CN111454998B - 一种手性羟基酸酯的生物制备方法 - Google Patents
一种手性羟基酸酯的生物制备方法 Download PDFInfo
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- CN111454998B CN111454998B CN202010316359.5A CN202010316359A CN111454998B CN 111454998 B CN111454998 B CN 111454998B CN 202010316359 A CN202010316359 A CN 202010316359A CN 111454998 B CN111454998 B CN 111454998B
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- chain dehydrogenase
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- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 13
- 229930195729 fatty acid Natural products 0.000 claims abstract description 13
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- 108010013347 alkene monooxygenase Proteins 0.000 claims abstract description 4
- 230000001590 oxidative effect Effects 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 241000588724 Escherichia coli Species 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
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- 241000894006 Bacteria Species 0.000 claims description 9
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Abstract
本发明公开了一种手性羟基酸酯的生物制备方法，包括以下步骤：（1）将脂肪酸经酮化酶球形烯单氧酶氧化后生成4‑氧代脂肪酸；（2）4‑氧代脂肪酸经短链脱氢酶PpYSDR‑M85Q/L136A不对称还原得到R‑羟基酸酯。本发明手性羟基酸酯的生物制备方法通过构建短链脱氢酶突变体，其较野生型对酮酸的活性、手性选择性大幅提高能提高该酶在催化酮酸制备手性内酯上的效率，以充分发掘其应用价值，同时酶法合成手性羟基酸酯具有工艺简单、环境友好、效益高等特点。
Description
技术领域
本发明属于生物化工技术领域，尤其涉及一种手性羟基酸酯的生物制备方法。
背景技术
手性化合物的两种对映异构体往往具有不同的功效或其作用效果相差甚远，因此，单一对映体的合成越来越受关注。光学活性手性内脂被广泛应用于手性天然香料。传统化学合成具有难度大、污染大的特点。脂肪酸经酮化酶氧化可得到酮酸。酮酸的不对称还原是制备光学活性手性羟基酸的重要方法，理论上能将 100％底物酮酸转化为单一对映体的手性羟基酸，具有很高的工业应用价值。 与化学法不对称还原相比，生物法催化法在化学选择性、区域选择性和立体选择性方面更具优势，产物光学纯度高；此外，生物催化通常在温和条件下进行，避免了剧烈化学反应过程中化合物的异构化、差向异构化、消旋和重排等现象的发生。因此，生物催化法合成光学活性手性内脂已成为最受瞩目的医药及精细化学品的绿色合成技术之一。
在具有催化不对称还原潜手性内脂活性的酶中，短链脱氢酶由于其催化底物谱广、热稳定性好以及有机溶剂耐受性强等特点而倍受关注。
发明内容
本发明目的就是为了弥补已有技术的缺陷，提供一种手性羟基酸酯的生物制备方法，两步酶法实现R-羟基酸酯的生物制备。
为了实现上述的目的，本发明提供以下技术方案：
