CN111778223B - 一种改造羰基还原酶立体选择性的方法、羰基还原酶突变体及应用 - Google Patents
一种改造羰基还原酶立体选择性的方法、羰基还原酶突变体及应用 Download PDFInfo
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Abstract
本发明公开了一种改造羰基还原酶立体选择性的方法、羰基还原酶突变体及应用,通过酶结合口袋的大小和形状鉴定羰基还原酶立体选择性,反Prelog酶的口袋外部形状接近于立方体,Prelog酶的口袋外部形状更类似于长方体,通过调控关键位点氨基酸可实现酶立体选择性的反转或提升。本发明制备的羰基还原酶突变体KmCR‑E87D/A129C/V239N的立体选择性(99.5%deP,R)较野生型KmCR(99.5%deP,S)得到反转,在10h能不对称合成35.4mM的6‑氰基‑(3R,5R)‑二羟基己酸叔丁酯。
Description
技术领域
本发明涉及一种羰基还原酶立体选择性改造方法,运用该方法构建立体选择性羰基还原酶突变体,及其在光学纯手性醇化合物制备方面的应用,具体涉及利用计算机辅助预测羰基还原酶立体选择性及关键位点,筛选获得羰基还原酶并应用于阿托伐他汀手性合成6-氰基-(3R,5R)-二羟基己酸叔丁酯及(S)-4-氯-3-羟基丁酸乙酯手性生物合成。
背景技术
羰基还原酶(Carbonyl reductase,EC 1.1.1.184)属于氧化还原酶,能够催化潜手性羰基化合物如脂肪酮、芳香酮,醛和醌等不对称还原生成相应的手性醇,具有广泛的底物谱、良好的催化活性与立体选择性。通常以NAD(H)(烟酰胺腺嘌呤二核苷酸)或NADP(H)(烟酰胺腺嘌呤二核苷酸磷酸)作为辅酶,普遍存在于自然界中。主要存在于短链脱氢酶/还原酶超家族(Short-chain dehydrogenase/reductases,SDRs),中链脱氢氢酶/还原酶超家族(Medium-chain dehydrogenases/reductases,MDRs)和醛酮还原酶超家族(Aldo-ketoreductases,AKRs)。SDR通常由250-350个氨基酸组成,MDR通常由约350个左右氨基酸组成,AKR通常由300个左右氨基酸组成。SDR与MDR具有保守的Rossmann折叠(αβα)结构,AKR则具有典型的(α/β)8桶状结构,其中仅MDR包含锌依赖性醇脱氢酶和相关蛋白。三种家族的蛋白结构相似度低,因此尽管底物特异性重叠,但不同羰基还原酶的结构存在较大差异。
天然的羰基还原酶多数遵循Prelog规则,遵循反Preolg规则的酶较少。因此可通过挖掘新型的反Prelog酶或者采用蛋白质工程对遵循Prelgo的酶进行立体选择性改造以丰富反Prelog的羰基还原酶酶源。目前已有不少研究报道成功精准调控羰基还原酶的立体选择性,如CpRCR、TeSADH、PpYSDR等。但是对羰基还原酶立体选择性的研究中,底物通常为芳基酮,例如苯乙酮类、双芳基酮类、α/β卤代芳基酮类等。对脂肪族酮酯的研究相对较少,因此相关的策略在改造羰基还原酶对脂肪族酮酯立体选择性的借鉴意义有限。同时由于不同酶的结构差异,特定的改造策略、突变位点仅适用于特定的酶或结构高度相似的酶。目前缺乏可用于指导大多数羰基还原酶对脂肪族酮酯类底物立体选择性调控的普适性的规律。
阿托伐他汀钙(7-[2-(4-氟苯基)-3-苯基-4-(苯胺基甲酰基)-5-(2-丙基)吡咯-1-基]-3,5-二羟基庚酸钙)属于第三代合成他汀类药物,具有高效的降脂功效,安全和长期临床益处,可降低心脑血管疾病的发病率和死亡率。阿托伐他汀钙最初由美国辉瑞公司研发,于1996年经FDA批准后以商品名“立普妥”上市,是历史上第一个年销售额超过百亿美元的降血脂药,阿托伐他汀钙的累计销售额已超过1000亿美元。
6-氰基-(3R,5R)-二羟基己酸叔丁酯是β,δ-二羟基戊酸结构的前体,作为关键手性中间体,可用于合成多种他汀类药物,例如阿托伐他汀。可通过化学法或化学-酶法得到6-氰基-(3R,5R)-二羟基己酸叔丁酯。譬如,经典的Paal-Knorr化学合成法以R-4-氰基-3-羟基丁酸乙酯为原料,通过LDA(二异丙基胺基锂)与乙酸叔丁酯经克莱森酯缩合反应生成6-氰基-(5R)-羟基-3-羰基己酸叔丁酯,在-90℃的条件下经NaBH4催化选择性还原生成6-氰基-(3R,5R)-二羟基己酸叔丁酯。化学法存在反应条件苛刻、反应选择性不强,产物光学纯度低等缺陷。生物催化法具有反应条件温和、环境友好及高选择性的优势,运用氧化还原酶不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯受到产学界的广泛重视。
随着蛋白质结晶技术的发展,越来越多的蛋白质结构被解析。蛋白质构效关系被挖掘和解析。在本发明中我们通过研究已报道的Prelog和反Prelog羰基还原酶的底物结合口袋信息与一级序列,总结出一种预测羰基还原酶立体选择性的方法和影响酶立体选择性的关键位点,并从一株耐热马克斯克鲁维酵母Kluyveromyces marxianus ZJB14056基因组中克隆出一种新的羰基还原酶(KmCR)编码基因kmcr。采用理性设计,通过同源建模、分子的对接确定KmCR中的相应关键位点,并构建突变文库,提高/反转KmCR的立体选择性。
发明内容
本发明的目的是提供一种改造羰基还原酶立体选择性的方法,运用该方法构建立体选择性羰基还原酶突变体及其在光学纯手性醇化合物制备方面的应用,采用计算机辅助预测羰基还原酶在不对称还原脂肪族酮酯时的立体选择性,及影响酶立体选择性的关键位点,成功构建对6-氰基-(5R)-羟基-3-羰基己酸叔丁酯、4-氯乙酰乙酸乙酯立体选择性反转的突变体,解决制备光学纯手性药物中间体的问题。
