CN110358804A - R-3-氨基正丁醇的酶法生产工艺 - Google Patents

R-3-氨基正丁醇的酶法生产工艺 Download PDF

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CN110358804A
CN110358804A CN201810313800.7A CN201810313800A CN110358804A CN 110358804 A CN110358804 A CN 110358804A CN 201810313800 A CN201810313800 A CN 201810313800A CN 110358804 A CN110358804 A CN 110358804A
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陶荣盛
王博
沈青
朱傅赟
孙梁栋
沈正权
郑云
潘震华
刘萍
王亚夫
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Huzhou Yi Hui Biotechnology Co Ltd
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Abstract

本发明公开了一种R‑3‑氨基正丁醇的酶法生产工艺，其以3‑氨基正丁醇外消旋体和丙酮酸为底物，通过微生物来源的ω‑转氨酶立体特异性地催化S‑3‑氨基正丁醇与丙酮酸反应，然后从反应体系中回收得到未参与反应的R‑3‑氨基正丁醇，得到高光学纯度的对映体R‑3‑氨基正丁醇，适用于工业化生产。

Description

R-3-氨基正丁醇的酶法生产工艺
技术领域
本发明属于生物催化技术领域，涉及一种R-3-氨基正丁醇的酶法生产工艺，具体地说，涉及一种通过ω-转氨酶催化拆分3-氨基正丁醇外消旋体来制备R-3-氨基正丁醇的方法。
背景技术
R-3-氨基正丁醇又称(R)-3-氨基丁醇、R-3-氨基-1-丁醇，是合成抗艾滋病整合酶抑制剂-度鲁特韦的重要中间体，与现有的HIV整合酶抑制剂雷特格韦、埃替格韦相比，该药物的安全性提高，与默沙东的抗HIV/AIDS药物拉替拉替韦相比，度鲁特韦不但在三期临床试验中达到了与其相匹敌的疗效，而其不需要与药物促进剂联合用药，具有非常强效的耐药属性，而其用药量为日服一次。度鲁特韦自上市以来销售额呈现飞跃式发展，R-3-氨基正丁醇的光学纯度和价格高低对于度鲁特韦的品质及生产成本有着重要影响。
目前R-3-氨基正丁醇的生产以化学合成方法为主，一种是采用动力学拆分法获得手性纯的3-氨基丁醇(Journal of Organic Chemistry,42,1650,1977)，但化学拆分法存在收率低，拆分剂消耗大以及反应条件苛刻等缺陷。另一种是以手性化合物为起始原料，D-丙氨酸或R型苯乙胺通过多步反应获得手性纯的3-氨基正丁醇(Tetrahedron,61,9031,2005和
CN101417954B)，这类方法中手性纯原料价格较贵，且反应步骤冗长，生产成本较高。
近些年来，生物法制备R-3-氨基正丁醇已经成为一个研究热点。与化学合成法相比，生物合成法具有反应条件温和、转化效率高和立体选择性强等优点。专利CN104131048A、CN104178533A和CN106754806A公开了利用转氨酶及其突变体不对称催化4-羟基-2-丁酮合成R-3-氨基丁醇的方法，但由于反应浓度低，转化不稳定等原因，尚未实现工业化应用。
发明内容
为了克服现有技术的不足，探索适用于工业化生产高光学纯度的R-3-氨基正丁醇的工艺，发明人对转氨酶催化法进行了大量的研究，意外地发现部分来源于微生物的ω-转氨酶(ω-TA)对于3-氨基正丁醇对映体具有高度的立体特异性，而且S-3-氨基正丁醇与丙酮酸反应时不存在转氨酶普遍存在的动态平衡问题，S-3-氨基正丁醇几乎能够反应完全，这一发现构成了本发明的基础。具体而言，本发明的技术方案如下所述。
一种制备R-3-氨基正丁醇的方法，包括如下步骤：以3-氨基正丁醇外消旋体和丙酮酸为底物，用ω-转氨酶为催化剂，立体特异性地催化S-3-氨基正丁醇与丙酮酸反应，然后从反应体系中回收未参与反应的对映体R-3-氨基正丁醇。反应式如下所述：
上述ω-转氨酶优选是来源于选自下组的微生物：Ochrobactrum anthropic(人苍白杆菌)、Bradyrhizobium sp.Ec3.3(慢生根瘤菌sp.Ec3.3)、Brucella neotomae(沙林鼠种布鲁氏菌)、Novosphingobium acidiphilum(嗜酸新鞘脂菌)、Ochrobactrumintermedium(中间苍白杆菌)、Pseudaminobacter salicylatoxidans(假氨基酸杆菌)、Brucella abortus(牛种布鲁氏菌)、Paracoccus denitrificans PD1222(脱氮副球菌PD1222)。更优选来源于Ochrobactrum anthropic、Brucella neotomae、Novosphingobiumacidiphilum、Brucella abortus。
优选地，上述ω-转氨酶选自下组：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:4、SEQID NO:7。
在一种优选的实施方式中，反应体系中还添加有磷酸吡哆醛(PLP)。磷酸吡哆醛作为辅酶可以促进转氨酶催化的氨基转移反应。磷酸吡哆醛在反应体系中的添加量为0.1-1mmol/L(即0.1-1mM)。
优选地，上述反应体系所用的反应溶液为水溶液、磷酸盐缓冲液、Tirs缓冲液或碳酸氢盐缓冲液。比如可以是10-100mM磷酸盐缓冲液、10-100mM Tirs缓冲液、或10-100mM碳酸氢盐缓冲液。
上述ω-转氨酶可以呈酶形式，也可以呈ω-转氨酶表达微生物形式，比如是表达ω-转氨酶的重组大肠杆菌。当采用重组大肠杆菌时，ω-转氨酶的表达宿主可以是大肠杆菌BL21(DE3)。
在一种实施方式中，反应体系为pH6.5-pH9.0、优选pH6.8-pH8.5、更优选pH7.0-pH8.0、更优选pH7.0-pH7.8、更优选pH7.0-pH7.5。
在一种实施方式中，反应温度为25-45℃、优选30-40℃、更优选32-40℃、更优选35-38℃、例如37℃。
优选地，从反应体系中回收R-3-氨基正丁醇的方法可以是转化液浓缩后用甲醇或乙醇将R-3-氨基正丁醇溶出，然后成盐结晶的方法，也可以通过柱层析进行回收，也可以采用有机溶剂比如乙酸乙酯或二氯甲烷在碱性条件下通过萃取法回收。
由于本发明是通过酶促拆分3-氨基丁醇外消旋体的方法来制备R-构型对映体的，底物的转化率越高，R-3-氨基正丁醇的光学纯度也就越高，因此可以通过控制底物的转化率来控制最终产物R-3-氨基正丁醇的光学纯度，使其达到符合预期要求的光学纯度。酶促拆分方法不仅进程可控，而且具有反应速度快、操作简便安全的优点。
同时，ω-转氨酶的反应底物3-氨基正丁醇和丙酮酸允许具有高浓度，可以根据化学平衡原理调节这两个反应底物的加料比，有利于降低成本，易于实现工业化生产。
具体实施方式
以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