一种手性羟基酸酯的生物制备方法，包括以下步骤：
（1）将脂肪酸经酮化酶球形烯单氧酶氧化后生成4-氧代脂肪酸；
（2）4-氧代脂肪酸经短链脱氢酶PpYSDR-M85Q/L136A不对称还原得到R-羟基酸酯。
进一步的，所述短链脱氢酶PpYSDR-M85Q/L136A由野生型短链脱氢酶PpYSDR经突变改造制得。
进一步的，所述野生型短链脱氢酶PpYSDR经突变改造制得短链脱氢酶PpYSDR-M85Q/L136A的步骤如下：
（1）突变体的构建：以含有突变点的寡核苷酸片段为引物分别进行PCR扩增反应，扩增含有PpYSDR基因的pET-30a重组质粒得到单点突变体后，再以该突变体的重组质粒为模板，另一突变体为引物，构建两点组合突变体；
（2）短链脱氢酶突变体的诱导表达：将步骤1中构建的工程菌接种至50μg/mL卡那霉素的 LB培养基中，37℃，200rpm培养过夜，再以1%接种量（v/v）接种至含50μg/mL卡那霉素的LB培养基中，37℃，200rpm培养至菌体浓度OD600至0.6左右，加入终浓度为0.1mMIPTG，26℃诱导培养6h后，4℃、8000rpm离心10min收集菌体，-80℃储存备用。
进一步的，所述步骤1中构建突变体所使用的基因工程菌为大肠杆菌，优选为E.coli BL21(DE3)大肠杆菌。
进一步的，所述步骤1中构建突变体所使用的基因工程菌菌体的质量用量以菌体湿重计为50-300g/L。
本发明的优点是：
常规短链脱氢酶在催化酮酸（如4-氧代十酸甲酯、4-氧代十一酸甲酯）活性较低，本发明手性羟基酸酯的生物制备方法通过构建短链脱氢酶突变体，其较野生型对酮酸的活性、手性选择性大幅提高能提高该酶在催化酮酸制备手性内酯上的效率，以充分发掘其应用价值，同时酶法合成手性羟基酸酯具有工艺简单、环境友好、效益高等特点。
具体实施方式
以下结合具体的实例对本发明的技术方案做进一步说明：
实施例1
一种手性羟基酸酯的生物制备方法，包括以下步骤：
（1）将脂肪酸经酮化酶球形烯单氧酶氧化后生成4-氧代脂肪酸；
（2）4-氧代脂肪酸经短链脱氢酶PpYSDR-M85Q/L136A不对称还原得到R-羟基酸酯。
其中酮化酶球形烯单氧酶为EC 1.14.15.9-spheroidene monooxygenase，含亚铁血红素，参与环状菌素和2，2'-双氧-螺菌黄素的生物合成，来源于Rhodobactercapsulatus，Rhodobacter sphaeroides，Rhodovulum sulfidophilum，Rubrivivaxgelatinosus。
其中步骤2反应如下式所示：
所述短链脱氢酶PpYSDR-M85Q/L136A由野生型短链脱氢酶PpYSDR经突变改造制得，具体包括以下过程：
突变体的构建：
以含有突变点的寡核苷酸片段为引物，具体引物序列如下：
M85Q-F：GGCGTCCAAGGCCCCCTGCCGCAAGACCT（SEQ ID NO.1，下划线处为突变位点）
M85Q-R：AGGGGGCCTTGGACGCCCGCATTGACGAAT（SEQ ID NO.2，下划线处为突变位点）
L136A-F：TCGATCGCTGGCAGCGTAACCATCCCCGA（SEQ ID NO.3，下划线处为突变位点）
L136A-R：GCTGCCAGCGATCGAACTCATGAAGGCCAG（SEQ ID NO.4，下划线处为突变位点）
将上述引物分别进行PCR扩增反应。采用QuickChangeTM方法（Stratagene,LaJolla，CA）扩增含有PpYSDR基因的pET-30a重组质粒得到单点突变体后，再以该突变体的重组质粒为模板，另一突变体为引物，构建两点组合突变体。
其中，野生型短链脱氢酶PpYSDR的基因序列如SEQ ID NO.5所示。
野生型短链脱氢酶PpYSDR的氨基酸序列如SEQ ID NO.6所示。
突变型短链脱氢酶PpYSDR-M85Q/L136A的基因序列如SEQ ID NO.7所示。
突变型短链脱氢酶PpYSDR-M85Q/L136A的氨基酸序列如SEQ ID NO.8所示。
