本文選題：酮泛解酸內(nèi)酯還原酶 + 釀酒酵母　； 參考：《浙江工業(yè)大學(xué)》2017年碩士論文

【摘要】：D-泛酸,俗稱維生素B5,可以維持頭發(fā),血液和皮膚的健康,同時它可以作為輔酶A的前體。目前,主要以D-泛解酸內(nèi)酯和β-丙氨酸為原料通過化學(xué)法合成D-泛酸。前體D-泛解酸內(nèi)酯主要通過化學(xué)法合成和水解酶動力學(xué)拆分獲得,但化學(xué)法步驟繁瑣,污染環(huán)境,而水解酶拆分過程中需要化學(xué)外消旋化和酸化內(nèi)酯化過程,增大了額外的生產(chǎn)成本。因此,開發(fā)全新的D-泛解酸內(nèi)酯合成工藝具有非常大的意義。本文以釀酒酵母基因組為模板,成功克隆出酮泛解酸內(nèi)酯還原酶基因SceCPR1,并成功構(gòu)建了SceCPR1與葡萄糖脫氫酶EsGDH偶聯(lián)的輔酶循環(huán)再生系統(tǒng),即“一菌雙酶”體系,用于高效不對稱還原酮泛解酸內(nèi)酯獲得D-泛解酸內(nèi)酯;SceCPR1和EsGDH蛋白分子大小分別為35 kDa和27 kDa。通過誘導(dǎo)條件優(yōu)化,最終單位濕菌體中SceCPR1和Es GDH的酶活分別為1179.2 U/g和442.8 U/g。SceCPR1酶學(xué)性質(zhì)表征發(fā)現(xiàn),其最適的pH為5.5,溫度45℃,并且酶在45℃下容易變性,但當(dāng)添加5 mM的NADPH可以保證其溫度穩(wěn)定性;其次,SceCPR1不屬于金屬離子依賴型還原酶,但是5mM的Fe3+以及大多數(shù)有機溶劑對酶活力有抑制作用;底物譜研究發(fā)現(xiàn)該酶對于酮泛解酸內(nèi)酯具有最高的活力,而對于酮泛解酸以及D-或L-泛解酸內(nèi)酯都無活力;在低底物濃度條件下,測定了SceCPR1的動力學(xué)參數(shù),SceCPR1對酮泛解酸內(nèi)酯和NADPH的Km值分別為0.164 mM和0.029 mM,反應(yīng)的Vmax分別為131.03 U/mg和137.81U/mg。以BL21(DE3)/pACYCDuet 1-SceCPR1/EsGDH的凍干細(xì)胞作為生物催化劑,優(yōu)化全細(xì)胞催化條件,確定最適反應(yīng)pH為5.5,溫度35℃,生物催化劑添加量為0.03 g/mL,輔底物葡萄糖與底物配比為1.5:1.0,攪拌速度400 rpm;通過對底物水解的研究,發(fā)現(xiàn)底物自發(fā)水解是影響產(chǎn)物得率的關(guān)鍵因素。因此,在最適催化條件下,將酮泛解酸內(nèi)酯和葡萄糖溶解于p H 2.5的溶液中,采用持續(xù)流加補料的方法,最終產(chǎn)物濃度達(dá)到475 mM,得率95%,產(chǎn)物光學(xué)純度e.e.p≥99.9%。
[Abstract]:D- pantothenic acid, commonly known as vitamin B 5, maintains healthy hair, blood and skin, and it acts as a precursor to coenzyme A. At present, D-pantothenic acid was synthesized by chemical method from D-panactone and 尾-alanine. The precursor D-panlytic acid lactone was mainly synthesized by chemical synthesis and kinetic resolution of hydrolase, but the steps of chemical method were tedious and polluted the environment, and the hydrolase resolution process needed chemical racemization and acidizing internal esterification. Additional production costs are increased. Therefore, it is of great significance to develop a new synthesis process of D-panalactone. Using Saccharomyces cerevisiae genome as template, the gene SceCPR1 was cloned successfully, and the system of coenzyme cycle regeneration coupled with glucose dehydrogenase (EsGDH) was successfully constructed, that is, "one bacterium double enzyme" system. The molecular sizes of SceCPR1 and EsGDH protein were 35 kDa and 27 kDa, respectively. The enzyme activity of SceCPR1 and es GDH was 1179.2 U / g and 442.8 U/g.SceCPR1, respectively. The optimum pH was 5.5, the temperature was 45 鈩，

本文編號：1978332