Heterologous Expression and Enzymatic Properties of Xylanase of GH11 Family from Trichoderma asperellum(棘孢木霉GH11家族木聚糖酶的异源表达及酶学性质)
摘要
A glycoside hydrolase (GH) 11 family xylanase gene (TaXyn11A) from Trichoderma asperellum was cloned and heterologously expressed in Escherichia coli. The gene had an open reading frame of 672 bp, encoding 223 amino acids. The deduced amino acid sequence shares the highest identity (85.2%) with the GH 11 family xynII (NCBI accession No. B5A7N4.1) from Trichoderma harzianum C4. The crude enzyme was purified by one-step to homogeneity by Ni-NTA with a molecular mass of 24.2 kDa. The recombinant xylanase (TaXyn11A) was biochemically characterized, and the results showed that TaXyn11A was an acidic and mesophilic enzyme, having optimal pH and temperature of pH 5.0 and 50℃, respectively. The stability of the enzyme was between the pH range of 4.0~10.0 and temperature lower than or equal to 45℃, and its half-life at 45℃ was 14.3h. The enzyme was activated by EDTA, Mn<sup>2+</sup> and Cr<sup>3+</sup>, while strongly inhibited by SDS and CTAB. Analyses for substrate specificities and hydrolysis properties indicated that TaXyn11A exhibited great sensitivity towards oat xylan, wheat arabinoxylan, beechwood xylan and birchwood xylan, with specific activities of 989.6,727.6,706.0 and 484.5U·mg-1, respectively, and the enzyme hydrolyzed verious xylans for 12h to mainly yield xylooligosaccharides with polymerization degree of 2~6. The favorable properties of TaXyn11A may make it a good candidate in xylooligosaccharides production in industry.(从棘孢木霉(Trichoderma asperellum)中克隆得到一个糖苷水解酶(glycoside hydrolase,GH)11家族木聚糖酶基因TaXyn11A,并在大肠杆菌中进行了异源表达。该基因含有一个672bp的开放阅读框,编码223个氨基酸,编码的蛋白与来自哈茨木霉C4(Trichoderma harzianum)的GH11家族木聚糖酶xynⅡ(NCBI accession No. B5A7N4.1)的同源性为85.2%。粗酶液经Ni-NTA亲和层析一步纯化得到电泳级纯酶(TaXyn11A),该酶分子质量为24.2kDa。研究了纯酶的酶学性质,结果表明:TaXyn11A为酸性中温木聚糖酶,最适pH值和最适温度分别是5.0和50℃,在pH值为4.0~10.0时和45℃及以下保持稳定,45℃时半衰期为14.3h。EDTA、Mn<sup>2+</sup>和Cr<sup>3+</sup>对该酶有激活作用,而SDS和CTAB对该酶有抑制作用。底物特异性及水解特性分析表明:TaXyn11A可特异性水解燕麦木聚糖、小麦阿拉伯木聚糖、榉木木聚糖和桦木木聚糖等木聚糖底物,比酶活力分别为989.6、727.6、706.0、484.5U·mg-1,水解12h后,产物以聚合度为2~6