CN115537405A - 一种酮还原酶及其在制备(s)-1-(3-氯苯基)-1,3-丙二醇中的应用 - Google Patents
一种酮还原酶及其在制备(s)-1-(3-氯苯基)-1,3-丙二醇中的应用 Download PDFInfo
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- CN115537405A CN115537405A CN202110741180.9A CN202110741180A CN115537405A CN 115537405 A CN115537405 A CN 115537405A CN 202110741180 A CN202110741180 A CN 202110741180A CN 115537405 A CN115537405 A CN 115537405A
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- ketoreductase
- ala
- val
- gly
- chlorphenyl
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Abstract
本发明公开了一种酮还原酶突变体及其在制备(S)‑1‑(3‑氯苯基)‑1,3‑丙二醇中的应用。本发明以3‑氯苯甲酸为底物，先制备3‑(3‑氯苯基)‑3‑羰基丙酸甲酯，然后在酮还原酶的作用下转化得到(S)‑3‑(3‑氯苯基)‑3‑羟基丙酸甲酯，进而还原得到(S)‑1‑(3‑氯苯基)‑1,3‑丙二醇。与现有技术相比，该方案反应条件温和，收率较高，手性选择性好，具有良好的工业应用价值。
Description
技术领域：
本发明属于生物催化技术领域，具体涉及一种酮还原酶突变体及其在制备(S)-1-(3-氯苯基)-1,3-丙二醇中的应用。
背景技术：
(S)-1-(3-氯苯基)-1,3-丙二醇是一种用于制备多种环磷酸酯类药物分子的重要中间体，CAS号：625095-57-0，结构如式I所示。
专利CN103333209A中公开(S)-1-(3-氯苯基)-1,3-丙二醇可以用于制备富马酸替诺福韦酯的前药化合物；专利CN110590839A中公开(S)-1-(3-氯苯基)-1,3-丙二醇可以用于制备乐伐替尼衍生物；专利CN1997377A中公开(S)-1-(3-氯苯基)-1,3-丙二醇可以用于制备治疗肝炎C病毒感染有效的化合物；文献中国海洋大学学报，2013，43(2)，055～059中报道(S)-1-(3-氯苯基)-1,3-丙二醇可以用于制备熊果酸环磷酸酯衍生物。未来，(S)-1-(3-氯苯基)-1,3-丙二醇可以用于制备更多的新药分子。
目前，制备(S)-1-(3-氯苯基)-1,3-丙二醇的方法多为化学法合成。专利WO2015154716中报道一种制备(S)-1-(3-氯苯基)-1,3-丙二醇的方法，如Scheme 1所示。该路线以3-氯苯乙酮与碳酸二甲酯为起始原料，需要经过有腐蚀性和刺激性的金属催化剂三氯化钌催化才能获得手性中心。
文献Journal of Medicinal Chemistry(2008),51(3),666-676和中国海洋大学学报，2013，43(2)，055～059中均报道一种制备(S)-1-(3-氯苯基)-1,3-丙二醇的方法，如Scheme 2所示。该路线选用3-氯苯乙酮制得1-(3-氯苯基)-1,3-丙二醇，然后在三氟甲磺酸三甲基硅酯(TMSOTf)催化下与六甲基二硅胺烷(HMDS)发生硅醚化反应，再与薄荷酮发生缩酮化反应得到2种非对映异构体，需要硅胶柱层析分离后再经盐酸催化水解得到(S)-1-(3-氯苯基)-1,3-丙二醇，操作繁琐，收率低，不适合工业化生产。
文献European Journal of Organic Chemistry,2014(24),5247-5255中报道一种酶法(S)-1-(3-氯苯基)-1,3-丙二醇的方法，如Scheme 3所示。该路线选用3-氯苯乙酮为原料，在脂肪酶的催化条件下，一锅法制得(S)-1-(3-氯苯基)-1,3-丙二醇，反应时间96小时，收率为74％，ee值仅为87％。
因此，需要开发一种高效的酶以及操作简单、收率高、选择性好、成本低、环境友好的制备(S)-1-(3-氯苯基)-1,3-丙二醇的方法。
发明内容：
本发明的目的在于针对现有技术的不足，提供一种酮还原酶突变体及其在制备(S)-1-(3-氯苯基)-1,3-丙二醇中的应用。
一方面，本发明提供一种酮还原酶突变体。
本发明利用基因工程手段，对野生型酮还原酶氨基酸进行了半理性设计，通过饱和突变，最后获得突变体Mut1(P41G/S58V/G141A/I 221V/G254H)。
进一步，所述野生型酮还原酶选自Novosphingobium aromaticivorans。
更进一步，所述野生型酮还原酶在NCBI的登录号为WP_011906790.1。
另一方面，本发明提供的酮还原酶突变体可以用于制备(S)-1-(3-氯苯基)-1,3-丙二醇。
本发明采用的技术方案如下：
本发明提供一种制备(S)-1-(3-氯苯基)-1,3-丙二醇的方法，具体包括如下步骤：以3-氯苯甲酸为底物，先制得3-(3-氯苯基)-3-羰基丙酸甲酯，然后在酮还原酶(KRED酶)的作用下转化得到化合物4，进而还原制得(S)-1-(3-氯苯基)-1,3-丙二醇。
进一步，以3-氯苯甲酸和丙二酸单甲酯钾盐为原料制得化合物3。
进一步，以化合物3为底物，在酮还原酶的作用下转化得到化合物4。
更进一步，所述酮还原酶以酮还原酶酶粉、酮还原酶酶液、含酮还原酶的细胞等形式参与催化反应。
更进一步，该步反应在反应温度为10～60℃条件下进行，优选40℃。
