CN115537406A - 一种酮还原酶及其在制备(s)-1-(4-吡啶基)-1,3-丙二醇中的应用 - Google Patents
一种酮还原酶及其在制备(s)-1-(4-吡啶基)-1,3-丙二醇中的应用 Download PDFInfo
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- CN115537406A CN115537406A CN202110743873.1A CN202110743873A CN115537406A CN 115537406 A CN115537406 A CN 115537406A CN 202110743873 A CN202110743873 A CN 202110743873A CN 115537406 A CN115537406 A CN 115537406A
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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‑(4‑吡啶基)‑1,3‑丙二醇中的应用。本发明以异烟酸为底物，先制得3‑氧代‑3‑(吡啶‑4‑基)丙酸甲酯，然后在酮还原酶的作用下转化得到(S)‑3‑羟基‑3‑(吡啶‑4‑基)丙酸甲酯，进而还原制得(S)‑1‑(4‑吡啶基)‑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-(4-吡啶基)-1,3-丙二醇中的应用。
背景技术：
(S)-1-(4-吡啶基)-1,3-丙二醇是一种制备多种膦酸二酯类药物分子的重要中间体，CAS号：685111-87-9，结构如式I所示。
专利CN100439387A中公开(S)-1-(4-吡啶基)-1,3-丙二醇可以用于制备阿糖胞苷单磷酸盐药物前体；专利CN1882327A中公开(S)-1-(4-吡啶基)-1,3-丙二醇可以用于制备含磷的新的拟甲状腺素药，在预防和/或治疗代谢病如肥胖症、NASH、高胆固醇血症和高脂血中有疗效；专利CN1997377A中公开(S)-1-(4-吡啶基)-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；专利CN107540710A中公开(S)-1-(4-吡啶基)-1,3-丙二醇可以用于制备一种新药分子，该药物可以治疗抗乙型肝炎病毒(HBV)、丁型肝炎病毒(HDV)和人类免疫缺陷病毒(HIV)及其引起的疾病中的应用。专利CN104558080B中公开(S)-1-(4-吡啶基)-1,3-丙二醇可以用于制备一种新化合物，该化合物为FXR和/或TGR5受体激活物，其具有激活FXR和/或TGR5受体活性，可用于制备治疗慢性肝病、代谢性疾病或门脉高压症的药物。未来，(S)-1-(4-吡啶基)-1,3-丙二醇可以用于制备更多的新药分子。
专利CN100439387 C，CN1997377 A，CN101252839 A中报道一种制备(S)-1-(4-吡啶基)-1,3-丙二醇的方法，如Scheme 1所示。该路线以4-吡啶甲醛为起始原料，需要经过手性钌催化剂催化才能获得手性中心ee值可达98％，但总收率不足50％。
专利CN109929005B中公开了另外一种制备(S)-1-(4-吡啶基)-1,3-丙二醇的方法，如Scheme 2所示。该路线操作繁琐，三步反应总收率不足20％，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-(4-吡啶基)-1,3-丙二醇的方法，如Scheme 3所示。该路线选用4-吡啶甲醛为原料，在脂肪酶的催化条件下，一锅法制得(S)-1-(4-吡啶基)-1,3-丙二醇，反应时间72小时，收率为74％，ee值仅为90％。
目前，制备(S)-1-(4-吡啶基)-1,3-丙二醇的方法存在收率低，选择性差，不适合工业化生产等问题。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-(4-吡啶基)-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-(4-吡啶基)-1,3-丙二醇中的应用。
一方面，本发明提供一种新的酮还原酶突变体。
本发明利用基因工程手段及分子对接技术，将底物和产物分别对接到野生型酮还原酶结构中，根据底物或产物在结构中的能量最小化原则，设计了多个定点突变体，经过对这些突变体的有效组合，获得了两个高催化活性的突变体菌株Var-1(E16V/H68S/A112G/G171P)和Var-2(E16V/H68S/A112G/A184N/R241A),
进一步，所述野生型酮还原酶选自Exiguobacterium sp.MH3。
更进一步，所述野生型酮还原酶在NCBI的登录号为WP_023468191.1。
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-(4-吡啶基)-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-(4-吡啶基)-1,3-丙二醇的方法，具体包括如下步骤：以异烟酸为底物，先制得3-氧代-3-(吡啶-4-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-(4-吡啶基)-1,3-丙二醇。
进一步，以异烟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，所述酮还原酶以菌体形式参与催化反应。
更进一步，该步反应在反应温度为10～60℃条件下进行，优选37℃。
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-(4-吡啶基)-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-(4-吡啶基)-1,3-丙二醇的工艺过程中，该酶催化底物的浓度能够达到310g/L，ee值为99.7％。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野生型酮还原酶的诱导表达
利用人工合成基因技术合成来源于Exiguobacterium sp.MH3的酮还原酶，通过内切酶NdeI和XhoI将目标基因连接到pET-28a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，获得重组野生型菌株。利用通用引物T7/T7-ter扩增目的基因，并鉴定基因的大小，结果见附图1，显示出所有单克隆的基因大小一致。
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0.2mM IPTG诱导，温度降低到25℃，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，蛋白的大小和氨基酸序列对应一致，且大部分为可溶性表达。
实施例2突变体的构建和组合
