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(shí)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(guò)化學(xué)法合成D-泛酸。前體D-泛解酸內(nèi)酯主要通過(guò)化學(xué)法合成和水解酶動(dòng)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(guò)程中需要化學(xué)外消旋化和酸化內(nèi)酯化過(guò)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(duì)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(guò)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(jī)溶劑對(duì)酶活力有抑制作用;底物譜研究發(fā)現(xiàn)該酶對(duì)于酮泛解酸內(nèi)酯具有最高的活力,而對(duì)于酮泛解酸以及D-或L-泛解酸內(nèi)酯都無(wú)活力;在低底物濃度條件下,測(cè)定了SceCPR1的動(dòng)力學(xué)參數(shù),SceCPR1對(duì)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(guò)對(duì)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(bǔ)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 B5, can maintain the health of hair, blood and skin. At the same time it can be used as a precursor of coenzyme A. Currently, D- pantothenic acid is synthesized by chemical method mainly by D- pantol and beta alanine. Precursor D- pan lactate is obtained by chemical synthesis and hydrolase kinetic resolution, but chemical process steps The process of chemical racemization and acidification in the process of hydrolysis of hydrolase requires the process of esterification of chemical racemization and acidification, which increases the extra cost of production. Therefore, it is very important to develop a new synthesis process of D- flooding acid lactone. This paper successfully cloned the ketone acid lactone reductase gene SceCPR1 by using the Saccharomyces cerevisiae genome as a template. SceCPR1 and glucose dehydrogenase EsGDH coupled coenzyme regeneration system was successfully constructed, that is, "one bacteria double enzyme" system, which is used for high efficiency unsymmetrical reducing ketone acid lactone to obtain D- flooding acid lactone. The molecular size of SceCPR1 and EsGDH protein molecules are 35 kDa and 27 kDa., respectively, by optimization of the inducement conditions, and finally the SceCPR1 in the unit wet mycelium and in the final unit. The enzyme activity of Es GDH was 1179.2 U/g and 442.8 U/g.SceCPR1 characterization respectively. The optimum pH was 5.5, the temperature was 45, and the enzyme was easily denatured at 45, but when the 5 mM NADPH was added to ensure its temperature stability; secondly, SceCPR1 did not belong to the metal ion dependent reductase, but 5mM Fe3+ and most organic solvents. The enzyme activity was inhibited by the enzyme, and the enzyme was found to have the highest activity for the ketone acid lactone, while the ketone flooding acid and D- or L- pan acid lactone were not active, and the kinetic parameters of SceCPR1 were measured under the low substrate concentration. The Km values of SceCPR1 to ketone and NADPH were 0.164 mM and 0.029 mM, respectively. The reaction Vmax is 131.03 U/mg and 137.81U/mg. using BL21 (DE3) /pACYCDuet 1-SceCPR1/EsGDH lyophilized cells as biocatalysts to optimize the whole cell catalytic conditions. The optimum reaction pH is 5.5, the temperature is 35, the addition of biocatalysts is 0.03 g/mL, the auxiliary substrate grape sugar and the substrate ratio is 1.5:1.0, and the stirring speed is 400 rpm; through the The study of substrate hydrolysis found that the spontaneous hydrolysis of the substrate was the key factor affecting the yield of the products. Therefore, under the optimum conditions, the solution of ketoacid lactone and glucose dissolved in P H 2.5 solution under the optimum catalytic condition, the final product concentration reached 475 mM, the yield was 95%, the optical purity of the product was E.E.P > 99.9%..
【學(xué)位授予單位】：浙江工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q55;Q78;O621.3

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