本发明采用的技术方案是:
本发明提供一种强化羰基还原酶立体选择性的改造方法,所述羰基还原酶立体选择性按如下方法改造(策略):1)通过蛋白质结晶或同源建模的方法获得待改造羰基还原酶的三维结构;2)利用计算机辅助方法(如Caver Analyst2.0等)测定待改造羰基还原酶底物结合口袋的大小与形状,确定组成结合口袋的关键位点及氨基酸残基;3)根据目的产物的构型确定待改造羰基还原酶的立体选择性,通过控制步骤2)组成结合口袋关键位点的氨基酸残基空间位阻或疏水性来调整羰基还原酶结合口袋大小与形状,若产物的构型为R型(酶的立体选择性为反Prelog偏好性),则将待改造羰基还原酶底物结合口袋形状调整为立方体,若产物的构型为S型(酶的立体选择性为Prelog偏好性),则将待改造羰基还原酶底物结合口袋形状调整为长方体,调整后的结合口袋大小均与产物匹配;4)根据步骤3)待调整位点的氨基酸残基设计突变文库,筛选获得立体选择性的羰基还原酶突变体。
进一步,步骤1)羰基还原酶优选具有Rossmann折叠且氨基酸序列长度为300-350个氨基酸的羰基还原酶。所述三维结构选择标准为:大于90%的氨基酸位于极可置信区域,不可信区域的氨基酸为0%,模型的G-factor在总平均值大于-0.5。
进一步,步骤2)所述底物为脂肪族酮酯。
进一步,步骤2)所述结合口袋大小与形状调整方法为:将组成酶底物结合口袋(非活性中心位点)的具有大空间位阻的氨基酸残基突变为具有小空间位阻的氨基酸残基,例如苯丙氨酸、色氨酸、脯氨酸、酪氨酸等突变为丙氨酸、缬氨酸、半胱氨酸等,可减小酶底物结合口袋的空间大小,使其偏向正方体;反之,则偏向长方体。结合底物羰基两侧基团的极性差异,调控酶底物结合口袋的疏水环境,便于底物在酶结合口袋中的构型的确定。若要极性增强,将疏水性氨基酸调整为亲水性氨基酸,例如丙氨酸、缬氨酸、亮氨酸等突变为甘氨酸、半胱氨酸、天冬酰胺等;若要疏水性增强,则反之。
进一步,所述设计突变文库采用定点突变方法。
本发明通过Caver Analyst 2.0对已报道具有蛋白质晶体结构的遵循Prelog或反Prelog羰基还原酶的酶底物口袋的体积和形状进行计算,发现所有报道的反Prelog酶的口袋空腔更类似于立方体,所有报道的Prelog酶的口袋空腔更类似于长方体(见图2)。通过多序列比对,确定影响酶立体选择性的关键位点(A1、A2、A3、B1、B2,见表1)。所有报道的反Prelog酶在A1位置为D或F,A2位置均具有较小的残基,如A、G等,在A3位置具有极性氨基酸如Q,而在B2位点具有较大位阻的F、Y、L等;所有报道的Prelog酶在B1位置具有较小位阻的氨基酸残基,如V。遵循该策略对羰基还原酶KmCR进行改造,实现立体选择性在Prelog规则和反Prelog规则之间的可控转换。
本发明通过理性设计,采用定点突变技术,对KmCR羰基还原酶基因(SEQ ID NO.1)进行突变,将获得的突变质粒以热击方式转入E.coli BL21(DE3)感受态细胞,对获得菌株进行接种、转接、诱导、菌体回收,利用重悬菌液催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯或4-氯乙酰乙酸乙酯不对称还原,测定突变体的立体选择性和活性。具体方法:通过SWISS-MODEL搜索与KmCR序列相似度最高的晶体结构,作为同源建模的模板,利用Modeller9.20进行同源建模,获得KmCR的三维结构,采用上述方法,选择A1、A2、A3、B1、B2对应的位点,设计突变引物,以pET28a(+)-kmcr为模板质粒,进行定点饱和突变,获得突变质粒,经转化、筛选,获得优势突变体。
本发明还涉及一种利用所述改造羰基还原酶立体选择性的方法获得的羰基还原酶突变体,所述突变体是将SEQ ID NO.1所示氨基酸序列第87位、第129位、第162位、第199位或第239位进行单突变或多突变获得的。
更进一步,所述羰基还原酶突变体为下列之一:(1)第87位谷氨酸突变为天冬氨酸(E87D)或苯丙氨酸(E87F);(2)第129位丙氨酸突变为甘氨酸(A129G)或半胱氨酸(A129C);(3)第239位缬氨酸突变为谷氨酰胺(V239Q)或天冬酰胺(V239N);(4)第162位苯丙氨酸突变为缬氨酸(F162V)或天冬酰胺(F162N);(5)第F199苯丙氨酸突变为亮氨酸(F199L)或酪氨酸(F199Y);(6)第87位谷氨酸突变为天冬氨酸,第129位丙氨酸突变为半胱氨酸且第239位缬氨酸突变为天冬酰胺(E87D/A129C/V239N)。
本发明突变体KmCR-E87D/A129C/V239N严格遵循反Prelog规则,氨基酸序列为SEQID NO.3所示。
本发明突变体KmCR-F162V严格遵循Prelog规则,氨基酸序列为SEQ ID NO.4所示。
由于氨基酸序列的特殊性,任何含有SEQ ID NO.3或SEQ ID NO.4所示氨基酸序列的肽蛋白的片段或其变体,如其保守性变体、生物活性片段或衍生物,只要该肽蛋白的片段或肽蛋白变体与前述氨基酸序列同源性在90%以上,均属于本发明保护范围之列。具体的所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换;其中对于变体的保守性改变,所替换的氨基酸具有与原氨基酸相似的化学结构或化学性质,如用亮氨酸替换异亮氨酸,变体也可具有非保守性改变,如用色氨酸替换甘氨酸。
