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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。
A strain of Pseudomonas sp.TN06 with high phytase activity was screened from the intestinal tract of Crabgrass. A new β-sheeting phytase gene (phyA06) was obtained by degenerate PCR and TAIL-PCR. The full-length open reading frame was 1 902 bp, encoding 633 amino acids and 1 stop codon. Amino acids are signal peptides containing two β-sheet barrel domains. The phyA06 gene was expressed in Escherichia coli and the recombinant protein was purified by nickel column and reached electrophoretic purity. The optimum pH of purified recombinant enzyme is 7.0, which has good stability under the condition of pH 6.0 ~ 10.0. The optimum temperature is 55 ℃ and the thermal stability is good. At the same time, the enzyme still maintain a certain degree of vitality at low temperatures, even 0 ℃ still active. This phytase has the potential to be used in the aquafeed industry.