更进一步，该步反应的反应体系中需要加入辅酶，所述辅酶选自NADP。
进一步，化合物4在硼氢化钠作用下还原为(S)-1-(3-氯苯基)-1,3-丙二醇。
本发明的有益效果在于，本发明提供了一种新的酮还原酶突变体，该突变体可以用于制备(S)-1-(3-氯苯基)-1,3-丙二醇的工艺过程中，该酶催化底物的浓度能够达到200g/L，ee值为99.6％。与现有方法相比，该方案反应条件温和，收率较高，选择性好，具有良好的工业应用价值。
附图说明
图1野生型酮还原酶的基因鉴定图
图2野生型酮还原酶的蛋白电泳图
图3全质粒扩增电泳图
具体实施方式
下面结合具体实施例对本发明的技术内容作进一步的阐述，其目的是为了更好的理解本发明的内容，但本发明的保护范围不限于此。
实施例1酮还原酶的构建和表达
利用人工合成基因技术合成来源于Novosphingobium aromaticivorans的酮还原酶，基因的N端和C端的密码子分别是ATG和TGA，两端不需要融合标签。通过内切酶NdeI和NcoI将目标基因连接到pET-24a上，转化到BL21(DE3)感受态细胞中，孵育1h后涂布于在含有Kan的平板上，37℃培养过夜，第二天获得重组野生型菌株。利用通用引物T7/T7-ter为结合载体上的上下游片段，扩增出目的基因，鉴定基因的大小，结果见附图1，明显获得有目的基因大小的条带。
转接平板上的单克隆到含有Kan+抗性的LB试管中，37℃下培养过夜。将种子液按照1％的接种量转接到2YT培养基中，继续在37℃下培养细菌，待生物量OD600值达到0.4～0.8之间，进行0.5mM IPTG诱导，温度降低到30℃，表达16h后收集菌体，并破碎细胞进行电泳分析，结果见附图2，蛋白的大小和预期的一致。
实施例2高通量突变建库和筛选
根据蛋白质和底物对接的模拟结构，选出热点氨基酸P41、T57、P95、G141、I221、G254等进行饱和突变，突变的引物设计如表1所示，所有待突变的氨基酸都利用NNK/NNM替换。
表1定点突变引物的核酸序列
利用PCR的常规技术，在高保真聚合酶PFU polymerase作用下，利用温度梯度控制体系下，实现全长质粒的扩增。对扩增的结果进行电泳检测(见附图3)，每对引物都能完好的扩增出全质粒片段。
PCR扩增产物在FD DpnI限制性内切酶的消化下，除去野生型的模板DNA序列，转入到BL21(DE3)感受态细胞中，涂布于含有100ug/mL的Kan+抗性LB平板上，37℃培养过夜，挑取单克隆用于后期的筛选鉴定。
实施例3化合物3的制备
氮气保护下将150g丙二酸单甲酯钾盐加入到300mL四氢呋喃中，室温搅拌15min。冰浴条件下，将100g 3-氯苯甲酸缓慢滴加到反应液中，室温搅拌1.5h。向反应液中加入300mL水及13mL稀盐酸溶液，搅拌15min，萃取，无水硫酸钠干燥，过滤蒸干得到130.5g化合物3。
实施例4化合物4的制备(野生型酮还原酶细胞的转化反应)
向500mL反应釜中加入160mL 100mM的磷酸盐缓冲液和40mL异丙醇，称量0.02gNADP和8g化合物3，一起投入反应釜中，边搅拌边添加，利用10％的Na2CO3溶液控制pH 7.0，最后加入10g野生型细胞(氨基酸序列如SEQ ID No.2)，在40℃下反应24h；反应结束后向反应液中加入一倍的乙腈溶液，混匀后在12000rpm下离心1min，去上清分析转化率和手性值；HPLC显示出转化率在99.5％，S手性ee值在85.2％。
实施例5化合物4的制备(突变体酮还原酶细胞的转化反应)
向500mL反应釜中加入160mL 100mM的磷酸盐缓冲液和40mL异丙醇，称量0.02gNADP固体和40g化合物3，一起投入反应釜中，边搅拌边添加，利用10％的Na2CO3溶液控制pH7.0，最后加入10g酮还原酶细胞(氨基酸序列如SEQ ID No.4)，在40℃下转化24h；反应结束后向反应液中加入一倍的乙腈溶液，混匀后在12000rpm下离心1min，去上清分析转化率和手性值；HPLC显示出转化率在99.5％，S手性ee值在99.6％。
实施例6化合物4的制备(突变体酮还原酶酶粉的转化反应)
向500mL反应釜中加入160mL 100mM的磷酸盐缓冲液和40mL异丙醇，称量0.02gNADP固体和40g化合物3，一起投入反应釜中，边搅拌边添加，利用10％的Na2CO3溶液控制pH7.0，最后加入10g酮还原酶酶粉(氨基酸序列如SEQ ID No.4)，在50℃下转化24h；反应结束后向反应液中加入一倍的乙腈溶液，混匀后在12000rpm下离心1min，去上清分析转化率和手性值；HPLC显示出转化率在99.4％，S手性ee值在99.5％。
实施例7化合物1的制备
氮气保护下将1.64g硼氢化钠及1mL水加入到37.5mL正丁醇中，冰浴下滴加10.4g化合物4的正丁醇溶液，滴毕搅拌0.5h，90℃反应4h。冷却至室温，加入20mL 10％碳酸钾溶液，搅拌10min。反应结束后萃取，无水硫酸钠干燥，过滤，蒸干得到7.72g化合物1。
序列表
<110> 尚科生物医药（上海）有限公司
<120> 一种酮还原酶及其在制备(S)-1-(3-氯苯基)-1,3-丙二醇中的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 780
<212> DNA/RNA
<213> Novosphingobium aromaticivorans
<400> 1
atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60
cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcg 120
ccctcggccg atgtcgaagg cgcggaccat tatctccagc acgacgtgac gagcgaggcc 180
