马氏副球菌W10173基因组测序及其酰胺酶基因的克隆与表达

发布时间：2019-03-12 11:41

【摘要】：酰胺酶可以将酰胺水解生成对应的羧酸和氨,在医药、食品、纺织皮革加工等方面具有极其重要的作用。L-天冬酰胺酶广泛被临床上用于儿童急性淋巴细胞白血病(ALL)的治疗;L-谷氨酰胺酶在谷氨酰胺分解过程中是关键酶和限速酶,与肿瘤的生长、肿瘤血管的生成及肿瘤免疫都有重要的关系;立体专一性哌嗪-2-甲酰胺酶可以催化水解产生一种手性药物中间体哌嗪-2-羧酸,其不同的单一对映体(R型和S型)衍生物具有多种不同的药理学活性。本实验室在前期工作中筛选得到的马氏副球菌(Paracoccus marcusii)W10173可立体选择性水解(R,S)-哌嗪-2-甲酰胺中的(S)-哌嗪-2-甲酰胺得到(S)-哌嗪-2-羧酸,本实验通过基因组测序技术获得马氏副球菌W10173基因组,为了进一步发掘菌株本身酰胺酶资源,分析得到5个酰胺酶基因并对其进行克隆及异源表达,为酰胺酶的生产和应用提供基础。通过分析基因组测序,马氏副球菌W10173编码1个谷氨酰胺酶(PM1509)、2个天冬酰胺酶(PM1383,PM3012)和2个潜在的哌嗪-2-甲酰胺酶(PM2486,PM3475)。PM1509,PM1383和PM3012成功克隆并构建pET28-a(+)-X/BL 21(DE3)原核重组表达载体,以IPTG为诱导剂,当OD600约为0.6时,加入0.8 mmol/L IPTG,18℃诱导5 h,目的蛋白的表达量分别占蛋白表达总量的30.5%,23.8%,20.3%。但目的蛋白都以包涵体形式存在,经超声破碎处理后将包涵体蛋白用Ni柱试剂盒纯化,纯化后蛋白的表达量分别为2.53 mg/mL,2.13 mg/mL,1.62 mg/mL,纯化蛋白复性后蛋白的表达量分别为0.41mg/mL,0.34 mg/mL,0.31 mg/mL,收率分别为16.2%,15.9%,19.1%。分别以L-天冬酰胺、L-谷氨酰胺为底物采用TLC法测定复性后的酶蛋白活性,催化水解可以得到相应的产物,证明PM1509表达的蛋白有L-谷氨酰胺酶酶活,PM1383和PM3012表达的蛋白有L-天冬酰胺酶活性。利用SOMPA软件、Predict Protein数据库对PM2486和PM3475基因进行编码蛋白的二级结构组成预测,结果显示β-折叠较少,而无规则卷曲较多且含有半胱氨酸可形成二硫键,这些因素导致了复性后的蛋白无活性的可能性较大,故选取巴斯德毕赤酵母GS115进行真核表达。筛选得到的转化子经过验证大多数为甲醇利用快型,以甲醇为诱导剂诱导蛋白表达,重组菌甲醇诱导表达后SDS-PAGE检测目的蛋白得到表达,发酵液上清和菌体中都有蛋白表达,以哌嗪-2-甲酰胺为底物进行酶活测定,结果显示PM2486表达的蛋白在发酵液上清和菌体中且都有活性,而PM3475表达的蛋白没有活性。对初筛发酵液上清所含蛋白含量较高的转化子PM2486-5进行诱导表达,通过对每天样品的检测可知随着诱导时间的延长,蛋白表达量在增加,Bradford法测定PM2486-5诱导表达6 d后发酵液上清蛋白含量为0.519 mg/mL。
[Abstract]:Amidase can hydrolyze amide to form corresponding carboxylic acids and ammonia, in medicine, food, L-asparaginase (L-asparaginase) has been widely used in the treatment of childhood acute lymphoblastic leukemia (ALL). The L-glutaminase is the key enzyme and the rate-limiting enzyme in the process of glutamine decomposition, and it has an important relationship with the growth of tumor, tumor angiogenesis and tumor immunity. The stereospecific piperazine-2-formamidase can catalyze the hydrolysis to produce a chiral drug intermediate piperazine-2-carboxylic acid. Different enantiomers (R-type and S-type) derivatives of piperazine-2-carboxylic acid have different pharmacological activities. (S)-piperazine-2-formamide from paracoccus martensii (Paracoccus marcusii) W10173 was stereoselective hydrolyzed (R, S)-piperazine-2-formamide to obtain (S)-piperazine-2-carboxylic acid. In this experiment, the genome of paracoccus martensii W10173 was obtained by genomic sequencing. In order to further explore the Amidase resources of the strain, five amidase genes were obtained and cloned and expressed heterologous. It provides the basis for the production and application of Amidase. By sequencing the genome, paracoccus martensii W10173 encodes one glutaminase (PM1509), two asparaginases (PM1383,PM3012) and two potential piperazine-2-formamidases (PM2486,PM3475). PM1509, PM1383 and PM3012 cloned and constructed the prokaryotic recombinant expression vector of pET28-a ()-X/BL 21 (DE3). When OD600 was about 0.6, the recombinant expression vector of pET28-a ()-X/BL 21 (DE3) was induced at 0.8 mmol/L IPTG,18 鈩，

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