本文選題：巧克力微桿菌　切入點(diǎn)：酯酶　出處：《上海應(yīng)用技術(shù)大學(xué)》2016年碩士論文

【摘要】：酯酶(Esterases,EC 3.1.1.1)是一類可以催化酯鍵水解和形成的酶,現(xiàn)在已經(jīng)成為一種重要的生物催化劑應(yīng)用于制藥、食品和生物降解等領(lǐng)域。我們課題組在前期的工作中,篩選出一株巧克力微桿菌SIT101,其所產(chǎn)酯酶能夠高立體選擇性的催化內(nèi)消旋的生物素中間體二甲酯不對稱水解為對應(yīng)的(4S,5R)構(gòu)型的半酯,它是合成D-生物素的重要手性中間體。由于野生菌產(chǎn)酶水平較低,將酯酶克隆表達(dá)于重組菌將有可能顯著提高其表達(dá)水平,提升其工業(yè)應(yīng)用潛力。本文首先在測定巧克力微桿菌SIT101基因組草圖的基礎(chǔ)上,采用基因組數(shù)據(jù)挖掘結(jié)合計(jì)算機(jī)輔助虛擬篩選的策略,預(yù)測了 5種可能具有催化活性的酯酶序列,通過酶基因的克隆表達(dá)以及活性和選擇性的測試確定了酯酶EstSIT101能夠催化生物素中間體二甲酯不對稱水解為相應(yīng)的(4S,5R)-半甲酯,對映體過量值高達(dá)99%。其次,從溫度、pH值、底物特異性、動(dòng)力學(xué)等方面對酯酶EstSIT101的酶學(xué)性質(zhì)進(jìn)行了表征,為進(jìn)一步應(yīng)用奠定了基礎(chǔ)。主要實(shí)驗(yàn)結(jié)論如下:(1)巧克力微桿菌SIT01酯酶的篩選、克隆表達(dá)與純化。首先應(yīng)用基因組挖掘的方法從Microbacterium chocolatum SIT 101基因組中預(yù)測了31個(gè)酯水解酶基因,采用Swiss-Model軟件對它們進(jìn)行了同源建模,并將其與底物分子進(jìn)行了分子對接,以二酯酯鍵021與活性位點(diǎn)組氨酸的咪唑的H原子之間的距離作為計(jì)算機(jī)輔助篩選的重要判據(jù),從中預(yù)測了五種酯酶可能會(huì)立體選擇性催化水解生物素中間體二甲酸,對結(jié)合能最低的三種酯酶進(jìn)行了克隆表達(dá)和活性測試。發(fā)現(xiàn)只有酯酶EstSIT01具有催化生物素中間體二甲酯水解生成(4S,5R)生物素半甲酯。經(jīng)過鎳柱純化和超濾離心后的純化酶濃度為3.56mg/mL,目標(biāo)酶占細(xì)胞總的可溶性蛋白的28.2%。SDS-PAGE結(jié)果顯示酯酶EstSIT01分子量約為39kDa,與理論分子量為39.35kDa相符。酯酶EstSIT01包含370個(gè)氨基酸,同源建模的結(jié)果表明,EstSIT01擁有典型的α/β水解酶折疊結(jié)構(gòu)和保守的催化三聯(lián)體Ser110-His330-Asp268。(2)酯酶EstSIT01酶學(xué)性質(zhì)的表征。酯酶EstSIT01傾向于水解短鏈酯類,而且該酶立體選擇性水解生物素二甲酯生成生物素半甲酯的產(chǎn)率和e.e值均高達(dá)99%。酯酶EstSIT01對對硝基苯酚乙酸酯(p-NPA)和生物素中間體二甲酯的最適反應(yīng)溫度分別為65℃和45℃,最適pH分別為9.5和10.0。在30℃下,EstSIT01比較穩(wěn)定,而過堿(pH10)不利于酶的穩(wěn)定性。并且以生物素二甲酯為底物,溫度和pH對e.e值沒有影響。當(dāng)溫度為30℃,pH 8.0時(shí),Km和Vmax分別為0.1468mM和8.856μM/min。酯酶EstSIT01高選擇性和高活力表明它是一種在制藥工業(yè)上有前景的生物催化劑。
[Abstract]:Esterasesus (EC 3.1.1.1) is a kind of enzyme which can catalyze the hydrolysis and formation of ester bonds. It has been used as an important biocatalyst in pharmaceutical, food and biodegradation fields.In our earlier work, a strain of microbacillus chocolate SIT101 was screened. The esterase produced by SIT101 can catalyze asymmetric hydrolysis of dimethyl ester, a biotinylated intermediate with high stereoselectivity, into a corresponding semi-ester with a 4S5R configuration, and the esterase produced by SIT101 can catalyze asymmetric hydrolysis of dimethyl ester, a biotin intermediate, with high stereoselectivity.It is an important chiral intermediate for the synthesis of D-biotin.Due to the low level of enzyme production by wild bacteria, it is possible to improve the expression level of esterase by cloning esterase in recombinant bacteria and enhance its potential for industrial application.In this paper, based on the determination of SIT101 genome sketches of microbacillus chocolate, five possible esterase sequences with catalytic activity were predicted by using genomic data mining combined with computer-aided virtual screening strategy.It was confirmed that esterase EstSIT101 could catalyze asymmetric hydrolysis of dimethyl ester, an intermediate of biotin, into corresponding semi-methyl ester by cloning, expression and activity of enzyme gene. The enantiomeric overdose of esterase EstSIT101 was as high as 99g.Secondly, the enzymatic properties of esterase EstSIT101 were characterized from temperature and pH value, substrate specificity and kinetics, which laid a foundation for further application.The main results are as follows: 1) screening, cloning, expression and purification of SIT01 esterase from microbacillus chocolate.First, 31 esterase genes were predicted from the genome of Microbacterium chocolatum SIT 101 by the method of genome mining, and the homologous modeling of them was carried out by Swiss-Model software, and they were docked with the substrate molecule.Using the distance between diester ester bond 021 and H atom of active site histidine imidazole as an important criterion of computer-aided screening, it is predicted that five esterases may catalyze stereoselective hydrolysis of biotin intermediate dicarboxylic acid.Three esterases with the lowest binding energy were cloned, expressed and tested for their activity.It was found that only esterase EstSIT01 could catalyze the hydrolysis of dimethyl ester, an intermediate of biotin, to form biotinyl hemimethyl ester.After purification by nickel column and ultrafiltration centrifugation, the concentration of purified enzyme was 3.56 mg / mL. The result of 28.2%.SDS-PAGE showed that the molecular weight of esterase EstSIT01 was about 39 kDa, which was consistent with the theoretical molecular weight of 39.35kDa.Esterase EstSIT01 tends to hydrolyze short chain esters, and the yield and e.e value of stereoselective hydrolysis of biotinyldimethyl ester to biotin hemimethyl ester are as high as 99e.The optimum reaction temperature of esterase EstSIT01 for p-nitrophenol acetate (p-NPA) and biotin intermediate dimethyl ester was 65 鈩，

本文編號(hào)：1700921