本发明提供一种所述羰基还原酶突变体在不对称还原脂肪族酮酯中的应用,所述脂肪族酮酯为6-氰基-(5R)-羟基-3-羰基己酸叔丁酯或4-氯乙酰乙酸乙酯,所述的应用方法为:将含羰基还原酶突变体基因的工程菌经发酵培养后的发酵液离心,收集湿菌体,以湿菌体或湿菌体经超声破碎且镍柱纯化的纯酶为催化剂,以含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体或湿菌体经超声破碎且镍柱纯化的纯酶为辅助催化剂,以脂肪族酮酯为底物,以葡萄糖为辅底物,以pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系,在35-40℃、400-600rpm条件下进行反应,反应结束,反应液分离纯化,获得手性化合物;当底物为6-氰基-(5R)-羟基-3-羰基己酸叔丁酯时,手性化合物为6-氰基-(3R,5R)-二羟基己酸叔丁酯;当底物为4-氯乙酰乙酸乙酯时,手性化合物为(S)-4-氯-3-羟基丁酸乙酯。
所述反应体系中,底物添加终浓度为10-20g/L,葡萄糖添加终浓度20-40g/L,所述催化剂用量以湿菌体干重计量,所述催化剂与辅助催化剂的湿菌体以干重比1.0-5.0:1混合成混合菌体形式加入,催化剂和辅助催化剂用量以混合菌体总量干重计为5-12.5g DCW/L。
本发明还提供一种所述羰基还原酶突变体在不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯中的应用,具体所述的应用方法为:将含羰基还原酶突变体基因的工程菌经发酵培养后的发酵液离心,收集湿菌体,以湿菌体或湿菌体经超声破碎且镍柱纯化的纯酶为催化剂,以含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体或湿菌体经超声破碎且镍柱纯化的纯酶为辅助催化剂,以6-氰基-(5R)-羟基-3-羰基己酸叔丁酯为底物,以葡萄糖为辅底物,以pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系,在35-40℃、400-600rpm条件下(优选35℃、600rpm)进行反应,反应结束,反应液分离纯化,获得6-氰基-(3R,5R)-二羟基己酸叔丁酯;所述反应体系中,底物添加终浓度为10-20g/L,葡萄糖添加终浓度20-40g/L,所述催化剂与辅助催化剂的湿菌体以干重比1.0-5.0:1(w/w),优选3.0-5.0:1混合成混合菌体形式加入,催化剂和辅助催化剂用量以混合菌体总量干重计为5-12.5g DCW/L(DCW:dry cell weight,干重)。
本发明还提供一种所述羰基还原酶突变体在不对称还原4-氯乙酰乙酸乙酯制备手性(S)-4-氯-3-羟基丁酸乙酯中的应用,所述的应用为:将含羰基还原酶突变体基因的工程菌经发酵培养后的发酵液离心,收集湿菌体,以湿菌体或湿菌体经超声破碎且镍柱纯化的纯酶为催化剂,以含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体或湿菌体经超声破碎且镍柱纯化的纯酶为辅助催化剂,以4-氯乙酰乙酸乙酯为底物,以葡萄糖为辅底物,以pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系,在35-40℃、400-600rpm(优选35℃、600rpm)条件下进行反应,反应结束,反应液分离纯化,获得(S)-4-氯-3-羟基丁酸乙酯。所述反应体系中,底物添加终浓度为10-20g/L,葡萄糖添加终浓度20-40g/L,所述催化剂与辅助催化剂的湿菌体以干重比1.0-5.0:1(w/w),优选3.0-5.0:1混合成混合菌体形式加入,催化剂和辅助催化剂用量以混合菌体总量干重计为5-12.5g DCW/L(DCW:dry cellweight,干重)。
本发明所述葡萄糖脱氢酶基因(GenBank NO.KM817194.1)来自Exiguobacteriumsibirium DSM 17290。
进一步,所述湿菌体按如下方法制备:将含羰基还原酶突变体基因的工程菌接种到含终浓度50μg/mL卡那霉素的LB液体培养基中,37℃培养10h,以体积浓度1.5-2.0%的接种量接种到新鲜的含终浓度50μg/mL卡那霉素的LB液体培养基中,37℃、200rpm培养2h,再向培养液中加入终浓度为0.10mM异丙基硫代半乳糖苷(Isopropylβ-D-thiogalactoside,IPTG),28℃培养12h后,4℃、8000rpm离心10min,获得含羰基还原酶突变体的湿菌体;所述含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体制备方法同含羰基还原酶基因的湿菌体。
本发明羰基还原酶突变体和葡萄糖脱氢酶基因工程菌的接种、转接、诱导、菌体回收,培养基可为本领域任何可使菌体生长并产生本发明的培养基,优选LB培养基:胰蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L,蒸馏水溶解,调节pH 7.0。培养方法和培养条件没有特殊的限制,培养方法和条件可以根据宿主类型和培养方法等因素的不同按本领域普通知识进行适当的选择。
与现有技术相比,本发明主要的有益效果主要体现在:本发明开发了一种基于计算机辅助的羰基还原酶对脂肪族酮酯类底物的立体选择性改造方法,该方法能大大减少筛选工作量,用于新酶制剂的挖掘和改造;并将此方法成功应用到一条来自K.marxianusZJB14056的羰基还原酶KmCR的分子改造中。改造获得的突变体首次用于催化合成6-氰基-(3R,5R)-二羟基己酸叔丁酯或(S)-4-氯-3-羟基丁酸乙酯。其中,野生型羰基还原酶KmCR遵循Prelog规则,活性高,但立体选择性相反且低(66.6%deP,S)。KmCR的突变体M-R3b(E87D-A129C-V239N)对6-氰基-(5R)-羟基-3-羰基己酸叔丁酯立体选择性反转成R-选择性,遵循反Prelog规则(99.5%deP,R),而且,能合成光学纯6-氰基-(3R,5R)-二羟基己酸叔丁酯(>99.5%deP);此外,还能催化4-氯乙酰乙酯合成手性4-氯-3-羟基丁酸乙酯转化率90.7%,ee值79.2%。
