【作者】 陈瑞东；

【导师】 周志刚；

【作者基本信息】 中国农业科学院 ， 动物营养与饲料科学， 2010， 硕士

【摘要】 N-酰基高丝氨酸内酯酶是一类特异性降解N-酰基高丝氨酸内酯类信号分子(AHLs)的金属蛋白水解酶,广泛存在于多种微生物中。近年来N-酰基高丝氨酸内酯酶作为一种新型抗菌策略(群体感应淬灭策略)的工具酶而成为研究的热点。目前主要是通过构建转基因植物、转基因细菌研究其在群体感应淬灭中的作用机制,而转基因植物或转基因细菌在实际应用中存在生物安全和有效性方面的问题。本研究的目的是利用毕赤酵母表达系统高效表达N-酰基高丝氨酸内酯并将该酶以酶制剂的形式来防治水产养殖中嗜水气单胞菌引起的疾病。本论文通过富集培养的方法使用以3-oxo-C6-HSL为唯一碳源的筛选培养基从天津武清池塘底泥分离出一株产N-酰基高丝氨酸内酯酶的菌株,命名为B546,经16s rDNA鉴定为芽孢杆菌。通过设计该种属N-酰基高丝氨酸内酯酶的简并引物,成功克隆了N-酰基高丝氨酸内酯酶基因aiiAB546。aiiAB546片段大小为753 bp,编码250个氨基酸,预测编码蛋白的分子量为28.14 kDa,等电点为4.64,在蛋白的N端预测有一个氮糖基化位点(Asn-Ser-Thr)。将aiiAB546连接pPIC9表达载体,转化毕赤酵母GS115感受态细胞并成功表达。通过根癌农杆菌KYC 55活性平板法筛选出高效表达的13号菌株。以3-oxo-C8-HSL为底物,在摇瓶水平上该菌株表达的N-酰基高丝氨酸内酯酶酶活为27.1±3.2 U/mL,通过3.7 L发酵罐高密度发酵,重组蛋白的表达量高达3,558.4±81.3 U/mL,而大肠杆菌表达的酶活仅为0.24 U/mL。毕赤酵母明显提高了N-酰基高丝氨酸内酯酶的表达量,实现了目的蛋白的高效表达。毕赤酵母表达的重组蛋白通过硫酸铵沉淀和阴离子柱纯化出33.6 kDa的单一条带。经质谱鉴定为目的蛋白,Endo H处理验证了氮糖基化的存在。纯化的重组AiiAB546的最适pH和温度分别为8.0和20°C,在pH 6.5–8.5范围内,酶活性均能维持在80%以上,在0°C,20°C~37°C,AiiAB546具有60%的酶活力。AiiAB546具有良好的pH和温度稳定性。在pH 6.5–12.0范围内处理60 min后,酶活性均高于100%,说明AiiAB546在中性和碱性条件下非常稳定。AiiAB546在60°C下处理30 min,酶活性基本维持不变,70°C处理15 min,剩余90%以上的酶活,说明AiiAB546在60°C和70°C条件下仍非常稳定。AiiAB546具有非常好的抗胰蛋白酶,α-糜蛋白酶,枯草杆菌蛋白酶,胶原蛋白酶和碱性蛋白酶的能力,AiiAB546与上述蛋白酶共处理60 min,其酶活性均大于100%。此外,大多数离子对AiiAB546无抑制作用。AiiAB546和嗜水气单胞菌共注射锦鲤可以明显降低嗜水气单胞菌致死锦鲤的平均累积死亡率为25%,延迟锦鲤的半数致死时间为18 h。单独注射AiiAB546,经长时间观察锦鲤生存状态良好。根据试验结果预测AiiAB546对鱼体无害并且可以降低嗜水气单胞菌致死能力,本试验为潜在机制的研究提供了重要的试验数据。本试验不仅在毕赤酵母中成功高效表达了AiiAB546,使低成本获得大量的AiiAB546成为可能,而且以酶制剂的形式通过活体注射的方式成功衰减嗜水气单胞菌在锦鲤中的致死能力。根据所知,这是首次在毕赤酵母中高效表达AiiAB546并通过注射AiiAB546衰减嗜水气单胞菌的毒力。本试验具有潜在的抗菌策略研究与水产养殖应用方面的价值。更多还原