借助蛋白质的模拟结构同底物和产物对接运算，理性设计出单个位点突变的信息E16V、H68S、A112G、S136T、G171P、A184N、R241A，根据突变信息，设计出定点突变引物，引物信息表1所示,所有待突变的氨基酸标记有下划线。
表1定点突变引物的核酸序列
利用PCR高温退火扩增技术，在高保真聚合酶PreimSTAR max作用下，实现全长质粒的扩增，扩增程序如下表所示。
表2全质粒扩增程序表
对扩增的结果进行电泳检测(见附图3)，每对引物都能清晰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和测试。以此类推，将每个突变体进行有序组合，获得最佳突变体Var-1(E16V/H68S/A112G/G171P)和Var-2(E16V/H68S/A112G/A184N/R241A)。
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异烟酸缓慢滴加到反应液中，室温搅拌2h。向反应液中加入300mL水及10mL稀盐酸溶液，搅拌0.5h，萃取，无水硫酸钠干燥，过滤蒸干得到138.2g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)
向反应釜中加入20mL 1M的磷酸盐缓冲液和140mL异丙醇，称量0.02g NADP固体和10g化合物3，一起投入反应釜中，同时补加40mL的纯化水，利用10％的Na2CO3溶液控制pH7.0，最后加入20g酮还原酶野生型菌体，在37℃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90％，S手性ee值为99.7％。
实施例5化合物4的制备(酮还原酶突变体Var-1)
向反应釜中加入20mL 1M的磷酸盐缓冲液和140mL异丙醇，称量0.02g NADP固体和60g化合物3，一起投入反应釜中，同时补加40mL的纯化水，利用10％的Na2CO3溶液控制pH7.0，最后加入20g酮还原酶突变体Var-1菌体，在37℃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％，S手性ee值为99.8％。
实施例6化合物4的制备(酮还原酶突变体Var-2)
向反应釜中加入20mL 1M的磷酸盐缓冲液和140mL异丙醇，称量0.02g NADP固体和62g化合物3，一起投入反应釜中，同时补加40mL的纯化水，利用10％的Na2CO3溶液控制pH7.0，最后加入20g酮还原酶突变体Var-2菌体，在37℃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值为99.7％。
实施例7化合物1的制备
氮气保护下将1.6g硼氢化钠及1mL水加入到35mL正丁醇中，冰浴下滴加10g化合物4(实施例6中获得)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7.19g化合物1，S手性ee值为99.7％。
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-(4-吡啶基)-1,3-丙二醇中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 750
<212> DNA/RNA
<213> Exiguobacterium sp. MH3
<400> 1
atgaagtaca ccgttattac cggcgcaagc agtggcattg gttatgaaac cgccaaactg 60
ctggccggta aaggcaaaag cctggtgctg gtggcccgtc gtaccagcga actggaaaaa 120
ctgcgtgatg aagttaaaca gattagtccg gatagcgatg ttattctgaa aagtgttgat 180
ctggccgata atcagaatgt tcatgatctg tatgaaggtc tgaaagaact ggatattgaa 240
acctggatta ataatgcagg ctttggcgat tttgatctgg ttcaggatat tgaactgggc 300
aaaattgaaa aaatgctgcg tctgaatatc gaagccctga ccattctgag cagcctgttt 360
gttcgcgatc atcatgatat tgaaggtaca accctggtta atattagcag cgccggtggt 420
tatcgcattg tgccgaatgc agtgacctat tgcgcaacca aattttatgt tagtgcatat 480
accgaaggtc tggcacagga actgcagaaa ggtggtgcca aactgcgtgc caaagttctg 540
gccccggccg caaccgaaac cgaatttgcc gatcgcagcc gcggcgaagc aggctttgat 600
tatagcaaaa atgttaaaaa gtaccacacc gcagccgaaa tggcaggttt tctgcatcag 660
ctgattgaaa gtgatgcaat tgttggcatt gttgatggtg aaacctatga atttgaactg 720
cgcggtccgc tgtttaatta tgcaggctaa 750
<210> 2
<211> 249
<212> PRT
<213> Exiguobacterium sp. MH3
<400> 2
Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Glu
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Thr Ala Lys Leu Leu Ala Gly Lys Gly Lys Ser Leu Val Leu Val Ala
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Arg Arg Thr Ser Glu Leu Glu Lys Leu Arg Asp Glu Val Lys Gln Ile
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Ser Pro Asp Ser Asp Val Ile Leu Lys Ser Val Asp Leu Ala Asp Asn
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Gln Asn Val His Asp Leu Tyr Glu Gly Leu Lys Glu Leu Asp Ile Glu
65 70 75 80
Thr Trp Ile Asn Asn Ala Gly Phe Gly Asp Phe Asp Leu Val Gln Asp
85 90 95
Ile Glu Leu Gly Lys Ile Glu Lys Met Leu Arg Leu Asn Ile Glu Ala
100 105 110
