CN113862234A - 转氨酶及其在制备(R)-α-甲基色胺类化合物的应用 - Google Patents
转氨酶及其在制备(R)-α-甲基色胺类化合物的应用 Download PDFInfo
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- CN113862234A CN113862234A CN202010630461.2A CN202010630461A CN113862234A CN 113862234 A CN113862234 A CN 113862234A CN 202010630461 A CN202010630461 A CN 202010630461A CN 113862234 A CN113862234 A CN 113862234A
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- transaminase
- indolylacetone
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- 102000003929 Transaminases Human genes 0.000 title claims abstract description 59
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- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 50
- -1 (R) -alpha-methyltryptamine compound Chemical class 0.000 claims abstract description 32
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 14
- ISYORFGKSZLPNW-UHFFFAOYSA-N propan-2-ylazanium;chloride Chemical compound [Cl-].CC(C)[NH3+] ISYORFGKSZLPNW-UHFFFAOYSA-N 0.000 claims abstract description 9
- WAGPFYGOLZGETK-UHFFFAOYSA-N 1-(1h-indol-2-yl)propan-2-one Chemical class C1=CC=C2NC(CC(=O)C)=CC2=C1 WAGPFYGOLZGETK-UHFFFAOYSA-N 0.000 claims abstract description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 33
- 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 24
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- 150000001875 compounds Chemical class 0.000 claims description 10
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- 125000003545 alkoxy group Chemical group 0.000 claims description 4
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Abstract
本发明提供了一种转氨酶，其氨基酸序列如SEQ ID NO.1所示。本发明还提供了其在制备(R)‑α‑甲基色胺类化合物中的应用。本发明的转氨酶的酶活性高，立体选择性高，工艺成本更低。将本发明的转氨酶用于催化3‑吲哚基丙酮类化合物制备(R)‑α‑甲基色胺类化合物时，转化率和ee值均较高，并且可用于无D‑丙氨酸的反应体系，例如使用异丙胺盐酸盐代替D‑丙氨酸作为氨基供体，后处理更简单。
Description
技术领域
本发明涉及生物催化技术领域，涉及一种转氨酶及其催化制备(R)-α-甲基色胺类化合物的方法以及应用。
背景技术
单萜吲哚生物碱是一类重要药用价值的天然产物，但是单萜吲哚生物碱在植物体内的含量极低(约占鲜重的0.0002％)，而且植物宿主多为木本，生长周期长，很难满足日益增长的市场需求。因此需要进行大量的化学或生物合成而满足市场的需求。
异胡豆苷是单萜吲哚生物碱合成的关键骨架化合物，由色胺和开环的马钱子苷汇聚合成。因此作为异胡豆苷合成途径中的重要原料色胺的制备方法就显得尤其重要。其中(R)-α-甲基色胺(结构如式一所示)可用作3-甲基异胡豆苷衍生物的重要原料，
目前已有(R)-α-甲基色胺的制备方法的相关报道，如Wolfgang Kroutil在《Stereoselective Cascade to C3-Methylated Strictosidine Derivatives EmployingTransaminases and Strictosidine Synthases》，ACS Catal.，2016，6，23-30上报道了来源于节杆菌(Arthrobacter sp.)的(R)-选择性的转氨酶KNK168能催化3-吲哚基丙酮制备(R)-α-甲基色胺，转化率可达98％，ee值可达98％以上，但是反应采用D-丙氨酸作为氨基供体，工业化生产成本较高，且D-丙氨酸会产生丙酮酸，丙酮酸会留在水里，后处理相对比较复杂。
