CN113801859B - 一种用于制备手性醇化合物的羰基还原酶突变体及其用途 - Google Patents
一种用于制备手性醇化合物的羰基还原酶突变体及其用途 Download PDFInfo
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Abstract
本发明涉及一种羰基还原酶突变体及其用途，所述羰基还原酶突变体是在对应于SEQ ID NO：2的氨基酸序列存在下述突变之一或者两种以上的组合：45位的V突变为I(V45I)，63位的K突变为Q(K63Q)，141位的G突变为A(G141A)，141位的G突变为V(G141V)，195位的I突变为L(I195L)，204位的A突变为V(A204V)。本发明羰基还原酶突变体用于手性醇合成时，底物浓度可达100g/L以上，并且有高达99.5％的的立体选择性，酶活性高，催化效率好。本发明方法与化学合成法相比，显著减少或消除了各类污染性化学品的使用，从而显著降低了环境污染风险。选择了特定的羰基还原酶突变体，酶活性高，立体选择性好，极大地提高生产效率，降低生产成本，适合大规模的工业生产。
Description
技术领域
本发明属于酶工程和有机合成领域，尤其涉及一种用于制备手性醇化合物的羰基还原酶突变体及其用途。
背景技术
手性双醇类化合物是靶向肝脏药物所使用的关键中间体，已在多个临床及临床前研究药物中使用。例如甲磺酸帕拉德福韦，该肝靶向前药目前由西安新通医药股份有限公司开发，已完成临床二期，目前正在开展临床三期。该关键双醇中间体均采用化学的合成路线。
在专利文献US20030225277中报道了制备方法，工艺路线如下：
该技术方案需要使用有机催化剂(+)-DIPCl，该催化剂价格昂贵，工艺操作繁琐，难以应用于规模化生产。使用该工艺路线制备的双醇化合物的手性纯度为98％，为了满足注册申报要求，需要对其手性纯度进行精制，进一步增加了生产成本，整体路线生产成本较高，因此迫切需要一条满足商业生产的制备方法。
现有技术中有很多使用羰基还原酶及其突变体对羰基进行还原的文献。羰基还原酶可以实现羰基的高立体选择性，在制备手性醇方面具有广泛的应用。需要解决的问题主要是高的酶活力和立体选择性。
CN112941043公开了一种羰基还原酶及其突变体，可以用于制备手性β’羟基-β-氨基酸；CN112626144A公开了一种用于制备替诺福韦中间体的生物合成方法，使用了羰基还原酶；CN112359028A公开了一种羰基还原酶，是产酶菌株Escherichia coli TM1908接种，发酵得到。
CN102482648A公开了一种制备α-氯代醇的立体选择性好的酮还原酶。其转化率和立体选择性都高，但是酶活力不足，还不能满足实际需求。特别是工业化大规模制备。
CN108753851A公开了一种以羰基还原酶为生物催化剂制备手性单羟基衍生化的的手性醇的方法，其利用的羰基还原酶为ChKRED12，其在NCBI数据库中的登录号为KC342012。
但是利用现有技术中已知的羰基还原酶在还原特定结构的底物时，还存在酶活性不足的情况，并不利于开展大规模的工业化制备。由于的酶的价格都比较昂贵，酶活性严重制约着是否能产业化。
发明内容
针对现有技术中的羰基还原酶制备手性双醇化合物，特别是帕拉德福韦中间体(S)-1-(3-卤代苯基)-1,3-丙二醇时，存在酶活性不足的缺陷，本发明提出了一种羰基还原酶突变体，其具有更高的酶活性和优异的立体选择性，为(3-卤代苯基)-1,3-丙二醇类的医药中间体的酶合成提供了产业化的便利。
本发明第一个目的是提供一种羰基还原酶突变体，是在对应于SEQ ID NO：2的氨基酸序列存在下述突变之一或者两种以上的组合：45位的V突变为I(V45I)，63位的K突变为Q(K63Q)，141位的G突变为A(G141A)，141位的G突变为V(G141V)，195位的I突变为L(I195L)，204位的A突变为V(A204V)。
进一步地，所述羰基还原酶突变体，是在对应于SEQ ID NO：2的氨基酸序列存在下述突变之一：
(i)141位的G突变为A(G141A)，且195位的I突变为L(I195L)，其氨基酸序列如SEQID No：4所示；
(ii)141位的G突变为V(G141V)，且195位的I突变为L(I195L)，其氨基酸序列如SEQID No：6所示。
(iii)45位的V突变为I(V45I)；141位的G突变为V(G141V，I195L)，195位的I突变为L(I195L)，其氨基酸序列如SEQ ID No：8所示；
(iv)45位的V突变为I(V45I)，141位的G突变为V(G141V，I195L)，195位的I突变为L(I195L)，204位的A突变为V(A204V)，其氨基酸序列如SEQ ID No：10所示；
(v)45位的V突变为I(V45I)，63位的K突变为Q(K63Q)，118位的L突变为M(L118M)，141位的G突变为V(G141V，I195L)，204位的A突变为V(A204V)，其氨基酸序列如SEQ ID No：12所示。
SEQ ID NO：2的氨基酸序列对应的羰基还原酶来源于Novosphingobiumaromaticivorans的羰基还原酶NaKRED，参考文献CN102482648，发明人通过酶库的筛选，预料不到地发现在对SEQ ID NO：2的氨基酸序列的WO03羰基还原酶的特定位点突变为特定氨基酸后得到的突变体，在利用式(IV)底物制备式(V)时，酶活性高，转化率高，立体选择型好。
