CN109943543B - 醇脱氢酶突变体及其制备方法和应用 - Google Patents
醇脱氢酶突变体及其制备方法和应用 Download PDFInfo
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- CN109943543B CN109943543B CN201910244434.9A CN201910244434A CN109943543B CN 109943543 B CN109943543 B CN 109943543B CN 201910244434 A CN201910244434 A CN 201910244434A CN 109943543 B CN109943543 B CN 109943543B
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Abstract
本发明公开了多个醇脱氢酶突变体及其在合成双芳基手性醇中的应用。本发明通过计算机辅助的理性设计，成功获得了针对双芳基酮底物具备高立体选择性的一系列突变体，可高效催化制备镇咳药及抗组胺药物的关键手性中间体(R)‑4‑氯苯基苯甲醇、(S)‑(4‑氯苯基)‑2‑吡啶甲醇及(R)‑4‑甲基苯基苯甲醇。本发明提供的产品制备方法与已有方法相比具备光学纯度高、操作简便、产物纯度高、成本低和不含重金属等优点，具备良好的应用前景。
Description
技术领域
本发明涉及酶及酶工程领域，尤其涉及一种醇脱氢酶突变体及其制备方法和应用。
背景技术
双芳基手性醇为很多手性药物的关键中间体。(R)-4-氯苯基苯甲醇是合成镇咳药左旋氯哌斯汀芬地柞酸的关键手性中间体。外消旋的氯哌斯汀本身就具有良好的生物活性和药物活性，为中枢性镇咳药，主要抑制咳嗽中枢而镇咳，也有微弱的抗组胺作用，无成瘾性及耐受性，临床上用于上呼吸道感染引起的咳嗽。而左旋氯哌斯汀芬地柞酸在临床上药物活性表现更高，疗效作用是外消旋氯哌斯汀的多倍，基本无副作用，尤其可以用作儿童用药，所以本产品市场需求大，前景好。
目前合成高光学纯度(R)-4-氯苯基苯甲醇的方法局限于化学法。如使用手性配体(R)-H8-binol催化合成(CN 105237469 A)，该路线缺点是反应温度需要降至-35℃，且手性配体结构复杂。因而该路线合成(R)-4-氯苯基苯甲醇成本及能耗较高。另有以外消旋4-氯二苯甲醇为起始原料。这种方法一般用到马钱子碱和二甲基马钱子碱拆分或是用D-酒石酸直接拆分。马钱子碱和二甲基马钱子碱这两种物质极毒，为植物提取物，价格昂贵不易得，同时以这两种物质作为拆分剂的，拆分收率只有20％左右，导致产品成本昂贵。用D-酒石酸直接拆分，得到左旋氯哌斯汀光学纯度不高，拆分收率低，产品质量不合格，这些均不利于产品的工业化生产。其他不经拆分的方法，反应过程中需添加贵金属钯炭、钌碳、氧化铂为催化剂，价格昂贵也难以购买，不利于工业化生产。
此外，(S)-(4-氯苯基)-2-吡啶甲醇是合成抗过敏药卡比沙明及倍他司汀的关键手性中间体(图2)。卡比沙明和倍他司汀为H1受体拮抗剂，抗组胺类药物，具有温和镇静作用。2013年4月，美国FDA批准了马来酸卡比沙明缓释剂用于儿童季节性和常年性过敏性鼻炎的治疗。研究表明，S构型的卡比沙明及倍他司汀活性远远优于其外消旋体。
目前合成高光学纯度(S)-(4-氯苯基)-2-吡啶甲醇的方法主要局限于化学法，大部分是手性催化剂作用下的不对称还原。如以trans-RuCl2[(R)-xylbinap][(R)-daipen]为催化剂，在压力为40-60psi充氮气条件下室温反应24h，还原1mM羰基底物，获得(S)-(4-氯苯基)-(吡啶-2-基)-甲醇，其ee值仅为60.6％，产率为98％。(C.Y.Chen,et al.,Org.Lett.,2003,5,5039-5042)。发明专利CN107827812A使用过渡金属络合物作为催化剂，在叔丁醇钾、氩气、氢气的作用下，在高压釜内催化不对称还原，转化率及ee值>98％。发明专利CN104774714使用手性三氮唑并噁嗪卡宾盐作为催化剂以及铑化合物，在氮气保护下以2-吡啶甲醛和对氯苯基硼酸为原料合成(S)-(4-氯苯基)-2-吡啶甲醇，ee值为91％-96％。由此可见，上述反应存在贵金属配位催化剂成本较高、底物浓度低、反应需要高压条件、操作步骤较多、物光学纯度低的问题，不能满足药物对光学纯度的要求，不利于工业化生产。
