立體選擇性酰胺酶的篩選及其動(dòng)力學(xué)拆分制備

1、浙江工業(yè)大學(xué)博士學(xué)位論文立體選擇性酰胺酶的篩選及其動(dòng)力學(xué)拆分制備S-(+)-2,2-二甲基環(huán)丙烷甲酰胺的研究摘 要手性生物催化已成為制備光學(xué)純化合物的經(jīng)典方法。腈轉(zhuǎn)化酶尤其是酰胺酶，底物范圍廣、立體選擇性專(zhuān)一，在制備手性藥物和農(nóng)用化學(xué)品中間體中發(fā)揮越來(lái)越重要的作用。S-(+)-2,2-二甲基環(huán)丙烷甲酰胺是腎二肽脫氫酶抑制劑西司他丁合成的關(guān)鍵中間體。通過(guò)R-立體選擇性酰胺酶動(dòng)力學(xué)拆分，制備S-(+)-2,2-二甲基環(huán)丙烷甲酰胺，反應(yīng)條件溫和、立體專(zhuān)一性好、環(huán)境友好，具有很好的工業(yè)化前景。本論文圍繞該工藝路線，就生物催化劑的發(fā)現(xiàn)、表征、制備與應(yīng)用等幾方面展開(kāi)研究。論文首先建立了一種新的立體選擇性酰

2、胺酶的篩選模型。該模型基于酰胺酶的酰基轉(zhuǎn)移活性，即在鹽酸羥胺存在的條件下，酰胺酶能夠催化酰胺生成相應(yīng)的氧肟酸。然后經(jīng)氧肟酸/Fe(III)復(fù)合物的顯色反應(yīng)，快速測(cè)定酰胺酶的活力及立體選擇性。為驗(yàn)證模型的準(zhǔn)確性，考察了酰胺酶催化的?；D(zhuǎn)移反應(yīng)和水解反應(yīng)在立體選擇性上的差異。通過(guò)該模型，從523株菌種中篩選到產(chǎn)酰胺酶的菌種8株，其中2株能夠R-型立體選擇性降解2,2-二甲基環(huán)丙烷甲酰胺。為了實(shí)現(xiàn)對(duì)生物拆分過(guò)程的監(jiān)測(cè)和控制，研究了外消旋2,2-二甲基環(huán)丙烷甲酰胺及相應(yīng)的甲酸在一根商品化的手性色譜柱BGB-175上的手性分離。根據(jù)動(dòng)力學(xué)拆分過(guò)程中的手性平衡原理，建立了一種基于底物和產(chǎn)物的對(duì)映體過(guò)量值(

3、ee)測(cè)定轉(zhuǎn)化液中4種對(duì)映異構(gòu)體濃度的新方法。由于該方法只引入相對(duì)量(ee)，因此可以有效避免樣品處理、進(jìn)樣方式等引入的誤差，提高準(zhǔn)確度。運(yùn)用形態(tài)學(xué)、生理生化試驗(yàn)、ATB自動(dòng)鑒定系統(tǒng)、16S rRNA序列及系統(tǒng)發(fā)育分析等手段，對(duì)由篩選模型得到的一株R-型酰胺酶產(chǎn)生菌ZJB-05174進(jìn)行鑒定。該菌株鑒定為Delftia tsuruhatensis，這是該種內(nèi)首次報(bào)道的產(chǎn)R-型酰胺酶的菌株。D. tsuruhatensis ZJB-05174在30 C下，水解外消旋2,2-二甲基環(huán)丙甲酰胺的平均對(duì)映體選擇率(E)為27；該菌株胞內(nèi)酰胺酶的熱穩(wěn)定性好，在30和40 C下的半衰期分別達(dá)到78.6和4

4、6.2 h；酰胺酶的常用抑制劑尿素對(duì)D. tsuruhatensis ZJB-05174酰胺酶的抑制效果不明顯。這可能是該酰胺酶活性位點(diǎn)結(jié)構(gòu)與其它酰胺酶不同引起的。驗(yàn)證了用以乙酰胺為底物的?；D(zhuǎn)移反應(yīng)替代直接水解外消旋2,2-二甲基環(huán)丙甲酰胺測(cè)定酰胺酶活力的可行性。通過(guò)單因素實(shí)驗(yàn)和正交試驗(yàn)，對(duì)D. tsuruhatensis ZJB-05174產(chǎn)酰胺酶的培養(yǎng)基組成進(jìn)行了優(yōu)化。確定較佳的培養(yǎng)基組成為(g/l)：葡萄糖8.4，乙酰胺3.56，酵母抽提物6.3，蛋白胨0.7， KH2PO41.0，K2HPO4 1.0，NaCl 1.0。D. tsuruhatensis ZJB-05174產(chǎn)酶的適宜培