附图说明
图1利用计算机理性设计羰基还原酶立体选择性的方法流程图。
图2报道的羰基还原酶底物催化口袋形状,其中A为Prelog规则和反Prelog规则酶的口袋形状,B为总结的规律。
图3鉴定酶的底物结合口袋的形状。
图4野生型羰基还原酶kmcr琼脂糖凝胶电泳图,泳道M为标准DNA分子量,泳道1、2、3均为kmcr基因片段。
图5野生型羰基还原酶KmCR基因重组载体pET28a(+)-kmcr图谱。
图6野生型羰基还原酶KmCR及其突变体琼脂糖凝胶电泳图谱,其中,泳道M为标准DNA分子量,泳道1为pET28a(+)-kmcr;泳道2为pET28a(+)-kmcr-E87D-A192C-V239N;泳道3为pET28a(+)-kmcr-F162V;泳道4为pET28a(+)-kmcr-E87D;泳道5为pET28a(+)-kmcr-A129C;泳道6为pET28a(+)-kmcr-V239N;泳道7为pET28a(+)-kmcr-E87D-A129C;泳道8为pET28a(+)-kmcr-E87D-V239N;泳道9为pET28a(+)-kmcr-A129C-V239N。
图7KmCR及突变体的底物结合口袋的形状与大小,其中A、B为KmCR-WT的口袋形状与关键位置的氨基酸残基,C、D为KmCR突变体的形状与关键位置的氨基酸残基。
图8重组羰基还原酶野生型与突变体的SDS-PAGE图,M:标准蛋白质分子;泳道1为KmCR上清液;泳道2为KmCR沉淀;泳道3为KmCR-E87D/A129C/V239N上清液;泳道4:KmCR-E87D/A129C/V239N沉淀;泳道5:KmCR-F162V上清液;泳道6:KmCR-F162V沉淀。
图9生物-化学法合成阿托伐他汀路线图,其中A总体合成路线图,B羰基还原酶与葡萄糖脱氢酶EsGDH偶联催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯不对称还原制备6-氰基-(3R,5R)-二羟基己酸叔丁酯的反应示意图,C羰基还原酶与葡萄糖脱氢酶EsGDH偶联催化4-氯乙酰乙酸乙酯不对称还原制备(S)-4-氯-3-羟基丁酸乙酯的反应示意图。
图10利用羰基还原酶突变体KmCR-E87D/A129C/V239N偶联EsGDH不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯时间进程图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述。
实施例1:建立预测羰基还原酶立体选择性的方法
参照图1,在PDB数据库中搜索羰基还原酶的晶体结构,将其三维模型导入CaverAnalyst 2.0,选择软件的口袋分析,将探头的大小设置为对酶的底物结合口袋大小和形状进行分析,结果如图2中(A)所示。已报道的遵循反Prelog规则的酶(PDB:3CTM、5ZFM、4R1S、5ZED、5TQW、3VDR和6NBR)的底物结合口袋的外部形状都像立方体(六个等边方形的规则实体),而遵循Prelog规则的酶更像长方体(PDB:2WDZ、4BMV、5GWT、3AWD、4PVC、4FN4和5ZEC)。将7个反Prelog酶的序列和7个Prelog酶的序列进行比对,结果如表1所示。通过对比后总结,反Prelog酶通常在A1位置为D或F,A2位置均具有较小的残基,如A、G等,在A3位置具有极性氨基酸如Q,而在B2位点具有较大位阻的F、Y、L等;所有报道的Prelog酶在B1位置具有较小位阻的氨基酸残基,如V。由此确定影响立体选择性的关键位点(A1、A2、A3、B1及B2)。7个反Prelog规则的羰基还原酶属于短链脱氢酶家族。其中3VDR、3CTM、5ZFM、2WDZ、4BMV、5GWT、4FN43、3AWD属于经典类型的SDR家族,氨基酸序列长度为250-270左右,5ZED、5ZEC、4R1S、5TQM、6NBR、4PVC属于拓展类型的SDR家族,氨基酸序列长度在300-350之间。由此,该方法适用于具有Rossmann折叠的序列长度为300-350个氨基酸的SDR。
表1多序列比对结果
实施例2:利用计算机预测11种未开发的羰基还原酶的立体选择性
根据实施例1的总结的规律,在NCBI数据库中检索与KmCR序列相似度在20-40%,氨基酸序列长度为320-350的未开发的短链脱氢酶。分别对其进行同源建模,观察酶底物结合口袋的大小、形状及关键位点的氨基酸残基。结果如图10所示,其中CCR1、CCR2、CCR3、CCR4、HP1、HP2的结合口袋的形状类似于正方体,而AR1、HP3、AR2、AR3和HP4的结合口袋形状类似于长方体。通过对比关键位点(A1、A2、A3、B1、B2)的氨基酸,所得结果如表2所示,6条序列满足反Prelog的规律,而5条序列满足Prelog的规律。
表2预测未开发的羰基还原酶对6-氰基-(5R)-羟基-3-羰基己酸叔丁酯的立体选择性
实施例3:野生型羰基还原酶KmCR基因克隆及其重组表达载体的构建
(1)目的基因克隆