醇脱氢酶参与的以潜手性酮为底物的不对称还原是制备手性醇药物中间体的替代途径之一，作用条件温和，在常温、中性和水等环境中完成反应，符合“可持续发展”、“绿色化学”、“环境友好制造”等工业发展的目标。与已存在的化学合成方法相比，使用醇脱氢酶将潜手性酮中的羰基进行不对称还原的反应具有反应条件温和，酶催化剂易于制备等优势。2009年，朱敦明等发现来源于Sporobolomyces salmonicolor的重组羰基还原酶SsCR及其突变体可以立体选择性还原不同双芳基酮底物。利用葡萄糖脱氢酶参与辅酶循环，还原底物生成(R)-4-氯苯基苯甲醇，转化率95％，对映体过量值78％。2018年，倪烨等通过蛋白质工程的手段对来自Kluyveromyces sp.CCTCCM2011385的醇脱氢酶进行定向改造，获得了活性提高的醇脱氢酶突变体运用于(S)-(4-氯苯基)-2-吡啶甲醇的制备，ee值达到75.5％ee，制备(R)-4-氯苯基苯甲醇ee值达到88.6％(CN 108384765 A)。
由于应用于药物合成的手性中间体光学纯度至少要达到ee>95％，而目前的醇脱氢酶路线仍然存在着立体选择性不高导致的光学纯度不高的问题，亟需开发具备工业应用前景的新型醇脱氢酶用于生产手性双芳基醇。
发明内容
本发明针对现有技术中醇脱氢酶催化制备双芳基醇立体选择性较低的问题，提供了一系列醇脱氢酶的突变体蛋白质、编码该突变体蛋白质的核酸序列，含有该核酸序列的重组表达载体和含有重组载体的宿主菌，以及突变体在催化制备重要手性中间体的具体条件及方法。
本发明的首要目的是提供一个人工设计的新型醇脱氢酶突变体序列，能够作为生物催化剂不对称还原制备双芳基手性醇，并且具备较高的底物浓度及光学纯度。
一种醇脱氢酶突变体，所述醇脱氢酶突变体的氨基酸序列是SEQ ID NO：1所示氨基酸序列发生突变的氨基酸序列，所述发生突变的氨基酸序列具有如下至少一个突变位点：第144位异亮氨酸(Ile)、第145位谷氨酸(Glu)、第147位苯丙氨酸(Phe)、第190位酪氨酸(Tyr)、第199位亮氨酸(Leu)或第206位甲硫氨酸(Met)。
优选的，所述醇脱氢酶突变体的氨基酸序列为SEQ ID NO：2所示的氨基酸序列；或者
所述醇脱氢酶突变体的氨基酸序列为与SEQ ID NO：2所示的氨基酸序列具有90％以上的同源性且功能性相同的的氨基酸序列。
更优选的，所述醇脱氢酶突变体的氨基酸序列为与SEQ ID NO：2所示的氨基酸序列具有95％以上的同源性且功能性相同的的氨基酸序列。
具体的，本发明提供的SEQ ID NO：2是将氨基酸序列SEQ ID No.1所示醇脱氢酶的第144位异亮氨酸替换为缬氨酸、第145位谷氨酸替换为半胱氨酸、第147位苯丙氨酸替换为亮氨酸、第190位酪氨酸替换为脯氨酸、第199位亮氨酸替换为缬氨酸、第206位甲硫氨酸替换为苯丙氨酸。
本发明提供的Mutant1(突变体1)是将氨基酸序列SEQ ID No.1所示醇脱氢酶的第190位酪氨酸替换为脯氨酸或半胱氨酸以及第144位异亮氨酸替换为缬氨酸或丙氨酸。
本发明提供的Mutant2是将Mutant1的第199位亮氨酸替换为丙氨酸或缬氨酸。
本发明提供的Mutant3是将氨基酸序列Mutant1的第145位谷氨酸替换为丙氨酸或甲硫氨酸或苯丙氨酸或酪氨酸或半胱氨酸或天冬酰胺或亮氨酸。
本发明提供的Mutant4是将氨基酸序列Mutant2的第145位谷氨酸替换为丙氨酸或甲硫氨酸或苯丙氨酸或酪氨酸或半胱氨酸或天冬酰胺或亮氨酸。
本发明提供的Mutant5是将氨基酸序列Mutant4的第206位甲硫氨酸或202位丙氨酸替换为苯丙氨酸。
