CN115433727B - L-苏氨酸醛缩酶及其制备方法与应用 - Google Patents
L-苏氨酸醛缩酶及其制备方法与应用 Download PDFInfo
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- CN115433727B CN115433727B CN202110614530.5A CN202110614530A CN115433727B CN 115433727 B CN115433727 B CN 115433727B CN 202110614530 A CN202110614530 A CN 202110614530A CN 115433727 B CN115433727 B CN 115433727B
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- threonine aldolase
- acid residue
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
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- 238000000034 method Methods 0.000 claims description 27
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- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 23
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000004471 Glycine Substances 0.000 claims description 18
- 238000005119 centrifugation Methods 0.000 claims description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
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- 229910052739 hydrogen Inorganic materials 0.000 abstract description 2
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- 238000001514 detection method Methods 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
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Abstract
本发明公开了一种L‑苏氨酸醛缩酶及其制备方法与应用。所述L‑苏氨酸醛缩酶的氨基酸序列如SEQ ID NO:36所示，其中：第10位氨基酸残基为S、F或K；和/或，第93位氨基酸残基为D、T或H；和/或，第310位氨基酸残基为R、V、T、H、F或Q；且所述L‑苏氨酸醛缩酶的氨基酸序列不为SEQ ID NO:1。本发明的L‑苏氨酸醛缩酶在制备(2S,3R)‑对甲砜基苯丝氨酸中的底物转化率可达66％，d.e.值均在80％以上，提高了现有L‑苏氨酸醛缩酶的底物转化率和d.e.值，具有产业化应用的价值。
Description
技术领域
本发明属于酶工程生物技术领域，具体涉及一种L-苏氨酸醛缩酶及其制备方法与应用。
背景技术
氟苯尼考又称氟甲砜霉素、氯砜尼可等，为新一代的动物专用广谱抗菌药，其结构类似于甲砜霉素，但其抗菌能力可达甲砜霉素的10倍之多。而且它抗菌谱广、杀菌作用强大、安全高效，不会造成再生障碍性贫血，无致畸、致癌和致突变作用，故其被广泛应用。目前，世界上已有20多个国家批准并允许其销售使用。在国内，氟苯尼考已被批准用于猪、禽、鱼等多种动物。
对甲砜基苯丝氨酸是β-氨基醇类广谱抗生素甲砜霉素和氟苯尼考合成的关键手性砌块，具有2个手性中心，可形成4种手性异构体，分别是：(2S,3R)-对甲砜基苯丝氨酸、(2S,3S)-对甲砜基苯丝氨酸、(2R,3R)-对甲砜基苯丝氨酸和(2R,3S)-对甲砜基苯丝氨酸。目前已报道的活性中间体为(2S,3R)-构型，其结构式如下式所示：
目前，国内外工业生产(2S,3R)-对甲砜基苯丝氨酸的主要方法是以对甲砜基苯甲醛和甘氨酸为原料，在硫酸铜和氨水存在的条件下，通过羟醛缩合反应生成对甲砜基苯丝氨酸，酯化得到对甲砜基苯丝氨酸乙酯，再经D-酒石酸拆分得到(2S,3R)-对甲砜基苯丝氨酸。该制备方法存在反应条件剧烈、使用强酸和强碱、需要昂贵的手性拆分试剂D-酒石酸以及废水的处理成本高等问题。
相较于化学合成法，生物催化法具有转化条件温和、环境友好以及立体选择性强等优点。苏氨酸醛缩酶(Threonine Aldolase，TA；EC4.1.2.5)属于甘氨酸依赖型醛缩酶，该类型醛缩酶最大的特点是在辅助因子磷酸吡哆醛(Pyridoxal phosphate；PLP)的存在下，能催化不同取代基的醛与甘氨酸进行羟醛缩合反应，直接形成不对称C-C键，从而得到β-羟基-α-氨基酸。但是常见的苏氨酸醛缩酶在形成的两个手性中心的α-碳具有高度的选择性，而β-碳的立体选择性较差。
专利CN110914288A公布了以一个来源于恶臭假单胞菌(Pseudomonas putida)的野生型醛缩酶为起点，经过基因工程的改造获得的工程化醛缩酶多肽，不对称地合成(2S,3R)-3-[对-(甲砜基)苯基]-3-羟基-2-氨基-丙酸)的方法，底物的总转化率≥65％，产物的d.e.≥90％。
专利CN110272856A提供了一种表达D-苏氨酸醛缩酶(氨基酸序列为该专利中的SEQ ID NO:2)的重组木糖氧化产碱杆菌(Alcaligenes xylosoxydans)，生产的D-苏氨酸醛缩酶可高效催化对甲砜基苯甲醛和甘氨酸合成(2S,3R)-对甲砜基苯丝氨酸，产率为65％以上，d.e.值为95％以上，该方法中底物浓度仅100mmol/L；且反应温度需要控制在较低温度，如果5-15度，当反应温度达到30度时，d.e.值明显地降低。但是，该专利未提供L-苏氨酸醛缩酶。
专利CN110577948A公开了一种来源于草状珊瑚状放线菌(Actinocoralliaherbida)的L-苏氨酸醛缩酶(GenBank NO:WP_123664127-1)，具有将对甲砜基苯甲醛和甘氨酸不对称催化合成(2S,3R)-对甲砜基苯丝氨酸的活性。但是，其产物d.e.值仅62.3％、底物转化率仅75.7％。