以马克斯克鲁维酵母(K.marxianus)ZJB14056(保藏中心编号:ATCC36534)基因组DNA为模板,通过上游引物:5’-TAAGAAGGAGATATACCATGGATGACATTTACAGTGGTGACAG-3’,下游引物5’-GTGGTGGTGGTGGTGCTCGAGTTACCCACGGTACGCGCCCA-3’进行PCR扩增,获得扩增产物。取5μL PCR扩增产物进行琼脂糖凝胶电泳检验。检测结果见图4泳道1、2、3,电泳条带清晰,大小在1000bp左右,与理论值(1038bp)相符合。PCR体系(总体积100μL):50μL 2×PhantaMax缓冲液、40μL双蒸水、2μL dNTP Mix2、2μL dNTP混合物(各10mM)、上下游引物各2μL,基因组DNA 2μL、Phanta Max Super-Fidelity DNA聚合酶2μL。PCR反应条件:95℃预变性5min;95℃变性30s,56℃~58℃退火30s,72℃延伸1min 20s(30cycle);72℃延伸10min。
(2)重组表达载体pET28a(+)-kmcr的构建
按照《AxyPrep PCR Cleanup Kit》试剂盒步骤回收步骤(1)中获得的PCR扩增产物。纯化后的基因片段记为kmcr(氨基酸序列为SEQ ID NO.1、核苷酸序列为SEQ ID NO.2)。用Nco I-Xho I分别对pET-28a(+)质粒和目的基因KmCR进行双酶切。双酶切体系(100μL):pET-28a(+)片段65μL,Nco I和Xho I酶各1μL,10×Tango Buffer 10μL,ddH2O 23μL。在37℃,200rpm条件下酶切6h,按照《AxyPrep PCR Cleanup Kit》回收酶切后的产物。将双酶切后的目的基因与线性化pET-28a载体进行连接重组反应。连接体系(20μL):线性化载体4μL,目的基因8μL,5×CE II缓冲液4μL,II 1μL,双蒸水3μL。连接体系在37℃下温育30min。反应完成后立即取出,并冰浴5min。构建重组表达载体pET28a(+)-kmcr,示意图见图5。将重组表达载体转化至E.coli BL21(DE3),在含有50μg/mL卡那霉素的LB平板上进行涂布并筛选阳性克隆子,测序并保存E.coli BL21(DE3)/pET28a(+)-kmcr。
实施例4:羰基还原酶立体选择性关键氨基酸的确定及突变体文库的构建
1)根据实施例1,对实施例3获得的野生型羰基还原酶KmCR(记为KmCR-WT)进行同源建模,得到多个建模结果,通过Procheck program进行评估打分,选择合理的三维模型(判断标准:大于90%的氨基酸位于极可置信区域,不可信区域的氨基酸为0%,模型的G-factor在总平均值大于-0.5);2)利用计算机辅助方法,将步骤1)三维模型导入CaverAnalyst 2.0,分析酶底物结合口袋大小与形状,结果如图7中(A、B)所示,KmCR-WT的口袋形状更类似于长方体,大小为确定影响酶立体选择性的组成结合口袋的关键位点A1、A2、A3、B1及B2,关键位点对应KmCR中的氨基酸残基为E87、A129、V239、F162及F199;3)根据产物的构型(6-氰基-(3R,5R)-二羟基己酸叔丁酯为R构型)判断所需要的酶的底物结合口袋形状应为正方体,而KmCR口袋形状更类似于长方体,偏好Prelog规则,因此通过调控步骤2)关键位点氨基酸残基的位阻控制酶结合口袋的大小和形状继而来调控酶的立体选择性,通过减小口袋的大小即增加关键位点氨基酸残基位阻可使酶的口袋结构更接近正方体。4)因此构建突变文库E87(D、F),A129(G、C),V239(Q、N),F162(V、N),F199(L、Y)。
羰基还原酶突变体文库的制备通过定点突变来实现,引物设计如表3,以E.coliBL21(DE3)/pET28a(+)-kmcr中载体pET28a(+)-kmcr为模板,经过PCR,将SEQ ID NO.1所示羰基还原酶KmCR氨基酸序列的第87位、第129位、第162位、第199位、第239位突变为相应氨基酸,并转化,涂平板。PCR反应体系(50μL):1μL正向引物(100μM),1μL反向引物(100μM),25μL2×Phanta Max缓冲液,1μL dNTP混合物(各10mM),1μL质粒模板,1μLDNA聚合酶和20μL双蒸水。PCR反应条件:95℃预变性5min;95℃变性30s,56~58℃退火30s,72℃延伸7min(30cycle);72℃延伸10min。取5μL PCR产物进行琼脂糖凝胶电泳检验,电泳图见图6。按照《AxyPrep PCR Cleanup Kit》试剂盒步骤回收剩余PCR产物。移取20μL电泳检测为阳性的PCR回收产物,加入1μL的Dpn I酶和1μL cutsmart缓冲液,37℃、160rpm,15min,消化原模板,将产物转化至E.coli BL21(DE3),在含有50μg/mL卡那霉素的LB平板上进行涂布、37℃过夜培养。随机挑取单菌落提取质粒进行测序。测序后对突变的阳性菌落,进行保菌。
表3羰基还原酶定点突变引物设计
实施例5:野生型羰基还原酶、突变体和葡萄糖脱氢酶的诱导表达
葡萄糖脱氢酶湿菌体的制备:所述E.coli BL21(DE3)/pET28b(+)-esgdh由GenBank中来自E.sibiriumDSM 17290葡萄糖脱氢酶基因(GenBank NO.KM817194.1)插入到pET28b(+)构建重组表达载体,并将此表达载体转入E.coli BL21(DE3)制得E coli BL21(DE3)/pET28b(+)-esgdh。
将实施例3出发菌株E.coli BL21(DE3)/pET28a(+)-kmcr和实施例4突变得到的羰基还原酶突变菌株以及E coli BL21(DE3)/pET28b(+)-esgdh分别接种到含有终浓度50μg/mL卡那霉素的LB液体培养基中,37℃培养10h,以体积浓度2.0%(v/v)的接种量接种到新鲜的含有终浓度50μg/mL卡那霉素的LB液体培养基中,37℃、180rpm培养2h,再向培养液中加入终浓度为0.10mM IPTG,28℃培养12h后,4℃、8000rpm离心10min,获得相应的湿菌体细胞。以上获得的细胞产有相应的蛋白,可用于蛋白纯酶液的制备,也可用于粗酶液催化不对称还原潜手性酮制备手性醇。
