1、浙江工业大学博士学位论文I立体选择性酰胺酶的筛选及其动力学拆分制备S-(+)-2,2-二甲基环丙烷甲酰胺的研究摘 要手性生物催化已成为制备光学纯化合物的经典方法。腈转化酶尤其是酰胺酶，底物范围广、立体选择性专一，在制备手性药物和农用化学品中间体中发挥越来越重要的作用。S-(+)-2,2-二甲基环丙烷甲酰胺是肾二肽脱氢酶抑制剂西司他丁合成的关键中间体。通过 R-立体选择性酰胺酶动力学拆分，制备 S-(+)-2,2-二甲基环丙烷甲酰胺，反应条件温和、立体专一性好、环境友好，具有很好的工业化前景。本论文围绕该工艺路线，就生物催化剂的发现、表征、制备与应用等几方面展开研究。论文首先建立了一种新的立体选
2、择性酰胺酶的筛选模型。该模型基于酰胺酶的酰基转移活性，即在盐酸羟胺存在的条件下，酰胺酶能够催化酰胺生成相应的氧肟酸。然后经氧肟酸/Fe(III)复合物的显色反应，快速测定酰胺酶的活力及立体选择性。为验证模型的准确性，考察了酰胺酶催化的酰基转移反应和水解反应在立体选择性上的差异。通过该模型，从 523 株菌种中筛选到产酰胺酶的菌种 8 株，其中 2 株能够 R-型立体选择性降解 2,2-二甲基环丙烷甲酰胺。为了实现对生物拆分过程的监测和控制，研究了外消旋 2,2-二甲基环丙烷甲酰胺及相应的甲酸在一根商品化的手性色谱柱 BGB-175 上的手性分离。根据动力学拆分过程中的手性平衡原理，建立了一种基
3、于底物和产物的对映体过量值(ee) 测定转化液中 4 种对映异构体浓度的新方法。由于该方法只引入相对量(ee)，因此可以有效避免样品处理、进样方式等引入的误差，提高准确度。运用形态学、生理生化试验、ATB 自动鉴定系统、16S rRNA 序列及系统发育分析等手段，对由筛选模型得到的一株 R-型酰胺酶产生菌 ZJB-05174 进行鉴定。该菌株鉴定为 Delftia tsuruhatensis，这是该种内首次报道的产 R-型酰胺酶的菌株。D. tsuruhatensis ZJB-05174 在 30 C 下，水解外消旋 2,2-二甲基环丙甲酰胺的平均对映体选择率(E)为 27；该菌株胞内酰胺酶的
4、热稳定性好，在 30和 40 C 下的半衰期分别达到 78.6 和 46.2 h；酰胺酶的常用抑制剂尿素对浙江工业大学博士学位论文IID. tsuruhatensis ZJB-05174 酰胺酶的抑制效果不明显。这可能是该酰胺酶活性位点结构与其它酰胺酶不同引起的。验证了用以乙酰胺为底物的酰基转移反应替代直接水解外消旋 2,2-二甲基环丙甲酰胺测定酰胺酶活力的可行性。通过单因素实验和正交试验，对 D. tsuruhatensis ZJB-05174 产酰胺酶的培养基组成进行了优化。确定较佳的培养基组成为(g/l)：葡萄糖 8.4，乙酰胺 3.56，酵母抽提物 6.3，蛋白胨 0.7， KH2PO
5、41.0， K2HPO4 1.0， NaCl 1.0。D. tsuruhatensis ZJB-05174 产酶的适宜培养条件为：温度 30 C，初始 pH 7.5，接种量 4% (v/v)，装液量 16%(v/v)。上述条件下，该菌株指数生长期的比生长速率 为 0.33 h-1，培养 20 h 后由酰基转移反应表示的酰胺酶的活力达到 1.51 U/ml 发酵液，是优化前的 2.65 倍。论文还研究了酶催化反应的(微)环境对该酰胺酶活力和选择性的影响。结果表明，该酰胺酶的适宜工作 pH 为 7.68.8。该酶在偏酸（ pH 5.4）或偏碱（pH 9.4）的环境下，表现出比中性条件更高选择性，E
6、 值分别达到 60 和 71。该酶在 41 C 时酶活最高，但其立体选择性随温度的升高而不断降低，直至立体选择性的完全反转。该酶催化反应过程的 2 个重要热力学参数，熵变和焓变在菌体热处理 (56 C) 前后发生改变，是造成立体选择性不可逆反转的根本原因。向反应体系中添加共溶剂乙醇和乙腈后，酶活力分别提高 2.7 和 2.2 倍，E值由 32 上升至 91 和 140。经反应条件优化后，酰胺酶的活力由 14.3 mol min-1 g-1 提高到 91.8 mol min-1 g-1，为原来的 6.2 倍。产物经分离、纯化，制备得到S-(+)-2,2-二甲基环丙烷甲酰胺样品，总收率达到 43.