综上，目前报道的苏氨酸醛缩酶对α-碳具有较高的选择性，ee(％)>99％，而对β-碳的选择性不高，d.e.不高。因此亟需研究出一种能够高选择性合成(2S,3R)-对甲砜基苯丝氨酸的L-苏氨酸醛缩酶，并且达到保证高底物转化率、高产物d.e.值、高底物浓度的综合优良效果。
发明内容
本发明所要解决的技术问题是为了克服现有技术通过苏氨酸醛缩酶生产(2S,3R)-对甲砜基苯丝氨酸中底物转化率不高、产物d.e.值不高的缺陷，提供一种L-苏氨酸醛缩酶及其制备方法与应用。本发明的L-苏氨酸醛缩酶在制备(2S,3R)-对甲砜基苯丝氨酸中的底物转化率可达66％，d.e.值均在80％以上。
本发明通过以下技术方案实现上述问题：
本发明的第一方面提供一种L-苏氨酸醛缩酶，其特征在于，所述L-苏氨酸醛缩酶的氨基酸序列如SEQ ID NO:36所示，其中：
第10位氨基酸残基为S、F或K；和/或，
第93位氨基酸残基为D、T或H；和/或，
第310位氨基酸残基为R、V、T、H、F或Q；
且所述L-苏氨酸醛缩酶的氨基酸序列不为SEQ ID NO:1。
在本发明一些较佳实施方案中，所述L-苏氨酸醛缩酶的氨基酸序列中：
第10位氨基酸残基S突变为F；和/或，
第93位氨基酸残基D突变为T；和/或，
第310位氨基酸残基R突变为F或Q。
在本发明一些更佳实施方案中，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F；或者，所述L-苏氨酸醛缩酶的氨基酸序列中第93位氨基酸残基为T；或者，所述L-苏氨酸醛缩酶的氨基酸序列中第310位氨基酸残基为F；或者，所述L-苏氨酸醛缩酶的氨基酸序列中第310位氨基酸残基为Q；或者，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F，第93位氨基酸残基为T；或者，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F，第310位氨基酸残基为F；或者，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F，第93位氨基酸残基为T，第310位氨基酸残基为F；或者，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F，第93位氨基酸残基为T，第310位氨基酸残基为Q。
在本发明一些具体实施方案中，编码所述L-苏氨酸醛缩酶的核苷酸序列如SEQ IDNO:3、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ IDNO:16或SEQ ID NO:17所示。
本发明的第二方面提供一种分离的核酸，所述核酸编码如第一方面所述的L-苏氨酸醛缩酶。
较佳地，所述核酸的核苷酸序列如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ IDNO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:17所示。
更佳地，所述核酸的核苷酸序列如SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:9、SEQID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:16或SEQ ID NO:17所示。
本发明的第三方面提供一种重组表达载体，所述重组表达载体包含如第二方面所述的分离的核酸。
本发明的第四方面提供一种转化体，所述转化体为包含如第二方面所述的分离的核酸或如第三方面所述的重组表达载体的宿主细胞。
所述宿主细胞可为本领域常规，较佳地为埃希氏大肠杆菌(Escherichia coli)。
本发明的第五方面提供一种制备如第一方面所述的L-苏氨酸醛缩酶的方法，所述L-苏氨酸醛缩酶通过以下步骤制备：
培养如第四方面所述的转化体，使其表达所述L-苏氨酸醛缩酶，即得。
所述转化体表达L-苏氨酸醛缩酶后，可采用本领域常规技术手段进行提取，例如可制备粗酶液，粗酶液制备后可进行常规的浓缩、置换，也可将粗酶液进一步经离子交换层析、亲和层析、疏水层析和分子筛层析等纯化步骤中的一种或多种以提纯所述L-苏氨酸醛缩酶。
所述转化体表达L-苏氨酸醛缩酶后，可采用本领域常规技术手段进行固定化，得到固定化酶。
较佳地，所述步骤具体包括：
(1)将含所述L-苏氨酸醛缩酶的转化体接种至含抗生素的培养基例如LB培养基中振荡培养，得种子液；
(2)将(1)中的种子液转接至含抗生素的培养基例如TB培养基中振荡培养；
(3)向(2)中的培养基中加入IPTG诱导过夜，离心后收集菌体；
(4)洗涤并重悬(3)中收集的菌体，破碎后离心，即得含所述L-苏氨酸醛缩酶的粗酶液。
更佳地，所述抗生素为50μg/mL卡纳霉素；和/或，
(1)中所述振荡培养的条件为37℃、12h；和/或，
(2)中所述种子液的接种量为2％体积；和/或，所述振荡培养的条件为37℃、直至OD600为0.75-0.85；和/或，
(3)中所述IPTG的终浓度为0.05-5mM，例如0.1mM IPTG；和/或，所述诱导过夜的温度为20～30℃例如25℃；和/或，所述离心的条件为4000-12000rpm例如10000rpm离心5-30min例如10min；和/或，
(4)中所述洗涤使用50mM pH 8.0磷酸缓冲液洗涤；和/或，所述重悬使用50mM pH8.0磷酸缓冲液，其与菌体的体积重量比为5-20:1例如10:1mL/g；和/或，所述破碎为使用超声破碎或高压均质机均质；和/或，所述离心的条件为8000-12000rpm例如10000rpm，离心5-20min例如10min。
本发明的第六方面提供一种(2S,3R)-对甲砜基苯丝氨酸的制备方法，包括以下步骤：在如第一方面所述的L-苏氨酸醛缩酶、磷酸吡哆醛和反应溶剂存在的条件下，使对甲砜基苯甲醛和甘氨酸进行醛缩反应，即得(2S,3R)-对甲砜基苯丝氨酸。
所述醛缩反应的反应式如下所示：
本发明所述的对甲砜基苯丝氨酸包括四种构型，分别为(2S，3R)构型、(2R，3S)构型、(2S，3S)构型和(2R，3R)构型，如下所示：
在本发明一较佳实施方案中，制备所述L-苏氨酸醛缩酶的方法如第五方面所述。
本发明的第七方面提供一种氟苯尼考的制备方法，包括以下步骤：在如第一方面所述的L-苏氨酸醛缩酶、磷酸吡哆醛和反应溶剂存在的条件下，使对甲砜基苯甲醛和甘氨酸进行醛缩反应，得(2S,3R)-对甲砜基苯丝氨酸；其后由(2S,3R)-对甲砜基苯丝氨酸制得所述氟苯尼考。
