黑曲霉糖化酶基因的克隆及其在毕赤酵母X33中的表达

目的]以毕赤酵母（Pichia pastoris）X33为宿主菌高效表达黑曲霉（Aspergillus niger）糖化酶,为进一步扩大糖化酶在工业上的应用奠定基础。方法]利用RT-PCR技术,提取黑曲霉总RNA进行反转录,以得到的cDNA为模板,根据NCBI数据库中A.nigerCBS513.88糖化酶的cDNA序列（glaA）设计引物,通过PCR扩增得到去除天然信号肽的糖化酶成熟肽编码基因glaAm,并将其克隆到pUC19载体中,序列分析表明,glaAm的开放阅读框由1 879个核苷酸组成,编码625个氨基酸。以此片段构建了pFLDα-glaAm重组表达载体,经NsiI线性化后,电击转化毕赤酵母X-33。摇瓶培养中通过添加终浓度为0.5%的甲醇诱导糖化酶的分泌。结果]SDS-PAGE和Starch-PAGE显示,糖化酶得到正确的分泌表达,且具有较高的生物活性,发酵上清液中酶活最高达到380.78 U/ml（发酵液）。重组糖化酶的最适反应温度和最适pH分别为6065℃和4.0,在pH2.55.5范围内均保持90%以上的酶活力。该酶具有较高的热稳定性,pH4.0条件下,该酶在50℃下稳定;60℃处理60 min,仍能保持90%以上的酶活力;65℃下的半衰期约为44 min。结论]黑曲霉糖化酶基因在毕赤酵母X33中得到了异源高效表达。

Cloning of Glucoamylase (glaA) Gene from A.niger and Its Expression in P.pastoris X33

Objective] The aim was to lay the foundation to further expand the application of glucoamylase in the industry for using P.pastoris X-33 as the host strain to express the glucoamylase gene from A.niger.Methods] Amplified gene of glucoamylase from total RNA of A.niger mycelium by RT-PCR technology.A pair of primers were designed and synthesized according to the cDNA sequence of the glaA gene from A.niger CBS 513.88 in NCBI database(GenBank Accession No.XM_001390493).Sequence analysis revealed that glaAm ha...