Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants
Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants
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DOI：
10.1007/s00253-004-1599-1
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发表时间：
2004-06-01
影响因子：
5
通讯作者：
Huan, LD
中科院分区：
文献类型：
--
作者：
Yuan, J;Zhang, ZZ;Huan, LD
为了研究链球菌素铰链区在Nisin中的作用，并获得具有改变或新的生物学活性和功能特性的突变体，我们用NisinZ结构基因(NisZ)进行定点突变，改变了铰链区的某些氨基酸。结果表明，NisinZ突变体对黄色微球菌NCIB8166和嗜热链球菌的抗菌活性降低。有趣的是，与野生nisinZ相比，突变体N20K nisinZ和M21K nisinZ对革兰氏阴性志贺氏菌、假单胞菌和沙门氏菌显示出抗菌活性，并且它们的溶解度高于野生型nisinZ。在pH值为8时，N20K NisinZ和M21K NisinZ的溶解度分别是NisinZ的3倍和5倍。突变体N20Q NisinZ和M21G NisinZ在较高的温度和中性或碱性的pH条件下比NisinZ更稳定。这些突变体提供的信息表明，NisinZ的中央铰链区在提供膜上抗菌活性所需的构象灵活性方面发挥着重要作用。我们的发现证明，很值得考虑构建对广泛的革兰氏阴性细菌具有不同抑制活性的新的Nisin突变体，并通过定点突变来改善功能特性。
To study the role of the hinge region in nisin and to obtain mutants that exhibit altered or new biological activities and functional properties, we changed certain amino acids in the hinge region by performing site-directed mutagenesis with the nisinZ structural gene (nisZ). The results showed that the nisinZ mutants had decreased antimicrobial activities against Micrococcus flavus NCIB8166 and Streptococcus thermophilus. Interestingly, compared with wild nisinZ, mutant N20K nisinZ and M21K nisinZ displayed antimicrobial activity against gram-negative Shigella, Pseudomonas and Salmonella; and they had a higher solubility than wild-type nisinZ. At pH 8, the solubilities of N20K nisinZ and M21K nisinZ were, respectively, three-fold higher and five-fold higher than that of nisinZ. Mutant N20Q nisinZ and M21G nisinZ were considerably more stable than nisinZ at higher temperatures and neutral or alkaline pH. These mutants provided information that the central hinge region in nisinZ plays an important role in providing the conformational flexibility required for the antimicrobial activity on the membrane. Our finding documented that it may well be worth considering the construction of the new nisin mutants with changed inhibitory activity against a wide range of gram-negative bacteria and the improvement of functional properties by site-directed mutagenesis.