为描述方便起见，在本文中有时将来源于Ochrobactrum anthropic的ω-转氨酶简称为OATA，其氨基酸序列为SEQ ID NO:1，编码基因为NCBI登录号：NC_009667；将来源于Bradyrhizobium sp.Ec3.3的ω-转氨酶简称为BSTA，其氨基酸序列为SEQ ID NO:2，编码基因为NCBI登录号：WP027523883；将来源于Brucella neotomae的ω-转氨酶简称为BNTA，其氨基酸序列为SEQ ID NO:3，编码基因为NCBI登录号：WP004687895；将来源于Novosphingobium acidiphilum的ω-转氨酶简称为NATA，其氨基酸序列为SEQ ID NO:4，编码基因为NCBI登录号：WP028641684；将来源于Ochrobactrum intermedium的ω-转氨酶简称为OITA，其氨基酸序列为SEQ ID NO:5，编码基因为NCBI登录号：WP006465915；将来源于Pseudaminobacter salicylatoxidans的ω-转氨酶简称为PSTA，其氨基酸序列为SEQ IDNO:6，编码基因为NCBI登录号：WP019171585；将来源于Brucella abortus的ω-转氨酶简称为BATA，其氨基酸序列为SEQ ID NO:7，编码基因为NCBI登录号：WP006212579；将来源于Paracoccus denitrificans PD1222的ω-转氨酶简称为PDTA，其氨基酸序列为SEQ ID NO:8，编码基因为NCBI登录号：ABL72050。
在本文中，为描述方便起见，有时将R-3-氨基正丁醇简称为R-对映体，将S-3-氨基正丁醇简称为S-对映体，将(R,S)-3-氨基正丁醇或3-氨基正丁醇简称为外消旋体。显而易见，本发明选用的ω-转氨酶的催化底物是S-对映体。
本发明中使用的ω-转氨酶结构明确，因此本领域技术人员能够容易地获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
当作为生物催化剂用于生产R-对映体时，本发明的ω-转氨酶可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶，包括纯化酶、粗酶、发酵液、载体固定的酶等。而且这些酶的分离纯化、包括固定化酶制备技术也是本领域技术人员所熟知的。所述菌体的形式包括存活菌体和死亡菌体。
在一种优选的实施方式中，反应体系可以添加有0.1-1mmol/L磷酸吡哆醛，作为ω-转氨酶的辅酶来促进拆分反应。具体加入量取决于酶量和底物加入量，可通过简单的实验予以确定。
实施例
材料和方法
本文中的全基因合成、引物合成及测序皆由南京金斯瑞生物科技有限公司完成。
本文中的分子生物学实验包括质粒构建、酶切、感受态细胞制备、转化等主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。可根据相关试剂盒说明书操作。必要时可以通过简单试验确定具体实验条件比如PCR条件。
TB培养基：24g/L酵母提取物、12g/L胰蛋白胨、16.43g/L K2HPO4.3H2O、2.31g/LKH2PO4、5g/L甘油，pH 7.0-7.5，121℃高温高压灭菌20min。
反应底物及产物的HPLC测定条件：
流动相组分A：0.05M醋酸钠水溶液
流动相组分B：甲醇
流动相：A:B＝70:30(v/v)
进样量：5.0μL
流速：1.0mL/min
采集时间：30min
色谱柱：Agilent C8(180×4.6)
柱温：30℃
检测波长：334nm
保留时间：丙氨酸3.8min
R-3-氨基正丁醇26.7min。
实施例1ω-转氨酶表达菌株的构建
从NCBI登录号分别为NC_009667、WP027523883、WP004687895、WP028641684、WP006465915、WP019171585、WP006212579、ABL72050获得编码ω-转氨酶OATA、BSTA、BNTA、NATA、OITA、PSTA、BATA和PDTA的多核苷酸序列。全基因合成这些ω-转氨酶基因序列，分别在两端设计限制性内切酶位点NdeI和BamHI，亚克隆到载体pET24a(购自Novagen)上相应位点，获得重组质粒pET24a。将构建好的重组质粒分别地用氯化钙法转化大肠杆菌表达宿主BL21(DE3)，得到分别表达各个ω-转氨酶的重组大肠杆菌。
实施例2转氨酶催化3-氨基正丁醇拆分能力的比较
2.1ω-转氨酶表达菌种的摇瓶发酵：配制液体培养基TB(pH7.0)，分装于500mL三角摇瓶，装液量100mL，然后在高压灭菌锅中于121℃加热灭菌20min。从ω-转氨酶表达菌种的平板上用接种环挑数环菌体接种于TB摇瓶中，接种前在TB培养基中加入100μg/mL卡那霉素，于37℃、220rpm摇床培养至OD600＝5-6，加入0.2mM IPTG(异丙基硫代半乳糖苷)于28℃诱导24h左右。
2.2粗酶液的制备：取发酵液50ml装入离心管中；8000rpm离心去上清液得到菌体，按菌体200g/L加纯化水重悬，悬浮菌体用冰浴冷却，进行超声破碎(电压400W，超声时间3s，间隔时间5s，工作次数80次)得粗酶液。
2.3酶促拆分反应：称取0.88g(0.4M)丙酮酸钠放置于摇瓶中，然后加入0.893g(0.5M)3-氨基正丁醇、0.1mm/L PLP、水10ml，用醋酸调PH至7.5，再加入20g/L破碎好的转氨酶粗酶液，加纯化水定容至20ml，37℃进行转化。分别转化1h和16h后取样，用体积比为1:1的0.1M盐酸终止反应，HPLC检测丙氨酸、R-3-氨基正丁醇和S-3-氨基正丁醇含量，并计算产物R-3-氨基正丁醇的ee值。
各ω-转氨酶催化3-氨基正丁醇拆分的结果列于表1和表2中。
表1多种微生物来源ω-转氨酶转化1h后的结果比较
表2多种微生物来源ω-转氨酶转化16h后的结果比较
由表1和表2可见，ω-转氨酶OATA、BATA、BNTA、NATA的催化活力相对更高。
实施例3转氨酶催化高浓度3-氨基正丁醇的拆分能力比较
3.1ω-转氨酶表达菌种的摇瓶发酵：配制液体培养基TB(pH7.0-7.5)，分装于500mL三角摇瓶，装液量100mL，然后在高压灭菌锅中于121℃加热灭菌20min。从ω-转氨酶OATA、BATA、BNTA和NATA表达菌种的平板上分别用接种环挑数环菌体接种于各个TB摇瓶中，接种前在TB培养基中加入100μg/mL卡那霉素，于37℃、220rpm摇床培养至OD600＝5-6，加入0.2mM IPTG于28℃诱导24h左右。
3.2粗酶液的制备：取发酵液50ml装入离心管中；8000rpm离心去上清液得到菌体，按菌体200g/L加纯化水重悬，悬浮菌体用冰浴冷却，进行超声破碎(电压400W，超声时间3s，间隔时间5s，工作次数80次)得粗酶液。。
3.3酶促拆分反应：称取1.76g(0.8M)丙酮酸钠放置于摇瓶中，然后加入2g(100g/L)3-氨基正丁醇、0.1mm/L PLP、水10ml，用醋酸调PH至7.5，再加入20g/L破碎好的转氨酶酶液，加水定容至20ml，37℃进行转化。分别在转化1h后和16h后取样，用体积比为1:1的0.1M盐酸终止反应，HPLC检测丙氨酸、R-3-氨基正丁醇和S-3-氨基正丁醇含量，并计算产物R-3-氨基正丁醇的ee值。
各ω-转氨酶催化3-氨基正丁醇拆分的结果列于表3中。
表3不同ω-转氨酶催化高浓度3-氨基正丁醇拆分的比较