本发明提供的Mutant6是将Mutant5的第147位苯丙氨酸替换为亮氨酸。
本发明第二个方面提供了一种核苷酸序列，所述核苷酸序列编码上述的醇脱氢酶突变体。
本发明第三个方面公开了一种重组载体，所述重组载体含有上述的核苷酸序列。
优选的，所述重组载体为重组质粒，在本发明的一些较佳实施例中，所述重组载体为pET21a(+)、pET21b或pET28a。
本发明第四个方面公开了一种重组菌，所述重组菌含有上述的重组载体。
优选的，所述重组菌的宿主为大肠杆菌。在本发明的一具体实施例中，所述宿主为大肠杆菌BL21。
应当理解，本发明中的重组菌的宿主并不限于大肠杆菌，在本发明的教导之下，本领域技术人员能够选择任何其他合适的宿主种类，并且在本发明的保护范围之内。
本发明第五个方面公开了一种构建上述的重组菌的方法，包括以下步骤：将编码上述的醇脱氢酶突变体的核苷酸序列克隆到重组载体中，将重组载体转化到宿主中即得到重组菌。
本发明第六个方面公开了一种制备醇脱氢酶突变体的方法，包括以下步骤：
S1：将上述的重组菌通过诱导剂进行诱导培养，得到含有胞内表达重组载体的菌体；
S2：破碎所述菌体得到粗酶液；
S3：将所述粗酶液进行分离纯化得到醇脱氢酶突变体。
应该理解，本发明不限于上述步骤，还可以包含其他的步骤，例如在步骤S1之前、步骤S1和S2之间、步骤S2和S3之间、步骤S3之后，还包含其他额外的步骤，而不超出本发明的保护范围。
优选的，在S1和S2之间还包括冷冻干燥步骤，即将S1中得到的菌体(湿菌体)冷冻干燥得到冻干菌体。
本发明第七个方面公开了上述方法得到的醇脱氢酶突变体。
本发明第七个方面公开了上述的醇脱氢酶突变体在制备双芳基手性醇中的应用。
优选的，上述应用包括所述醇脱氢酶突变体作为催化剂，催化制备手性中间体，其中反应底物为双芳基酮底物。
优选的，上述应用中，所述手性中间体包括(R)-4-氯苯基苯甲醇、(S)-(4-氯苯基)-2-吡啶甲醇或(R)-4-甲基苯基苯甲醇中的任意一种。
优选的，上述应用中，包括以下步骤：
将反应体系加入辅酶循环系统中，进行不对称还原反应得到反应液，再通过萃取的方法从反应液中提取双芳基手性醇；其中，所述反应体系包括醇脱氢酶突变体。在本发明的一具体实施例中，反应体系：羰基底物浓度为10-500mM，上述脱氢酶突变体用量为1-10kU/L，NADP+用量为0.1～0.25mM，加入辅酶循环系统，辅酶循环系统中含有葡萄糖脱氢酶GDH和D-葡萄糖，其中葡萄糖脱氢酶GDH用量为1～10kU/L，D-葡萄糖用量为底物当量的1.2-1.5倍，Tris-HCl缓冲液的浓度为0.1-0.2M；助溶剂为20％-30％的甲基叔丁基醚或DMSO；在30～35℃，pH 6～8的条件下反应1～24h，不对称还原反应结束后，按照有机溶剂萃取方法从反应液中提取手性醇产物。
在符合本领域常识的基础上，上述各优选条件，可任意组合，而不超出本发明的构思与保护范围。
本发明的有益效果为：
本研究从对双芳基酮类底物没有活性的野生型(SEQ ID NO：1)出发，通过晶体结构及计算机辅助设计，首先使野生型产生活性，再通过多个突变进一步提高其活性、立体选择性及稳定性。最终获得了适宜于工业生产的六点突变体。
本发明提供的突变体对多个双芳基酮底物表现出活性与立体选择性，其中(R)-4-氯苯基苯甲醇、(S)-(4-氯苯基)-2-吡啶甲醇、(R)-4-甲基苯基苯甲醇有极佳的立体选择性，ee值分别为>98％，>99％和>96％。超过目前已知的酮还原酶不对称还原方法。
使用本发明提供的醇脱氢酶突变体作为生物催化剂制备双芳基手性醇，其中，得到的(R)-4-氯苯基苯甲醇的产物浓度可以达到500mM，即>108g/L、(S)-(4-氯苯基)-2-吡啶甲醇的产物浓度可以达到400mM，即>80g/L。本发明所获得的醇脱氢酶突变体还适用于其他潜在的双芳基酮的不对称还原反应，具有良好的工业应用前景。
附图说明
图1为醇脱氢酶野生型与突变体的转化率及立体选择性差异图；
