mg。在最适反应温度60℃,最适反应pH 9.0,45-65℃和pH 8.5-9.5范围具较高的酶活力。在40℃以下,pH 7.0-10.6的碱性范围,酶具有较高的稳定性。结论：PelB蛋白对高度甲酯化的果胶底物具有高解聚能力,在碱性条件下稳定,提示其具有良好的应用价值。

Objective： To heterologously express and purify alkaline polygalacturonate lyase that specifically depolymerize substrate with high methyl esterification degree and to investigate its enzymatic properties, and provide the experimental basis for its development and application. Methods： Alkaline polygalacturonate lyase structural gene, pelB,was amplified from Bacillus subtilis 7-3-3 genome by PCR and discovered that it has close genetic relationship with PelB from Bacillus subtilis BSn5 by constructing phylogenetic tree with its homology proteins. Signal peptide deleted pelB gene was cloned between the Sna BI and Not I sites of p PIC9 K, a methanol induced expressing plasmidand a recombinant plasmid, p PIC9K-pelB, was obtained. The recombinant plasmid was transformed to Pichia pastoris GS115 for expressing PelB and its glycosylation proteins. The enzymatic properties of PelB were determined. Results： PelB and its glycosylation proteins were purified from crude enzyme. Deglycosylation experiments showed that PGL activity of PelB was not affected by glycosylation. PelB has high depolymerization ability for pectin with 85% methyl esterification degree. The optimum temperature and pH of the PelB was 60 ℃ and 9.0, respectively. PelB had high activity at the range of 45 ℃ to 65 ℃and pH 8.5-9.5. It was stable at temperature below 40 ℃ and pH 7.0 to 10.6, respectively. Conclusions： PelB has high depolymerization ability for pectin with high methyl esterification degree, and it stable under alkaline conditions. It shows that good potential of PelB in industrial application.