实验结果表明，采用本发明的ω-转氨酶催化拆分3-氨基丁醇外消旋体允许反应底物3-氨基正丁醇具有高浓度，例如可以达到100g/L，而且反应速度快，有利于降低R-3-氨基正丁醇生产成本，因而具有工业化前景。
序列表
<110> 湖州颐辉生物科技有限公司
<120> R-3-氨基正丁醇的酶法生产工艺
<130> SHPI810302
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 456
<212> PRT
<213> Ochrobactrum anthropic
<400> 1
Met Thr Ala Gln Pro Asn Ser Leu Glu Ala Arg Asp Ile Arg Tyr His
1 5 10 15
Leu His Ser Tyr Thr Asp Ala Val Arg Leu Glu Ala Glu Gly Pro Leu
20 25 30
Val Ile Glu Arg Gly Asp Gly Ile Tyr Val Glu Asp Val Ser Gly Lys
35 40 45
Arg Tyr Ile Glu Ala Met Ser Gly Leu Trp Ser Val Gly Val Gly Phe
50 55 60
Ser Glu Pro Arg Leu Ala Glu Ala Ala Ala Arg Gln Met Lys Lys Leu
65 70 75 80
Pro Phe Tyr His Thr Phe Ser Tyr Arg Ser His Gly Pro Val Ile Asp
85 90 95
Leu Ala Glu Lys Leu Val Ser Met Ala Pro Val Pro Met Ser Lys Ala
100 105 110
Tyr Phe Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Val Val Lys Leu
115 120 125
Ile Trp Tyr Arg Ser Asn Ala Leu Gly Glu Pro Glu Arg Lys Lys Ile
130 135 140
Ile Ser Arg Lys Arg Gly Tyr His Gly Val Thr Ile Ala Ser Ala Ser
145 150 155 160
Leu Thr Gly Leu Pro Asn Asn His Arg Ser Phe Asp Leu Pro Ile Asp
165 170 175
Arg Ile Leu His Thr Gly Cys Pro His Phe Tyr Arg Glu Gly Gln Ala
180 185 190
Gly Glu Ser Glu Glu Gln Phe Ala Thr Arg Leu Ala Asp Glu Leu Glu
195 200 205
Gln Leu Ile Ile Ala Glu Gly Pro His Thr Ile Ala Ala Phe Ile Gly
210 215 220
Glu Pro Val Met Gly Ala Gly Gly Val Val Val Pro Pro Lys Thr Tyr
225 230 235 240
Trp Glu Lys Val Gln Ala Val Leu Lys Arg Tyr Asp Ile Leu Leu Ile
245 250 255
Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Leu Phe Gly
260 265 270
Ser Gln Thr Phe Asp Met Lys Pro Asp Ile Leu Val Met Ser Lys Gln
275 280 285
Leu Ser Ser Ser Tyr Leu Pro Ile Ser Ala Phe Leu Ile Asn Glu Arg
290 295 300
Val Tyr Ala Pro Ile Ala Glu Glu Ser His Lys Ile Gly Thr Leu Gly
305 310 315 320
Thr Gly Phe Thr Ala Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Ala Ile Ile Glu Glu Arg Asp Leu Val Ala Asn Ala Arg
340 345 350
Asp Arg Gly Thr Tyr Met Gln Lys Arg Leu Arg Glu Leu Gln Asp His
355 360 365
Pro Leu Val Gly Glu Val Arg Gly Val Gly Leu Ile Ala Gly Val Glu
370 375 380
Leu Val Thr Asp Lys Gln Ala Lys Thr Gly Leu Glu Pro Thr Gly Ala
385 390 395 400
Leu Gly Ala Lys Ala Asn Ala Val Leu Gln Glu Arg Gly Val Ile Ser
405 410 415
Arg Ala Met Gly Asp Thr Leu Ala Phe Cys Pro Pro Leu Ile Ile Asn
420 425 430
Asp Gln Gln Val Asp Thr Met Val Ser Ala Leu Glu Ala Thr Leu Asn
435 440 445
Asp Val Gln Ala Ser Leu Thr Arg
450 455
<210> 2
<211> 460
<212> PRT
<213> Bradyrhizobium sp. Ec3.3
<400> 2
Met Thr Met Leu Pro Asn Ser Gln Glu Ala Arg Asp Val Ala Tyr Gln
1 5 10 15
Leu His Pro Tyr Thr Asn Ala Arg Thr His Gln Gln Asn Gly Pro Leu
20 25 30
Val Ile Glu Arg Gly Glu Gly Pro Tyr Val Phe Asp Ala Ala Gly Lys
35 40 45
Arg Tyr Phe Glu Ala Met Ala Gly Leu Trp Ser Val Gly Leu Gly Phe
50 55 60
Asn Glu Lys Arg Leu Val Glu Ala Ala Tyr Arg Gln Met Gln Ala Leu
65 70 75 80
Pro Phe Tyr His Thr Phe Ser Ala Lys Ser His Gly Pro Ser Ile Asp
85 90 95
Leu Ala Glu Lys Leu Val Ala Leu Ala Pro Val Pro Met Ser Lys Val
100 105 110
Phe Phe Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Val Leu Lys Leu
115 120 125
Ile Ala Tyr Arg Ser Asn Ala Leu Gly Gln Pro Gln Arg Lys Lys Val
130 135 140
