CN109913511B - 一种酶法合成阿魏酸甲酯的方法 - Google Patents
一种酶法合成阿魏酸甲酯的方法 Download PDFInfo
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- CN109913511B CN109913511B CN201910261291.2A CN201910261291A CN109913511B CN 109913511 B CN109913511 B CN 109913511B CN 201910261291 A CN201910261291 A CN 201910261291A CN 109913511 B CN109913511 B CN 109913511B
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- methyl ester
- acid methyl
- ferulic acid
- leu
- methyltransferase
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- 238000000034 method Methods 0.000 title claims abstract description 51
- AUJXJFHANFIVKH-UHFFFAOYSA-N methyl cis-ferulate Natural products COC(=O)C=CC1=CC=C(O)C(OC)=C1 AUJXJFHANFIVKH-UHFFFAOYSA-N 0.000 title claims abstract description 44
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Abstract
本发明公开了一种酶法合成阿魏酸甲酯的方法，该方法以川芎咖啡酸‑O‑甲基转移酶为催化剂，以咖啡酸甲酯和S‑腺苷甲硫氨酸为反应物，再加入缓冲液、还原剂配置成反应体系，制得阿魏酸甲酯。本发明提供的酶法合成阿魏酸甲酯的方法，以咖啡酸甲基转移酶为催化剂、以咖啡酸甲酯和S‑腺苷甲硫氨酸为反应物制备阿魏酸甲酯，生产工艺步骤少，反应条件温和，绿色环保。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种酶法合成阿魏酸甲酯的方法。
背景技术
阿魏酸甲酯是一种紫外吸收剂，其对280nm～360nm波长的紫外线有良好的吸收能力且吸收率高，具有抗氧化、美白与消炎等功效。比如，在化妆品加入阿魏酸甲酯，不仅能够起到美白作用，长期使用还能够使皮肤具有绢质感。并且，阿魏酸甲酯还可作为强效美白剂阿魏酸异辛酯的合成中间体。最后，阿魏酸甲酯也是一个重要的医葯中间体。
目前阿魏酸甲酯的合成方法主要有以下几种。第一种是Knoevenagel两步法工艺：即第1步，香兰素与丙二酸经Knoevenagel缩合反应制得阿魏酸；第2步，阿魏酸与甲醇酯化反应得到阿魏酸甲酯。其中Knoevenagel缩合常用吡啶作溶剂，哌啶作催化剂，毒性较大。该方法缺点是步骤多，所有试剂如吡啶、甲醇毒性较大。
酯化方法包括酰氯法、DCC法、酸催化法等，这些方法或者使用毒性大的试剂，或者收率较低。
亦有文献报道采用“一锅合成法”和“微波辅助阳离子交换树脂催化法”合成阿魏酸甲酯的方法。其中，“一锅合成法”以香兰素、丙二酸二乙酯为主要原料，以甘氨酸为催化剂,无水甲醇为溶剂，经水解、酸化和Knoevenagel缩合等反应一锅式合成了阿魏酸甲酯。该方法缺点是转化率低，步骤多，所用试剂毒性大、污染环境。“微波辅助阳离子交换树脂催化法”以甲醇和阿魏酸为原料，以强酸性阳离子交换树脂为催化剂，在微波辐射下合成阿魏酸甲酯。该方法缺点是甲醇毒性大且污染环境)。总结现有的阿魏酸甲酯制备方法，均是利用有机合成的方法，存在生产工艺步骤较多、毒性大与污染严重等缺点。
发明内容
本发明的目的是提供一种步骤简单、反应条件温和、环境友好且转化效率较高的生物催化剂(酶法)合成阿魏酸甲酯的方法。
为解决上述技术问题，本发明的技术方案如下：一种酶法合成阿魏酸甲酯的方法，以咖啡酸-O-甲基转移酶为催化剂，以咖啡酸甲酯和S-腺苷甲硫氨酸为反应物，再加入缓冲液、还原剂配置成反应体系，制得阿魏酸甲酯；
上述过程反应式表示为：