【Abstract】 N-Acyl homoserine lactonase, widely identified in a range of bacterial species, is a group of metal-hydrolases that can specially hydrolyze N-Acyl homoserine lactone. In recent years, N-Acyl homoserine lactone lactonase has been attracted much attention as a new enzyme in antimicrobial therapeutics (quorum quenching strategy). Transgenic plants and bacteria are used to study the mechanism of quorum quenching; however, application of transgenic plants and bacteria is limited due to problems of safety and efficacy. The objective of this study was to overexpress AHL-lactonase in Pichia pastoris and attenuate the Aeromonas hydrophila virulence with AHL-lactonase to control A. hydrophila-related diseases.A strain, denoted as B546, producing AHL-lactonase was isolated from the mud of a fish pond at Wuqing, Tianjin, China using minimal medium containing 3-oxo-C6-HSL as the sole carbon source and was identified as Bacillus sp. by its 16S rDNA sequence. Based on the conserved amino acid sequences of AHL-lactonases from Bacillus sp., a degenerate primer set was designed. Using the degenerate primers, the full-length 753-bp AHL-lactonase gene, aiiAB546, was cloned from Bacillus sp. B546. aiiAB546 encoded a 250-amino acid polypeptide with a calculated molecular mass of 28.14 kDa and a pI of 4.64. One potential N-glycosylation site (Asn-Ser-Thr) was identified at the N terminus.The recombinant plasmid pPIC9-aiiAB546 was constructed and transformed into P. pastoris GS115 competent cells. Positive transformants were screened using well-diffusion assays with reporter strain Agrobacterium tumefaciens KYC 55, and clone 13 with highest enzyme activity was selected for further assay. In the shake-flask level, AHL-lactonase activity was up to 27.1±3.2 U/mL. In the 3.7 L fermenter, the expression level of recombinant AiiAB546 reached 3,558.4±81.3 U/mL after induction, which was significantly higher than that expressed in Escherichia coli (0.24 U/mL). Expression of aiiAB546 in P. pastoris enhanced the production of AiiAB546 significantly.Recombinant AiiAB546 in P. pastoris was purified to electrophoretic homogeneity by ammonium sulfate precipitation and anion exchange chromatography. The molecular weight of the purified recombinant AiiAB546 was 33.6 kDa. Further LC-ESI-MS/MS analysis and deglycosylation of AiiA with Endo H confirmed the identity of the objective band and the occurrence of N-glycosylation. Purified recombinant AiiAB546 showed optimal activity at pH 8.0 and 20°C, retained more than 80% of the maximum activity at pH 6.5–8.5. At 0 and 20–37°C, the enzyme maintained more than 60% of the highest activity. AiiAB546 exhibited excellent pH stability, retaining more than 100% activity after pre-incubation at 37°C, pH 6.5–12.0 for 1 h. AiiAB546 was thermostable at 60°C and 70°C, retaining more than 100% and 90% of the initial activity after pre-incubation at 60°C for 30 min and 70°C for 15 min, respectively. The purified recombinant AiiAB546 was protease-resistant. After incubation with trypsin, subtilisin A, collagenase and proleather at 37°C for 60 min, the enzyme retained almost all of the enzymatic activity. In addition, AiiAB546 was also resistant to many metal ions.Co-injection of AiiAB546 and A. hydrophila decreased the mortality rate of common carp by nearly 25%. Compared with the fish injected with A. hydrophila, the LT50 of the fish with co-injection of AiiAB546 and A. hydrophila was delayed 18 h. After injection with AiiAB546 alone, the fish displayed no signs of stress or disease and no mortalities were observed. The results indicate that AiiAB546 is safe for carp and can attenuate A. hydrophila virulence, and provide more novel data for further study.This study not only achieves overexpression of AiiAB546 in P. pastoris and makes it possible to mass-produce AHL-lactonase at low cost, but also opens up a promising foreground of application of AHL-lactonase in fish to control A. hydrophila disease by regulating its virulence. To our knowledge, this is the first report on heterologous expression of AHL-lactonase in P. pastoris and attenuating A. hydrophila virulence by co-injection with AHL-lactonase. This study has potential values for antimicrobial strategies and aquaculture applications.更多还原