在本发明一较佳实施方案中，制备所述L-苏氨酸醛缩酶的方法如第五方面所述。
在本发明一更佳实施方案中，所述对甲砜基苯甲醛和甘氨酸的摩尔比为1:0.8-1:10，优选地为1:1-1:5，例如为1:4；和/或，
所述对甲砜基苯甲醛的浓度为5-100g/L，例如为50g/L；和/或，
所述粗酶液中的蛋白的浓度为1-5g/L，优选地为2-3g/L，例如为2.5g/L；和/或，
所述磷酸吡哆醛与所述对甲砜基苯甲醛的质量比为1:10-1:10000；和/或，
所述反应溶剂为水、乙醇、甲醇或DMSO；优选为DMSO；和/或，
所述醛缩反应的温度为25-45℃，优选为40℃。
本发明第八方面提供一种如第一方面所述的L-苏氨酸醛缩酶在制备氟苯尼考或(2S,3R)-对甲砜基苯丝氨酸中的应用。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的L-苏氨酸醛缩酶在制备(2S,3R)-对甲砜基苯丝氨酸中的底物转化率可达60％以上(优选方案可达66％以上)，d.e.值均在80％以上(优选方案可达90％以上)，提高了现有L-苏氨酸醛缩酶的底物转化率和d.e.值，具有产业化应用的价值。
附图说明
图1为原料对甲砜基苯甲醛对照品图谱。
图2为产物对照品图谱。
图3为四种构型对照品图谱；
其中：A为(2S，3R)构型的图谱，B为(2R，3S)构型的图谱，C为(2R，3R)构型的图谱，D为(2S，3S)构型的图谱，E为消旋体的图谱。
图4为反应转化率检测图谱。
图5为产物d.e.值检测图谱。
具体实施方式
下面通过实施例的方式进一步说明本发明，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。
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参考J.萨姆布鲁克等编的《分子克隆实验指南》。
实施例中使用的生物材料和试剂如下表1所示：
表1生物材料和试剂
| 名称 | 供应商 | 货号 |
| Primer Star DNA聚合酶 | TaKaRa | R045A |
| pET28a | Novagen | 69864 |
| E.coli BL21(DE3) | Novagen | 69450 |
| DpnI酶 | Thermo Fisher | FD1703 |
| NdeI酶 | Thermo Fisher | FD0584 |
| HindIII酶 | Thermo Fisher | FD0504 |
| 蛋白Marker | Thermo Fisher | 26617 |
| 质粒提取试剂盒 | Axygen | AP-MN-P-50 |
| DNA纯化回收试剂盒 | Axygen | D2500 |
实施例中的基因合成、引物合成与基因测序工作由上海生工生物工程有限公司完成。
实施例中的对甲砜基苯甲醛采购自上海毕得医药科技有限公司；甘氨酸采购自上海麦克林生化科技有限公司；4种构型对甲砜基苯丝氨酸对照品由本实验室参考Asymmetric Synthesis of Florfenicol by Dynamic Reductive Kinetic Resolutionwith Ketoreductase,Eur.J.Org.Chem.2018 5044–5053合成。
实施例中产物的手性分析及原料转化率通过高效液相色谱(High PerformanceLiquid Chromatography，HPLC)进行，具体的分析方法如下：
转化率分析色谱条件：色谱柱Agilent Eclipse plus C18(150mm*4.6mm*3.5μm)；流动相A：0.1％TFA+H2O；流动相B：0.1％TFA+CAN(乙腈)；梯度洗脱：10％B(0.01min)，100％B(10min)，100％B(11min)，10％B(11.50min)，10％B(16min)；流速1mL/min；柱温35℃；进样体积10μL；检测波长254nm。
原料对甲砜基苯甲醛对照品的保留时间为：5.504min；产物对甲砜基苯丝氨酸对照品的保留时间为：2.308min。
实施例中产物的手性分析及浓度分析通过柱前衍生化高效液相色谱进行，具体的分析方法如下：
(1)色谱条件：Diamonsil C18(250mm*4.6mm*5.0μm)；缓冲液：0.1％TEA+H2O(pH＝2.5，磷酸调节)；流动相A：缓冲液:甲醇＝90:10；流动相B：缓冲液:甲醇＝10:90；梯度洗脱：40％B(0.01min)，70％B(15min)，70％B(20min)，40％B(21min)，40％B(28min)；流速：0.8mL/min；检测波长：340nm；进样体积10μL；柱温：35℃。
(2)衍生化试剂：Marfey试剂
(3)衍生反应：称取50mg供试品于25mL容量瓶中，加15mL稀释液(纯水:乙腈＝50:50)并以300W超声5min，再加纯化水稀释至刻度，混合均匀。移取上述溶液1mL于5mL容量瓶中，加入1mL的Marfey试剂溶液和0.1mL碳酸氢钠(1M)溶液，盖上盖子于50℃烘箱中避光加热1小时，反应结束后再加入0.1mL盐酸溶液，混合均匀。
移取1mL上述混合溶液，再加入4mL的稀释液，混合均匀即可倒入进样瓶。进样10μL进行分析。
d.e.值用面积归一化法计算。
四种构型对甲砜基苯丝氨酸对照品的保留时间分别为(2S,3R)8.9min；(2R,3S)9.3min；(2R,3R)9.6min；(2S,3S)10.2min。
实施例1L-苏氨酸醛缩酶的设计
从NCBI检索到来源于恶臭假单胞菌(Pseudomonas putida)的苏氨酸醛缩酶，NCBI登录号WP_016497628.1，其氨基酸序列如SEQ ID NO:1所示，核苷酸序列如SEQ ID NO:2所示，针对SEQ ID NO:1的第10、93、310位点进行突变的突变体文库构建所设计的引物序列如表2所示：
表2引物序列
根据氨基酸序列SEQ ID NO:1，进行大肠杆菌密码子优化，其多核苷酸序列为SEQID NO:2。由上海生工生物工程有限公司进行基因合成，并构建至pET28a载体的NdeⅠ、HindⅢ酶切位点间，命名为pET28a-TAs。
单点突变以质粒pET28a-TAs质粒为模板，使用待突变位点的正向、反向引物，采用全质粒PCR的方法，获得突变基因。
两点突变以已构建好的单点突变质粒为模板，用另一位点的正向、反向引物，采用全质粒PCR的方法，获得突变基因。