其中，PCR程序：（1）在98℃下预变性1min；（2）98℃，10s； 55℃，10s； 72℃，7min进行温度循环，循环20次后冷却至4℃。PCR产物经清洗后，利用特异性识别甲基化位点的限制性内切酶Dpn Ⅰ进行消化以降解甲基化模板质粒。酶切反应体系及条件：17μL经清洗处理的PCR产物，2.0μL 10×缓冲液，1.0μL限制性内切酶Dpn Ⅰ，37℃保温1h。将上述经酶切处理的PCR产物转化至E.coli BL21（DE3）中，得到相应的重组大肠杆菌，涂布于含卡那霉素的固体平板上，37℃下培养过夜，随机挑取单菌落进行菌落PCR鉴定和测序验证，结果表明含有羰基还原酶突变体基因的重组表达载体成功转化至表达宿主E.coli BL21（DE3）中。
短链脱氢酶突变体的诱导表达：
将上述构建的工程菌接种至50μg/mL卡那霉素的 LB培养基中，37℃，200rpm培养过夜，再以1%接种量（v/v）接种至含50μg/mL卡那霉素的LB培养基中，37℃，200rpm培养至菌体浓度OD600至0.6左右，加入终浓度为0.1mM IPTG，26℃诱导培养6h后，4℃、8000rpm离心10min收集菌体，-80℃储存备用。
短链脱氢酶突变体的发酵罐培养：
将上述构建的工程菌接种至50μg/mL卡那霉素的 LB培养基中，37℃，200rpm培养过夜，再以2%接种量（v/v）接种至含50μg/mL卡那霉素的培养基中，37℃，200rpm培养，在对数中期时以10%接种量（v/v）接种至15L含50μg/mL卡那霉素的发酵培养基的发酵罐中，37℃，培养14h左右（对数中后期），加入乳糖进行诱导20h后，利用管式离心机离心收集菌体备用。
短链脱氢酶突变体PpYSDR-M85Q/L136A催化酮酸制备手性羟基酸酯：
反应体系（10.0mL）：含有短链脱氢酶PpYSDR及其突变体编码基因的基因工程菌湿菌体细胞2g，10mM 4-氧代壬酸甲酯或4-氧代癸酸甲酯或4-氧代十一酸甲酯，0.75M葡萄糖，0.2mM NADP+，Na2HPO4-NaH2PO4缓冲液（100mM，pH7.4）加至10mL，于37℃，200rpm条件下反应，并每隔半小时用碳酸钠溶液调节PH至7.4，每隔2h取样，用乙酸乙酯萃取，取上清加适量无水硫酸钠，气相色谱检测反应效果，4h后反应基本完全，加酸调PH至2-3，使酯化以得到最终产物。当反应完成后，4-氧代壬酸甲酯转化为（R）-（+）-γ-壬酸内酯的转化率为99%，光学纯度91%；4-氧代癸酸甲酯转化为R-（+）-γ-癸内酯的转化率为99%，光学纯度93%；4-氧代十一酸甲酯转化为（R）-γ-十一烷丁酸内酯的转化率为99%，光学纯度97%。
短链脱氢酶突变体PpYSDR-M85Q/L136A催化高浓度酮酸制备手性羟基酸酯：
反应体系（10.0mL）：含有短链脱氢酶PpYSDR及其突变体编码基因的基因工程菌湿菌体细胞2g，500mM 4-氧代十酸甲酯或4-氧代十一酸甲酯，0.75M葡萄糖，0.2mM NADP+，Na2HPO4-NaH2PO4缓冲液（100 mM，pH 7.4）加至10mL，于37℃，200rpm条件下反应，并每隔半小时用碳酸钠溶液调节PH至7.4，每隔2h取样，用乙酸乙酯萃取，取上清加适量无水硫酸钠，气相色谱检测反应效果，10h后反应基本完全，加酸调PH至2-3，使酯化以得到最终产物。当反应完成后，4-氧代壬酸甲酯转化为（R）-（+）-γ-壬酸内酯的转化率为97%，光学纯度89%；4-氧代癸酸甲酯转化为R-（+）-γ-癸内酯的转化率为95%，光学纯度91%；4-氧代十一酸甲酯转化为（R）-γ-十一烷丁酸内酯的转化率为89%，光学纯度95%。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 黄山科宏生物香料股份有限公司
<120> 一种手性羟基酸酯的生物制备方法
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cccggtgccc tggcggacgt gcccggcgtg cgcatcgaac agctggaaat gaacgacacc 180
gcccaactcg atgggctgaa acaacgcctg caaggccagg tgttcgacct ggtattcgtc 240
aatgcgggcg tcatgggccc cctgccgcaa gacctggaga cggttcagaa caaggacatc 300
