Cloning of phytase gene from Aspergillus niger 563 and its expression in E.coli system

S. Geetha, K. Kumar, L. Arul, E. Kokiladevi, P. Balasubramanian, D. Sudhakar
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Abstract

A phyA was cloned from Aspergillus niger by reverse transcription polymerase chain reaction. The amplified 1404 bp fragment was cloned in a T/A cloning vector and confirmed by sequencing. The isolated phytase gene showed 99.0 % sequence identity at nucleotide and protein level with Aspergillus niger CBS 513.88 phytase. The amino acid sequence from the phyA cDNA contained the consensus motifs, RHGXRXP and HD which are conserved among histidine acid phosphatases. The phytase cDNA was subcloned in pET28a(+) expression vector and expressed in E. coli. The expression of the gene was confirmed through SDS-PAGE analysis. A 52 kDa protein as per the calculated molecular mass of the translational product of phytase gene was observed in crude lysate of E. coli culture induced with IPTG. The protein was purified using nickel based His-bind resin column and checked on SDS-PAGE. The purified recombinant enzyme showed a single band of 52 kDa protein. The phytase activity of pET-phyA IPTG induced E. coli culture and purified phytase was 383.5 U/ml and 826.33 U/ml, respectively.
黑曲霉563植酸酶基因的克隆及其在大肠杆菌系统中的表达
利用逆转录聚合酶链反应从黑曲霉中克隆了一株phyA。将扩增的1404 bp片段克隆到T/ a克隆载体上,并进行测序验证。分离得到的植酸酶基因在核苷酸和蛋白水平上与黑曲霉CBS 513.88植酸酶序列同源性达99.0%。phyA cDNA氨基酸序列包含在组氨酸酸磷酸酶中保守的共识基序、RHGXRXP和HD。植酸酶cDNA在pET28a(+)表达载体上亚克隆,并在大肠杆菌中表达。通过SDS-PAGE分析证实了该基因的表达。在IPTG诱导的大肠杆菌粗裂解液中,植酸酶基因翻译产物的分子量为52 kDa。采用镍基his - binding树脂柱纯化,SDS-PAGE检测。纯化后的重组酶显示一个52 kDa的单带蛋白。pET-phyA IPTG诱导大肠杆菌培养物和纯化物植酸酶活性分别为383.5 U/ml和826.33 U/ml。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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