粟酒裂殖酵母麦芽糖酶基因的克隆及在大肠杆菌内的表达

Cloning of Maltase Gene from Schizosaccharomyces Pombe and Overexpression of the Gene in Escherichia Coli

利用PCR的方法,以粟酒裂殖酵母菌的染色体DNA为模板,扩增得到粟酒裂殖酵母的结构基因,其开放阅读框1740 bp的序列,可以编码579氨基酸,分子量为67.7 kD.利用NCBI对它的蛋白序列进行比对,发现这个蛋白序列与Aspergillus oryza、Candida albicans、Hansenula polymorpha、Saccharomycescerevisiae、Saccharomyces pastorianus的麦芽糖酶相比分别具有40.6%、41.6%、37.9%、34.7%、34.5%的相似性.而与粟酒裂殖酵母的α-葡萄糖苷酶具有99.8%的相似性,由此可以得出克隆得到的结构基因应该是粟酒裂殖酵母的麦芽糖酶结构基因.将克隆的粟酒裂殖酵母的麦芽糖酶基因克隆到pQE30表达载体中在大肠杆菌中进行诱导表达,然后测定其麦芽糖酶的活力,其酶的比活力是野生菌株的21倍,诱导条件下麦芽糖酶的比活力是非诱导条件下的10倍.

The Schizosaccharomyces pombe maltase structural gene （SPMAL） is amplified from a genomic DNA of S. pombe by PCR. An open reading frame of 1740 bp encoding 579 amino-acid protein with calculated molecular weight of 67. 7 kD is characterized in the genomic DNA insert of plasmid pQE30. The deduced protein shows 40.6% identity with maltase from Aspergillus oryza, 41.6% identity with maltase from Candida albicans, 37.9% idendity with maltase from Hansenula polymorpha, 34.7% identity with maltase from Saccharomyces cerevisiae, 34.5% identity with maltase from Saccharomyces pastorianus and 99. 8% identity with α-glucosidase from S. pombe. The specific maltase activity in the induced transformants is 21 times higher than that in wild-type and the specific maltase activity in the transformants which are not induced by IPTG is over ten times higher than that in wild-type XL-blue.