本发明提供的羰基还原酶突变体还包括以下范围：对SEQ ID No:4、6、8、10、或12所示的氨基酸序列在保持酶活性范围内，进行一个或多个氨基酸的替换、缺失、改变、插入或增加，所得到的氨基酸序列；或
对SEQ ID No:4、6、8、10、或12所示的氨基酸序列在保持酶活性范围内，在序列的N端或C端进行一个或多个氨基酸的插入，所述插入的氨基酸残基个数为1-20个；优选1-10个，更优选1-5个。
对于酶的制备方法，将实现辅酶再生的醇脱氢酶或葡萄糖脱氢酶或甲酸脱氢酶与目的基因构建在同一质粒pET28a(+)载体上，然后导入表达宿主大肠杆菌，通过诱导表达，获得含有目的酶的菌体。可直接使用离心获得菌体，将其破壁获得粗酶液或粗酶粉，用于后续的生物转化反应。
本发明的第二个目的是提供一种手性双醇类化合物的制备方法，包括以下步骤：
以式Ⅳ化合物为底物，在辅酶存在下，在上述羰基还原酶催化下，进行不对称还原反应，得到式V化合物；
其中R基团选自
中的至少一种；X选自F、Cl、Br、I。
所述羰基还原酶的加入形式包括菌体、粗酶液、粗酶粉或纯酶。
优选地，所述反应体系中，所述底物，即式Ⅵ化合物的浓度为50～200g/L；更优选为100～150g/L。
优选地，羰基还原酶的用量与底物质量比率优选为1wt％～6wt％，比如当以湿菌体加入时，加入湿菌体的质量与底物的质量比率为30wt％～100wt％，优选50wt％-70wt％。
进一步地，所述的反应体系中，还存在共底物，所述的共底物选自下组：异丙醇、葡萄糖、甲酸铵中的至少一种；优选地，共底物的浓度为底物浓度的100-200％，优选共底物的浓度为底物浓度的140-170％。
进一步地，制备过程中在磷酸缓冲盐体系中进行，pH为6-9，较佳地6.5-8.5，更佳地7.0-7.5；和/或
反应温度为10℃-50℃，较佳地20℃-40℃，更佳地25℃-35℃；和/或
反应时间为0.1-240小时，较佳地0.5-120小时，更佳地1-72小时，又更佳地3-10小时。
进一步地，所述的辅酶指能够实现氧化还原反应中电子传递的辅酶；包括还原型辅酶、氧化型辅酶中的至少一种；所述还原型辅酶选自NADH、NADPH、或其组合；所述氧化型辅酶选自NAD+、NADP+或其组合。
更进一步地，所述辅酶用量与底物用量比率为0.01wt％～1.0wt％、优选为0.01wt％～0.5wt％。
进一步地，所述的反应体系中，羰基还原酶为游离形式的酶、固定化酶、或菌体形式的酶。
进一步地，所述羰基还原酶和/或用于辅酶再生的酶的基因构建在表达载体上。
进一步地，所述的反应体系中，还存在用于辅酶再生的酶，具体选自醇脱氢酶，甲酸脱氢酶、葡萄糖脱氢酶、或其组合。
在本发明的羰基还原酶催化下，式(IV)的底物化合物转变为产物式(V)化合物的转化率在70％以上，优选在85％以上，更优选在95％以上，最优选在99％以上；式V化合物ee值在90％以上，更优选95％以上，更优选在99％以上。
在另一优选例中，在步骤(b)中，所述的分离包括：加入异丙醇，离心菌体，部分浓缩，甲叔醚萃取，浓缩有机层。
本发明的主要优点在于：
(1)本发明适合工业化生产具有高化学纯度和高光学纯度的式V化合物，然后进一步进行后续反应，制备(S)-1-(3-氯苯基)-1,3-丙二醇。
(2)本发明方法和催化还原反应体系有高达99.5％的的立体选择性，以及对有机溶剂的高耐受性、对底物的耐受性，高催化活性，底物浓度可达100g/L以上，从而可以在较高的底物浓度下，进行大规模生产。
(3)本发明方法与化学合成法相比，显著减少或消除了各类污染性化学品的使用，从而显著降低了环境污染风险。选择了特定的羰基还原酶突变体，酶活性高，立体选择性好，极大地提高生产效率，降低生产成本。本发明方方法后处理仅需萃取操作，操作简单。
附图说明
图1是式V化合物的消旋体的手性图谱；
图2是实施例1以SEQ ID NO：4的氨基酸序列的羰基还原酶突变体制备式V化合物的手性图谱；
图3实施例3所得产物的1HNMR图谱。
具体实施方式
以下以具体实施方式对本发明保护内容进行进一步地解释说明，但是应该理解，具体实施方式的内容并不应理解为是对本发明保护内容的限制。
制备例羰基还原酶工程菌及羰基还原酶同源突变库的构建
将来自WO03羰基还原酶酶基因及来自其突变体基因的5种羰基还原酶酶基因克隆入pET28a(+)载体，然后导入宿主大肠杆菌BL21，通过诱导表达，获得羰基还原酶的重组基因工程菌。
以野生型的羰基还原酶WO03(其核酸序列如SEQ NO.1所示，其氨基酸序列如SEQNO.2所示)为模板，利用随机点突变试剂盒(II Site-DirectedMutagenesis Kit)进行易错PCR，或通过定向进化将野生型的羰基还原酶基因进行突变，获得包含进化的羰基还原酶基因的质粒文库。构建后的质粒文库转入大肠杆菌BL21(DE3)(货号：康为世纪CW0809S)中，于37℃烘箱中，在含50μg/mL卡那霉素的LB固体培养基上培养过夜。将单菌落挑选至含400μL LB液体培养基(含50μg/mL卡那霉素)的96孔板中，37℃，200rpm过夜培养，得种子液。然后将10μL种子液转移至含400μL发酵培养基(含50μg/mL卡那霉素)的96深孔板中，37℃培养至OD600值>0.8。然后加入终浓度为1mM的异丙基硫代半乳糖苷(IPTG)，降温至28℃诱导突变体表达，继续培养20h。发酵结束后，通过4000g，30min离心收集菌体，然后再用200μL裂解缓冲液(含有1000U溶菌酶的0.1M磷酸缓冲液，pH 7.0)重新悬浮菌体，并在30℃裂解1h。裂解结束后，于4℃，4000g，离心30min，澄清的上清液用于测定突变体活性。将190μL反应液(含0.4mM底物、1mMNADH、40μL二甲基亚砜)加入新的96孔板中，再加入10μL的上清液后，在340nm检测NADH的变化，NADH的消耗量反应突变体酶活的高低，筛选得到如下表1所示的羰基还原酶突变体。
