The objective of this study was supplement to determine the enzymatic properties of QsdA-like AHLases and evaluate their application potentials. The geneqsda-rh5 was amplified from theRhodococcus erythropolis BLJF-1genomevia PCR technique. After the recombinant vector pET28a/qsda-rh5 was transformed into E. coli BL21 (DE3), transformants with N-acyl-homoserine lactonase activity were screened. The purified QsdA-RH5 was obtained with Ni-NTA column. Both N-acyl-homoserine lactonase activities of QsdA-RH5 were additional characterized. The results showed that an AHL lactonase gene,qsda-rh5, of 972 base pairs, was identified fromRhodococcus erythropolis BFXJ-1. Deduced QsdA-RH5 was a metallo dependent hydrolase belonging to Phosphotriesterase (PTE) superfamily. Recombinant QsdA-RH5 was successfully expressed inEscherichia coli BL21 and purified to electrophoretic homoge-neity. The enzyme maintained≥80% of its activity at 10–40℃, pH 8.0. QsdA-RH5 had significant resistance toproteo-lytic digestion. Interestingly the enzyme conferred the ability to inactivate AHLs with an acyl chain lengths ranging from six to twelve carbon atoms, without substitution at carbon three. We were the expression and characteristics of the QsdA-RH5, indicate the QsdA-RH5 has specificity of substrate. These properties make QsdA-RH5 an outstanding quo-rum-quenching tool for environment.%通过研究 QsdA 型 N-酰基高丝氨酸内酯酶酶学性质来评估其饲用潜力.研究通过提取红球菌(Rhodococcus erythropolis)BLJF-1的基因组,利用 PCR技术克隆得到N-酰基高丝氨酸内酯酶基因qsdA- rh5.构建重组表达载体pET28a/qsdA-rh5转化大肠杆菌BL21(DE3),筛选得到具有N-酰基高丝氨酸内酯酶活性的转化子即为重组菌株, 随后经Ni-NTA柱纯化得到的重组蛋白QsdA-RH5进行补充酶学性质的研究.结果表明,克隆得到972 bp的目的基因.构建重组载体,筛选得到重组菌株经诱导表达后得到具有N-酰基高丝氨酸内酯酶活性的目的蛋白即QsdA-RH5,经分析表明,该蛋白的理论分子量为36 kD,属于金属依赖性水解酶PET超家族.酶学性质研究表明:其最适作用 pH为 8.0,作用温度为 35℃,在 pH 6—11内能够稳定的存在,在10—40℃,酶活性能够维持在 80%以上,且该酶对多种金属离子、化合物具有很好的抗性.该融合蛋白具有较为专一的底物特异性, 只对没有取代基团的底物具有水解作用, 以 C7-HSL 为底物时的Km值为0. 0125 mmol/L.实验经酶学性质研究表明,该酶具有较为专一的底物特异性,因此可具有针对性的控制外源性病原菌毒性效应对维护畜禽 (水产)消化道健康方面具有一定的应用前景.
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