【摘要】：生物催化法由于具有立體選擇性強、反應條件溫和、環(huán)境友好等優(yōu)點而深受青睞，酶立體選擇性的定向調(diào)控在生物催化中具有重要意義。通過對酶不對稱催化選擇性的定向調(diào)控，可以控制反應進行的方向與程度，獲得高光學純度的、具有特殊結(jié)構(gòu)的目標產(chǎn)物。本文以磷脂酶B（PLB）和氧化還原酶為研究對象，探討其立體選擇性的定向調(diào)控策略，主要研究結(jié)果如下： （1）對菌株Candida albicans MYA-2876產(chǎn)磷脂酶B的發(fā)酵條件進行了優(yōu)化，確定了最適發(fā)酵溫度30℃，初始pH6.5，接種量為8%，培養(yǎng)時間為72h。初始純化酶條件為30%濃度飽和硫酸銨，80%濃度沉淀目的蛋白，經(jīng)過透析得到濃縮酶液，酶活可達201.36U/mg-protein。 （2）考察了Candida albicans MYA-2876PLB的酶學性質(zhì)，結(jié)果表明其最適反應溫度是30℃，最適pH為7.8。溫度與pH對酶活影響較大，0.1mM Mg2+與Ca2+對酶活有促進作用，分別提高了23.5%與18.8%，PLB對卵磷脂乳化液的底物適應性較好。 （3）考察反應條件(反應溫度、pH以及金屬離子)、介質(zhì)工程（有機溶劑、離子液體、兩相體系）、化學修飾與固定化酶對磷脂酶B的催化位點選擇性的影響。結(jié)果表明，離子液體對磷脂酶B的催化位點選擇性的調(diào)控效果較好，在[Omim][D-HL]作用下磷脂酶B的Sn-1位點催化選擇性CSS1得到較大提高（接近100%），而在[Emim]Cl作用下Sn-2位點催化選擇性CSS2提高到47.08%；兩相體系對磷脂酶B的催化位點選擇性的調(diào)控效果一般，對產(chǎn)率影響較大，大部分相比水相提高3倍以上。正己烷-水（體積比1:1）兩相系統(tǒng)CSS1提高最大至84.78%，2-庚醇-水（體積比1:1）兩相系統(tǒng)CSS2提高最大至43.77%。 （4）考察了乙醇脫氫酶ADH-A3263，ADH-A7011以及大腸桿菌重組酮還原酶E-Ketoreductase與八個底物2-辛酮、2-庚酮、苯乙酮、1-苯丙酮、1-苯乙醇、1-苯丙醇、2-庚醇和2-辛醇間的酶催化反應。結(jié)果表明，此三種酶均在30℃到35℃時酶活較高；ADH-A3263、ADH-A7011酶活最適pH偏堿性，E-Ketoreductase酶活最適pH偏中性；利用自動旋光儀測旋光率通過考察反應溫度與pH，初步篩選出更合適的酶催化底物苯乙酮、1-苯丙酮、1-苯乙醇1-苯丙醇。 （5）考察反應系統(tǒng)(反應溫度、pH以及金屬離子)、介質(zhì)工程（有機溶劑、離子液體、兩相體系）、酶的化學修飾、固定化酶以及輔酶再生對氧化還原酶的對映體選擇性的影響。結(jié)果表明，本實驗中有機溶劑對E-Ketoreductase催化1-苯丙酮的對映體選擇性調(diào)控有較好效果，在異辛烷（含水量1%）中反應S型對映體選擇率（E）高達30.885，，而在正己烷（含水量1%）中反應時卻為R型選擇性，E值為3.207；離子液體對E-Ketoreductase催化1-苯丙酮的對映體選擇性調(diào)控有十分明顯的效果，產(chǎn)物發(fā)生構(gòu)象翻轉(zhuǎn)，反應體系添加[Emim][HL]后R型E值為6.769，而添加[Bmim]PF6時卻為S型選擇性，E值高達到200以上；兩相體系對E-Ketoreductase催化1-苯丙酮的對映體選擇性調(diào)控有很好的效果，且與有機溶劑體系相比，轉(zhuǎn)化率普遍都有一定提高，在異辛烷-水（體積比1:1）中反應S型對映體選擇率E最大達到333.448，而在辛酸乙酯-水（體積比1:1）中反應時R型E最大達到7.890。
[Abstract]:Biocatalytic method is very popular because of its strong stereoselectivity, mild reaction conditions and friendly environment. The directional regulation of enzyme stereoselectivity is of great significance in biocatalysis. In this paper, phospholipase B (PLB) and oxidoreductase were studied to investigate the stereoselective directional regulation strategies. The main results are as follows:
(1) The optimum fermentation conditions for the production of phospholipase B by Candida albicans MYA-2876 were optimized. The optimum fermentation temperature was 30 C, initial pH 6.5, inoculation amount was 8%, culture time was 72 h. The initial purified enzyme conditions were 30% saturated ammonium sulfate, 80% precipitated the target protein, and the concentrated enzyme solution was obtained after dialysis. The enzyme activity was 201.36 U/mg-protein.
(2) The enzymatic properties of Candida albicans MYA-2876 PLB were investigated. The results showed that the optimum reaction temperature was 30 C and the optimum pH was 7.8. Temperature and pH had a great influence on the enzyme activity. 0.1 mMg2+ and Ca2+ promoted the enzyme activity by 23.5% and 18.8% respectively. PLB had a better substrate adaptability to lecithin emulsion.
(3) The effects of reaction conditions (reaction temperature, pH and metal ions), medium engineering (organic solvents, ionic liquids, two-phase system), chemical modification and immobilized enzyme on the site selectivity of phospholipase B were investigated. Catalytic selectivity of CSS1 at the Sn-1 site of enzyme B was greatly improved (nearly 100%) while the selectivity of CSS2 at the Sn-2 site was increased to 47.08% under the action of [Emim] Cl. The two-phase system had a general effect on the selectivity of catalytic site of phospholipase B, and had a greater effect on the yield, most of which was more than three times higher than that in the aqueous phase. The phase system CSS1 increased to the maximum of 84.78%, and the CSS2 of 2- heptanol water (volume ratio 1:1) two-phase system increased to the maximum of 43.77%..
(4) The enzymatic reactions of alcohol dehydrogenase ADH-A3263, ADH-A7011, recombinant ketone reductase E-Ketoreductase with eight substrates, 2-octanone, 2-heptanone, acetophenone, 1-phenylacetone, 1-phenylethanol, 1-phenylpropanol, 2-heptanol and 2-octanol, were investigated. The optimum pH of enzyme activity was alkaline, and the optimum pH of E-Ketoreductase activity was neutral. The optimum substrate for enzyme catalysis was acetophenone, 1-phenylacetone and 1-phenylethanol-1-phenylpropanol by measuring the optical rotation with automatic polarimeter.
(5) The effects of reaction system (reaction temperature, pH and metal ions), medium engineering (organic solvent, ionic liquid, two-phase system), chemical modification of enzyme, immobilized enzyme and coenzyme regeneration on the enantioselectivity of redox enzyme were investigated. The results showed that the enantioselectivity of 1-phenylacetone catalyzed by E-Ketoreductase in organic solvents was improved. The selectivity of S-type enantiomers (E) was as high as 30.885 in isooctane (water content 1%) and R-type enantiomers (E value 3.207) in n-hexane (water content 1%). Ionic liquids had a very obvious effect on the enantioselective regulation of 1-phenylacetone catalyzed by E-Ketoreductase. When [Emim] [HL] was added, the R-type E value was 6.769, but when [Bmim] PF6 was added, it was S-type selectivity, and the E value was above 200. The two-phase system had a good effect on enantioselective regulation of 1-phenylacetone catalyzed by E-Ketoreductase, and the conversion of 1-phenylacetone was generally higher than that of organic solvent system. The maximum enantioselectivity E of S-type in the medium reaction was 333.448, and that of R-type E in the ethyl octanoate-water (volume ratio 1:1) was 7.890.
【學位授予單位】：北京理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：TQ032.4