表1
实施例1
以化合物Ⅳ为底物，对羰基还原酶进行筛选，筛选方法与筛选结果如下；
反应转化率的检测方法：phenomenex Gemini C18 4.6*250mm 5μm)；流动相A为10％乙腈，流动相B为乙腈，按下表进行梯度洗脱；流速为每分钟1.0ml；柱温为30℃；检测波长为220nm。
化合物V的手性监测方法如下：色谱柱：大赛璐IB-3,5μm,4.6*250mm；流动相：异丙醇∶正己烷＝10:90；流速1.0mL/min；运行时间：20min；柱温30℃；检测波长：220nm。S构型10min,R构型13min。
结果如下表2所示：
表2
| 酶编号 | 转化率 | e.e.值 | 构型 |
| WO03(SEQ ID No.2)[a] | 98.9％ | 99.80％ | S |
| LSADH[b] | 21.3％ | 93.78％ | R |
| LK[b] | 12.4％ | 38.52％ | S |
注：羰基还原酶LSADH来源于Leifsonia sp.Strain S749，登记号为AB213459，羰基还原酶LK来源于Lactobacillus kefir，参考文献WO 2010/025238。
反应条件:(a)1g IV化合物,0.2g湿菌体,0.001g NADP+,0.1g GDH湿菌体，1.5g葡萄糖和10ml磷酸缓冲液(100mM,pH 7.0)于30℃、220rpm摇床反应24h；(b)1g IV化合物,0.2g湿菌体,0.001g NAD+,20％异丙醇和10ml磷酸缓冲液(100mM,pH 7.0)于30℃、220rpm摇床反应24h。
可以看出，上述羰基还原酶，除WO03(氨基酸序列如SEQ ID No:2所示，其编码基因如SEQ ID No:1所示。)的转化率和立体选择性都不能满足工业化生产的要求，不适合大规模的生产关键中间体(S)-1-(4-氯苯基)-1,3-丙二醇，但羰基还原酶WO03的酶活性还不够高，因此，本发明以WO03的羰基还原酶作为基础，对其进行突变，筛选出酶活性，转化率和立体选择性皆优异的羰基还原酶突变体。
反应体系中可以用上述羰基还原酶的湿菌体、粗酶液、粗酶粉或者纯酶等。为获得较高的转化效率，优选使用粗酶液或者湿菌体。羰基还原酶的用量与底物质量比率优选为1wt％～6wt％或者休止细胞菌体的质量与底物的质量比率为10wt％～100wt％。
实施例2:
将制备例1中保存于甘油中的重组基因工程菌，接种到含100ug/ml氨茉霉素的LB液体培养基中，37C，220rpm，培养12-16h，得到种子培养基，将此种子液按1.5％的比例接种到含100ug/ml氨茉抗性的液体培养基中，然后37℃，220rpm培养至OD600值>2.0，加入终浓度为1.0％乳糖，降温至25C，继续培养2h，加入终浓度为0.5％的乳糖，培养20h，放罐，离心得菌体，用于生物转化的催化剂。按照实施例1的反应条件(a)，即：1g IV化合物,0.3g湿菌体,0.001g NADP+,0.1g GDH湿菌体，0.2g葡萄糖和10ml磷酸缓冲液(100mM,pH 7.0)于30℃、220rpm摇床反应24h制备化合物V。对所得羰基还原酶同源突变库库进行筛选，结果如下表3所示：
表3
由于密码子的简并性，如上羰基还原酶突变体的核酸序列不局限于表3中所列出核酸序列。本领域技术人员可以通过适当引入替换、缺失、改变、插入或增加来获得该碱基序列的同系物，本发明涵盖这些同系物，只要其表达的重组酶保持对Ⅳ化合物的催化还原活性即可。本发明中多聚核扞酸的同系物可以通过对碱基序列的一个或多个碱基在保持酶活性范围内进行替换、缺失或增加来制得。
优选地，本发明提供了氨基酸序列如SEQ ID No:4、6、8、10、或12所示的羰基还原酶突变体，其相对于野生型酶WO03均具有明显改善的酶活性，对S构型的立体选择性均可达到99％以上。
酶活性的定义：在50ml离心管内反应，称取底物底物0.5g，加入异丙醇1.5ml，加入pH6.0-6.5磷酸缓冲液3.5ml，称取辅酶NAD 0.025g，将以上体系在30℃水浴锅预热15min称取0.5g菌体，加入反应体系，放入30℃恒温室摇床150rpm，开始计时1h。结束后，在摇匀的状态下快速用移液枪移去0.1ml反应液，加入10ml离心管中，然后加入4.9ml异丙醇，用移液枪充分混匀。用离心机离心10min，3500rpm。将上清液倒入取样管中，检测转化率，即为酶活性。
实施例3、化合物V(R＝Et)的生物催化制备
称取100g化合物Ⅳ(R＝Et)于3L四口烧瓶中，加入0.1mol/L pH7.0磷酸缓冲液1.0L，加入葡萄糖158.4g，搅拌均匀，加入GDH湿菌体10g，52.3g羰基还原酶突变体WO08的的湿菌体，NAD+ 0.2g，置于35℃水浴中，220rpm机械搅拌下反应，半小时后，测得反应液pH值约5.6，用饱和碳酸钠溶液调节pH值至7.2，再过半小时，测得反应液pH值约6.4，用饱和碳酸钠溶液调节pH至7.1，随后继续监测pH值，基本不变，保持6.9～7.0。反应10h，HPLC监测原料剩余0.55％，终止反应，加入1.5L乙酸乙酯，搅拌约10min，随后分液，水层用乙酸乙酯萃取，500mL×2，合并乙酸乙酯，用饱和NaCl洗涤500mL×2，有机相经无水硫酸钠干燥后，浓缩得淡黄色油状物97.1g，收率95.4％，HPLC纯度为99.1％，ee值99.9％。在相同的情况下，使用相同量的野生型羰基还原酶WO03湿菌体，收率为74.8％。产物式V化合物的1H NMR如图3所示：(600MHz,Chloroform-d)δ7.39(s,1H),7.26(dq,J＝15.9,7.7Hz,3H),5.24–4.94(m,1H),4.18(q,J＝7.1Hz,2H),3.52(s,1H),2.82–2.52(m,2H),J＝7.2Hz,3H).13C NMR(151MHz,Chloroform-d)δ172.20,144.60,134.44,129.82,125.95,123.81,69.65,61.04,43.19,14.13.