Ile Ser Arg Met Arg Ala Tyr His Gly Val Thr Ile Ala Ser Ala Ser
145 150 155 160
Leu Thr Gly Leu Pro Asn Asn His Arg Ser Phe Asp Leu Pro Leu Pro
165 170 175
Asn Ile Leu His Thr Gly Ser Pro His Phe Tyr Lys Asp Gly Gln Ala
180 185 190
Gly Glu Ser Glu Glu Ala Phe Ala Thr Arg Arg Ala Glu Glu Leu Glu
195 200 205
Ala Leu Ile Val Lys Glu Gly Pro Asp Thr Ile Ala Ala Phe Phe Gly
210 215 220
Glu Pro Val Met Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr Tyr
225 230 235 240
Trp Asp Lys Val Gln Gln Val Leu Lys Lys Tyr Asp Ile Leu Leu Val
245 250 255
Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Lys Met Phe Gly
260 265 270
Cys Asp Thr Tyr Asn Ile Lys Pro Asp Ala Ile Val Val Ser Lys Gln
275 280 285
Ile Thr Ser Ser Tyr Phe Pro Phe Ser Ala Val Leu Met Asn Asp Arg
290 295 300
Met Phe Glu Pro Ile Ala Asp Glu Ser Asn Lys Ile Gly Val Leu Gly
305 310 315 320
His Gly Phe Thr Ala Gly Gly His Pro Val Gly Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Lys Ile Ile Glu Glu Arg Gly Leu Val Ala His Ala Ala
340 345 350
Glu Thr Gly Ala His Leu Gln Ala Arg Leu Arg Glu Leu Ser Ser His
355 360 365
Pro Leu Val Gly Glu Val Arg Gly Val Gly Met Ile Ala Ala Ile Glu
370 375 380
Leu Val Leu Asp Lys Gln Arg Lys Thr Ala Ala Ala Thr Pro Gly Ala
385 390 395 400
Val Gly Gly Ile Ala Ala Arg Met Leu Gln Glu Arg Gly Val Ile Ser
405 410 415
Arg Asn Met Leu Glu Ala Ile Ala Ile Cys Pro Pro Leu Ile Val Thr
420 425 430
Lys Ser Gln Ile Asp Glu Leu Val Gly Thr Ile Ala Gly Val Leu Asp
435 440 445
Asp Met Lys Thr Glu Ala Ala Lys Leu Thr Pro Ala
450 455 460
<210> 3
<211> 456
<212> PRT
<213> Brucella neotomae
<400> 3
Met Thr Ala Gln Pro Asn Ser Leu Glu Ala Arg Asp Ile Arg Tyr His
1 5 10 15
Leu His Ser Tyr Thr Asp Ala Val Arg Leu Glu Ala Glu Gly Pro Leu
20 25 30
Val Ile Glu Arg Gly Asp Gly Ile Tyr Val Glu Asp Ile Ala Gly Lys
35 40 45
Arg Tyr Ile Glu Ala Met Ser Gly Leu Trp Ser Val Gly Val Gly Phe
50 55 60
Ser Glu Gln Arg Leu Ala Glu Ala Ala Ala Arg Gln Met Lys Lys Leu
65 70 75 80
Pro Phe Tyr His Thr Phe Ser Tyr Arg Ser His Gly Pro Val Ile Asp
85 90 95
Leu Ala Glu Lys Leu Val Ala Met Ala Pro Val Pro Met Ser Lys Ala
100 105 110
Tyr Phe Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Val Val Lys Leu
115 120 125
Ile Trp Tyr Arg Ser Asn Ala Leu Gly Glu Pro Glu Arg Lys Lys Ile
130 135 140
Ile Ser Arg Lys Arg Gly Tyr His Gly Val Thr Ile Ala Ser Ala Ser
145 150 155 160
Leu Thr Gly Leu Pro Asn Asn His Arg Ser Phe Asp Leu Pro Ile Asp
165 170 175
Arg Ile Leu His Thr Gly Cys Pro His His Tyr His Asp Ala Leu Pro
180 185 190
Gly Glu Ser Glu Glu Gln Phe Thr Thr Arg Leu Ala Asn Glu Leu Glu
195 200 205
Gln Leu Ile Leu Ala Glu Gly Pro His Thr Ile Ala Ala Phe Ile Gly
210 215 220
Glu Pro Val Met Gly Ala Gly Gly Val Val Val Pro Pro Lys Thr Tyr
225 230 235 240
Trp Glu Lys Val Gln Ala Val Leu Gly Arg Tyr Asn Ile Leu Leu Val
245 250 255
Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Leu Phe Gly
260 265 270
Cys Gln Thr Phe Asp Ile Lys Pro Asp Ile Leu Val Met Ser Lys Gln
275 280 285
Leu Ser Ser Ser Tyr Leu Pro Ile Ser Ala Phe Leu Ile Asn Glu Arg
290 295 300
Val Tyr Ala Pro Ile Ala Glu Glu Ser His Lys Ile Gly Thr Leu Gly
305 310 315 320
Thr Gly Phe Thr Ala Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Ala Ile Ile Glu Glu Arg Asp Leu Val Ala Asn Ala Arg