ggctggaagg cggtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240
cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300
cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360
ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420
ggcggtctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480
atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540
tccgtgcatc cgggcggcat cgataccccg atgctcggct cgatcatgga caagtacgtc 600
gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
atcggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcgagct tcgtcacctg cacggaattc gtgatggacg gcggcttcag ccaggtctga 780
<210> 2
<211> 259
<212> PRT
<213> Novosphingobium aromaticivorans
<400> 2
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
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Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
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Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp Val Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
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Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
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Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Gly Gly Leu Arg
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Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
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Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Ile Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
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Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Ile Gly Arg Met
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Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
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Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp Gly Gly Phe
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Ser Gln Val
<210> 3
<211> 780
<212> DNA/RNA
<213> Novosphingobium aromaticivorans
<400> 3
atgacgattg ctctcaacaa tgtggtcgcc gtcgtcaccg gcgcggcggg aggcatcggc 60
cgcgaactgg tcaaggcgat gaaggccgcc aacgccatcg tcatcgccac cgacatggcg 120
ggctcggccg atgtcgaagg cgcggaccat tatctccagc acgacgtgac ggtcgaggcc 180
ggctggaagg cggtcgcggc gctggcccag gaaaagtacg ggcgcgtcga tgcgctggtg 240
cacaacgcgg gcatctcgat cgtcacgaag ttcgaagaca ctccgctgtc cgatttccac 300
cgcgtgaaca cggtcaacgt cgattccatc atcatcggta cgcaggtcct gctgccgctg 360