三点突变以已构建好的两点突变质粒为模板，用另一位点的正向、反向引物，采用全质粒PCR的方法，获得突变基因。
PCR扩增体系如表3所示：
表3PCR扩增体系
| 试剂 | 用量(μL) |
| 2×Primer Star Mix | 12.5 |
| 正向引物(F) | 1 |
| 反向引物(R) | 1 |
| 模板 | 0.5 |
| 去离子水 | 10 |
PCR扩增程序如表4所示：
表4PCR扩增程序
对PCR产物进行DpnI酶消化，37℃消化2小时。反应完成转化至E.coli BL21(DE3)感受态细胞，涂布在含有50μg/mL卡纳霉素的LB固体培养基上，37℃培养过夜，挑单菌落测序。Sanger法测序得到正确的L-苏氨酸醛缩酶转化体，其序列如表5所示。
表5L-苏氨酸醛缩酶序列
所述野生型的氨基酸序列如SEQ ID NO:1所示。
实施例2粗酶液的制备
将L-苏氨酸醛缩酶转化体接种至含50μg/mL卡纳霉素的5mL LB液体培养基试管中，37℃震荡培养12h。按2％体积接种量转接至150mL同样含50μg/mL卡纳霉素的新鲜TB液体培养基中，37℃震荡至OD600达到0.8左右时，加0.1mM IPTG于25℃过夜诱导后，将培养液10000rpm离心10min，弃上清液，收集菌体，置于-20℃超低温冰箱中保存，待用。
取培养结束后收集到的菌体3g，用50mM pH 8.0磷酸缓冲液洗涤菌体两次，之后将菌体重悬于30mLpH 8.0的磷酸缓冲液中，超声破碎，破碎液10000rpm离心10min去除沉淀，得到的上清液为含L-苏氨酸醛缩酶的粗酶液。
上清粗酶液用Bradford试剂盒(采购自上海捷瑞生物工程有限公司)测定蛋白浓度，测定结果如下表6所示：
表6L-苏氨酸醛缩酶野生型及实施例1中L-苏氨酸醛缩酶的蛋白浓度
| 酶编号 | L-苏氨酸醛缩酶 | 蛋白浓度mg/mL |
| 1 | 野生型 | 24.6 |
| 2 | S10F | 23.3 |
| 3 | S10K | 23.5 |
| 4 | D93T | 24.2 |
| 5 | D93H | 25.3 |
| 6 | R310V | 24.9 |
| 7 | R310T | 22.7 |
| 8 | R310F | 23.8 |
| 9 | R310H | 24.5 |
| 10 | R310Q | 24.6 |
| 11 | S10F-D93T | 22.2 |
| 12 | S10F-R310F | 23.6 |
| 13 | D93T-R310H | 24.7 |
| 14 | D93H-R310Q | 25.1 |
| 15 | S10F-D93T-R310F | 24.3 |
| 16 | S10F-D93T-R310Q | 24.7 |
实施例3L-苏氨酸醛缩酶在水-乙醇溶液中制备(2S,3R)-对甲砜基苯丝氨酸
定量称取1.0g对甲砜基苯甲醛、1.6g甘氨酸到100mL反应器中，加入120μL的1％(m/m)磷酸吡哆醛母液、40mL乙醇、10mL实施例2方法获得的野生型醛缩酶或实施例1中L-苏氨酸醛缩酶的粗酶液，用100mM pH 8.0磷酸缓冲溶液定容到100mL。通过水浴控制反应温度为30℃，磁力搅拌，反应16h。反应完成后，取反应液用HPLC检测产物含量，计算转化率。反应完成后，取反应液调酸，离心除酶，再调中性，用Marfey试剂衍生，用手性柱测(2S,3R)-对甲砜基苯丝氨酸d.e.值。结果如表7所示。
表7L-苏氨酸醛缩酶野生型及实施例1中的L-苏氨酸醛缩酶在水-乙醇体系中的d.e.值
| 酶编号 | L-苏氨酸醛缩酶 | 转化率 | d.e.值％ |
| 1 | 野生型 | 63.5％ | 76.47 |
| 2 | S10F | 64.2％ | 87.69 |
| 3 | S10K | 59.7％ | 83.79 |
| 4 | D93T | 63.8％ | 85.18 |
| 5 | D93H | 55.7％ | 82.07 |
| 6 | R310V | 58.9％ | 80.02 |
| 7 | R310T | 62.7％ | 84.37 |
| 8 | R310F | 65.1％ | 84.53 |
| 9 | R310H | 59.2％ | 82.04 |
| 10 | R310Q | 63.7％ | 84.20 |
| 11 | S10F-D93T | 65.6％ | 92.23 |
| 12 | S10F-R310F | 65.5％ | 91.54 |
| 13 | D93T-R310H | 61.5％ | 93.19 |
| 14 | D93H-R310Q | 62.3％ | 90.86 |
| 15 | S10F-D93T-R310F | 66.3％ | 96.12 |
| 16 | S10F-D93T-R310Q | 64.2％ | 94.63 |
实施例4L-苏氨酸醛缩酶及三点突变体在不同溶液中制备(2S,3R)-对甲砜基苯丝氨酸
按实施例2的方法培养能够表达L-苏氨酸醛缩酶的工程菌及其三点突变体(L-苏氨酸醛缩酶15、16)并获得粗酶液。定量称取5.0g对甲砜基苯甲醛、8.2g甘氨酸到100mL反应器中，加入120μL的1％磷酸吡哆醛母液、40mL甲醇或20mL DMSO、10mL野生型醛缩酶或三点突变体的粗酶液，用100mM pH 8.0磷酸缓冲溶液定容到100mL。通过水浴控制反应温度为40℃，磁力搅拌，反应6h。反应完成后，取反应液用HPLC检测产物含量，计算转化率(对甲砜基苯甲醛对照品、产物对照品和四种构型对照品的图谱分别如图1、图2和图3所示)。取反应液调酸，离心除酶，再调中性，用Marfey试剂衍生，用手性柱测(2S，3R)-对甲砜基苯丝氨酸d.e.值。结果如表8所示。其中L-苏氨酸醛缩酶15在DMSO中的转化率图谱如图4所示，L-苏氨酸醛缩酶15的d.e.值图谱如图5所示。
表8L-苏氨酸醛缩酶在不同溶剂中的转化率、d.e.值
综上，本发明公开了一种L-苏氨酸醛缩酶、制备(2S,3R)-对甲砜基苯丝氨酸的方法。所述L-苏氨酸醛缩酶由野生型L-苏氨酸醛缩酶突变所得，能够以甘氨酸和对甲砜基苯甲醛为底物，以磷酸吡哆醛为辅酶，催化缩合反应生成(2S,3R)-对甲砜基苯丝氨酸。其中L-苏氨酸醛缩酶15在水-乙醇、水-甲醇、水-DMSO或其它助溶剂体系中，对甲砜基苯甲醛浓度可达50g/L或更高，甘氨酸物质的量浓度为对甲砜基苯甲醛的1-20倍，pH为5-10，反应温度15℃-60℃，反应时间0-24h，在优选的方案中最终转化率>66％，(2S,3R)-对甲砜基苯丝氨酸d.e.值>96％。
SEQUENCE LISTING
<110> 弈柯莱生物科技（上海）股份有限公司