WO2019245974也在Scheme E中公布了转氨酶可以催化3-吲哚基丙酮制备(R)-α-甲基色胺的路径，其中提及了所用的转氨酶为TA-P2-A01、TA-P2-A07(Codexis)或ATA12等等，并未给出使用转氨酶进行催化的具体技术实施方案。
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中转氨酶催化3-吲哚基丙酮类化合物制备(R)-α-甲基色胺类化合物需要使用D-丙氨酸作为氨基供体、生产成本高、后处理比较复杂等缺陷，本发明提供了一种转氨酶、将其用于催化3-吲哚基丙酮类化合物制备(R)-α-甲基色胺类化合物的方法及其应用。本发明的转氨酶的酶活性高，立体选择性高，工艺成本更低。将本发明的转氨酶用于催化3-吲哚基丙酮类化合物制备(R)-α-甲基色胺类化合物时，转化率和ee值均较高，并且可用于无D-丙氨酸的反应体系，例如使用异丙胺盐酸盐代替D-丙氨酸作为氨基供体，后处理更简单。
本发明人经过大量实验，发现将很多转氨酶用于催化3-吲哚基丙酮制备(R)-α-甲基色胺时都无法进行有效的催化，而本发明人通过多次尝试意外发现催化3-吲哚基丙酮类化合物制备(R)-α-甲基色胺类化合物时，特别是在不使用D-丙氨酸作为氨基供体(例如使用异丙胺或其盐作为氨基供体)的反应体系中时，使用本发明的转氨酶能够获得较高的转化率和ee值。
为了解决上述技术问题，本发明第一方面提供了一种转氨酶，所述转氨酶的氨基酸序列如SEQ ID NO.1所示。
为了解决上述技术问题，本发明第二方面提供了一种分离的核酸，其编码如本发明第一方面所述的转氨酶。
为了解决上述技术问题，本发明第三方面提供了一种包含如本发明第二方面所述的核酸的重组表达载体。
较佳地，所述重组表达载体的骨架为质粒pET21a。
为了解决上述技术问题，本发明第四方面提供了一种包含如本发明第二方面所述的核酸或如本发明第三方面所述的重组表达载体的转化体。
较佳地，所述转化体的宿主为大肠杆菌；优选为大肠杆菌BL21(DE3)。
为了解决上述技术问题，本发明第五方面提供了一种(R)-α-甲基色胺类化合物的制备方法，其包括在氨基供体存在时，在反应溶剂中用如本发明第一方面所述的转氨酶催化如式I所示的3-吲哚基丙酮类化合物得(R)-α-甲基色胺类化合物的步骤，所述的如式I所示的3-吲哚基丙酮类化合物为：
其中，R1、R2、R3独立地为H、OH、C1～C3的烷基或C1～C3的烷氧基。
较佳地，所述的式I中，所述的C1～C3的烷基为甲基、乙基、正丙基或异丙基，所述的C1～C3的烷氧基为甲氧基、乙氧基、正丙氧基或异丙氧基。
更佳地，所述的式I中，R1为H、OH或甲基，R2和R3独立地为H。
更佳地，所述的式I中，R2为甲氧基，R1和R3独立地为H。
更佳地，所述的式I中，R3为甲基，R1和R2为H。
在某一较佳实施例中，所述的如式I所示的3-吲哚基丙酮类化合物为下述任一化合物：
较佳地，所述反应溶剂为水。
较佳地，所述的3-吲哚基丙酮类化合物为经过助溶剂助溶的3-吲哚基丙酮类化合物。其中，所述的助溶剂优选自DMSO、N,N二甲基甲酰胺、乙醇、异丙醇吐温-80和吐温-60中的一种或多种。此外，所述助溶剂还可以优选占所述制备方法的反应体系总体积的0～20％。
较佳地，所述的氨基供体为异丙胺或其盐，所述的盐优选为异丙胺盐酸盐。
较佳地，所述制备方法的反应体系中还包括转氨酶的辅助因子例如吡哆醛磷酸，所述转氨酶的辅助因子与所述的如式I所示的3-吲哚基丙酮类化合物的质量比为1:1000～1:10；优选为1:100。
较佳地，所述氨基供体与所述3-吲哚基丙酮类化合物的摩尔比为1:1～6:1，例如3:1～6:1。
较佳地，所述3-吲哚基丙酮类化合物的浓度为10～50g/L，优选为15～34g/L，例如19g/L或25g/L。
较佳地，所述转氨酶与所述3-吲哚基丙酮类化合物的质量比为6:1～12:1，例如为9:1。
较佳地，所述反应的pH为7.5-8.5，例如为8。
较佳地，所述反应的温度为35-45℃。
为了解决上述技术问题，本发明第六方面提供了一种如本发明第一方面所述的转氨酶在制备(R)-α-甲基色胺类化合物中的用途。
较佳地，所述用途包括在氨基供体例如异丙胺或其盐存在时，在反应溶剂中用如本发明第一方面所述的转氨酶催化如式I所示的3-吲哚基丙酮类化合物得(R)-α-甲基色胺类化合物的步骤；
所述的如式I所示的3-吲哚基丙酮类化合物为：
其中，R1、R2、R3独立地为H、OH、C1～C3的烷基或C1～C3的烷氧基。
较佳地，所述的式I中，所述的C1～C3的烷基为甲基、乙基、正丙基或异丙基，所述的C1～C3的烷氧基为甲氧基、乙氧基、正丙氧基或异丙氧基。
更佳地，所述的式I中，R1为H、OH或甲基，R2和R3独立地为H。
更佳地，所述的式I中，R2为甲氧基，R1和R3独立地为H。
更佳地，所述的式I中，R3为甲基，R1和R2为H。
在某一较佳实施例中，所述的如式I所示的化合物为下述任一化合物：
较佳地，上述应用的具体条件如本发明第五方面所述。
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：本发明的转氨酶的酶活性高，立体选择性高，工艺成本更低。将本发明的转氨酶用于催化3-吲哚基丙酮类化合物制备(R)-α-甲基色胺类化合物时，转化率和ee值均较高，并且可用于无D-丙氨酸的反应体系，例如使用异丙胺盐酸盐代替D-丙氨酸作为氨基供体，后处理更简单。
附图说明
图1为实施例5转化率的HPLC图谱。
图2为底物3-吲哚基丙酮对照品的图谱。
图3为产物甲基色胺(消旋体)对照品的图谱。
图4为产物ee值检测图谱。
图5为产品甲基色胺(消旋体)对照品的图谱。
