黑曲霉糖化酶基因的克隆及其在毕赤酵母X33中的表达  

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

作　　者：曹慕琛[1,2] 徐健勇 罗立超[3] 张同存[1] 孙劭靖[1,2] 宋诙 

机构地区：[1]天津科技大学生物工程学院,教育部工业微生物重点实验室,天津市工业微生物重点实验室,天津300457 [2]天津工业生物技术研究所,基因工程与微生物应用技术研究组,天津300308 [3]中国农业大学生物学院,天津300308

出　　处：《安徽农业科学》2011年第14期8226-8230,8306,共6页Journal of Anhui Agricultural Sciences

摘　　要：[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。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.XM001390493）.Sequence analysis revealed that glaAm had an open reading frame of 1 879 bp,which encoded a putative polypeptide of 625 a mino acids and the theoretical molecular mass was 67 kD.The amplified fragment was cloned into pUC19 to generate the recombinant expression vector pFLDα-glaAm containing the mature glucoamylase encoding gene free of the signal peptide.The recombinant plasmid pFLDα-glaAm was transformed into P.pastoris X33 through electroporation after linearized by Nsi I digestion.The recombinant P.pastoris X33/pFLDα-glaAm were screened in 2% soluble starch plates,and identified by PCR.In shaking culture condition,methanol was added to a final concentration of 0.5% to induce the secretion of glucoamylase.[Result]The SDS-PAGE and Starch-PAGE analysis revealed that the recombinant glucoamylase was secreted and expressed correctly in P.pastoris X33,and the expressed product had the normal bioactivity.The enzyme activity in the medium reached 380.78 U/ml after being induced by methanol for 96 h.The recombinant glucoamylase exhibited optimum catalytic activity at pH 4.0 and 60-65 ℃ respectively.It was thermostable at 50 ℃ and remained more than 90% of its original activity after 60 min at 60 ℃.The half-life was about 44 min at 65 ℃.[Conclusion] These results confirmed that A.niger glucoamylase was highly-effectively expressed in P.pastoris X33.

关 键 词：毕赤酵母 糖化酶 热稳定性 异源表达 

分 类 号：S188.1[农业科学—农业基础科学]