米根霉α-淀粉酶基因的克隆与表达

Gene Cloning and Expression of Rhizopus or yzae α-Amylase

摘要:

以博德研究公布的米根霉基因组信息为基础设计引物,分别从两株米根霉中克隆得到大小为1 386 bp的α-淀粉酶编码基因。通过比对分析发现该淀粉酶基因内部不含内含子,两条基因的相似度为95.54%,基因编码产物相似度为97.84%。基因编码产物属于淀粉酶家族13成员,具有淀粉酶家族所特有的4个典型的高度保守区域。以质粒载体pET-28a(+)为基础构建相关表达载体,实现了两个淀粉酶在E coli中的功能性表达。两个淀粉酶在E coliBL21(DE3)codon-Plus宿主中的表达量相对较高,分别为1.3 U/mL和0.5 U/mL。其中一个淀粉酶经Ni亲和柱纯化后,用于进行(马铃薯)淀粉水解实验,终产物中葡萄糖质量浓度约为10 g/dL,麦芽糖质量浓度约为74 g/dL,其它低聚糖含量较少。

Abstract:

Based on Rhizopus oryzae complete genome sequence published by Broad Institute,two 1389-bp gene sequences coding a-amylase were cloned from two R.oryzae strains.Sequences alignment indicated that there was no intron existed in the two amylase genes,and the two genes have a similarity of 95.54% and the similarity between the gene products was 97.84%.The gene products belongs to the amylase family 13 and have four classical highly conserved regions that observed in most a-amylases.Recombinant plasmids for expression of the cloned a-amylase genes were constructed based on the plasmid vector pET-28a(+).a-Amylases displayed higher expression levels in E coli BL21(DE3) codon plus strain,the a-amylase activity were achieved at 1.3 U/mL and 0.5 U/mL,respectively.One of the a-amylases was purified through Ni affinity column and subsequently used for hydrolyzation of(potato) starch,and there was approximately 10% glucose,74% maltose and negligible maltotriose or other oligosaccharides remained in the end-products.