实施例6:突变文库筛选
对获得的突变体进行优势突变体的筛选,筛选条件如下:将实施例5诱导表达的突变株湿菌体及葡萄糖脱氢酶湿菌体以菌体总干重5g/L(羰基还原酶突变体和葡萄糖脱氢酶菌体干重比5.0:1(w/w))的量加入pH 7.0的PBS(100mM)中重悬细胞,获得突变株混合菌液。同样条件下,用对照菌株E.coli BL21(DE3)/pET28a(+)-kmcr替换突变体菌株湿菌体制备对照株混合菌液。分别将突变株混合菌液和对照株混合菌液作为催化剂,以6-氰基-(5R)-羟基-3-羰基己酸叔丁酯为底物,以葡萄糖为辅助底物,不添加外源性NADPH或NADP+,运用菌体内源型NADPH,建立起辅酶循环系统。
催化剂加入量为5mL,6-氰基-(5R)-羟基-3-羰基己酸叔丁酯加入浓度为20g/L,葡萄糖的加入浓度为30g/L,反应介质为pH 7.0、100mM的PBS,构成反应体系5mL,在35℃、600rpm条件下反应10min,取反应液500μL,加入500μL无水乙醇沉淀蛋白,即反应液稀释2倍,-20℃过夜,12000rpm离心3min,取上清,过0.22μm微滤膜,滤液作为液相样品,通过HPLC检测6-氰基-(3R,5R)-二羟基己酸叔丁酯、6-氰基-(3S,5R)-二羟基己酸叔丁酯浓度,筛选优势突变体。筛选获得优势菌株,并保存在-80℃冰箱。
以优势单突变菌株的质粒作为模板,引物设计如表3,按照单突变相同的操作方法,进行组合突变,实验结果示于表4。
液相检测条件:色谱柱C18柱(4.6×250mm,Acchrom,中国),流动相乙腈:水体积比为1:3(v/v),流速1.0mL/min,检测波长210nm,进样量10μL,柱温40℃。6-氰基-(5R)-羟基-3-羰基己酸叔丁酯、6-氰基-(3R,5R)-二羟基己酸叔丁酯、6-氰基-(3S,5R)-二羟基己酸叔丁酯保留时间分别为:13.25min、8.28min、7.90min。
表4KmCR及其突变体的催化性能和立体选择性
通过优势菌株筛选获得羰基还原酶突变体KmCR-E87D/A129C/V239N(即SEQ IDNO.1所示氨基酸第87位的谷氨酸突变为天冬氨酸,第129位的丙氨酸突变为半胱氨酸且第239位的缬氨酸突变为天冬酰胺,氨基酸序列为SEQ ID NO.3)和KmCR-F162V(即SEQ IDNO.1所示氨基酸第162位的苯丙氨酸突变为缬氨酸,氨基酸序列为SEQ ID NO.4)。突变体的结合口袋图见图7中(C、D)。
实施例7:羰基还原酶母本及其突变体的纯化
将实施例6获得的优势突变体(表4中KmCR-E87D/A129C/V239N,KmCR-F162V),根据实施例5所述方法获得羰基还原酶突变体湿菌体,分别在8000rpm,4℃下离心10min收集菌体,并用0.9%(w/v)盐水洗涤两次。按照菌体总量25g/L的量加入pH 7.0、100mM PBS缓冲液中重悬,在冰水混合物上超声破碎6min,超声破碎条件:功率为400W,破碎1s、暂停1s,获得突变株粗酶液。通过在8000rpm,4℃下离心10min收集上清液(电泳图见图8泳道1、3、5所示),将其通过0.45μm膜微过滤后,采用镍亲和柱纯化突变体蛋白。
使用镍亲和柱(1.6×10cm,Bio-Rad公司,美国)纯化突变体蛋白,具体操作如下:①用缓冲液A(含0.30M NaCl、20mM咪唑的pH 8.0、20mM PBS缓冲液)进行预平衡。②用缓冲液A以1.0mL/min的流速洗去未结合的杂质,直至电导率稳定。③然后用缓冲液B(含0.3MNaCl、500mM咪唑的pH 8.0、20mM PBS缓冲液)洗脱目的蛋白。将收集的洗脱液用20mM PBS缓冲液(pH7.0)透析过夜,取截留液即为纯酶液。所有纯化步骤均在4℃下进行。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定蛋白质大小。对照菌株E.coli BL21(DE3)/pET28a(+)-kmcr的羰基还原酶纯酶采用相同条件收集。
实施例8:野生型羰基还原酶KmCR及其突变体的动力学参数测定
将实施例7中制备的野生型羰基还原酶KmCR及其突变体KmCR-E87D/A129C/V239N,KmCR-F162V纯酶液在35℃温浴5min,获得预处理的酶液。总反应体系为1mL,以6-氰基-(5R)-羟基-3-羰基己酸叔丁酯为底物,浓度分别为0.5、1、2、5、10mM,NADPH的浓度为1mM,加入终浓度1g/L的纯酶液,35℃、pH 7.0、600rpm条件下反应5min,加入2μL浓盐酸终止反应,取样,经过0.22μm膜微过滤后通过实施例6所述HPLC检测产物浓度。
以NADPH为底物,浓度分别为0.5、1、2、5、10mM,6-氰基-(5R)-羟基-3-羰基己酸叔丁酯浓度为2mM,加入终浓度1g/L的纯酶液,反应、检测条件同上。拟合反应曲线,根据米氏方程分别计算6-氰基-(5R)-羟基-3-羰基己酸叔丁酯和NADPH的亲和常数Km和最大反应速率vmax及周转数kcat。
KmCR酶活检测标准条件:10mM 6-氰基-(5R)-羟基-3-羰基己酸叔丁酯,1mMNADPH,适量酶液,35℃、pH 7.0、600rpm条件下反应5min,样品处理后进行HPLC检测分析。
酶活定义:在标准条件下,每分钟每生成1μmol的6-氰基-(3,5)-二羟基-己酸叔丁酯所需的酶量定义为一个酶活单位U。
比酶活定义:每毫克蛋白所具有的酶活单位数,记为U/mg。