Leu Thr Ile Leu Ser Ser Leu Phe Val Arg Asp His His Asp Ile Glu
115 120 125
Gly Thr Thr Leu Val Asn Ile Ser Ser Ala Gly Gly Tyr Arg Ile Val
130 135 140
Pro Asn Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Tyr
145 150 155 160
Thr Glu Gly Leu Ala Gln Glu Leu Gln Lys Gly Gly Ala Lys Leu Arg
165 170 175
Ala Lys Val Leu Ala Pro Ala Ala Thr Glu Thr Glu Phe Ala Asp Arg
180 185 190
Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys Lys Tyr
195 200 205
His Thr Ala Ala Glu Met Ala Gly Phe Leu His Gln Leu Ile Glu Ser
210 215 220
Asp Ala Ile Val Gly Ile Val Asp Gly Glu Thr Tyr Glu Phe Glu Leu
225 230 235 240
Arg Gly Pro Leu Phe Asn Tyr Ala Gly
245
<210> 3
<211> 750
<212> DNA/RNA
<213> Exiguobacterium sp. MH3
<400> 3
atgaagtaca ccgttattac cggcgcaagc agtggcattg gttatgttac cgccaaactg 60
ctggccggta aaggcaaaag cctggtgctg gtggcccgtc gtaccagcga actggaaaaa 120
ctgcgtgatg aagttaaaca gattagtccg gatagcgatg ttattctgaa aagtgttgat 180
ctggccgata atcagaatgt tagtgatctg tatgaaggtc tgaaagaact ggatattgaa 240
acctggatta ataatgcagg ctttggcgat tttgatctgg ttcaggatat tgaactgggc 300
aaaattgaaa aaatgctgcg tctgaatatc gaaggcctga ccattctgag cagcctgttt 360
gttcgcgatc atcatgatat tgaaggtaca accctggtta atattagcag cgccggtggt 420
tatcgcattg tgccgaatgc agtgacctat tgcgcaacca aattttatgt tagtgcatat 480
accgaaggtc tggcacagga actgcagaaa cctggtgcca aactgcgtgc caaagttctg 540
gccccggccg caaccgaaac cgaatttgcc gatcgcagcc gcggcgaagc aggctttgat 600
tatagcaaaa atgttaaaaa gtaccacacc gcagccgaaa tggcaggttt tctgcatcag 660
ctgattgaaa gtgatgcaat tgttggcatt gttgatggtg aaacctatga atttgaactg 720
cgcggtccgc tgtttaatta tgcaggctaa 750
<210> 4
<211> 249
<212> PRT
<213> Exiguobacterium sp. MH3
<400> 4
Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Val
1 5 10 15
Thr Ala Lys Leu Leu Ala Gly Lys Gly Lys Ser Leu Val Leu Val Ala
20 25 30
Arg Arg Thr Ser Glu Leu Glu Lys Leu Arg Asp Glu Val Lys Gln Ile
35 40 45
Ser Pro Asp Ser Asp Val Ile Leu Lys Ser Val Asp Leu Ala Asp Asn
50 55 60
Gln Asn Val Ser Asp Leu Tyr Glu Gly Leu Lys Glu Leu Asp Ile Glu
65 70 75 80
Thr Trp Ile Asn Asn Ala Gly Phe Gly Asp Phe Asp Leu Val Gln Asp
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Ile Glu Leu Gly Lys Ile Glu Lys Met Leu Arg Leu Asn Ile Glu Gly
100 105 110
Leu Thr Ile Leu Ser Ser Leu Phe Val Arg Asp His His Asp Ile Glu
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Gly Thr Thr Leu Val Asn Ile Ser Ser Ala Gly Gly Tyr Arg Ile Val
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Pro Asn Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Tyr
145 150 155 160
Thr Glu Gly Leu Ala Gln Glu Leu Gln Lys Pro Gly Ala Lys Leu Arg
165 170 175
Ala Lys Val Leu Ala Pro Ala Ala Thr Glu Thr Glu Phe Ala Asp Arg
180 185 190
Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys Lys Tyr
195 200 205
His Thr Ala Ala Glu Met Ala Gly Phe Leu His Gln Leu Ile Glu Ser
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Asp Ala Ile Val Gly Ile Val Asp Gly Glu Thr Tyr Glu Phe Glu Leu
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Arg Gly Pro Leu Phe Asn Tyr Ala Gly
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<210> 5
<211> 750
<212> DNA/RNA