ggcgacctgt tcatgaccaa tgccgtgtcg cccatccgcg tggcccgccg cctggtcggc 360
caggtgcgcg agggcagcgg cgtgctggcc ttcatgagtt cgatcctggg cagcgtaacc 420
atccccgacg ggggcgaaat ttgcctgtac aaggccagca aggcagcgct caactcgatg 480
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ggctgggtga aaaccgacat gggcggcgaa aacgccgaaa tcgacgtgtt caccagcacc 600
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cccggtgccc tggcggacgt gcccggcgtg cgcatcgaac agctggaaat gaacgacacc 180
gcccaactcg atgggctgaa acaacgcctg caaggccagg tgttcgacct ggtattcgtc 240
aatgcgggcg tccaaggccc cctgccgcaa gacctggaga cggttcagaa caaggacatc 300
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Gly Glu Ile Cys Leu Tyr Lys Ala Ser Lys Ala Ala Leu Asn Ser Met
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Ile Asn Ser Phe Val Val Glu Gln Gln Arg Pro Asp Leu Cys Val Leu
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Glu Ile Asp Val Phe Thr Ser Thr Arg Gly Met Leu Asp Gln Ile Asn
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Claims (4)
1.一种手性羟基酸酯的生物制备方法，其特征在于，包括以下步骤：
（1）将脂肪酸经酮化酶球形烯单氧酶氧化后生成4-氧代脂肪酸；
（2）4-氧代脂肪酸经短链脱氢酶PpYSDR-M85Q/L136A不对称还原得到R-羟基酸酯；
所述短链脱氢酶PpYSDR-M85Q/L136A由野生型短链脱氢酶PpYSDR经突变改造制得；
野生型短链脱氢酶PpYSDR经突变改造制得短链脱氢酶PpYSDR-M85Q/L136A的步骤如下：
S1、突变体的构建：以含有突变点的寡核苷酸片段为引物分别进行PCR扩增反应，扩增含有PpYSDR基因的pET-30a重组质粒得到单点突变体后，再以该突变体的重组质粒为模板，另一突变体为引物，构建两点组合突变体；
S2、短链脱氢酶突变体的诱导表达：将步骤S1中构建的工程菌接种至50μg/mL卡那霉素的 LB培养基中，37℃，200rpm培养过夜，再以1%接种量（v/v）接种至含50μg/mL卡那霉素的LB培养基中，37℃，200rpm培养至菌体浓度OD600至0.6左右，加入终浓度为0.1mM IPTG，26℃诱导培养6h后，4℃、8000rpm离心10min收集菌体，-80℃储存备用；
所述的短链脱氢酶PpYSDR-M85Q/L136A的基因序列如SEQ ID NO.7所示；
所述的短链脱氢酶PpYSDR-M85Q/L136A的氨基酸序列如SEQ ID NO.8所示。
2.根据权利要求1所述的手性羟基酸酯的生物制备方法，其特征在于，所述步骤S1中构建突变体所使用的基因工程菌为大肠杆菌。
3.根据权利要求1所述的手性羟基酸酯的生物制备方法，其特征在于，所述步骤S1中构建突变体所使用的基因工程菌为E.coli BL21(DE3)大肠杆菌。
4.根据权利要求1所述的手性羟基酸酯的生物制备方法，其特征在于，所述步骤S1中构建突变体所使用的基因工程菌菌体的质量用量以菌体湿重计为50-300g/L。
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