<120> L-苏氨酸醛缩酶及其制备方法与应用
<130> P21011547C
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<210> 5
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 93T
<400> 5
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactactg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggcgt ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 6
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 93H
<400> 6
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactcatg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggcgt ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 7
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 310V
<400> 7
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactgatg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttgggtt ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 8
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 310T
<400> 8
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactgatg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggact ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 9
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 310F
<400> 9
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactgatg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggttt ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 10
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 310H
<400> 10
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactgatg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggcat ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 11
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 310Q
<400> 11
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactgatg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggcaa ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 12
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 10F-93T
<400> 12
atgactgata aatcgcagca attcgctttt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactactg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggcgt ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 13
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 10F-310F
<400> 13
atgactgata aatcgcagca attcgctttt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactgatg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggttt ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 14
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 93T-310H
<400> 14
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactactg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggcat ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 15
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 93H-310Q
<400> 15
atgactgata aatcgcagca attcgctagt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactcatg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggcaa ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 16
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 10F-93T-310F
<400> 16
atgactgata aatcgcagca attcgctttt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactactg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggttt ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 17
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> 10F-93T-310Q
<400> 17
atgactgata aatcgcagca attcgctttt gataactata gcggcatctg cccggaagcg 60
tgggcagcga tggaaaaagc taaccacggc catgataggg cgtacggtga tgaccagtgg 120
actgaacgtg cttctgaata cttccgtaac ctgtttgaaa ctgactgtga agtgttcttc 180
gcgtttaacg gtaccgccgc taatagtcta gccctagcta gtctctgcca gtcctatcat 240
agcgtgatct gtagtgaaac cgcgcacgtt gagactactg aatgcggcgc tcctgagttc 300
ttctccaacg gtagtaaact gctgaccgcg gcgagcgtta acggtaagct gaccccgcag 360
tctatccgcg aagttgccct gaaacgccag gatatccatt atccaaagcc tcgtgtggtt 420
actattaccc aggctaccga agttggtacc gtttaccgtc cggatgaact gaaagcgatc 480
agcgcgacct gtaaagaact ggggctgaac ctgcacatgg atggtgcacg tttcactaac 540
gcgtgtgcgt tcctgggttg ctctccggcg gaactgacct ggaaggctgg cgtggacgtg 600
ctgtgcttcg gcggcaccaa aaacggtatg gcagttggcg aagcgatcct gtttttcaat 660
cgccaactgg ctgaagactt cgactaccgt tgcaaacagg cgggccagct ggcatccaaa 720
atgcgtttcc tgtctgcgcc gtgggttggc ctgctggaag atggcgcgtg gctgcgtcat 780
ggtaaccacg ccaaccactg cgcgcagctg ctggcgctgc tggtttccga cctgccgggt 840
gttgaactga tgttcccggt tgaagcgaac ggcgttttcc tgcagatgcc tgaacacgcg 900
atcgaagcgc tgcgtgcgaa aggttggcaa ttctacacct tcatcggttc tggcggtgcg 960
cgtttcatgt gctcttggga taccgaagaa gaacgtgttc gtgaactggc ggctgatatc 1020
cgtagcatca tcaccgcg 1038
<210> 18
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 10F-F
<400> 18
tcgcagcaat tcgcttttga taactatagc ggc 33
<210> 19
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 10F-R
<400> 19
gccgctatag ttatcaaaag cgaattgctg cga 33
<210> 20
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 10K-F
<400> 20
tcgcagcaat tcgctaaaga taactatagc ggc 33
<210> 21
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 10K-R
<400> 21
gccgctatag ttatctttag cgaattgctg cga 33
<210> 22
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 93T-F
<400> 22
gcgcacgttg agactactga atgcggcgct cct 33
<210> 23
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 93T-R
<400> 23
aggagcgccg cattcatgag tctcaacgtg cgc 33
<210> 24
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 93H-F
<400> 24
gcgcacgttg agactcatga atgcggcgct cct 33
<210> 25
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 93H-R
<400> 25
aggagcgccg cattcatgag tctcaacgtg cgc 33
<210> 26
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310V-F
<400> 26
cgtgcgaaag gttgggtttt ctacaccttc atc 33
<210> 27
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310V-R
<400> 27
gatgaaggtg tagaaaaccc aacctttcgc acg 33
<210> 28
