CN111996222A - 采用酶突变技术制备药物中间体(r)-2-(氨乙基)-4-氯苯酚的方法 - Google Patents

采用酶突变技术制备药物中间体(r)-2-(氨乙基)-4-氯苯酚的方法 Download PDF

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CN111996222A
CN111996222A CN202010939533.1A CN202010939533A CN111996222A CN 111996222 A CN111996222 A CN 111996222A CN 202010939533 A CN202010939533 A CN 202010939533A CN 111996222 A CN111996222 A CN 111996222A
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傅荣昭
谢志军
宋鹏
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Shenghong Enterprise Management Center Of Ganjiang New Area LP
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Abstract

本发明属于生物制药领域，具体涉及一种采用酶突变技术制备药物中间体的方法，采用定点突变型ω‑转氨酶，具有SEQ ID NO.4所示的氨基酸序列，对药物中间体(R)‑2‑(氨乙基)‑4‑氯苯酚进行催化。采用生物酶合成法替代传统的纯化学合称法有效的降低了三废的排放，符合绿色化学趋势。酶突变技术制得的突变型ω‑转氨酶具有较好的酶活性与热稳定性。点突变得到的ω‑转氨酶还原效率高，收率高、且与纯化学法相比更加节约反应时间、能耗。

Description

采用酶突变技术制备药物中间体(R)-2-(氨乙基)-4-氯苯酚 的方法
技术领域
本发明属于生物制药领域，具体涉及一种采用酶突变技术制备药物
中间体的方法。
背景技术
手性胺广泛存在于自然界中，是很多重要生物活性分子的结构单元，是合成天然产物和手性药物的重要中间体，很多手性胺还可成为重要的手性助剂和手性拆分试剂。所以，手性胺化合物的制备有很重要的经济意义。
现阶段，手性胺的制备主要采用化学还原的方法，利用前手性酮制备得到光学活性的胺。在Pd/C及喹宁的催化作用下，将手性酮与甲酸以及无机氨/有机伯胺进行反应生成手性胺；另有研究者以钌配合物为催化剂，通过前手性酮不对称胺化还原得到手性胺(Angewandte Chemie International Edition.2003,42(44),5472-5474)，此类反应中金属催化剂是非常关键的因素，且对金属催化剂要求苛刻，反应需要在高压条件下完成，操作设备要求高，同时金属催化剂价格昂贵，对环境污染也较大(Journal of the AmericanChemical Society.2002,124(23),6508-6509)。中国科学院天津工业生物技术研究所朱敦明研究员和吴洽庆研究员带领的生物催化反应机理及应用团队针对新ω-转氨酶的生物催化进行了一系列的研究。从基因数据库中筛选鉴定出一种新的(S)立体选择性ω-转氨酶((S)-TA)和五种新的(R)立体选择性ω-转氨酶((R)-TA)，并测定了其酶学性质，进行了水相和有机相催化应用试验。
随着环保政策的收紧，纯化学法合成手性胺将变得十分的困难，因此选择更为绿色环保的手性胺合成工艺将成为时代发展的主要潮流。在此基础上，利用生物酶进行羰基的还原胺化成为手性胺发展的主要方向。氨基转移酶，也称为转氨酶，可以催化一个氨基与羰基互换的过程。转氨酶属于转氨酶之一，但有少许不同。转氨酶是指一类酶，只要在其催化的转氨反应中，反应的底物或产物中不含有α-氨基酸，就可以称该酶为转氨酶。ω-转氨酶可以利用酮类化合物为原料，通过立体选择性地转氨基作用，高效生产手性胺。因其底物相对廉价、产物纯度高的特点，受到研究人员越来越多的关注。由于底物结合区域特殊的空间结构，野生型ω-转氨酶在合成大位阻手性胺方面的应用受到了限制。此外，在立体选择性和稳定性方面这一类酶也存在一些不足，目前满足工业应用需求的ω-转氨酶仍较为有限。
定点突变是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组，也可以是质粒)中引入所需变化(通常是表征有利方向的变化)，包括碱基的添加、删除、替换等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征，是基因研究工作中一种非常有用的手段，现有技术中尚没有将ω-转氨酶突变用来制备药物中间体(R)-2-(氨乙基)-4-氯苯酚技术的报道。
发明内容
基于背景技术中提到的问题，本发明拟公开采用酶突变技术制备药物中间体(R)-2-(氨乙基)-4-氯苯酚的方法，以克服纯化学法合成手性胺带来的污染问题，采用酶突变技术筛选出一种还原效果好的突变型ω-转氨酶。本发明是通过以下技术方案实现的：
采用酶突变技术制备药物中间体(R)-2-(氨乙基)-4-氯苯酚的方法：所述突变酶为定点突变型ω-转氨酶，具有SEQ ID NO.10所示的氨基酸序列。
SEQ ID NO.10所示的氨基酸序列，由以下方式制备得到：
(1)从突变文库中筛选ω-转氨酶突变体，筛选得到酶活性高的ω-转氨酶突变体SEQ ID NO.1和热稳定性高的ω-转氨酶突变体SEQ ID NO.2；
(2)将SEQ ID NO.1中点突变引入SEQ ID NO.2获得SEQ ID NO.3～5，将SEQ IDNO.2中点突变引入SEQ ID NO.1获得SEQ ID NO.6～11；