本实施例所得产物化合物V的手性图谱如图2所示，数据列于如下表4所示：
表4
| 编号 | 保留时间(min) | 峰面积 | 相对含量 |
| 1(S构型) | 9.978 | 577.918 | 99.90 |
| 2(R构型) | 13.095 | 0.571 | 0.10 |
| 总 | 578.489 | 100.00 |
实施例4、化合物V(R＝Me)的生物催化制备
称取100g化合物Ⅳ(R＝Me)于3L四口烧瓶中，加入0.1mol/L pH7.0磷酸缓冲液1.0L，加入异丙醇200ml(密度0.7855g/mL)，搅拌均匀，54.7g羰基还原酶突变体WO07的湿菌体，NAD+ 0.2g,置于35℃水浴中，220rpm机械搅拌下反应，半小时后，测得反应液pH值约5.8，用饱和碳酸钠溶液调节pH值至7.0，再过半小时，测得反应液pH值约6.4，用饱和碳酸钠溶液调节pH至7.3，随后继续监测pH值，基本不变，保持6.9～7.0。反应第3h取样TLC监测原料点基本消失，HPLC监测原料剩余0.52％，终止反应，加入1.0L乙酸乙酯，搅拌约10min，随后分液，水层用500mL×2乙酸乙酯萃取，合并乙酸乙酯，用饱和NaCl洗涤500mL×2，有机相经无水硫酸钠干燥后，浓缩得淡黄色油状物92.9g，收率91.4％，HPLC纯度为99.3％，ee值99.8％。
序列表
<110> 山东寰酶生物制药有限公司
<120> 一种用于制备手性醇化合物的羰基还原酶突变体及其用途
<160> 12
<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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tccgtgcatc cgggcggcat cgataccccg atgctcggct cgatcatgga caagtacgtc 600
gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780
<210> 2
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Gly Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Ile Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
<210> 3
<211> 780
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60
cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120
ccctcggccg atgtcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180
ggctggaagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240
cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300
cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360
ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420
gcgggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480
atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540
tccgtgcatc cgggcggcat cgataccccg atgctcggct cgctcatgga caagtacgtc 600
gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780
<210> 4
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Ala Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
<210> 5
<211> 780
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60
cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120
ccctcggccg atgtcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180
ggctggaagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240
cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300
cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360
ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420
gtcggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480
atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540
tccgtgcatc cgggcggcat cgataccccg atgctcggct cgctcatgga caagtacgtc 600
gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780
<210> 6
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Val Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
<210> 7
<211> 780
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60
cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120
ccctcggccg atatcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180
ggctggaagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240
cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300
cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360
ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420
gtcggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480
atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540
tccgtgcatc cgggcggcat cgataccccg atgctcggct cgctcatgga caagtacgtc 600
gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780
<210> 8
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Ile Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Val Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
<210> 9
<211> 780
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60
cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120
ccctcggccg atatcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180
ggctggaagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240
cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300
cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360
ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420
gtcggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480
atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540
tccgtgcatc cgggcggcat cgataccccg atgctcggct cgctcatgga caagtacgtc 600
gaactcggcg tcgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780
<210> 10
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Ile Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Val Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Val Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
<210> 11
<211> 780
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60
cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcc 120
ccctcggccg atatcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180
ggctggcagg ccgtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240
cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300
cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gatgccgctg 360
ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420
gtcggcctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480
atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540
tccgtgcatc cgggcggcat cgataccccg atgctcggct cgctcatgga caagtacgtc 600
gaactcggcg tcgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcaagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780
<210> 12
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Ile Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Gln Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Met Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Val Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Val Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
245 250 255
Ser Gln Val
Claims (16)
1.一种羰基还原酶突变体，其特征在于，在对应于SEQ ID NO：2的氨基酸序列存在下述突变：
45位的V突变为I(V45I)，63位的K突变为Q(K63Q)，118位的L突变为M(L118M)，141位的G突变为V(G141V，I195L)，195位的I突变为L(I195L)，204位的A突变为V(A204V)，其氨基酸序列如SEQ ID No：12所示。
2.一种手性双醇类化合物的制备方法，其特征在于，包括以下步骤：
以式Ⅳ化合物为底物，在辅酶存在下，在权利要求1所述羰基还原酶催化下，进行不对称还原反应，得到式Ⅴ化合物；
；
其中R基团选自,/>,/>,/>,/>,,/>,/>,/>,/>,,/>,/>中的至少一种；X选自F、Cl、Br、I。
3.根据权利要求2所述的制备方法，其特征在于，所述羰基还原酶的加入形式包括菌体、粗酶液、粗酶粉或纯酶。
4.根据权利要求2所述的制备方法，其特征在于，式IV化合物的浓度为50~200g/L；羰基还原酶的用量与底物质量比率为1wt% ~ 6wt% 。
5.根据权利要求4所述的制备方法，其特征在于，式IV化合物的浓度为100~150g/L。
6.根据权利要求2所述的制备方法，其特征在于，除式IV化合物外，还存在共底物，所述的共底物选自下组：异丙醇、葡萄糖、甲酸铵中的至少一种。
7.根据权利要求6所述的制备方法，其特征在于，共底物的质量浓度为底物浓度的100-200%。
8.根据权利要求7所述的制备方法，其特征在于，共底物的浓度为底物质量浓度的140-170%。
9.根据权利要求2所述的制备方法，其特征在于，反应在磷酸缓冲盐体系中进行，pH 为6-9；和/或
反应温度为 10℃-50℃；和/或
反应时间为0.1-240小时。
10.根据权利要求9所述的制备方法，其特征在于，反应在磷酸缓冲盐体系中进行，pH为6.5-8.5；和/或
反应温度为20℃-40℃；和/或
反应时间为0.5-120 小时。
11.根据权利要求9所述的制备方法，其特征在于，反应在磷酸缓冲盐体系中进行，pH为7.0-7.5；和/或
反应温度为25℃-35℃；和/或
反应时间为1-72小时。
12.根据权利要求2所述的制备方法，其特征在于，所述的辅酶指能够实现氧化还原反应中电子传递的辅酶；包括还原型辅酶、氧化型辅酶中的至少一种；所述还原型辅酶选自NADH、NADPH、或其组合；所述氧化型辅酶选自NAD+、NADP+或其组合。
13.根据权利要求12所述的制备方法，其特征在于，所述辅酶用量与底物用量比率为0.01wt%~1.0wt%。
14.根据权利要求12所述的制备方法，其特征在于，所述辅酶用量与底物用量比率0.01wt%~0.5wt%。
15.根据权利要求2所述的制备方法，其特征在于，还存在用于辅酶再生的酶。
16.根据权利要求15所述的制备方法，其特征在于，用于辅酶再生的酶选自醇脱氢酶，甲酸脱氢酶、葡萄糖脱氢酶或其组合。
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