红球菌来源QsdA型N-酰基高丝氨酸内酯酶水产养殖饲用潜力分析  

AN ANALYSIS OF THE POTENTIAL FOR AQUACULTURE OF N-ACYL HOMOSERINE LACTONASE FROM RHODOCOCCUS ERYTHROPOLIS BFXJ-1

作　　者：张美超[1] 杨雅麟[1] 宋水山[2] 徐俐[1] 姚斌[1] 周志刚[1] 

机构地区：[1]中国农业科学院饲料研究所,农业部饲料生物技术重点试验室,北京100081 [2]河北省科学院生物研究所,石家庄050081

出　　处：《水生生物学报》2015年第3期540-548,共9页Acta Hydrobiologica Sinica

基　　金：“十二五”国家科技支撑计划项目(2012 BAD2 5 B02;2013BAD10B01-2);现代农业产业技术体系北京市鲟鱼鳟鱼创新团队(SCGWZJ 20141104-4)资助

摘　　要：通过研究Qsd A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qsd Arh5。构建重组表达载体p ET28a/qsd A-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Qsd A-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Qsd A-RH5,经分析表明,该蛋白的理论分子量为36 k D,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p H为8.0,作用温度为35℃,在p H 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。The objective of this study was supplement to determine the enzymatic properties of Qsd A-like AHLases and evaluate their application potentials. The gene qsda-rh5 was amplified from the Rhodococcus erythropolis BLJF-1genomevia PCR technique. After the recombinant vector p ET28a/qsda-rh5 was transformed into E. coli BL21（DE3）, transformants with N-acyl-homoserine lactonase activity were screened. The purified Qsd A-RH5 was obtained with Ni-NTA column. Both N-acyl-homoserine lactonase activities of Qsd A-RH5 were additional characterized. The results showed that an AHL lactonase gene, qsda-rh5, of 972 base pairs, was identified from Rhodococcus erythropolis BFXJ-1. Deduced Qsd A-RH5 was a metallo dependent hydrolase belonging to Phosphotriesterase（PTE） superfamily. Recombinant Qsd A-RH5 was successfully expressed in Escherichia coli BL21 and purified to electrophoretic homogeneity. The enzyme maintained≥80% of its activity at 10–40℃, p H 8.0. Qsd A-RH5 had significant resistance toproteolytic 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 Qsd A-RH5, indicate the Qsd A-RH5 has specificity of substrate. These properties make Qsd A-RH5 an outstanding quorum-quenching tool for environment.

关 键 词：红球菌 N-酰基高丝氨酸内酯酶 重组表达 酶学性质 

分 类 号：S963.7[农业科学—水产养殖]