嗜热子囊菌原变种Thermoascus aurantiacus var.aurantiacus热稳定几丁质酶的克隆表达及活性研究

研究通过RT-PCR和Tail-PCR技术从嗜热子囊菌原变种Thermoascus aurantiacus var.aurantiacus中克隆了一个几丁质酶同源基因。该基因全长1,253bp,包含一个由1,197个碱基构成的开放阅读框,编码398个氨基酸。序列比对分析表明,该基因编码蛋白属于糖苷水解酶18家族的几丁质酶。利用基因重组的方法构建酵母分泌型表达载体,并转化毕赤酵母。在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,达到0.433g/L,酶活力为28.96U/mg,同时对表达的几丁质酶进行了纯化,SDS-PAGE检测该蛋白的分子量为43.9kDa。该几丁质酶的最适反应温度为60℃,最适反应pH值为8.0,70℃处理30min仍有45%的相对酶活,具有较好的热稳定性及工业应用价值。

Cloning, expression and characterization of a thermostable chitinase from Thermoascus aurantiacus var. aurantiacus

A chitinase gene was isolated from Thermoascus aurantiacus var. aurantiacus through RT-PCR and Tail-PCR methods. The full-length cDNA of 1,253bp contains an ORF of 1,197bp encoding 398 amino acids. Analysis of the deduced amino acid from nucleotide sequence revealed high homology with the catalytic domains of the glycoside hydrolase family 18. The chitinase gene was expressed in Pichia pastoris GS115. After induction for six days using methanol the transformed strain reached the highest production level and the expression amount was 0.433g protein per litter. The activity of expressed protein was 28.96U/mg. The molecular mass of a single band of the enzyme was estimated to be 43.9kDa by SDS-PAGE analysis. The optimum temperature of the enzyme was 60℃ and it maximal activity was at pH 8.0. The enzyme was thermostable at 50℃ and 60℃ and could still keep 45% activity at 70℃ after 30min, thus showing a potential for commercial uses.