图6为产品R构型((R)-α-甲基色胺)对照品的图谱。
图7为产品甲基色胺(消旋体)的HNMR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》。
pET21a和蛋白抽提试剂(bugbuster protein extraction reagent)购买自Novagen公司；DpnI酶购买自英潍捷基(上海)贸易有限公司；NdeI酶、HindIII酶购买自Thermo Fisher公司，E.coli BL21(DE3)感受态细胞购买自北京鼎国昌盛生物技术有限责任公司。3-吲哚基丙酮采购自上海毕得医药科技有限公司；甲基色胺(消旋体)由本实验室根据WO2009132921A1自行合成，鉴定图谱为图7；(R)-α-甲基色胺对照品为本实验室以消旋体为原料根据文献J.Am.Chem.Soc.1989，111，3095-3096自行拆分获得。
实施例1
根据如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3(表1)所示的转氨酶的酶基因序列，全基因合成转氨酶酶基因，在NdeI、HindIII位点连入pET21a载体，经过测序验证基因成功连接到相应的载体上。基因合成和测序的公司为生工生物工程有限公司(上海)股份(上海市松江区香闵路698号)。
将含有转氨酶基因的载体转化宿主大肠杆菌BL21(DE3)感受态细胞，得到分别含有各转氨酶的工程菌株。将分别含有转氨酶基因的工程菌在经平皿划线活化后，挑单菌落接种至含100μg/mL氨苄抗生素的5mL LB液体培养基中，37℃震荡培养12h。按2％接种量转接至150mL同样含100μg/mL氨苄抗生素的新鲜LB液体培养基中，37℃震荡至OD600达到0.8左右时，加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)至其终浓度为0.5mM，25℃诱导培养16h。培养结束后，将培养液10000rpm离心10min，弃上清液，收集菌体(菌泥)，置于-20℃超低温冰箱中保存，待用。
表1
| 酶编号 | 来源 | NCBI登录号 | 氨基酸序列 |
| Enz.01 | \ | \ | SEQ ID NO:1 |
| Enz.02 | Arthrobacter sp.KNK168 | BAK39753.1 | SEQ ID NO:2 |
| Enz.03 | \ | \ | SEQ ID NO:3 |
实施例2转氨酶的筛选
将实施例1所得的三种转氨酶的菌泥和三乙醇胺缓冲液按质量比1:4均质后得到转氨酶酶液，以备使用。
反应容器中加入625μLpH为7.0的200mM PBS(磷酸盐缓冲溶液)，1mL的转氨酶酶液，100μL的50mM的磷酸吡哆醛(PLP)，62.5μL的4M异丙胺盐酸盐，加入212.5μL水，加入底物(3-吲哚基丙酮)DMSO溶液(0.0086g底物溶于500μL DMSO中)，30℃下进行反应。定时取样，HPLC测转化率和ee值。
实验结果如下表2所示。
表2
表中的\代表(因为转化率过低)未检测。
由表中可以看出，在本实施例的反应体系中，转氨酶Enz.01在催化底物3-吲哚基丙酮获得产物(R)-α-甲基色胺的转化效果明显优于Arthrobacter sp.KNK168来源的转氨酶Enz.02和Enz.03，因此选取Enz.01继续后续实验。
实施例3转氨反应中的pH优化
将实施例1所得的Enz.01的菌泥和三乙醇胺缓冲液按质量比1:4均质后得到转氨酶酶液，以备使用。
反应容器中加入不同pH值的0.8mL三乙醇胺缓冲液，60mg转氨酶均质后的酶液，12μL的16mg/mL磷酸吡哆醛(PLP)、异丙胺盐酸盐33mg，pH分别调到如表3所示pH值后搅拌，加入底物(3-吲哚基丙酮)DMSO溶液(20mg底物溶于0.4ml DMSO中)，35℃下进行反应，同时控pH。定时取样，HPLC测转化率。
实验结果如下表3所示。
表3
| pH | 3h转化率％ | 6h转化率％ | 16h转化率％ |
| 7.5 | 20 | 31.3 | 40.4 |
| 8.0 | 21 | 28.9 | 38.8 |
| 8.5 | 20 | 28.8 | 38 |
| 9.0 | 18 | 15.6 | 32 |
由表中可以看出，反应pH在7.5-8.5之间的底物转化率相对较高。
实施例4转氨反应中的温度优化
将实施例1所得的Enz.01的菌泥和三乙醇胺缓冲液按质量比1:4均质后得到转氨酶酶液，以备使用。
反应容器中加入pH为8.0的0.8mL三乙醇胺缓冲液，60mg转氨酶均质后的酶液，12μL的16mg/mL磷酸吡哆醛(PLP)、异丙胺盐酸盐33mg，调pH到8.0搅拌，加入底物(3-吲哚基丙酮)DMSO溶液(20mg底物溶于0.4mL DMSO中)，不同温度进行反应，同时控pH。定时取样，HPLC测转化率。
实验结果如下表4所示。
表4
| 温度℃ | 3h转化率％ | 6h转化率％ | 16h转化率％ |
| 30 | 9 | 17 | 28 |
| 35 | 21 | 28.9 | 38.8 |
| 45 | 35 | 42 | 47 |
由表中可以看出，反应温度在35-45℃之间的底物转化率相对较高。
实施例5转氨反应中的投酶量优化
将实施例1所得的Enz.01的菌泥和三乙醇胺缓冲液按质量比1:4均质后得到转氨酶酶液，以备使用。
反应容器中加入不同量的pH为7.5的三乙醇胺缓冲液(0.7mL、0.4mL、0.1mL)，不同量的转氨酶均质后的酶液，12μL的16mg/mL的磷酸吡哆醛(PLP)，异丙胺盐酸盐33mg，调pH到7.5搅拌，加入底物(3-吲哚基丙酮)DMSO溶液(20mg底物溶于0.4mL DMSO中)，45℃下进行反应，同时控pH。定时取样，HPLC测转化率。