5、養(yǎng)條件為：溫度 30 C，初始pH 7.5，接種量4% (v/v)，裝液量16%(v/v)。上述條件下，該菌株指數(shù)生長(zhǎng)期的比生長(zhǎng)速率為0.33 h-1，培養(yǎng)20 h后由?；D(zhuǎn)移反應(yīng)表示的酰胺酶的活力達(dá)到1.51 U/ml發(fā)酵液，是優(yōu)化前的2.65倍。論文還研究了酶催化反應(yīng)的(微)環(huán)境對(duì)該酰胺酶活力和選擇性的影響。結(jié)果表明，該酰胺酶的適宜工作pH為7.68.8。該酶在偏酸（pH 5.4）或偏堿（pH 9.4）的環(huán)境下，表現(xiàn)出比中性條件更高選擇性，E值分別達(dá)到60和71。該酶在41 C時(shí)酶活最高，但其立體選擇性隨溫度的升高而不斷降低，直至立體選擇性的完全反轉(zhuǎn)。該酶催化反應(yīng)過(guò)程的2個(gè)重要熱力學(xué)參數(shù)，

6、熵變和焓變?cè)诰w熱處理 (56 C) 前后發(fā)生改變，是造成立體選擇性不可逆反轉(zhuǎn)的根本原因。向反應(yīng)體系中添加共溶劑乙醇和乙腈后，酶活力分別提高2.7和2.2倍，E值由32上升至91和140。經(jīng)反應(yīng)條件優(yōu)化后，酰胺酶的活力由14.3 mol min-1 g-1提高到91.8 mol min-1 g-1，為原來(lái)的6.2倍。產(chǎn)物經(jīng)分離、純化，制備得到S-(+)-2,2-二甲基環(huán)丙烷甲酰胺樣品，總收率達(dá)到43.6%。樣品經(jīng)旋光儀、紅外光譜和核磁共振表征，表明其化學(xué)純度和光學(xué)純度均達(dá)到99%以上。論文最后考察了D. tsuruhatensis ZJB-05174酰胺酶催化的立體選擇性?；D(zhuǎn)移反應(yīng)。結(jié)果表明

7、，添加共溶劑會(huì)加快副反應(yīng)酰胺水解的速率，對(duì)酰基轉(zhuǎn)移反應(yīng)的初速率沒(méi)有影響。但隨著副反應(yīng)的加劇，底物消耗量增加，?；D(zhuǎn)移反應(yīng)的速率隨之降低。相反地，增加反應(yīng)體系中羥胺的濃度，可以有效降低副反應(yīng)的發(fā)生。當(dāng)羥胺和底物的濃度比達(dá)到10:1時(shí)，?；D(zhuǎn)移反應(yīng)和水解反應(yīng)的速度相當(dāng)。對(duì)該酶催化的?；D(zhuǎn)移反應(yīng)的動(dòng)力學(xué)研究表明，其催化過(guò)程屬于雙底物乒乓機(jī)制。其動(dòng)力學(xué)參數(shù)如下：表觀最大反應(yīng)速率：129.9 mol min-1 g-1，對(duì)羥胺的米氏常數(shù)：150 mM，對(duì)酰胺的米氏常數(shù)：=10 mM。關(guān)鍵詞：酰胺酶，高通量篩選，手性分離，手性平衡，動(dòng)力學(xué)拆分，立體選擇性反轉(zhuǎn)，S-(+)-2,2-二甲基環(huán)丙烷甲酰胺，共溶劑

8、，?；D(zhuǎn)移反應(yīng)，氧肟酸SCREENING FOR ENANTIOSELECTIVE AMIDASE: KINETIC RESOLUTION OF RACEMATE TO S-(+)-2,2-DIMETHYLCYCLOPROPANE CARBOXAMIDEABSTRACTChiral biocatalysis has already been a classical method in preparation enantiomerically pure compounds. Nitrile-converting enzymes, especially amidase, with wide sub

9、strate spectrum and strict stereospecificity, are playing more and more important roles in industrial biotransformations for production of optically pure pharmaceuticals and agrochemicals. S-(+)-2,2-dimethylcyclopropane carboxamide is a key intermediate in the synthesis of cilastatin, a renal dehydr

10、opeptidase inhibitor. Preparation of S-(+)-2,2-dimethylcyclopropane carboxamide by R-enantioselective amidase catalyzed kinetic resolution proceeds under mild conditions with excellent enantioselectivity and has great potential for industrialization. A novel enantioselective amidase screening system