蛋白浓度用二喹啉甲酸蛋白测定试剂盒(南京凯基生物科技发展有限公司,南京)测定。
由表5和表6可看出,KmCR和突变体KmCR-F162V对NADPH的Km和vmax数值接近,而突变体KmCR-E87D/A129C/V239N明显降低。
表5羰基还原酶及其突变体对6-氰基-(5R)-羟基-3-羰基己酸叔丁酯的动力学参数
表6羰基还原酶及其突变体对NADPH的动力学参数
实施例9:羰基还原酶突变体KmCR-E87D/A129C/V239N不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯
根据实施例5的描述,通过发酵获得羰基还原酶突变体KmCR-E87D/A129C/V239N湿菌体和葡萄糖脱氢酶EsGDH湿菌体。在建立的双酶偶联体系中(示意图见图9),将湿菌体KmCR-E87D/A129C/V239N和葡萄糖脱氢酶EsGDH湿菌体以干重比4.0:1(w/w)混合成混合菌体,先将混合菌体用pH7.0、100mM的PBS缓冲液重悬,转化体系中混合菌体加入干重量为12.5g DCW/L,底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯的投料量为10g/L,葡萄糖浓度为20g/L,pH 7.0、100mM PBS缓冲液为反应介质构成转化体系50mL,35℃、600rpm反应,反应进程曲线如图10所示,在10h能转化(>99.5%)合成35.4mM 6-氰基-(3R,5R)-二羟基己酸叔丁酯(>99.5%deP)。
实施例10:羰基还原酶及其突变体不对称还原4-氯乙酰乙酸乙酯制备手性4-氯-3-羟基丁酸乙酯
根据实施例5的描述,通过发酵获得羰基还原酶及突变体湿菌体和葡萄糖脱氢酶EsGDH湿菌体。在建立的双酶偶联体系中,将羰基还原酶湿菌体和葡萄糖脱氢酶EsGDH湿菌体按菌体干重比5.0:1(w/w)混合成混合菌体,先将混合菌体用pH 7.0、100mM的PBS缓冲液重悬,转化体系中混合菌体加入干重量为5g DCW/L,底物4-氯乙酰乙酸乙酯的投料量为15g/L,葡萄糖浓度为30g/L,pH 7.0、100mM PBS缓冲液为反应介质构成转化体系10mL,35℃、600rpm反应3h,反应结果如表7所示。突变体KmCR-E87D/A129C/V239N能在3h能不对称合成产物(S)-4-氯-3-羟基丁酸乙酯,浓度为63.5mM(79.9%,ee值)。
表7羰基还原酶及其突变体不对称还原4-氯乙酰乙酸乙酯
序列表
<110> 浙江工业大学
<120> 一种改造羰基还原酶立体选择性的方法、羰基还原酶突变体及应用
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Lys Tyr Ser Leu Thr Ser Val Glu His Ile Val Tyr Thr Ser Ser Ile
115 120 125
Cys Ala Ser Ser Leu Glu Ser Glu Phe Thr Asp Pro Thr Leu Val Val
130 135 140
Ser Glu Asp Ser Trp Asn Pro Gln Gly Leu Glu Glu Ala Lys Thr Glu
145 150 155 160
Phe Phe Thr Ala Tyr Ser Tyr Ser Lys Lys Ile Ala Glu Lys Thr Met
165 170 175
Trp Asp Phe Val Glu Glu Tyr Lys Gly Thr Glu His Glu Ile Lys Leu
180 185 190
Thr Thr Val Asn Pro Cys Phe Asn Ile Gly Pro Gln Ala Tyr Glu Ala
195 200 205
Asp Val Thr Glu Thr Met Asn Phe Thr Ala Glu Leu Ile Asn His Val
210 215 220
Val Lys Ser Lys Val Gly Asp Pro Leu Pro Pro Thr Arg Ile Asn Pro
225 230 235 240
Tyr Val Asp Val Arg Asp Thr Ala Arg Ala His Val Asp Ala Leu Lys
245 250 255
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260 265 270
Ser Ser Gln Gln Ile Tyr Asp Ile Val Asn Glu Arg Phe Pro Gln Leu
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Arg Gly Lys Ile Ala Arg Gly Glu Pro Gly Ser Asp Leu Asp Pro Ala
290 295 300
Lys Leu Ala Lys Phe Asp His Ala Arg Thr Thr Gln Ala Leu Gly Trp
305 310 315 320
Glu Phe Thr Pro Ile Glu Lys Ala Ile Ala Asp Glu Val Ala Gln Ile
325 330 335
Leu Arg Val Gly Ala Tyr Arg Gly
340
<210> 4
<211> 344
<212> PRT
<213> 未知(Unknown)
<400> 4
Met Thr Phe Thr Val Val Thr Gly Ala Asn Gly Tyr Ile Ala Lys His
1 5 10 15
