来源于云斑天牛肠道细菌Pseudomonas sp. TN06的β-折叠桶植酸酶基因phyA06的克隆、表达和酶学性质研究

从云斑天牛肠道中筛选到一株产植酸酶活性较高的假单胞菌Pseudomonas sp.TN06。采用简并PCR和TAIL-PCR的方法获得一个新的β-折叠桶植酸酶基因(phyA06),开放阅读框全长1 902 bp,编码633个氨基酸和1个终止密码子,预测其前23个氨基酸为信号肽,含有两个β-折叠桶结构域。将phyA06基因在大肠杆菌中表达,重组蛋白经镍柱纯化后达到电泳纯。纯化后的重组酶最适pH为7.0,在pH 6.0~10.0的条件下具有很好的稳定性;最适温度为55℃,热稳定性良好。同时,该酶在低温下仍保持一定活力,甚至0℃仍有活性。该植酸酶具有应用于水产饲料行业的潜在价值。

Cloning,Expression and Characterization of a BPP Phytase Gene from Insect Symbiotic Bacterium Pseudomonas sp. TN06

A Pseudomonas sp. TN06 strain producing phytase was isolated from the gut of Batocera horsfieldi. A novel β\|propellar phytase gene (phyA06) was cloned by degenerate PCR and TAIL\|PCR. The ORF of this gene is 1 902 bp, encoding 633 amino acids and 1 terminator codon, containing a putative signal peptide of initiative 23 residues and 2 β\|propellar phytase domains. The phyA06 gene was transformed into Escherichia coli for expression. The recombinant enzyme of PhyA06 was purified to homogeneity by Ni\|NTA affinity chromatography. The enzymological properties of recombinant phytase were assayed. The optimum pH was 7.0，and the optimum reaction temperature was 55℃. The recombinant enzyme showed high stability at pH 6.0~10.0， and had good thermal stability. It still remained partial activity at low temperature，even at 0℃. PhyA06 has potential value for applying in aquatic feed.