实验结果如下表5所示。
表5
| 酶量 | 2h转化率％ | 16h转化率％ |
| 120mg | 55 | 80 |
| 180mg | 67 | 80 |
| 240mg | 73 | 79 |
由表中可以看出，转氨酶与底物的质量比范围为6:1～12:1时底物的转化率达到79％以上。
实施例6转氨反应放大实验
转氨反应：在2L三口瓶中加入150mL异丙胺(4M)，加入20mL三乙醇胺缓冲液，加入90g Enz.01菌体均质后的酶液450mL，控制温度45℃下搅拌。加入9mL的16mg/mL的磷酸吡哆醛PLP，调pH到8.0控温搅拌，加入底物DMSO溶液(15g底物溶于150mLDMSO)开始反应，同时控pH。反应16小时后停止反应。HPLC检测转化率85％，检测图谱如图1，底物3-吲哚基丙酮对照品图谱为图2，产物甲基色胺(消旋体)对照品图谱为图3。
转化率HPLC检测方法：
色谱柱：Agilent Eclipse plus C18(3.5μm，150×4.6mm)；流动相A：0.1％TFA水溶液；流动相B：0.1％TFA乙腈溶液；梯度洗脱：90％A+10％B(0min)，100％B(10min)，100％B(11min)，90％A+10％B(11.5min)，90％A+10％B(16min)；流速：1mL/min；柱温：30℃；检测波长：254nm。
后处理操作：反应液室温加入10mL盐酸，调节pH到2，加入硅藻土50g，搅拌0.5h，过滤。硅藻土用200mL水打浆一次，过滤，合并滤液。滤液用乙酸异丙酯(250mL+250mL)萃取2次，收集水相，水相调pH到10，搅拌0.5h。用乙酸异丙酯(250mL+250mL)萃取2次。收集有机相。有机相再用200mL水洗一次。有机相用无水硫酸钠干燥后，旋干。得8g产品，收率53.3％。HPLC进行ee值测定。ee值为98.9％，检测图谱如图4，甲基色胺(消旋体)对照品图谱如图5，R构型((R)-α-甲基色胺)对照品图谱如图6。可知该反应制得了正确结构的(R)-α-甲基色胺。
光学纯度的HPLC检测方法：
采用Marfey试剂衍生后进行测量的方法。色谱柱：Agilent Eclipse plus C18(3.5μm，150×4.6mm)；缓冲液：0.1％三乙胺水溶液(磷酸调节pH至2.5)，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：90％A+10％B(0min)，50％A+50％B(5min)，40％A+60％B(10min)，100％B(12min)，100％B(13min)，90％A+10％B(14min)，90％A+10％B(20min)；流速：1mL/min；柱温：30℃；检测波长：340nm。
SEQUENCE LISTING
<110> 上海弈柯莱生物医药科技有限公司
<120> 转氨酶及其在制备(R)-α-甲基色胺类化合物的应用
<130> P20012193C
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.01
<400> 1
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
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Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
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Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
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Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
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Val Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
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Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
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Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
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Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Thr Val Thr Ile Thr
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Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
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Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg
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Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
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Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
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Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly
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Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
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Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
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Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Ala Gly Gly Val Trp
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Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
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Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
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<210> 2
<211> 330
<212> PRT
<213> Arthrobacter sp. KNK168
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Met Ala Phe Ser Ala Asp Thr Ser Glu Ile Val Tyr Thr His Asp Thr
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Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
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Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp
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Val Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
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Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Met Arg Ile
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Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Ser Val Ser Ile Thr
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Arg Gly Tyr Ser Ser Thr Pro Gly Glu Arg Asp Ile Thr Lys His Arg
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Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe
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Asp Arg Ile Arg Asp Gly Val His Ala Met Val Ala Gln Ser Val Arg
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Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
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Gly Asp Leu Ile Arg Ala Val Gln Glu Thr His Asp Arg Gly Phe Glu
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Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
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Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp
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Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly
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Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr
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<210> 3
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.03
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Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
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Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe
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Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
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Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
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Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
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Claims (10)
1.一种转氨酶，其特征在于，所述转氨酶的氨基酸序列如SEQ ID NO.1所示。
2.一种分离的核酸，其特征在于，其编码如权利要求1所述的转氨酶。
3.一种包含如权利要求2所述的核酸的重组表达载体。
4.如权利要求3所述的重组表达载体，其特征在于，所述重组表达载体的骨架为质粒pET21a。
5.一种包含如权利要求2所述的核酸或如权利要求3或4所述的重组表达载体的转化体。
6.如权利要求5所述的转化体，其特征在于，所述转化体的宿主为大肠杆菌；优选大肠杆菌BL21(DE3)。
7.一种(R)-α-甲基色胺类化合物的制备方法，其特征在于，其包括在氨基供体存在时，在反应溶剂中用如权利要求1所述的转氨酶催化如式I所示的3-吲哚基丙酮类化合物得(R)-α-甲基色胺类化合物的步骤，所述的如式I所示的3-吲哚基丙酮类化合物为：
其中，R1、R2、R3独立地为H、OH、C1～C3的烷基或C1～C3的烷氧基。
8.如权利要求7所述的制备方法，其特征在于，所述反应溶剂为水；
和/或，所述的3-吲哚基丙酮类化合物为经过助溶剂助溶的3-吲哚基丙酮类化合物，所述助溶剂优选自DMSO、N,N二甲基甲酰胺、乙醇、异丙醇吐温-80和吐温-60中的一种或多种；所述助溶剂优选占所述制备方法的反应体系的总体积的0～20％；
和/或，所述的氨基供体为异丙胺或其盐，所述的盐优选为异丙胺盐酸盐；
和/或，所述制备方法的反应体系中还包括转氨酶的辅助因子例如吡哆醛磷酸，所述转氨酶的辅助因子与所述如式I所示的3-吲哚基丙酮类化合物的质量比为1:1000～1:10，优选为1:100；
和/或，所述的式I中，所述的C1～C3的烷基为甲基、乙基、正丙基或异丙基，所述的C1～C3的烷氧基为甲氧基、乙氧基、正丙氧基或异丙氧基；
较佳地，所述的如式I所示的3-吲哚基丙酮类化合物为下述任一化合物：
9.如权利要求7或8所述的制备方法，其特征在于，所述氨基供体与所述3-吲哚基丙酮类化合物的摩尔比为1:1～6:1，例如3:1～6:1；
和/或，所述3-吲哚基丙酮类化合物的浓度为10g/L～50g/L，优选为15～34g/L，例如19g/L；
和/或，所述转氨酶与所述3-吲哚基丙酮类化合物的质量比为6:1～12:1，例如为9:1；
和/或，所述反应的pH为7.5-8.5，例如为8；
和/或，所述反应的温度为35-45℃。
10.一种如权利要求1所述的转氨酶在制备(R)-α-甲基色胺类化合物中的用途；
较佳地，所述用途包括在氨基供体例如异丙胺或其盐存在时，在反应溶剂中用如权利要求1所述的转氨酶催化如式I所示的3-吲哚基丙酮类化合物得(R)-α-甲基色胺类化合物的步骤，所述的如式I所示的3-吲哚基丙酮类化合物为：
其中，R1、R2、R3独立地为H、OH、C1～C3的烷基或C1～C3的烷氧基；
更佳地，所述的式I中，所述的C1～C3的烷基为甲基、乙基、正丙基或异丙基，所述的C1～C3的烷氧基为甲氧基、乙氧基、正丙氧基或异丙氧基；
进一步更佳地，所述的如式I所示的化合物为下述任一化合物：
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| CN114958938B (zh) * | 2022-05-13 | 2023-12-08 | 金达威生物技术(江苏)有限公司 | 一种稠合二环脯氨酸甲酯盐酸盐的制备方法 |
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