(3)对得到的SEQ ID NO.3～11进行酶活性测试和热稳定性测试，得到酶活性与热稳定性都提高的SEQ ID NO.10。
所述酶活性高的ω-转氨酶突变体具有SEQ ID NO.1所示的氨基酸序列。
所述热稳定性高的ω-转氨酶突变体具有SEQ ID NO.2所示的氨基酸序列。
采用酶突变技术制备药物中间体(R)-2-(氨乙基)-4-氯苯酚的方法具体由以下步骤组成：
(1)以2-羟基-5-氯苯乙酮为原料加入三口瓶中，然后加入乙醇和的磷酸氢二钾的缓冲溶液搅拌20min；
(2)加入经突变型R型ω-转氨酶粗酶液；
(3)然后开始滴加质量分数为28％的氨水，滴完后继续在室温下继续搅拌2h；
(4)取样检测，采用HPLC检测，原料含量低于0.10％后，蒸出乙醇，在0℃下保温2h，过滤，收集固体；
(5)所得固体再用乙醇/水重结晶即可得到(R)-2-(氨乙基)-4-氯苯酚。
所述SEQ ID NO.10所示氨基酸在制备药物中间体手性胺化合物的应用。
如本领域技术人员所知：所述编码基因或氨基酸序列在不影响表达功能的前提下可以适当引入替换、缺失、改变、插入或增加碱基来制得。
本发明的有益效果
1、采用生物酶合成法替代传统的纯化学合称法有效的降低了三废的排放，符合绿色化学趋势。
2、采用酶突变技术制得的突变型ω-转氨酶具有较好的酶活性与热稳定性。
3、本发明点突变得到的ω-转氨酶还原效率高、收率高、且与纯化学法相比更加节约反应时间及能耗。
具体实施方式
以下实施例及其说明用于解释本发明，但并不构成对本发明的不当限定。
本申请中下述实施例中所使用的方法如无特殊说明均为常规方法，如《分子克隆实验指南》(J.萨姆布鲁克，D.W.拉塞尔著，黄培堂，汪嘉玺，朱厚础，等译。第3版，北京：科学出版社，2002)中所述的方法进行。同时，本发明中的氨基酸无特别说明外均用其缩写或代号标明。
实施例1
SEQ ID NO.10突变体的获得：突变体文库构建及高通量筛选方法：
突变体文库的构建：
为了提高野生型ω-转氨酶酶的活力，以重组表达载体PET-28a(+)-BS为DNA模板，通过易错PCR的方法构建一个随机突变体文库，并通过调整易错PCR反应体系中Mg2+和Mn2+浓度以及dCTP和dTTP寡核苷酸浓度，使该突变体文库的碱基错配率为千分之十，即保证一个突变体有2到6个氨基酸发生突变，构建突变体文库的具体过程如下。易错PCR反应体系和条件：
易错PCR反应体系：
Figure BDA0002673144580000051
Figure BDA0002673144580000061
易错PCR反应条件是：先95℃预变性5min；然后94℃变性30s，55℃退火1min，72℃1.5min，共30个循环；最后72℃延伸10min。
将上述得到的易错PCR产物切胶回收纯化，与原核表达载体pET28a(+)连接后转化重组基因工程菌，即得到一个库容量大的突变体文库。
A:第一轮突变
从上述突变子文库中筛选了约20000个克隆，选择10个酶活增加最明显的突变子。接着对这10个突变子进行摇瓶筛选。具体过程为：将这10个突变子接种于含100ml LB培养基的500ml摇瓶中进行发酵及诱导，并通过HPLC法测定活力，最终获得1个比对照酶活力高2-5倍的突变子，命名为SEQ ID NO.1，经过测序结果显示其在第63、396位点氨基酸发生了改变，由S突变成了G、P突变成了S。
从上述突变子文库中筛选了约20000个克隆，选择10个热稳定性增加最明显的突变子。接着对这10个突变子进行摇瓶筛选。具体过程为：将这10个突变子接种于含100ml LB培养基的500ml摇瓶中进行发酵及诱导，在45℃检测热稳定性，最终获得1个比对照热稳定性提高10％-20％的突变子，命名为SEQ ID NO.2，经过测序结果显示其在第25、158、271位点氨基酸发生了改变，由H突变成了D、Y突变成了F、R突变成了T。
B:定点突变
通过全基因合成将SEQ ID NO.1中两个点突变单独或全部引入SEQ ID NO.2获得SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5，分别连接PET28a(+)、转化BL21(DE3)发酵后，按照上述方法提取酶液验证功能。
同样方法将SEQ ID NO.2中三个点突变单独或两位点组合后引入SEQ ID NO.1获得SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10和SEQ ID NO.11。
C:测试SEQ ID NO.3～11突变子的酶活性与热稳定性：酶活检测采用连续监测法，热稳定性检测方式为：将酶液置于45℃孵育3h，检测酶活，以原始酶液做对照。
确定SEQ ID NO.10突变子酶活性和稳定性均获得提高，与原始酶相比原始酶活3.5U/ml，SEQ ID NO.10酶活性5.8U/ml。原始酶在45℃保存1h酶活剩余48％，SEQ ID NO.10剩余76％。
实施例2
以170.59g 2-羟基-5-氯苯乙酮为原料加入三口瓶中，然后加入300g乙醇和300g5mM pH＝9.6的磷酸氢二钾的缓冲溶液搅拌20min；加入经突变型R型ω-转氨酶粗酶液10g，酶活为2547.16U·g-1；然后开始滴加质量分数为28％的氨水150g，滴完后继续在室温下继续搅拌2h；取样检测，采用HPLC检测，原料含量低于0.10％后，蒸出乙醇，在0℃下保温2h，过滤，收集固体，所得固体再用乙醇/水重结晶即可得到(R)-2-(氨乙基)-4-氯苯酚。得到(R)-2-(氨乙基)-4-氯苯酚165g，收率96％，ee值98.50％。
序列表
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Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Phe His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Thr Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Pro Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 4