ctcaaggaag gcggcaaggc gcgcgcaggg ggcgcctcgg tggtcaactt ctccagcgtc 420
gcgggtctgc gcggcgcggc gttcaatgcg gcctattgca ccagcaaggc ggcggtgaag 480
atgctctcga agtgcctcgg cgcggaattc gcggcgctcg gctacaacat ccgcgtcaac 540
tccgtgcatc cgggcggcat cgataccccg atgctcggct cgatcatgga caagtacgtc 600
gaactcggcg ctgccccctc gcgcgaggtg gcccaggccg cgatggaaat gcgccacccg 660
gtgggtcgca tgggtcgccc tgccgaaatg ggcggcggcg tggtctatct ctgctccgac 720
gcagcgagct tcgtcacctg cacggaattc gtgatggacc acggcttcag ccaggtctga 780
<210> 4
<211> 259
<212> PRT
<213> Novosphingobium aromaticivorans
<400> 4
Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val Thr Gly Ala Ala
1 5 10 15
Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys Ala Ala Asn Ala
20 25 30
Ile Val Ile Ala Thr Asp Met Ala Gly Ser Ala Asp Val Glu Gly Ala
35 40 45
Asp His Tyr Leu Gln His Asp Val Thr Val Glu Ala Gly Trp Lys Ala
50 55 60
Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val Asp Ala Leu Val
65 70 75 80
His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu Asp Thr Pro Leu
85 90 95
Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp Ser Ile Ile Ile
100 105 110
Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly Gly Lys Ala Arg
115 120 125
Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val Ala Gly Leu Arg
130 135 140
Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys Ala Ala Val Lys
145 150 155 160
Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala Leu Gly Tyr Asn
165 170 175
Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp Thr Pro Met Leu
180 185 190
Gly Ser Ile Met Asp Lys Tyr Val Glu Leu Gly Ala Ala Pro Ser Arg
195 200 205
Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro Val Gly Arg Met
210 215 220
Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr Leu Cys Ser Asp
225 230 235 240
Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met Asp His Gly Phe
245 250 255
Ser Gln Val
Claims (7)
1.一种酮还原酶突变体，其特征在于，以SEQ ID No.2所示的野生型酮还原酶的氨基酸序列为参考序列，所述酮还原酶突变体的氨基酸序列的突变位点为第41位的脯氨酸突变为甘氨酸，第58位的丝氨酸突变为缬氨酸，第141位的甘氨酸突变为丙氨酸，第221位的异亮氨酸突变为缬氨酸，第254位的甘氨酸突变为组氨酸。
2.如权利要求1所述的酮还原酶突变体，其特征在于，所述酮还原酶突变体氨基酸序列如SEQ ID No.4所示。
3.如权利要求1所述的酮还原酶突变体，其特征在于，所述酮还原酶突变体基因核苷酸序列如SEQ ID No.3所示。
4.如权利要求1所述的酮还原酶突变体，其特征在于，所述酮还原酶表达于基因工程菌。
5.如权利要求4所述的酮还原酶突变体，其特征在于所述的基因工程菌选自大肠杆菌或酵母菌。
6.如权利要求1所述的酮还原酶突变体，其特征在于，所述酮还原酶可以用于制备(S)-1-(3-氯苯基)-1,3-丙二醇。
7.如权利要求6所述的酮还原酶突变体，其特征在于，所述酮还原酶可以先将3-(3-氯苯基)-3-羰基丙酸甲酯转化为(S)-3-(3-氯苯基)-3-羟基丙酸甲酯，然后还原为(S)-1-(3-氯苯基)-1,3-丙二醇。
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