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310T-F
<400> 28
cgtgcgaaag gttggacttt ctacaccttc atc 33
<210> 29
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310T-R
<400> 29
gatgaaggtg tagaaagtcc aacctttcgc acg 33
<210> 30
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310F-F
<400> 30
cgtgcgaaag gttggttttt ctacaccttc atc 33
<210> 31
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310F-R
<400> 31
gatgaaggtg tagaaaaacc aacctttcgc acg 33
<210> 32
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310H-F
<400> 32
cgtgcgaaag gttggcattt ctacaccttc atc 33
<210> 33
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310H-R
<400> 33
gatgaaggtg tagaaatgcc aacctttcgc acg 33
<210> 34
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310Q-F
<400> 34
cgtgcgaaag gttggcaatt ctacaccttc atc 33
<210> 35
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 310Q-R
<400> 35
gatgaaggtg tagaattgcc aacctttcgc acg 33
<210> 36
<211> 346
<212> PRT
<213> Artificial Sequence
<220>
<223> L-苏氨酸醛缩酶
<220>
<221> MUTAGEN
<222> (10)..(10)
<223> Xaa为Ser或Phe或Lys
<220>
<221> MUTAGEN
<222> (93)..(93)
<223> Xaa为Asp或Thr或His
<220>
<221> MUTAGEN
<222> (310)..(310)
<223> Xaa为Arg或Val或Thr或His或Phe或Gln
<400> 36
Met Thr Asp Lys Ser Gln Gln Phe Ala Xaa Asp Asn Tyr Ser Gly Ile
1 5 10 15
Cys Pro Glu Ala Trp Ala Ala Met Glu Lys Ala Asn His Gly His Asp
20 25 30
Arg Ala Tyr Gly Asp Asp Gln Trp Thr Glu Arg Ala Ser Glu Tyr Phe
35 40 45
Arg Asn Leu Phe Glu Thr Asp Cys Glu Val Phe Phe Ala Phe Asn Gly
50 55 60
Thr Ala Ala Asn Ser Leu Ala Leu Ala Ser Leu Cys Gln Ser Tyr His
65 70 75 80
Ser Val Ile Cys Ser Glu Thr Ala His Val Glu Thr Xaa Glu Cys Gly
85 90 95
Ala Pro Glu Phe Phe Ser Asn Gly Ser Lys Leu Leu Thr Ala Ala Ser
100 105 110
Val Asn Gly Lys Leu Thr Pro Gln Ser Ile Arg Glu Val Ala Leu Lys
115 120 125
Arg Gln Asp Ile His Tyr Pro Lys Pro Arg Val Val Thr Ile Thr Gln
130 135 140
Ala Thr Glu Val Gly Thr Val Tyr Arg Pro Asp Glu Leu Lys Ala Ile
145 150 155 160
Ser Ala Thr Cys Lys Glu Leu Gly Leu Asn Leu His Met Asp Gly Ala
165 170 175
Arg Phe Thr Asn Ala Cys Ala Phe Leu Gly Cys Ser Pro Ala Glu Leu
180 185 190
Thr Trp Lys Ala Gly Val Asp Val Leu Cys Phe Gly Gly Thr Lys Asn
195 200 205
Gly Met Ala Val Gly Glu Ala Ile Leu Phe Phe Asn Arg Gln Leu Ala
210 215 220
Glu Asp Phe Asp Tyr Arg Cys Lys Gln Ala Gly Gln Leu Ala Ser Lys
225 230 235 240
Met Arg Phe Leu Ser Ala Pro Trp Val Gly Leu Leu Glu Asp Gly Ala
245 250 255
Trp Leu Arg His Gly Asn His Ala Asn His Cys Ala Gln Leu Leu Ala
260 265 270
Leu Leu Val Ser Asp Leu Pro Gly Val Glu Leu Met Phe Pro Val Glu
275 280 285
Ala Asn Gly Val Phe Leu Gln Met Pro Glu His Ala Ile Glu Ala Leu
290 295 300
Arg Ala Lys Gly Trp Xaa Phe Tyr Thr Phe Ile Gly Ser Gly Gly Ala
305 310 315 320
Arg Phe Met Cys Ser Trp Asp Thr Glu Glu Glu Arg Val Arg Glu Leu
325 330 335
Ala Ala Asp Ile Arg Ser Ile Ile Thr Ala