图2为醇脱氢酶突变体催化不对称还原4-氯苯基-苯基甲酮反应进程曲线；
图3为醇脱氢酶突变体应用于左旋氯哌斯汀芬地柞酸关键手性中间体(R)-4-氯苯基苯甲醇的合成；
图4为醇脱氢酶突变体应用于卡比沙明及倍他司汀的关键手性中间体(S)-(4-氯苯基)-2-吡啶甲醇的合成。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细描述，但并不因此将本发明限制在所述的实施例范围之中。
下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。本发明所用试剂和原料均市售可得。
实施例1突变体的构建
根据野生型蛋白的晶体结构(PDB ID 4RF2；SEQ ID NO：1，该野生型蛋白来源于Lactobacillus kefir(凯夫氏乳杆菌))、醇脱氢酶的反应机理、双芳基底物的空间位阻及疏水特性、酶活性口袋大小、分子对接与分子动力学实验结果，分别选择了构成活性口袋的P190，I144，E145，L199，M206作为进行改造的位点。具体的，以含有SEQ ID NO：1核酸序列的pET21b为PCR模板，利用突变引物设计网站https://www.agilent.com/store/primerDesignProgram.jsp设计互补引物，即可得到用于构建各种氨基酸突变的引物。PCR反应体系如表1所示，PCR反应循环温度如表2所示。
表1 PCR反应体系
表2 PCR反应循环温度
经过以上PCR反应得到突变质粒产物，使用DpnI酶切掉模板质粒后转化至E.coliDH5α。在37℃的条件下，在氨苄抗性LB固体培养基平板培养16h，得到包含突变体的转化子。挑取转化子测序验证是否为预期突变。将突变体培养后提取质粒转化至E.coli BL21(DE3)中。在固体LB平板上涂布得到突变体单克隆。
实施例2突变体的筛选
将实施例1中得到的E.coli BL21(DE3)的突变体单克隆接种至含有3ml氨苄抗性的LB液体培养基中，生长至OD0.6左右，加入IPTG使其终浓度为0.1mM，在20℃下诱导突变体蛋白表达。20h后，将菌液离心收集，使用生理盐水洗涤后用400μl的100mM Tris-HCL(pH8.0)重新悬浮。反应体系：400μl以上悬浮菌液，加入50μl的200mM 4-氯苯基-苯基甲酮(溶解于异丙醇中)，50μl的5mM NADP，构成总体积500μl的反应体系。
在30℃下反应0.5-2h后，加入1ml异丙醇终止反应，离心后所得样品溶液通过手性HPLC分析。转化测定及手性分析方法如下：大赛璐液相手性分析柱OB-H，流动相使用正己烷/异丙醇＝90/10，R构型的保留时间为15min，S构型的保留时间为24min，底物保留时间为13.2min。通过比较野生型与突变体的转化率及立体选择性差异，考察突变体活性或立体选择性的提高，实验结果如图1所示，通过理性设计，发明人获得了催化活性从无到有，立体选择性与转化率逐步提高的多个突变体。
序列如SEQ ID NO：2所示的醇脱氢酶突变体对双芳基酮底物的立体选择性如下表3所示：
表3
实施例3醇脱氢酶突变体催化4-氯苯基-苯基甲酮不对称还原制备500mM(R)-4-氯苯基苯甲醇
本实施例包括以下步骤：
一、突变体湿菌体制备：在培养皿上挑取突变体单克隆(含序列如如SEQ ID NO：2所示的醇脱氢酶突变体)接种于3ml含有Amp抗性的LB培养基中37℃培养16h，以1％的接种量接种新鲜培养基，继续培养约2.5h，OD达到0.6-0.8。加入终浓度为0.1mM的IPTG，20℃继续培养20h，获得突变体静息细胞(湿菌体)。以同样方式制备含有葡萄糖脱氢酶的静息细胞(湿菌体)。
50ml反应体系包括：2.4g突变体静息细胞(湿菌体)，0.8g葡萄糖脱氢酶湿菌体，32.5ml pH8.0 Tris-HCl，2.5ml NADP(终浓度0.125mM)，4.87g 4-氯苯基-苯基甲酮溶解于15ml甲基叔丁基醚，5g D-葡萄糖。使用2M Na2CO3溶液调节pH，使反应过程中的pH维持在6.5-8。反应温度30℃，搅拌转速150rpm。该反应体系催化不对称还原4-氯苯基-苯基甲酮反应进程曲线如图2所示。