7、6%。样品经旋光仪、红外光谱和核磁共振表征，表明其化学纯度和光学纯度均达到 99%以上。论文最后考察了 D. tsuruhatensis ZJB-05174 酰胺酶催化的立体选择性酰基转移反应。结果表明，添加共溶剂会加快副反应酰胺水解的速率，对酰基转移反应的初速率没有影响。但随着副反应的加剧，底物消耗量增加，酰基转移反应的速率随之降低。相反地，增加反应体系中羟胺的浓度，可以有效降低副反应的发生。当羟胺和底物的浓度比达到 10:1 时，酰基转移反应和水解反应的速度相当。对该酶催化的酰基转移反应的动力学研究表明，其催化过程属于双底物乒乓机制。其动力学参数如下：表观最大反应速率： 129.9 mol
8、 mVmin-1 g-1，对羟胺的米氏常数： 150 mM，对酰胺的米氏常数：OHNK2浙江工业大学博士学位论文III=10 mM。amideK关键词：酰胺酶，高通量筛选，手性分离，手性平衡，动力学拆分，立体选择性反转，S-(+)-2,2-二甲基环丙烷甲酰胺，共溶剂，酰基转移反应，氧肟酸浙江工业大学博士学位论文IVSCREENING FOR ENANTIOSELECTIVE AMIDASE: KINETIC RESOLUTION OF RACEMATE TO S-(+)-2,2-DIMETHYLCYCLOPROPANE CARBOXAMIDEABSTRACTChiral biocatalysi
9、s has already been a classical method in preparation enantiomerically pure compounds. Nitrile-converting enzymes, especially amidase, with wide substrate spectrum and strict stereospecificity, are playing more and more important roles in industrial biotransformations for production of optically pure
10、 pharmaceuticals and agrochemicals. S-(+)-2,2-dimethylcyclopropane carboxamide is a key intermediate in the synthesis of cilastatin, a renal dehydropeptidase inhibitor. Preparation of S-(+)-2,2-dimethylcyclopropane carboxamide by R-enantioselective amidase catalyzed kinetic resolution proceeds under
11、 mild conditions with excellent enantioselectivity and has great potential for industrialization. A novel enantioselective amidase screening system was developed and proved to be efficient and accurate. This screening system employed acyl transfer activity of amidase in the presence of hydroxylamine
12、, leading to the formation of hydroxamic acids, followed by spectrophotometric quantification of hydroxamic acid/iron (III) complex. To prove the accuracy of the screening system, the difference between enantioselectivity of acyl transfer reaction and that of hydrolysis reaction was evaluated. With
13、this method, we obtained eight microorganism strains with enantioselective amidase from 523 isolates, two of which showed R-stereospecific activity for (R,S)-2,2-dimethylcyclopropane carboxamide.In order to monitor and control the bioconversion process, enantioseparation of 2,2-dimethylcyclopropanec
14、arboxamide and corresponding acid was performed on a commercial chiral column BGB-175. Based on chiral balance in kinetic resolution progress, a novel method, employed enantiomeric excess of both substrate and product, was developed for determination of concentration of enantiomers in 浙江工业大学博士学位论文Vb
15、ioconversion broth. Since only relative quantity (ee) was required in the proposed method, calibration and cumbersome quantitative sample handling can be avoided and analytical accuracy can be greatly improved.Strain ZJB-05174, capable of R-enantioselective degradation of 2,2-dimethylcyclopropane ca
16、rboxamide, was isolated through the screening system. Based on morphology, physiological tests, ATB system and the 16S rRNA sequence, this strain was identified as Delftia tsuruhatensis. This is the first report on strains in this species with R-amidase activity. D. tsuruhatensis ZJB-05174 catalyzed
17、 hydrolysis of 2,2-dimethylcyclopropane carboxamide with an enantiomeric ratio (E value) of 27 at 30 C. The intracellular amidase exhibited excellent thermostability 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 amidas
18、e 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 composition and culture conditions on the amidase activity of D. tsuruhatensis ZJB-05174 were evaluated experimentally. The acyl transfer a
19、ctivity catalyzed by amidase with acetamide as substrate was firstly proved to be in accordance with correspondence hydrolysis activity. The optimized medium composition 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
20、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 ratio, 30% (v/v). Under these conditions, D. tsuruhatensis ZJB-05174 multiplied with growth rate () of 0.33 h-1. When D. tsuruhatensis ZJB-05174
21、 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 optimization.Influences of reaction conditions on amidase activity and enantioselectivity were also investigated. Results indicated that opt
22、imal 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 (pH 9.4) conditions than it under neutral 浙江工业大学博士学位论文VIconditions. The amidase showed highest activity at 41 C, but its enantiomeric ratio
23、decreased with increase of temperature, until reversal of stereospecificity was observed. Two thermodynamic parameters of the reaction, activation enthalpy and activation entropy, changed dramatically after cell suspensions pre-incubated at 56 C, which was the reason why the reversal of stereospecci
24、ficity was irreversible. Addition of cosolvent, ethanol and acetonitrile, had significant effect not only on enzyme activity but also on enantioselectivity. The enzyme activity was 2.7 and 2.2 times higher, and E-value increased from 32 to 91 and 140, respectively. After optimization of the reaction
25、 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 mixture with total yield of 43.6%. The sample was characterized by polarimeter, IR and 1H NMR analysis, and the results demonstrat
26、ed 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
27、of acyl transfer reaction. However, acyl transfer reaction then slowed down because substrate concentration dropped sharply due to accelerated side reaction. On the contrary, increasing hydroxylamine concentration effectively inhibited hydrolysis reaction. When concentration ratio of hydroxylamine a
28、nd substrate reached 10:1, reaction rate of acyl transfer and hydrolysis was almost the same. Moreover, kinetic studies of the amidase catalyzed acyl transfer reaction demonstrated that the reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constant were as follows: 129.9 mol min-1 g-1, 150 mM, =10 mM。mVOHNK2 amideKKEY WORDS: amidase, high-throughput screening, chiral balance, chiral separation, kinetic resolution, reversal of stereospecificity, S-(+)-2,2-dimethylcyclopropane carboxamide, cosolvent, acyl transfer reaction, hydroxamic acid