<213> Exiguobacterium sp. MH3
<400> 5
atgaagtaca ccgttattac cggcgcaagc agtggcattg gttatgttac cgccaaactg 60
ctggccggta aaggcaaaag cctggtgctg gtggcccgtc gtaccagcga actggaaaaa 120
ctgcgtgatg aagttaaaca gattagtccg gatagcgatg ttattctgaa aagtgttgat 180
ctggccgata atcagaatgt tagtgatctg tatgaaggtc tgaaagaact ggatattgaa 240
acctggatta ataatgcagg ctttggcgat tttgatctgg ttcaggatat tgaactgggc 300
aaaattgaaa aaatgctgcg tctgaatatc gaaggcctga ccattctgag cagcctgttt 360
gttcgcgatc atcatgatat tgaaggtaca accctggtta atattagcag cgccggtggt 420
tatcgcattg tgccgaatgc agtgacctat tgcgcaacca aattttatgt tagtgcatat 480
accgaaggtc tggcacagga actgcagaaa ggtggtgcca aactgcgtgc caaagttctg 540
gccccggcca ataccgaaac cgaatttgcc gatcgcagcc gcggcgaagc aggctttgat 600
tatagcaaaa atgttaaaaa gtaccacacc gcagccgaaa tggcaggttt tctgcatcag 660
ctgattgaaa gtgatgcaat tgttggcatt gttgatggtg aaacctatga atttgaactg 720
ggcggtccgc tgtttaatta tgcaggctaa 750
<210> 6
<211> 249
<212> PRT
<213> Exiguobacterium sp. MH3
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Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Val
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Thr Ala Lys Leu Leu Ala Gly Lys Gly Lys Ser Leu Val Leu Val Ala
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Arg Arg Thr Ser Glu Leu Glu Lys Leu Arg Asp Glu Val Lys Gln Ile
35 40 45
Ser Pro Asp Ser Asp Val Ile Leu Lys Ser Val Asp Leu Ala Asp Asn
50 55 60
Gln Asn Val Ser Asp Leu Tyr Glu Gly Leu Lys Glu Leu Asp Ile Glu
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Thr Trp Ile Asn Asn Ala Gly Phe Gly Asp Phe Asp Leu Val Gln Asp
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Ile Glu Leu Gly Lys Ile Glu Lys Met Leu Arg Leu Asn Ile Glu Gly
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Leu Thr Ile Leu Ser Ser Leu Phe Val Arg Asp His His Asp Ile Glu
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Gly Thr Thr Leu Val Asn Ile Ser Ser Ala Gly Gly Tyr Arg Ile Val
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Pro Asn Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Tyr
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Thr Glu Gly Leu Ala Gln Glu Leu Gln Lys Gly Gly Ala Lys Leu Arg
165 170 175
Ala Lys Val Leu Ala Pro Ala Asn Thr Glu Thr Glu Phe Ala Asp Arg
180 185 190
Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys Lys Tyr
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His Thr Ala Ala Glu Met Ala Gly Phe Leu His Gln Leu Ile Glu Ser
210 215 220
Asp Ala Ile Val Gly Ile Val Asp Gly Glu Thr Tyr Glu Phe Glu Leu
225 230 235 240
Ala Gly Pro Leu Phe Asn Tyr Ala Gly
245
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16位的谷氨酸突变为缬氨酸，第68位的组氨酸突变为丝氨酸，第112位的丙氨酸突变为甘氨酸，第171位的甘氨酸突变为脯氨酸；或者第16位的谷氨酸突变为缬氨酸，第68位的组氨酸突变为丝氨酸，第122位的丙氨酸突变为甘氨酸，第184位的丙氨酸突变为天冬酰胺，第241位的精氨酸突变为丙氨酸。
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和6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和5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-(4-吡啶基)-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-氧代-3-(吡啶-4-基)丙酸甲酯转化为(S)-3-羟基-3-(吡啶-4-基)丙酸甲酯，然后还原为(S)-1-(4-吡啶基)-1,3-丙二醇。
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