通过液相分析监测反应进程，反应停止后加入乙酸乙酯萃取三次，离心分离取有机相，去离子水洗涤有机相除盐，使用无水硫酸钠脱水，过滤后减压除去有机溶剂即得到ee>98％的(R)-4-氯苯基苯甲醇，如图3所示，(R)-4-氯苯基苯甲醇是合成左旋氯哌斯汀芬地柞酸的关键中间体。
实施例4：醇脱氢酶催化(4-氯苯基)(吡啶-2-基)甲酮不对称还原制备400mM(S)-(4-氯苯基)-2-吡啶甲醇
以实施例3的方法制备醇脱氢酶突变体及葡萄糖脱氢酶的静息细胞。
50ml反应体系包括：2.4g突变体静息细胞(湿菌体)，0.8g葡萄糖脱氢酶湿菌体，32.5ml pH8.0 Tris-HCl，2.5ml NADP(终浓度0.125mM)，4.35g(4-氯苯基)(吡啶-2-基)甲酮，5ml甲醇，5.4g D-葡萄糖。使用2M Na2CO3溶液调节pH，使反应过程中的pH维持在6.5-8。反应温度30℃，搅拌转速150rpm。通过手性HPLC监测反应进程，转化测定及手性分析方法如下：大赛璐液相手性分析柱OB-H，流动相使用正己烷/异丙醇＝95/5，S构型的保留时间为15.8min，R构型的保留时间为17.1min，底物保留时间为14min。
30摄氏度反应24小时后完全转化，加入乙酸乙酯萃取三次，离心分离取有机相，去离子水洗涤有机相除盐，使用无水硫酸钠脱水，过滤后减压除去有机溶剂，得到ee>97％的(S)-(4-氯苯基)-2-吡啶甲醇3.55g，收率为81％。如图4所示，其中(S)-(4-氯苯基)-2-吡啶甲醇是生产卡比沙明和倍他司汀的关键手型中间体。
本实施例提供的使用醇脱氢酶突变体进行双芳基手性醇合成的方法，与现有技术相比，具有化学纯度高、操作简便、产物纯度高和成本低和不含重金属等优点，具有良好的应用前景。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 上海健康医学院
<120> 醇脱氢酶突变体及其制备方法和应用
<160> 2
<170> PatentIn version 3.5
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<213> 高加索酸奶乳杆菌（Lactobacillus kefir）
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Claims (8)
1.一种醇脱氢酶突变体，其特征在于，所述醇脱氢酶突变体的氨基酸序列为SEQ IDNO：2所示的氨基酸序列；所述醇脱氢酶突变体作为催化剂，催化制备手性中间体，其中反应底物为4-氯苯基-苯基甲酮、(4-氯苯基)(吡啶-2-基)甲酮或4-甲基苯基苯甲酮。
2.一种核苷酸序列，其特征在于，所述核苷酸序列编码权利要求1所述的醇脱氢酶突变体。
3.一种重组载体，其特征在于，所述重组载体含有权利要求2所述的核苷酸序列。
4.根据权利要求3所述的重组载体，其特征在于，所述重组载体为pET21a(+)、pET21b或pET28a。
5.一种重组菌，其特征在于，所述重组菌含有权利要求3或4所述的重组载体。
6.根据权利要求5所述的重组菌，其特征在于，所述重组菌的宿主为大肠杆菌。
7.一种构建权利要求5所述的重组菌的方法，其特征在于，包括以下步骤：将权利要求2所述的核苷酸序列克隆到重组载体中，将重组载体转化到宿主中即得到所述重组菌。
8.一种制备醇脱氢酶突变体的方法，其特征在于，包括以下步骤：
S1：将权利要求5所述的重组菌通过诱导剂进行诱导培养，得到含有胞内表达重组载体的菌体；
S2：破碎所述菌体得到粗酶液；
S3：将所述粗酶液进行分离纯化得到所述醇脱氢酶突变体。
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