芽孢杆菌中性植酸酶基因的原核表达及酶学性质分析

植酸酶作为饲料添加剂能够有效提高动物对饲料中磷的利用率及减少粪便中磷排放对环境的污染,并降低植酸的抗营养作用。为了获得性能稳定的高活性植酸酶,采用PCR扩增芽孢杆菌(Bacillus sp.)中性植酸酶基因phyC(GenBank登录号:FJ986327)的成熟肽编码序列,将其克隆进原核表达载体pET-28a(+),并转化E.coli BL21(DE3)进行表达。在37℃条件下以0.5 mmol/L IPTG诱导4 h能够获得大量包涵体蛋白,在25℃条件下以0.5 mmol/L IPTG诱导6 h有利于可溶性蛋白的获得。利用Ni-NTA亲和层析柱纯化重组植酸酶产物,获得的中性植酸酶的部分酶学特性为:耐热性较好,最适反应温度55℃,在70℃处理10 min可保持20%以上的酶活性;耐酸、碱能力较强,最适pH 6.0～7.0,pH 5.5～9.0时能保持80%以上的酶活性,pH 5.0～10.0时处理60 min仍能保持70%以上的酶活性,在pH 2.0～4.0时能保持40%以上的酶活性。利用构建的切除芽孢杆菌中性植酸酶基因phyC信号肽编码序列的原核表达载体及优化的诱导表达条件,能够在大肠杆菌中高量表达性能稳定的芽孢杆菌中性植酸酶。

Prokaryotic Expression and Enzymological Property Analysis of Neutral Phytase Gene from Bacillus sp.

When used as feedstuff additive,phytase can effectively improve the utilization of phosphorus in feedstuff,reduce environmental pollution caused by fecal phosphorus excretion,and reduce anti-nutritional effect of phytic acid.In order to obtain highly active phytase with stable performance,the mature peptide coding sequence of neutral phytase gene phyC(GenBank accession number: FJ986327) was amplified from Bacillus sp.by PCR and cloned into prokaryotic expression vector pET-28a(+).The recombinant plasmid was subsequently transformed into E.coli BL21(DE3) for expression.Large amount of inclusion body protein was obtained after induction with 0.5 mmol/L IPTG for 4 h at 37 ℃,and the soluble protein could be easily obtained by induction with 0.5 mmol/L IPTG for 6 h at 25 ℃.After being purified with Ni-NTA affinity chromatography,enzymological properties of the recombinant phytase were assayed.The results showed that the obtained phytase had favorable heat tolerance.The optimum reaction temperature was 55 ℃,and more than 20% of enzyme activity was maintained after incubation at 70 ℃ for 10 min.The enzyme also had high resistance to acid and alkali.The optimum pH value was 6.0 to 7.0,and more than 80%,70% and 40% of enzyme activity was maintained when incubated at pH 5.5 ~9.0,pH 5.0~10.0 and pH 2.0 ~4.0 for 60 min,respectively.Using the constructed prokaryotic expression vector containing coding sequence of Bacillus neutral phytase gene phyC of which signal peptide sequence had been removed,neutral phytase with stable performance could be highly expressed in E.coli under the optimized induced expression condition.