11、 was developed and proved to be efficient and accurate. This screening system employed acyl transfer activity of amidase in the presence of hydroxylamine, leading to the formation of hydroxamic acids, followed by spectrophotometric quantification of hydroxamic acid/iron (III) complex. To prove the a

12、ccuracy of the screening system, the difference between enantioselectivity of acyl transfer reaction and that of hydrolysis reaction was evaluated. With this method, we obtained eight microorganism strains with enantioselective amidase from 523 isolates, two of which showed R-stereospecific activity

13、 for (R,S)-2,2-dimethylcyclopropane carboxamide.In order to monitor and control the bioconversion process, enantioseparation of 2,2-dimethylcyclopropanecarboxamide and corresponding acid was performed on a commercial chiral column BGB-175. Based on chiral balance in kinetic resolution progress, a no

14、vel method, employed enantiomeric excess of both substrate and product, was developed for determination of concentration of enantiomers in bioconversion broth. Since only relative quantity (ee) was required in the proposed method, calibration and cumbersome quantitative sample handling can be avoide

15、d and analytical accuracy can be greatly improved.Strain ZJB-05174, capable of R-enantioselective degradation of 2,2-dimethylcyclopropane carboxamide, was isolated through the screening system. Based on morphology, physiological tests, ATB system and the 16S rRNA sequence, this strain was identified

16、 as Delftia tsuruhatensis. This is the first report on strains in this species with R-amidase activity. D. tsuruhatensis ZJB-05174 catalyzed hydrolysis of 2,2-dimethylcyclopropane carboxamide with an enantiomeric ratio (E value) of 27 at 30 C. The intracellular amidase exhibited excellent thermostab

17、ility with half-life (t1/2) of 78.6 and 46.2 h at 30 and 40 C, respectively. Urea, regular inhibitor of amidase, was not effective to amidase from D. tsuruhatensis ZJB-05174, which suggested that this amidase might have a different active site structure with other reported ones.The effects of medium

18、 composition and culture conditions on the amidase activity of D. tsuruhatensis ZJB-05174 were evaluated experimentally. The acyl transfer activity catalyzed by amidase with acetamide as substrate was firstly proved to be in accordance with correspondence hydrolysis activity. The optimized medium co

19、mposition was as follows (g/l): glucose 8.4, acetamide 3.56, yeast extraction 6.3, peptone 0.7, KH2PO41, K2HPO4 1, NaCl 1. The satisfactory fermentation conditions for cell growth and formation of amidase were as follows: initial pH value, 7.0; 30 C; inoculum volume, 4% (v/v); medium volumetric rati

20、o, 30% (v/v). Under these conditions, D. tsuruhatensis ZJB-05174 multiplied with growth rate () of 0.33 h-1. When D. tsuruhatensis ZJB-05174 was cultivated for 20 h, the enzyme activity, expressed as acyl transfer activity, reached 1.51 U/ml of culture broth, which was 1.65 times higher than before

21、optimization.Influences of reaction conditions on amidase activity and enantioselectivity were also investigated. Results indicated that optimal working pH of the amidase ranged from 7.6 to 8.8. Moreover, the amidase exhibited stricter stereospecificity under partial acid (pH 5.4) or partial alkali

22、(pH 9.4) conditions than it under neutral conditions. The amidase showed highest activity at 41 C, but its enantiomeric ratio decreased with increase of temperature, until reversal of stereospecificity was observed. Two thermodynamic parameters of the reaction, activation enthalpy and activation ent

23、ropy, changed dramatically after cell suspensions pre-incubated at 56 C, which was the reason why the reversal of stereospeccificity was irreversible. Addition of cosolvent, ethanol and acetonitrile, had significant effect not only on enzyme activity but also on enantioselectivity. The enzyme activi

24、ty was 2.7 and 2.2 times higher, and E-value increased from 32 to 91 and 140, respectively. After optimization of the reaction conditions, amidase activity increased from 14.3 mol min-1 g-1 to 91.8 mol min-1 g-1. S-(+)-2,2-dimethylcyclopropane carboxamide was isolated and purified from reaction mixt

25、ure with total yield of 43.6%. The sample was characterized by polarimeter, IR and 1H NMR analysis, and the results demonstrated that chemical and optical purity of the sample were both above 99%.Further, enantioselective acyl transfer reaction catalyzed by D. tsuruhatensis ZJB-05174 was investigated. Results showed that addition of cosolvent accelerated initial reaction rate of amide hydrolysis, but had no effect on that of acy