大豆根部两株产植酸酶菌的分离鉴定及中性植酸酶基因的克隆

Isolation and Identification of Two Phytase-producing Strains from Soybean Rhizosphere Soil and Cloning of Neutral Phytase Gene

从大豆根部土壤中筛选到两株产植酸酶的细菌菌株,对其进行形态学分析和16S rRNA鉴定,确定两个菌株为枯草芽孢杆菌,并对其植酸酶的酶学性质进行初步研究。通过PCR方法从该芽孢杆菌基因组中克隆了植酸酶基因phy(ycD)和phy(ycE)。与NCBI已报道的植酸酶序列分别通过BLASTn和BLASTp程序进行比对,确定phy(ycD)和phy(ycE)属于β-螺旋桨植酸酶家族。两个植酸酶核酸序列同源性达到98%。两个基因的开放阅读框(ORF)均为1149个核苷酸,编码382个氨基酸。N端26个氨基酸为信号肽序列,整个酶分子有5个氨基酸不同,运用SWI SS-MODEL服务器进行同源建模,使用Spdbv软件对其三维结构评估,这5个氨基酸中的4个分别处在酶分子不同二级结构部位。β-螺旋桨植酸酶基因phy(ycD)和phy(ycE)的获得,为进一步探讨中性植酸酶的结构与功能以及该类植酸酶的产业化应用提供实验数据。

Two strains capable of enhanced phytase production were isolated from soybean rhizosphere soil. According to morphological observation and 16S rRNA sequence analysis, both were identified as Bacillus subtilis. From both stains, phytase-encoding genes, phy（ycD） and phy（ycE）, were cloned by polymerase chain reaction. Sequence analysis was conducted with BLASTn and BLASTp programs, available on the National Center for Biotechnology Information database （NCBI）. The results revealed that Phy（ycD） and phy（ycE） genes shared 98% identity at nucleic acid level. Each of them contained an open reading frame of 1149 bp encoding 382 amino acids. The deduced polypeptide phy^vcD） and phy（ycE） belong to beta-propeller phytases （BPPs）. A signal peptide consisting of 26 residues was found at the N- terminus. The two sequences were distinguished from each other by five amino acid substitutions. Homology modeling of these phytases was carired out using SWISS-MODEL. The software Spdbv was used to evaluate the constructed model. The results showed that four amino acid substitutions were located in different secondary structures. The cloning ofphy（ycD） andphy（ycE） can lay a good foundation for basic research and industrial application of natural phytase.