【摘要】：酰胺酶可以將酰胺水解生成對(duì)應(yīng)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(xì)胞白血病(ALL)的治療;L-谷氨酰胺酶在谷氨酰胺分解過(guò)程中是關(guān)鍵酶和限速酶,與腫瘤的生長(zhǎng)、腫瘤血管的生成及腫瘤免疫都有重要的關(guān)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(duì)映體(R型和S型)衍生物具有多種不同的藥理學(xué)活性。本實(shí)驗(yàn)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(shí)驗(yàn)通過(guò)基因組測(cè)序技術(shù)獲得馬氏副球菌W10173基因組,為了進(jìn)一步發(fā)掘菌株本身酰胺酶資源,分析得到5個(gè)酰胺酶基因并對(duì)其進(jìn)行克隆及異源表達(dá),為酰胺酶的生產(chǎn)和應(yīng)用提供基礎(chǔ)。通過(guò)分析基因組測(cè)序,馬氏副球菌W10173編碼1個(gè)谷氨酰胺酶(PM1509)、2個(gè)天冬酰胺酶(PM1383,PM3012)和2個(gè)潛在的哌嗪-2-甲酰胺酶(PM2486,PM3475)。PM1509,PM1383和PM3012成功克隆并構(gòu)建pET28-a(+)-X/BL 21(DE3)原核重組表達(dá)載體,以IPTG為誘導(dǎo)劑,當(dāng)OD600約為0.6時(shí),加入0.8 mmol/L IPTG,18℃誘導(dǎo)5 h,目的蛋白的表達(dá)量分別占蛋白表達(dá)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(dá)量分別為2.53 mg/mL,2.13 mg/mL,1.62 mg/mL,純化蛋白復(fù)性后蛋白的表達(dá)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(cè)定復(fù)性后的酶蛋白活性,催化水解可以得到相應(yīng)的產(chǎn)物,證明PM1509表達(dá)的蛋白有L-谷氨酰胺酶酶活,PM1383和PM3012表達(dá)的蛋白有L-天冬酰胺酶活性。利用SOMPA軟件、Predict Protein數(shù)據(jù)庫(kù)對(duì)PM2486和PM3475基因進(jìn)行編碼蛋白的二級(jí)結(jié)構(gòu)組成預(yù)測(cè),結(jié)果顯示β-折疊較少,而無(wú)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(dǎo)致了復(fù)性后的蛋白無(wú)活性的可能性較大,故選取巴斯德畢赤酵母GS115進(jìn)行真核表達(dá)。篩選得到的轉(zhuǎn)化子經(jīng)過(guò)驗(yàn)證大多數(shù)為甲醇利用快型,以甲醇為誘導(dǎo)劑誘導(dǎo)蛋白表達(dá),重組菌甲醇誘導(dǎo)表達(dá)后SDS-PAGE檢測(cè)目的蛋白得到表達(dá),發(fā)酵液上清和菌體中都有蛋白表達(dá),以哌嗪-2-甲酰胺為底物進(jìn)行酶活測(cè)定,結(jié)果顯示PM2486表達(dá)的蛋白在發(fā)酵液上清和菌體中且都有活性,而PM3475表達(dá)的蛋白沒(méi)有活性。對(duì)初篩發(fā)酵液上清所含蛋白含量較高的轉(zhuǎn)化子PM2486-5進(jìn)行誘導(dǎo)表達(dá),通過(guò)對(duì)每天樣品的檢測(cè)可知隨著誘導(dǎo)時(shí)間的延長(zhǎng),蛋白表達(dá)量在增加,Bradford法測(cè)定PM2486-5誘導(dǎo)表達(dá)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(hào)：2438743