Proteolytic activity of Vibrio harveyi YeaZ is related with resuscitation on the viable but non‐culturable state,Letters in Applied Microbiology

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Proteolytic activity of Vibrio harveyi YeaZ is related with resuscitation on the viable but non‐culturable state
Letters in Applied Microbiology ( IF 2.0 ) Pub Date : 2020-05-22 , DOI: 10.1111/lam.13304
R Zhao ₁ , J Chen ₁ , Y Wang ₂ , Y Li ₃ , X Kong ₁ , Y Han ₁

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The YeaZ protein of Vibrio harveyi was expressed in Escherichia coli and purified. The purified recombinant protein YeaZ exhibited the protease activity. The proteolytic activities with azocasein as substrate were 39 130 U mg−1. The mutation of the amino acid in active sites such as Asp88, Ser185 and Trp169 was performed. The enzyme activities of the purified mutant proteins with Asp88‐Ala, Ser185‐Leu and Trp169‐Glu were decreased to 24·28, 35·27 and 41·66%, respectively. The mutant protein with two amino acid residues (Asp88‐Ala/Ser185‐Leu) lost the protease activity completely. Addition of the purified recombinant YeaZ increased resuscitation of the viable but non‐culturable state (VBNC) cells to culturable state, and the culturable cell count increased from 1·35 × 102 to 3·10 × 106 CFU per ml. While addition of the mutant YeaZ without protease activities did not show obvious promoting effect on resuscitation of VBNC cells. Moreover, the purified YeaZ also showed lower muralytic activity, and the activities of proteins with single amino acids mutation (Thr71 and Asp112) were reduced from 7·05 to 4·75 and 2·50 U mg−1, the resuscitation‐promoting effect on VBNC cells was not affected by these mutant proteins. These results implied that resuscitation‐promoting effect of YeaZ on VBNC cell was partly related to its protease activities, but not with the muralytic activity.

中文翻译：

Vibrio harveyi YeaZ 的蛋白水解活性与存活但不可培养状态的复苏有关

哈维弧菌的 YeaZ 蛋白在大肠杆菌中表达并纯化。纯化的重组蛋白 YeaZ 表现出蛋白酶活性。以偶氮酪蛋白为底物的蛋白水解活性为 39 130 U mg-1。进行了 Asp88、Ser185 和 Trp169 等活性位点的氨基酸突变。纯化的含有 Asp88-Ala、Ser185-Leu 和 Trp169-Glu 的突变蛋白的酶活性分别降低至 24·28、35·27 和 41·66%。具有两个氨基酸残基（Asp88-Ala/Ser185-Leu）的突变蛋白完全丧失了蛋白酶活性。添加纯化的重组 YeaZ 增加了存活但不可培养状态 (VBNC) 细胞到可培养状态的复苏，可培养细胞计数从 1·35 × 102 增加到 3·10 × 106 CFU/ml。而加入没有蛋白酶活性的突变体YeaZ对VBNC细胞的复苏没有显示出明显的促进作用。此外，纯化的 YeaZ 还显示出较低的溶胞活性，单氨基酸突变蛋白（Thr71 和 Asp112）的活性从 7·05 降低到 4·75 和 2·50 U mg-1，促进复苏的作用VBNC 细胞上的基因不受这些突变蛋白的影响。这些结果表明，YeaZ 对 VBNC 细胞的复苏促进作用部分与其蛋白酶活性有关，但与胞壁溶解活性无关。VBNC 细胞的复苏促进作用不受这些突变蛋白的影响。这些结果表明，YeaZ 对 VBNC 细胞的复苏促进作用部分与其蛋白酶活性有关，但与胞壁溶解活性无关。VBNC 细胞的复苏促进作用不受这些突变蛋白的影响。这些结果表明，YeaZ 对 VBNC 细胞的复苏促进作用部分与其蛋白酶活性有关，但与胞壁溶解活性无关。

更新日期：2020-05-22

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