340 345
Claims (19)
1.一种L-苏氨酸醛缩酶，其特征在于，所述L-苏氨酸醛缩酶的氨基酸序列如SEQ IDNO: 36所示，其中：
所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F；
或者，所述L-苏氨酸醛缩酶的氨基酸序列中第93位氨基酸残基为T；
或者，所述L-苏氨酸醛缩酶的氨基酸序列中第310位氨基酸残基为F；
或者，所述L-苏氨酸醛缩酶的氨基酸序列中第310位氨基酸残基为Q；
或者，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F，第93位氨基酸残基为T；
或者，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F，第310位氨基酸残基为F；
或者，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F，第93位氨基酸残基为T，第310位氨基酸残基为F；
或者，所述L-苏氨酸醛缩酶的氨基酸序列中第10位氨基酸残基为F，第93位氨基酸残基为T，第310位氨基酸残基为Q。
2.一种分离的核酸，其特征在于，所述核酸编码如权利要求1所述的L-苏氨酸醛缩酶。
3. 如权利要求2所述的核酸，其特征在于，所述核酸的核苷酸序列如SEQ ID NO: 3、SEQ ID NO: 5、SEQ ID NO: 9、SEQ ID NO: 11、SEQ ID NO: 12、SEQ ID NO: 13、SEQ IDNO: 16或SEQ ID NO: 17所示。
4.一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求2或3所述的分离的核酸。
5.一种转化体，其特征在于，所述转化体为包含如权利要求2或3所述的分离的核酸或如权利要求4所述的重组表达载体的宿主细胞。
6. 如权利要求5所述的转化体，其特征在于，所述宿主细胞为埃希氏大肠杆菌（Escherichia coli）。
7.一种制备如权利要求1所述的L-苏氨酸醛缩酶的方法，其特征在于，所述L-苏氨酸醛缩酶通过以下步骤制备：
培养如权利要求5或6所述的转化体，使其表达所述L-苏氨酸醛缩酶，即得。
8.如权利要求7所述的方法，其特征在于，所述步骤具体包括：
（1）将如权利要求5或6所述的转化体接种至含抗生素的培养基中振荡培养，得种子液；
（2）将（1）中的种子液转接至含抗生素的培养基中振荡培养；
（3）向（2）中的培养基中加入IPTG诱导过夜，离心后收集菌体；
（4）洗涤并重悬（3）中收集的菌体，破碎后离心，即得含所述L-苏氨酸醛缩酶的粗酶液。
9.如权利要求8所述的方法，其特征在于，
（1）中所述培养基为LB培养基；
（2）中所述培养基为TB培养基。
10. 如权利要求8所述的方法，其特征在于，所述抗生素为50 μg/mL卡纳霉素；
和/或，
（1）中所述振荡培养的条件为37℃、12 h；
和/或，
（2）中所述种子液的接种量为2%体积；和/或，（2）中所述振荡培养的条件为37℃、直至OD600为0.75-0.85；
和/或，
（3）中所述IPTG的终浓度为0.05-5 mM；和/或，（3）中所述诱导过夜的温度为20-30℃；和/或，（3）中所述离心的条件为4000-12000 rpm离心5-30 min；
和/或，
（4）中所述洗涤使用50 mM pH 8.0磷酸缓冲液；和/或，（4）中所述重悬使用50 mM pH8.0磷酸缓冲液，其与菌体的体积重量比为5-20:1 mL/g；和/或，（4）中所述破碎为使用超声破碎或高压均质机均质；和/或，（4）中所述离心的条件为8000-12000 rpm，离心5-20 min。
11. 如权利要求10所述的方法，其特征在于，
（3）中所述IPTG的终浓度为0.1 mM IPTG；和/或，（3）中所述诱导过夜的温度为25℃；和/或，（3）中所述离心的条件为10000 rpm离心5-30 min，或者，所述离心的条件为4000-12000 rpm离心10 min，或者，所述离心的条件为10000 rpm离心10 min；和
（4）中所述磷酸缓冲液与菌体的体积重量比为10:1 mL/g；和/或，（4）中所述离心的条件为10000 rpm，离心5-20 min，或者，所述离心的条件为8000-12000 rpm离心10 min，或者，所述离心的条件为10000 rpm离心10 min。
12.一种(2S,3R)-对甲砜基苯丝氨酸的制备方法，其特征在于，所述制备方法包括以下步骤：在如权利要求1所述的L-苏氨酸醛缩酶、磷酸吡哆醛和反应溶剂存在的条件下，使对甲砜基苯甲醛和甘氨酸进行醛缩反应，即得(2S,3R)-对甲砜基苯丝氨酸。
13.如权利要求12所述的制备方法，其特征在于，制备所述L-苏氨酸醛缩酶的方法如权利要求7-11任一项所示。
14.一种氟苯尼考的制备方法，其特征在于，所述制备方法包括以下步骤：在如权利要求1所述的L-苏氨酸醛缩酶、磷酸吡哆醛和反应溶剂存在的条件下，使对甲砜基苯甲醛和甘氨酸进行醛缩反应，得(2S,3R)-对甲砜基苯丝氨酸；其后由(2S,3R)-对甲砜基苯丝氨酸制得所述氟苯尼考。
15.如权利要求14所述的制备方法，其特征在于，制备所述L-苏氨酸醛缩酶的方法如权利要求7-11任一项所示。
16.如权利要求12-13或14-15任一项所述的制备方法，其特征在于，所述对甲砜基苯甲醛和甘氨酸的摩尔比为1:0.8-1:10；和/或，
所述对甲砜基苯甲醛的浓度为5-100 g/L；和/或，
所述L-苏氨酸醛缩酶为通过如权利要求8所述的方法制备得到的含所述L-苏氨酸醛缩酶的粗酶液，所述L-苏氨酸醛缩酶的粗酶液的蛋白的浓度为1-5 g/L；和/或，
所述磷酸吡哆醛与所述对甲砜基苯甲醛的质量比为1:10-1:10000；和/或，
所述反应溶剂为水、乙醇、甲醇或DMSO；和/或，
所述醛缩反应的温度为25-45℃。
17.如权利要求16所述的制备方法，其特征在于，所述对甲砜基苯甲醛和甘氨酸的摩尔比为1:1-1:5；和/或，
所述对甲砜基苯甲醛的浓度为50 g/L；和/或，
含所述L-苏氨酸醛缩酶的粗酶液中的蛋白的浓度为2-3 g/L；和/或，
所述反应溶剂为DMSO；和/或，
所述醛缩反应的温度为40℃。
18.如权利要求17所述的制备方法，其特征在于，所述对甲砜基苯甲醛和甘氨酸的摩尔比为1:4。
19.一种如权利要求1所述的L-苏氨酸醛缩酶在制备氟苯尼考或(2S,3R)-对甲砜基苯丝氨酸中的应用。
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