【參考文獻】

相關期刊論文 前10條

1 李康;尹春華;喬世偉;張淵洲;張聰;閆海;;酸酐修飾脂肪酶的研究[J];安徽農(nóng)業(yè)科學;2011年31期

2 劉建忠,宋海燕,翁麗萍,計亮年;化學修飾改進酶的催化特性研究進展[J];分子催化;2002年06期

3 李彥鋒,李軍榮,伏蓮娣;固定化酶的制備及應用[J];高分子通報;2001年02期

4 楊朝芬;楊俊;朱艷琴;孫曉東;陳華;;L-脯氨酸修飾的銥催化苯乙酮及其衍生物不對稱加氫反應[J];分子催化;2013年06期

5 諶容;王秋巖;殷曉浦;魏東芝;謝恬;;醇脫氫酶不對稱還原制備手性醇的研究進展[J];化工進展;2011年07期

6 姜芳燕;王金梅;戴大章;李春;;產(chǎn)磷脂酶菌株的篩選鑒定及其應用[J];化工學報;2012年03期

7 張榮珍;徐巖;耿亞維;王珊珊;孫瑩;;羰基還原酶與甲酸脫氫酶偶聯(lián)催化制備(R)-苯基乙二醇[J];過程工程學報;2009年04期

8 王金梅;姜芳燕;戴大章;李春;;磷脂酶高產(chǎn)菌株的篩選、誘變及其在豆油脫膠中的應用[J];環(huán)境科學研究;2010年07期

9 許建和;胡英;;生物轉(zhuǎn)化中的有機介質(zhì)系統(tǒng)[J];化學世界;1991年07期

10 曹淑桂;有機溶劑中酶催化研究的新進展─—酶催化活性和選擇性的控制與調(diào)節(jié)[J];化學通報;1995年05期

相關博士學位論文 前1條

1 楊光;脂肪酶固定化的新方法研究及其應用[D];浙江大學;2009年

本文編號：2198506