340 345 350
Glu Arg Gly Ala Arg Met Gln Lys Arg Leu Arg Glu Leu Gln Asp His
355 360 365
Pro Leu Val Gly Glu Val Arg Gly Val Gly Leu Ile Ala Gly Val Glu
370 375 380
Leu Val Ile Asp Lys Glu Ala Lys Thr Gly Leu Glu Gln Pro Gly Ala
385 390 395 400
Leu Gly Ala Arg Ala Asn Ala Ala Leu Gln Glu Arg Gly Val Ile Ser
405 410 415
Arg Ala Met Gly Asp Thr Leu Ala Phe Cys Pro Pro Leu Ile Ile Asn
420 425 430
Asp Gln Gln Val Asp Thr Met Val Ser Ala Leu Glu Ala Ala Leu Asn
435 440 445
Asp Val Gln Ala Ser Leu Gly Lys
450 455
<210> 4
<211> 460
<212> PRT
<213> Novosphingobium acidiphilum
<400> 4
Met Leu Asp Ala Pro Asn Ser Ala Glu Ala Arg Asp Ile Arg Phe His
1 5 10 15
Leu His Ser Tyr Thr Asn Ala Arg Leu His Glu Gln Val Gly Pro Leu
20 25 30
Val Ile Glu Gly Gly Asp Gly Ile Tyr Val Ile Asp Ser Glu Gly Arg
35 40 45
Arg Tyr Ile Glu Ala Met Ser Gly Leu Trp Ser Val Gly Val Gly Phe
50 55 60
Ser Glu Lys Arg Leu Val Ala Ala Ala Thr Glu Gln Met Asn Lys Leu
65 70 75 80
Pro Phe Tyr His Thr Phe Thr His Lys Ser His Gly Pro Ala Ile Asp
85 90 95
Leu Ala Glu Lys Leu Val Thr Leu Ala Ser Pro Leu Thr Pro Gly Gly
100 105 110
Met Ser Lys Ala Phe Phe Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr
115 120 125
Ala Ile Lys Leu Ile Trp Tyr Arg Ser Asn Ala Leu Gly Lys Pro Glu
130 135 140
Lys Lys Lys Leu Ile Ser Arg Val Arg Ala Tyr His Gly Val Thr Leu
145 150 155 160
Ala Ala Ala Ser Leu Thr Gly Leu Pro Asn Asn His Arg Ser Phe Asp
165 170 175
Leu Pro Met Pro Gly Val Leu His Thr Gly Ser Pro His Tyr Trp Arg
180 185 190
Glu Gly Leu Pro Gly Glu Ser Glu Glu Ala Phe Ala Thr Arg Arg Ala
195 200 205
Glu Glu Leu Asp Ala Leu Ile Gln Ala Glu Gly Pro Asp Thr Ile Ala
210 215 220
Ala Phe Phe Ala Glu Pro Val Met Gly Ala Gly Gly Val Val Val Pro
225 230 235 240
Pro Ala Thr Tyr Trp Asp Lys Ile Gln Ala Val Leu Ala Lys Tyr Asp
245 250 255
Ile Leu Phe Val Ala Asp Glu Val Ile Asn Gly Phe Gly Arg Thr Gly
260 265 270
Thr Met Phe Ala Cys Glu Thr Tyr Gly Ile Arg Pro Asp Ile Leu Ile
275 280 285
Val Ser Lys Gln Leu Ser Ser Ser Tyr Met Pro Ile Ala Ala Leu Ile
290 295 300
Ala Asn Asp Arg Val Leu Gly Pro Val Met Asp Glu Ser Ala Arg Ile
305 310 315 320
Gly Thr Leu Gly His Gly Tyr Thr Ala Gly Gly His Pro Val Ala Thr
325 330 335
Ala Val Ser Leu Glu Asn Leu Arg Ile Ile Glu Glu Asp Gly Leu Val
340 345 350
Ala Asn Cys Ala Ile Gln Gly Ala Arg Leu Arg Ala Gly Leu Ala Ala
355 360 365
Leu Ser His His Pro Leu Val Gly Glu Val Arg Gly Val Gly Met Ile
370 375 380
Ala Ala Ile Glu Leu Val Thr Asp Lys Ala Gly Lys Val Ala Leu Asp
385 390 395 400
Val Pro Gly Lys Leu Gly Gly Met Val Asn Lys Ala Leu Gln Asp Asn
405 410 415
Gly Val Ile Cys Arg Ala Val Val Asp Ala Ile Cys Phe Cys Pro Pro
420 425 430
Met Ile Ile Thr Ala Asp Gln Ile Asp Asp Leu Leu Ala Ala Val Ser
435 440 445
Ala Ser Leu Asp Arg Val Ala Ala Asp Leu Gly Leu
450 455 460
<210> 5
<211> 456
<212> PRT
<213> Ochrobactrum intermedium
<400> 5
Met Thr Ala Gln Pro Asn Ser Leu Glu Ala Arg Asp Ile Arg Tyr His
1 5 10 15
Leu His Ser Tyr Thr Asp Ala Val Arg Leu Glu Ala Glu Gly Pro Leu
20 25 30
Val Ile Glu Arg Gly Asp Gly Ile Tyr Val Glu Asp Val Thr Gly Lys
35 40 45
Arg Tyr Ile Glu Ala Met Ser Gly Leu Trp Ser Val Gly Val Gly Phe
