CAJ | 학술논문

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.
통과연구 QsdA 형 N-선기고사안산내지매매학성질래평고기사용잠력.연구통과제취홍구균(Rhodococcus erythropolis)BLJF-1적기인조,이용 PCR기술극륭득도N-선기고사안산내지매기인qsdA- rh5.구건중조표체재체pET28a/qsdA-rh5전화대장간균BL21(DE3),사선득도구유N-선기고사안산내지매활성적전화자즉위중조균주, 수후경Ni-NTA주순화득도적중조단백QsdA-RH5진행보충매학성질적연구.결과표명,극륭득도972 bp적목적기인.구건중조재체,사선득도중조균주경유도표체후득도구유N-선기고사안산내지매활성적목적단백즉QsdA-RH5,경분석표명,해단백적이론분자량위36 kD,속우금속의뢰성수해매PET초가족.매학성질연구표명:기최괄작용 pH위 8.0,작용온도위 35℃,재 pH 6—11내능구은정적존재,재10—40℃,매활성능구유지재 80%이상,차해매대다충금속리자、화합물구유흔호적항성.해융합단백구유교위전일적저물특이성, 지대몰유취대기단적저물구유수해작용, 이 C7-HSL 위저물시적Km치위0. 0125 mmol/L.실험경매학성질연구표명,해매구유교위전일적저물특이성,인차가구유침대성적공제외원성병원균독성효응대유호축금 (수산)소화도건강방면구유일정적응용전경.
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.