<211> 452
<212> PRT
<213> SEQ ID NO：3(人工序列)
<400> 4
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala Asp Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Gly Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Phe His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Thr Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Pro Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 5
<211> 452
<212> PRT
<213> SEQ ID NO ：4(人工序列)
<400> 5
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala Asp Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Ser Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Phe His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Thr Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Ser Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 6
<211> 452
<212> PRT
<213> SEQ ID NO ：5(人工序列)
<400> 6
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala Asp Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Gly Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Phe His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Thr Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Ser Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 7
<211> 452
<212> PRT
<213> SEQ ID NO ：6(人工序列)
<400> 7
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala Asp Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Gly Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Tyr His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Arg Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Ser Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 8
<211> 452
<212> PRT
<213> SEQ ID NO：7(人工序列)
<400> 8
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala His Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Gly Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Phe His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Arg Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Ser Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 9
<211> 452
<212> PRT
<213> SEQ ID NO：8(人工序列)
<400> 9
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala His Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Gly Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Tyr His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Thr Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Ser Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 10
<211> 452
<212> PRT
<213> SEQ ID NO ：9(人工序列)
<400> 10
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala Asp Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Gly Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Phe His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Arg Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Ser Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 11
<211> 452
<212> PRT
<213> SEQ ID NO：10(人工序列)
<400> 11
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala Asp Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Gly Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Tyr His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Thr Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Ser Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450
<210> 12
<211> 452
<212> PRT
<213> SEQ ID NO：11(人工序列)
<400> 12
Met Thr Asp Ile Thr Thr Ala Ala Gln Gln Asp Asp Thr Thr Ile Arg
1 5 10 15
Thr Asp Ala Ala Trp Leu Asp Ala His Trp Met Pro Phe Thr Ala Asn
20 25 30
Arg Gln Phe Lys Ala Asp Pro Arg Met Ile Val Ser Gly Gln Gly Ala
35 40 45
Tyr Tyr Thr Asp Ala Glu Gly Arg Lys Ile Phe Asp Gly Leu Gly Gly
50 55 60
Leu Trp Cys Thr Gly Leu Gly His Gly Arg Thr Glu Ile Val Glu Ala
65 70 75 80
Val Ser Arg Gln Val Ala Gln Leu Asp Tyr Ala Pro Ala Phe Gln Phe
85 90 95
Gly His Pro Lys Ser Phe Glu Leu Ala Asn Lys Ile Lys Asp Leu Thr
100 105 110
Pro Ala Gly Leu Asp Tyr Val Phe Phe Thr Gly Ser Gly Ser Glu Ala
115 120 125
Ala Asp Thr Ser Leu Lys Leu Ala Arg Ala Tyr Trp Arg Ala Lys Gly
130 135 140
Lys Gly Thr Lys Thr Arg Leu Ile Gly Arg Glu Lys Gly Phe His Gly
145 150 155 160
Val Asn Phe Gly Gly Ile Ser Val Gly Gly Ile Gly Pro Asn Arg Lys
165 170 175
Leu Phe Gly Gln Gly Leu Asp Ala Asp Phe Leu Pro His Thr Gln Leu
180 185 190
Ala Glu Asn Lys Phe Ser Arg Gly Met Pro Glu His Gly Ala Glu Leu
195 200 205
Ala Asp Arg Leu Leu Asp Leu Ile Ala Leu His Asp Ala Ser Asn Ile
210 215 220
Ala Ala Val Ile Val Glu Pro Phe Ser Gly Ser Ala Gly Val Val Val
225 230 235 240
Pro Pro Lys Gly Tyr Leu Lys Arg Leu Arg Asp Ile Cys Thr Ala His
245 250 255
Asp Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Thr Ala
260 265 270
Gly Ala Met Thr Gly Ala Asp Ala Phe Gly Val Thr Pro Asp Ile Leu
275 280 285
Asn Phe Ala Lys Gln Val Thr Asn Gly Val Gln Pro Leu Gly Gly Val
290 295 300
Ile Ala Thr Lys Glu Ile Tyr Asp Thr Phe Met Ala Ala Gly Gly Pro
305 310 315 320
Glu Tyr Met Leu Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro
325 330 335
Val Ala Cys Ala Ala Gly Val Ala Ala Leu Asp Leu Leu Val Lys Glu