50 55 60
Ser Glu Pro Arg Leu Ala Glu Ala Ala Ala Arg Gln Met Lys Lys Leu
65 70 75 80
Pro Phe Tyr His Thr Phe Ser Tyr Arg Ser His Gly Pro Val Ile Asp
85 90 95
Leu Ala Glu Lys Leu Val Ser Met Ala Pro Val Pro Met Ser Lys Ala
100 105 110
Tyr Phe Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Val Ile Lys Leu
115 120 125
Ile Trp Tyr Arg Ser Asn Ala Leu Gly Glu Pro Glu Arg Lys Lys Ile
130 135 140
Ile Ser Arg Lys Arg Gly Tyr His Gly Val Thr Ile Ala Ser Ala Ser
145 150 155 160
Leu Thr Gly Leu Pro Asn Asn His Arg Ser Phe Asp Leu Pro Ile Asp
165 170 175
Arg Ile Leu His Ala Gly Cys Pro His His Tyr Arg Glu Gly Gln Ala
180 185 190
Gly Glu Thr Glu Glu Gln Phe Ala Thr Arg Leu Ala Asp Glu Leu Glu
195 200 205
Gln Leu Ile Leu Ala Glu Gly Pro His Thr Ile Ala Ala Phe Ile Gly
210 215 220
Glu Pro Val Met Gly Ala Gly Gly Val Val Val Pro Pro Arg Thr Tyr
225 230 235 240
Trp Glu Lys Val Gln Ala Val Leu Lys Arg Tyr Asp Ile Leu Leu Val
245 250 255
Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Leu Phe Gly
260 265 270
Ser Gln Thr Phe Asp Ile Lys Pro Asp Ile Leu Val Met Ser Lys Gln
275 280 285
Leu Ser Ser Ser Tyr Leu Pro Ile Ser Ala Phe Leu Ile Asn Glu Arg
290 295 300
Val Tyr Ala Pro Ile Ala Glu Glu Ser His Lys Ile Gly Thr Leu Gly
305 310 315 320
Thr Gly Phe Thr Ala Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Ala Ile Ile Glu Glu Arg Gln Leu Val Ala Asn Ala Arg
340 345 350
Asp Arg Gly Ala Tyr Met Gln Lys Arg Leu Arg Asp Leu Lys Asp His
355 360 365
Pro Leu Val Gly Glu Val Arg Gly Val Gly Leu Ile Ala Gly Val Glu
370 375 380
Leu Val Thr Asp Lys Gln Ala Lys Thr Gly Leu Glu Gln Ala Gly Ala
385 390 395 400
Leu Gly Ala Gln Ala Asn Ala Ala Leu Gln Asp Arg Gly Val Ile Ser
405 410 415
Arg Ala Met Gly Asp Thr Leu Ala Phe Cys Pro Pro Leu Ile Ile Asn
420 425 430
Asp Gln Gln Ile Asp Thr Met Val Ser Ala Leu Glu Ala Ala Leu Asn
435 440 445
Asp Val Gln Ala Ser Leu Ala Arg
450 455
<210> 6
<211> 456
<212> PRT
<213> Pseudaminobacter salicylatoxidans
<400> 6
Met Asp Thr Gln Pro Asn Ser Pro Glu Ala Arg Asp Ile Arg Tyr Asn
1 5 10 15
Leu His Ala Tyr Thr Asn Ala Arg Arg His Gln Glu Ala Gly Pro Leu
20 25 30
Ile Ile Glu Lys Gly Glu Gly Ile Tyr Val Glu Asp Asn Ser Gly Lys
35 40 45
Arg Tyr Ile Glu Ala Met Ala Gly Leu Trp Ser Val Ala Val Gly Phe
50 55 60
Ser Glu Gln Arg Leu Val Asp Ala Ala Thr Lys Gln Met Ser Lys Leu
65 70 75 80
Pro Tyr Tyr His Ser Phe Thr His Lys Ala His Ser Pro Leu Ile Asp
85 90 95
Leu Ala Glu Lys Leu Val Gln Met Ala Pro Val Pro Met Ser Lys Ala
100 105 110
Phe Phe Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Ala Met Lys Met
115 120 125
Ile Trp Tyr Arg Ser Asn Ala Leu Gly Gln Pro Gln Arg Lys Lys Ile
130 135 140
Ile Ser Arg Gln Arg Gly Tyr His Gly Val Thr Ile Ala Ser Ala Ser
145 150 155 160
Leu Thr Gly Leu Pro Asn Asn Gln Ile Ser Phe Asp Leu Pro Ile Ala
165 170 175
Asn Val Leu His Thr Ala Ser Pro His Tyr Trp Arg Glu Ala Arg Pro
180 185 190
Gly Glu Thr Glu Glu Gln Phe Ser Thr Arg Leu Ala Glu Glu Leu Glu
195 200 205
Lys Leu Ile Leu Ala Glu Gly Pro Glu Thr Val Ala Ala Phe Ile Gly
210 215 220
Glu Pro Val Met Gly Ala Gly Gly Val Val Val Pro Pro Ala Gly Tyr
225 230 235 240
Trp Glu Lys Ile Gln Ala Val Leu Lys Lys Tyr Asp Ile Leu Leu Ile
245 250 255
Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Met Phe Gly
260 265 270
Ser Gln Thr Tyr Gly Leu Gln Pro Asp Ile Met Val Met Ser Lys Gln
275 280 285
Leu Ser Ser Ser Tyr Leu Pro Ile Ser Ala Leu Leu Ile Asn Asp Lys
290 295 300
Val Phe Glu Pro Leu Ala Asp Glu Ser Asn Arg Ile Gly Thr Phe Gly
305 310 315 320
His Gly Phe Thr Ala Gly Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Arg Leu Ile Glu Glu Arg Asp Leu Val Gly Asn Ala Arg
340 345 350
Glu Val Gly Ala Tyr Met Gln Gln Arg Leu Arg Glu Leu Ser Ser His
355 360 365
Glu Leu Val Gly Glu Val Arg Gly Ile Gly Leu Ile Ala Ala Ile Glu
370 375 380
Leu Val Ala Asp Lys Ala Thr Lys Ala Pro Trp Gly Gln Pro Gly Ala
385 390 395 400
Leu Gly Ala Leu Thr Asn Gly Leu Leu Gln Gln Asn Gly Val Ile Ser
405 410 415
Arg Asn Met Gly Asp Ala Leu Ala Phe Cys Pro Pro Leu Ile Ile Thr
420 425 430
Arg Ala Gln Val Asp Glu Ile Ile Ser Ala Leu Lys Thr Ser Leu Asp
435 440 445
Glu Ala Leu Lys Gln Val Arg Ala
450 455
<210> 7
<211> 456
<212> PRT
<213> Brucella abortus
<400> 7
Met Thr Ala Gln Pro Asn Ser Leu Glu Ala Arg Asp Ile Arg Tyr His
1 5 10 15
Leu His Ser Tyr Thr Asp Ala Val Arg Leu Glu Ala Glu Gly Pro Leu
20 25 30
Val Ile Glu Arg Gly Asp Gly Ile Tyr Val Glu Asp Ile Ala Gly Lys
35 40 45
Arg Tyr Ile Glu Ala Met Ser Gly Leu Trp Ser Val Gly Val Gly Phe
50 55 60
Ser Glu Gln Arg Leu Ala Glu Ala Ala Ala Arg Gln Met Lys Lys Leu
65 70 75 80
Pro Phe Tyr His Thr Phe Ser Tyr Arg Ser His Gly Pro Val Ile Asp
85 90 95
Leu Ala Glu Lys Leu Val Ala Met Ala Pro Val Pro Met Ser Lys Ala
100 105 110
Tyr Phe Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Val Val Lys Leu
115 120 125
Ile Trp Tyr Arg Ser Asn Ala Leu Gly Glu Pro Glu Arg Lys Lys Ile
130 135 140
Ile Ser Arg Lys Arg Gly Tyr His Gly Val Thr Ile Ala Ser Ala Ser
145 150 155 160
Leu Thr Gly Leu Pro Asn Asn His Arg Ser Phe Asp Leu Pro Ile Asp
165 170 175
Arg Ile Leu His Thr Gly Cys Pro His His Tyr His Asp Ala Leu Pro
180 185 190
Gly Glu Ser Glu Glu Gln Phe Ala Thr Arg Leu Ala Asn Glu Leu Glu
195 200 205
Gln Leu Ile Leu Ala Glu Gly Pro His Thr Ile Ala Ala Phe Ile Gly
210 215 220
Glu Pro Val Met Gly Ala Gly Gly Val Val Val Pro Pro Lys Thr Tyr
225 230 235 240
Trp Glu Lys Val Gln Ala Val Leu Gly Arg Tyr Asn Ile Leu Leu Val
245 250 255
Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Leu Phe Gly
260 265 270
Cys Gln Thr Phe Asp Ile Lys Pro Asp Ile Leu Val Met Ser Lys Gln
275 280 285
Leu Ser Ser Ser Tyr Leu Pro Ile Ser Ala Phe Leu Ile Asn Glu Arg
290 295 300
Val Tyr Ala Pro Ile Ala Glu Glu Ser His Lys Ile Gly Thr Leu Gly
305 310 315 320
Thr Gly Phe Thr Ala Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Ala Ile Ile Glu Glu Arg Asp Leu Val Ala Asn Ala Arg
340 345 350
Glu Arg Gly Ala Arg Met Gln Lys Arg Leu Arg Glu Leu Gln Asp His
355 360 365
Pro Leu Val Gly Glu Val Arg Gly Val Gly Leu Ile Ala Gly Val Glu
370 375 380
Leu Val Ile Asp Lys Glu Ala Lys Thr Gly Leu Glu Gln Pro Gly Ala
385 390 395 400
Leu Gly Ala Arg Ala Asn Ala Ala Leu Gln Glu Arg Gly Val Ile Ser
405 410 415
Arg Ala Met Gly Asp Thr Leu Ala Phe Cys Pro Pro Leu Ile Ile Asn
420 425 430
Asp Gln Gln Val Asp Thr Met Val Ser Ala Leu Glu Ala Ser Leu Asn
435 440 445
Asp Val Gln Ala Ser Leu Gly Lys
450 455
<210> 8
