乙酰木聚糖酯酶基因的克隆、表达及酶学性质研究

Gene Cloning, Expression and Enzymatic Characterization of Acetyl Xylan Esterase

乙酰木聚糖酯酶可以水解乙酰化木聚糖的O-乙酰基,消除木聚糖酶水解木聚糖时该基团产生的空间阻碍作用。以宇佐美曲霉（Aspergillus usamii）E001菌株的总RNA为模板,利用RT-PCR技术扩增了乙酰木聚糖酯酶成熟肽的编码基因（Auaxe）,并构建了重组表达质粒p PIC9K^M-Auaxe,SalⅠ线性化后电转入毕赤酵母（Pichia pastoris）GS115中,经G418抗性筛选及甲醇诱导表达72 h,获得了重组乙酰木聚糖酯酶（reAuAxe）,其酶活性为35.6 U/m L。发酵液经纯化获得电泳纯reAuAxe,其表观相对分子质量约为3.4×10^4,比酶活性为390.5 U/mg。纯化后reAuAxe的最适反应温度为50℃,在45℃及以下稳定;其最适反应pH为6.0,在pH 4.5-7.0范围内稳定;所测的多数金属离子及EDTA对其酶活性影响不大。

Acetyl xylan esterase（Axe） can hydrolyze O-acetyl located in acetylated xylan. Thus,the steric hindrance effects of O-acetyl can be eliminated when xylan is hydroyzed by xylanase. In this study,a mature peptide gene,Auaxe,encoding an acetyl xylan esterase（AuAXE）,was amplified by RT-PCR technique using the total RNA from Aspergillus usamii E001 as template. The recombinant expressing plasmid p PIC9K^M-Auaxe was constructed and linearized with Sal Ⅰ,followed by transforming it into Pichia pastoris GS115 by electroporation. The recombinant P.pastoris was screened by G418,induced by methanol for 72 h,and then the recombinant acetyl xylan esterase（reAuAxe） was obtained. The activity of crude re Au Axe reached 35.6 U/m L. After purification,the re Au Axe was purified to electrophoretic homogeneity with the activity of 390.5 U/mg. SDS-PAGE analysis showed that the apparent molecular mass of purified re Au Axe was about34.0 k Da. Additionally,the enzymatic properties of purified re Au Axe were also analyzed. Its optimal temperature and p H were 50 ℃ and 6.0,respectively. It was stable at a temperature of 45 ℃ or below,and at a p H range of 4.5-7.0. Its enzymatic acvitity was not signficantly affected by an array of tested metal ions and EDTA. This paper laid a solid theoretical foundation for this enzyme＇s study in depth and application.