340 345 350
Asp Ala Val Ala Arg Val Arg Asp Leu Ala Pro His Phe Glu Ala Ala
355 360 365
Val His Gly Leu Lys Gly Gln Arg His Ile Thr Asp Ile Arg Asn Tyr
370 375 380
Gly Leu Ala Ala Gly Leu Thr Ile Ala Ala Leu Ser Gly Glu Pro Ala
385 390 395 400
Arg Arg Pro Tyr Glu Ile Ala Met Arg Cys Trp Ala Lys Gly Phe Tyr
405 410 415
Val Arg Tyr Gly Gly Asp Thr Ile Gln Leu Ala Pro Pro Phe Ile Ser
420 425 430
Glu Lys Arg Glu Ile Asp Asn Leu Val Asn Ala Leu Ser Asp Ala Leu
435 440 445
Asn Glu Val Asp
450

Claims (6)

1.采用酶突变技术制备药物中间体(R)-2-(氨乙基)-4-氯苯酚的方法，其特征在于：所述突变酶为定点突变型ω-转氨酶，具有SEQ ID NO.10所示的氨基酸序列。
2.根据权利要求1所述的氨基酸序列，其特征在于：由以下方式制备得到：
(1)从突变文库中筛选ω-转氨酶突变体，筛选得到酶活性高的ω-转氨酶突变体SEQID NO.1和热稳定性高的ω-转氨酶突变体SEQ ID NO.2；
(2)将SEQ ID NO.1中点突变引入SEQ ID NO.2获得SEQ ID NO.3～5，将SEQ ID NO.2中点突变引入SEQ ID NO.1获得SEQ ID NO.6～11；
(3)对得到的SEQ ID NO.3～11进行酶活性测试和热稳定性测试，得到酶活性与热稳定性都提高的SEQ ID NO.10。
3.根据权利要求2所述的制备方法，其特征在于：酶活性高的ω-转氨酶突变体具有SEQID NO.1所示的氨基酸序列。
4.根据权利要求2所述的制备方法，其特征在于：热稳定性高的ω-转氨酶突变体具有SEQ ID NO.2所示的氨基酸序列。
5.权利要求1所述(R)-2-(氨乙基)-4-氯苯酚的制备方法，其特征在于，具体由以下步骤组成：
(1)以2-羟基-5-氯苯乙酮为原料加入三口瓶中，然后加入乙醇和的磷酸氢二钾的缓冲溶液搅拌20min；
(2)加入经突变型R型ω-转氨酶粗酶液；
(3)然后开始滴加质量分数为28％的氨水，滴完后继续在室温下继续搅拌2h；
(4)取样检测，采用HPLC检测，原料含量低于0.10％后，蒸出乙醇，在0℃下保温2h，过滤，收集固体；
(5)所得固体再用乙醇/水重结晶即可得到(R)-2-(氨乙基)-4-氯苯酚。
6.权利要求1所述SEQ ID NO.10所示氨基酸在制备药物中间体手性胺化合物的应用。
CN202010939533.1A 2020-09-09 2020-09-09 采用酶突变技术制备药物中间体(r)-2-(氨乙基)-4-氯苯酚的方法 Pending CN111996222A (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107058256A (zh) * 2017-05-04 2017-08-18 浙江科技学院 ω‑转氨酶突变体及其制备方法和应用
CN109486778A (zh) * 2018-10-22 2019-03-19 浙江科技学院 一种基于共进化网络的ω-转氨酶突变体以及制备方法和应用
CN111518783A (zh) * 2020-04-10 2020-08-11 浙江工业大学 新型重组(R)-ω-转氨酶、突变体及其在制备西他列汀中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107058256A (zh) * 2017-05-04 2017-08-18 浙江科技学院 ω‑转氨酶突变体及其制备方法和应用
CN109486778A (zh) * 2018-10-22 2019-03-19 浙江科技学院 一种基于共进化网络的ω-转氨酶突变体以及制备方法和应用
CN111518783A (zh) * 2020-04-10 2020-08-11 浙江工业大学 新型重组(R)-ω-转氨酶、突变体及其在制备西他列汀中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NCBI: "aspartate aminotransferase family protein [Burkholderia stabilis]", 《NCBI》 *
ZHENGXU S.HAN等: "Design and Synthesis of Chiral Oxathiozinone Scaffolds: Efficient Synthesis of Hindered Enantiopure Sulfinamides and Sulfinyl Ketimines", 《ANGEWANDTE CHEMIE INTERNATIONAL EDITION》 *
ZHENGXU S.HAN等: "Efficient Asymmetric Synthesis of P-Chiral Phosphine Oxides via Properly Designed and Activated Benzoxazaphosphinine-2-oxide Agents", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 *

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