<211> 453
<212> PRT
<213> Paracoccus denitrificans PD1222
<400> 8
Met Asn Gln Pro Gln Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu
1 5 10 15
Tyr Gly Phe Thr Asp Met Pro Ser Val His Gln Arg Gly Thr Val Val
20 25 30
Val Thr His Gly Glu Gly Pro Tyr Ile Val Asp Val His Gly Arg Arg
35 40 45
Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp
50 55 60
His Lys Gly Leu Ile Glu Ala Ala Lys Ala Gln Tyr Asp Arg Phe Pro
65 70 75 80
Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gln Thr Val Met Leu
85 90 95
Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Asn Gly Arg Val Phe
100 105 110
Tyr Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Met Val Lys Met Leu
115 120 125
Trp Phe Leu His Ala Ala Glu Gly Lys Pro Gln Lys Arg Lys Ile Leu
130 135 140
Thr Arg Trp Asn Ala Tyr His Gly Val Thr Ala Val Ser Ala Ser Met
145 150 155 160
Thr Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe
165 170 175
Ile His Leu Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu Glu Gly Glu
180 185 190
Thr Glu Ala Gln Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Asp Thr
195 200 205
Ile Thr Arg Glu Gly Ala Asp Thr Ile Ala Gly Phe Phe Ala Glu Pro
210 215 220
Val Met Gly Ala Gly Gly Val Ile Pro Pro Ala Lys Gly Tyr Phe Gln
225 230 235 240
Ala Ile Leu Pro Ile Leu Arg Lys Tyr Asp Ile Pro Met Ile Ser Asp
245 250 255
Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Leu
260 265 270
Thr Tyr Asp Phe Met Pro Asp Ala Ile Ile Ser Ser Lys Asn Leu Thr
275 280 285
Ala Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Asp Leu Ala
290 295 300
Lys Arg Val Glu Ala Ala Val Glu Ala Ile Glu Glu Phe Pro His Gly
305 310 315 320
Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala Ile Ala Leu Lys Ala
325 330 335
Ile Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg Leu
340 345 350
Ala Pro Arg Phe Glu Ala Gly Leu Lys Arg Ile Ala Asp Arg Pro Asn
355 360 365
Ile Gly Glu Tyr Arg Gly Ile Gly Phe Met Trp Ala Leu Glu Ala Val
370 375 380
Lys Asp Lys Pro Thr Lys Thr Pro Phe Asp Ala Asn Leu Ser Val Ser
385 390 395 400
Glu Arg Ile Ala Asn Thr Cys Thr Asp Leu Gly Leu Ile Cys Arg Pro
405 410 415
Leu Gly Gln Ser Ile Val Leu Cys Pro Pro Phe Ile Leu Thr Glu Ala
420 425 430
Gln Met Asp Glu Met Phe Glu Lys Leu Glu Lys Ala Leu Asp Lys Val
435 440 445
Phe Ala Glu Val Ala
450

Claims (10)

1.一种制备R-3-氨基正丁醇的方法，包括如下步骤：
以3-氨基正丁醇外消旋体和丙酮酸为底物，用ω-转氨酶为催化剂，立体特异性地催化S-3-氨基正丁醇与丙酮酸反应，然后从反应体系中回收未参与反应的对映体R-3-氨基正丁醇。
2.如权利要求1所述的方法，其特征在于，所述ω-转氨酶来源于选自下组的微生物：Ochrobactrum anthropic、Bradyrhizobium sp.Ec3.3、Brucella neotomae、Novosphingobium acidiphilum、Ochrobactrum intermedium、Pseudaminobactersalicylatoxidans、Brucella abortus、Paracoccus denitrificans PD1222。
3.如权利要求1所述的方法，其特征在于，所述ω-转氨酶选自下组：SEQ ID NO:1、SEQID NO:3、SEQ ID NO:4、SEQ ID NO:7。
4.如权利要求1所述的方法，其特征在于，反应体系中还添加有磷酸吡哆醛。
5.如权利要求4所述的方法，其特征在于，磷酸吡哆醛在反应体系中的添加量为0.1-1mmol/L。
6.如权利要求1所述的方法，其特征在于，所述ω-转氨酶呈酶形式或者表达微生物形式。
7.如权利要求6所述的方法，其特征在于，所述表达微生物是表达ω-转氨酶的重组大肠杆菌。
8.如权利要求1所述的方法，其特征在于，所述反应体系所用的反应溶液为水溶液、磷酸盐缓冲液、Tirs缓冲液或者碳酸氢盐缓冲液。
9.如权利要求1所述的方法，其特征在于，反应体系为pH6.5-pH9.0。
10.如权利要求1所述的方法，其特征在于，反应温度为25-45℃。
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* Cited by examiner, † Cited by third party
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