【摘要】：酰胺酶可以將酰胺水解生成對應的羧酸和氨,在醫(yī)藥、食品、紡織皮革加工等方面具有極其重要的作用。L-天冬酰胺酶廣泛被臨床上用于兒童急性淋巴細胞白血病(ALL)的治療;L-谷氨酰胺酶在谷氨酰胺分解過程中是關鍵酶和限速酶,與腫瘤的生長、腫瘤血管的生成及腫瘤免疫都有重要的關系;立體專一性哌嗪-2-甲酰胺酶可以催化水解產(chǎn)生一種手性藥物中間體哌嗪-2-羧酸,其不同的單一對映體(R型和S型)衍生物具有多種不同的藥理學活性。本實驗室在前期工作中篩選得到的馬氏副球菌(Paracoccus marcusii)W10173可立體選擇性水解(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,本實驗通過基因組測序技術獲得馬氏副球菌W10173基因組,為了進一步發(fā)掘菌株本身酰胺酶資源,分析得到5個酰胺酶基因并對其進行克隆及異源表達,為酰胺酶的生產(chǎn)和應用提供基礎。通過分析基因組測序,馬氏副球菌W10173編碼1個谷氨酰胺酶(PM1509)、2個天冬酰胺酶(PM1383,PM3012)和2個潛在的哌嗪-2-甲酰胺酶(PM2486,PM3475)。PM1509,PM1383和PM3012成功克隆并構建pET28-a(+)-X/BL 21(DE3)原核重組表達載體,以IPTG為誘導劑,當OD600約為0.6時,加入0.8 mmol/L IPTG,18℃誘導5 h,目的蛋白的表達量分別占蛋白表達總量的30.5%,23.8%,20.3%。但目的蛋白都以包涵體形式存在,經(jīng)超聲破碎處理后將包涵體蛋白用Ni柱試劑盒純化,純化后蛋白的表達量分別為2.53 mg/mL,2.13 mg/mL,1.62 mg/mL,純化蛋白復性后蛋白的表達量分別為0.41mg/mL,0.34 mg/mL,0.31 mg/mL,收率分別為16.2%,15.9%,19.1%。分別以L-天冬酰胺、L-谷氨酰胺為底物采用TLC法測定復性后的酶蛋白活性,催化水解可以得到相應的產(chǎn)物,證明PM1509表達的蛋白有L-谷氨酰胺酶酶活,PM1383和PM3012表達的蛋白有L-天冬酰胺酶活性。利用SOMPA軟件、Predict Protein數(shù)據(jù)庫對PM2486和PM3475基因進行編碼蛋白的二級結構組成預測,結果顯示β-折疊較少,而無規(guī)則卷曲較多且含有半胱氨酸可形成二硫鍵,這些因素導致了復性后的蛋白無活性的可能性較大,故選取巴斯德畢赤酵母GS115進行真核表達。篩選得到的轉化子經(jīng)過驗證大多數(shù)為甲醇利用快型,以甲醇為誘導劑誘導蛋白表達,重組菌甲醇誘導表達后SDS-PAGE檢測目的蛋白得到表達,發(fā)酵液上清和菌體中都有蛋白表達,以哌嗪-2-甲酰胺為底物進行酶活測定,結果顯示PM2486表達的蛋白在發(fā)酵液上清和菌體中且都有活性,而PM3475表達的蛋白沒有活性。對初篩發(fā)酵液上清所含蛋白含量較高的轉化子PM2486-5進行誘導表達,通過對每天樣品的檢測可知隨著誘導時間的延長,蛋白表達量在增加,Bradford法測定PM2486-5誘導表達6 d后發(fā)酵液上清蛋白含量為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，

本文編號：2438743