NisinZ的定点突变及突变体性质的研究

以本实验室构建的含nisZ基因的质粒pHJ2 0 1为模板 ,采用定点突变技术将乳链菌肽Z分子中B环第 8位Thr突变为Ser(T8S)、将第 2位Dhb突变为Dha和第 31位His突变为Lys(T2S H31K)以及将第 2 7位Asn突变为Lys和第 31位His突变为Lys(N2 7K H31K) ,以pMG36e为载体 ,电击转化乳酸乳球菌 (L .lactis)NZ980 0进行表达。对表达产物性质的研究结果表明 ,3个突变体的抑菌谱和溶解度未发生变化 ,其抑菌活性略有下降 ,但它们的稳定性表现各不相同 :N2 7K H31K的稳定性与NisinZ几乎一致 ,而T8S和T2S H31K的稳定性有明显提高 ,在pH9条件下10 0℃加热 5min仍不丧失抑菌活性。

Site-directed Mutagenesis of NisinZ and Properties of NisinZ Mutants

According to the knowledge gained from engineering of nisinZ, using plasmid pHJ201 DNA as template, NisinZ was mutated by site-directed mutagenesis, NisinZ mutant T8S contains Serine at position 8 instead of Threonine, NisinZ mutant N27K/H31K contains Lysine at position 27 and 31, respectively, instead of Asparagine and Histidine and NisinZ mutant T2S/ H31K contains dehydrobutyrine and Lysine at position 2 and 31 instead of dehydroalanine and Histidine. They are cloned into pMG36e and expressed in L. Lactis NZ9800, the expression products of these mutants purified by Sephadex CM-25 and Sephadex G-25 chromatography, some properties of NisinZ mutants (T8S, T2S/H31K and N27K/H31K) were studied. The results showed that the spectrum of antimicrobial activity and solubility of these mutants had not been changed, their antimicrobial activities were found to be slightly lower than that of the wild-type NisinZ. but mutants T8S and T2S/H31K showed higher stability, which were significantly more stable than wild-type NisinZ at 55 approximately 100 degrees C and pH7 approximately 9.