Ile Leu Lys Ser Leu Leu Glu Asp Gly His Arg Val Ile Gly Thr Val
20 25 30
Arg Asn Ser Lys Lys Ala Glu Glu Leu Lys Arg Thr Val Asn Asp Glu
35 40 45
Asn Leu Ile Val Glu Leu Val Pro Asp Met Leu Val Glu Asn Ala Phe
50 55 60
Asp Glu Leu Phe Lys Lys Tyr Asn Thr Gln Ile Lys Tyr Val Phe His
65 70 75 80
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100 105 110
Lys Tyr Ser Leu Thr Ser Val Glu His Ile Val Tyr Thr Ser Ser Ile
115 120 125
Ala Ala Ser Ser Leu Glu Ser Glu Phe Thr Asp Pro Thr Leu Val Val
130 135 140
Ser Glu Asp Ser Trp Asn Pro Gln Gly Leu Glu Glu Ala Lys Thr Glu
145 150 155 160
Phe Val Thr Ala Tyr Ser Tyr Ser Lys Lys Ile Ala Glu Lys Thr Met
165 170 175
Trp Asp Phe Val Glu Glu Tyr Lys Gly Thr Glu His Glu Ile Lys Leu
180 185 190
Thr Thr Val Asn Pro Cys Phe Asn Ile Gly Pro Gln Ala Tyr Glu Ala
195 200 205
Asp Val Thr Glu Thr Met Asn Phe Thr Ala Glu Leu Ile Asn His Val
210 215 220
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225 230 235 240
Tyr Val Asp Val Arg Asp Thr Ala Arg Ala His Val Asp Ala Leu Lys
245 250 255
Asn Glu Lys Leu Ala Phe Gln Arg Leu Leu Val Val Gly Pro Phe Leu
260 265 270
Ser Ser Gln Gln Ile Tyr Asp Ile Val Asn Glu Arg Phe Pro Gln Leu
275 280 285
Arg Gly Lys Ile Ala Arg Gly Glu Pro Gly Ser Asp Leu Asp Pro Ala
290 295 300
Lys Leu Ala Lys Phe Asp His Ala Arg Thr Thr Gln Ala Leu Gly Trp
305 310 315 320
Glu Phe Thr Pro Ile Glu Lys Ala Ile Ala Asp Glu Val Ala Gln Ile
325 330 335
Leu Arg Val Gly Ala Tyr Arg Gly
340
Claims (6)
1.一种羰基还原酶突变体,其特征在于所述羰基还原酶突变体氨基酸序列为SEQ IDNO.3所示。
2.一种权利要求1所述羰基还原酶突变体在不对称还原脂肪族酮中的应用,其特征在于所述脂肪族酮酯为6-氰基-(5R)-羟基-3-羰基己酸叔丁酯或4-氯乙酰乙酸乙酯。
3.如权利要求2所述的应用,其特征在于所述的应用方法为:将含羰基还原酶突变体基因的工程菌经发酵培养后的发酵液离心,收集湿菌体,以湿菌体或湿菌体经超声破碎且镍柱纯化的纯酶为催化剂,以含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体或湿菌体经超声破碎且镍柱纯化的纯酶为辅助催化剂,以脂肪族酮酯为底物,以葡萄糖为辅底物,以pH 7.0、100 mM的PBS缓冲液为反应介质构成转化体系,在35-40℃、400-600 rpm条件下进行反应,反应结束,反应液分离纯化,获得手性化合物;当底物为6-氰基-(5R)-羟基-3-羰基己酸叔丁酯时,手性化合物为6-氰基-(3R, 5R)-二羟基己酸叔丁酯;当底物为4-氯乙酰乙酸乙酯时,手性化合物为(S)-4-氯-3-羟基丁酸乙酯。
4.如权利要求3所述的应用,其特征在于所述反应体系中,底物添加终浓度为10-20 g/L,葡萄糖添加终浓度20-40 g/L,所述催化剂与辅助催化剂的湿菌体以干重比1.0-5.0 : 1混合成混合菌体形式加入,催化剂和辅助催化剂用量以混合菌体总量干重计为5-12.5 gDCW/L。
5.如权利要求3所述的应用,其特征在于所述湿菌体按如下方法制备:将含羰基还原酶突变体基因的工程菌接种到含终浓度50 μg/mL卡那霉素的LB液体培养基中,37℃培养10h,以体积浓度1.5-2.0%的接种量接种到新鲜的含终浓度50 μg/mL卡那霉素的LB液体培养基中,37℃、200 rpm培养2 h,再向培养液中加入终浓度为0.10 mM异丙基硫代半乳糖苷,28℃培养12 h后,4℃、8000 rpm离心10 min,获得含羰基还原酶突变体的湿菌体。
6.如权利要求3所述的应用,其特征在于所述葡萄糖脱氢酶基因的核苷酸序列如GenBank NO. KM817194.1所示。
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