安徽农业科学2011，Vol.39Issue(14)：8226-8230,8306,6.
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
曹慕琛 1徐健勇 2罗立超 2张同存 3孙劭靖 1宋诙1
作者信息
- 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,北京,100093
- 折叠
摘要
Abstract
[ Objective ] The aim was to lay the foundation to further expand the application of glucoamylase in the industry for using P. pa storis 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 had an open reading frame of 1879 bp,which encoded a putative polypeptide of 625 amino 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Key words
P. pastoris/ Glucoamylase/ Thermostability/ Heterologous expression分类
农业科技引用本文复制引用
曹慕琛,徐健勇,罗立超,张同存,孙劭靖,宋诙..黑曲霉糖化酶基因的克隆及其在毕赤酵母X33中的表达[J].安徽农业科学,2011,39(14):8226-8230,8306,6.
