芽孢杆菌植酸酶基因在毕赤酵母中的分泌表达

Secretory expression of Bacillus subtilis phytase phyC in Pichia pastoris

根据枯草芽孢杆菌WHNB02植酸酶phyC基因全序列设计一对引物,采用PCR法从含有该基因的pUC18-phyC质粒上获得不含有信号肽序列的phyC基因表达片段,亚克隆到毕赤巴斯德表达载体pPIC9K的多克隆位点,并电转化毕赤巴斯德酵母GS115.经MD和MM平板筛选、酶活性测定,获得了阳性转化子PP9KC.首次在毕赤巴斯德酵母中实现了有生物学活性芽孢杆菌植酸酶的分泌表达,去糖基化试验表明该植酸酶的分子量为53.5 kD和50.9 kD糖基化程度不同的两种糖蛋白.用麦芽汁半合成培养基对PP9KC进行诱导培养,培养48 h后酶活可达到2453 U/mL,表达量为出发菌株的10.5倍.酶学性质分析表明,其酶促反应最适pH值为7.0,最适反应温度为65℃,经90℃处理10 min,残留酶活性为37℃时的78.2%.

The Bacillus phytase is very suitable to be used in animal feed particularly in carp feed, because its optimum pH is near to the neutral with the excellent thermostability. The phytase phyC fragment without signal peptide sequence from B. subtilis was amplified, sequenced and cloned into pPIC9K expression vector of P. pastoris. The pPIC9KphyC plasmid was linearized and transformed into Pichia Pastoris GS115 strain by electroporation. Positive strain was screened and purified by culturing on MD plate and MM plate. Furthermore, the positive strain was cultured and induced by methanol. The results show that the protein was expressed secretively with phytase activity. And it＇s a kind of glycoprotein which showed two molecular weight 53. 5 kD and 50. 9 kD because of the different glycosylation level. Additionally, in wort semi synthetic medium, the phytase yield came to 2453 U/mL after 48 hours induction, 10. 5 times of original B. subtilis strain. The optimum reaction pH and temperature for this enzyme activity were found to be 7.0 and 65 ℃, respectively. Moreover, the thermostability experiment showed its residual activity was 78.2% after treatment at 90℃ for lOmin.