本技术方案利用高纯度的咖啡酸-O-甲基转移酶(Caffeic acid O-methyltransferase，以下均缩写为COMT)为催化剂，以咖啡酸甲酯和S-腺苷-L-甲硫氨酸(S-adenosyl-L-methionine，以下均缩写为SAM)为反应物，“一步反应”生成阿魏酸甲酯和S-腺苷-L-高半胱氨酸(S-adenosyl-L-homocysteine，以下均缩写为SAH)；
上述酶法合成阿魏酸甲酯的方法中，COMT优选课题组通过基因工程方法制备的川芎咖啡酸-O-甲基转移酶(Ligusticum chuanxiong caffeic acid O-methyltransferase，以下均缩写为LcCOMT)。COMT属于O-甲基转移酶(OMT)家族成员，是植物苯丙烷类化合物，如类黄酮与木质素生物合成途径中的关键酶，其甲基基团的供体是SAM。目前尚未有研究报道COMT能够催化咖啡酸甲酯形成阿魏酸甲酯。因此，以LcCOMT为催化剂，催化咖啡酸甲酯与SAM反应形成阿魏酸甲酯，是保证本发明得以实施的关键技术点，也是本发明的重要创新点之一。
本发明中使用的LcCOMT为本课题组采用基因工程方法制备，包括将LcCOMT基因转入大肠杆菌BL21，IPTG诱导表达及纯化LcCOMT等方法，具体步骤如下：
S1、制备LcCOMT：
S11、构建生产转化菌株：
S111、用分别带有EcoR1和BamH1双酶切位点的引物从川芎cDNA中通过PCR扩增出LcCOMT基因的全长阅读框；
S112、将扩增出来的LcCOMT基因片段和pET28a分别用EcoR1和BamH1双酶切消化，连接获得重组质粒；
S113、重组质粒转化大肠杆菌BL21感受态细胞并筛选阳性克隆；
S114、用菌落PCR和质粒双酶切鉴定后，经测序以确定阅读框的正确，得到预期的重组菌株；
S12、发酵扩大重组菌株：
S121、发酵培养基：蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L；溶解后，高温高压灭菌；
S122、发酵条件：37℃，200rpm将重组菌株培养至OD600＝0.6～0.8；加入IPTG，使其终浓度为1mM/L，25～30℃培养8～10h；
S13、制取LcCOMT的混合酶液
S131、将步骤S122获得的发酵液，离心收集菌体；
S132、用Tris-HCl缓冲液重悬菌体，超声裂解菌体，离心去除细胞碎片，收集上清液；
S14、提纯LcCOMT溶液
S141、将上清液加入镍离子亲和层析柱，结合2h后使其流出；
S142、用含有咪唑的Tris-HCl缓冲液洗脱，得到LcCOMT溶液；
S143、将LcCOMT溶液在透析液中透析3次，获得纯化的LcCOMT。
S2、制备阿魏酸甲酯
S21、反应体系中包括咖啡酸甲酯，SAM，二硫苏糖醇，Tris-HCl缓冲液及LcCOMT重组酶。
S22、反应1h后，萃取并旋蒸，使溶质析出，再用溶液(甲醇：甲酸＝95：5)溶解溶质及定容；
S23、HPLC检测产物即阿魏酸甲酯。
上述酶法合成阿魏酸甲酯的方法中，咖啡酸甲酯对环境友好，其分子式是C10H10O4，分子量为194.184。SAM带有1个活性甲基，在甲基转移反应中作为甲基供体，存在于所有的真核细胞中。
上述酶法合成阿魏酸甲酯的方法中，还原剂优选二硫苏糖醇，其主要作用为反应体系提供还原性，保持LcCOMT的活性。缓冲溶液的作用是维持反应体系一定的pH，优选Tris-HCl缓冲液。对于保持酶活性的pH值，是本领域技术人员所掌握的常规技术。
上述酶法合成阿魏酸甲酯的方法中，需要说明的是，本发明中S1步骤中各反应的试剂量是本领域内常规技术，为本领域内技术人员所常规熟知剂量。在S2步骤催化反应过程中，对各原料的具体量并无特殊限制，对于本发明而言，试剂量也并不是本发明所涉及反应特异的。配制10ml反应体系，其中：咖啡酸甲酯为200μM，S-腺苷甲硫氨酸为1mM，还原剂为1mM，pH 7.0的三羟甲基氨基甲烷-盐酸缓冲液为200mM，咖啡酸-O-甲基转移酶为40μg/mL，反应温度优选为37℃。
本发明的有益效果是：本发明提供的酶法合成阿魏酸甲酯的方法，首次以LcCOMT为催化剂，采用对环境友好的反应物一步合成阿魏酸甲酯，生产工艺步骤少，反应条件温和，转化率高，绿色环保。
附图说明
图1是本发明的工艺流程图；
图2是重组质粒pET28a-LcCOMT的质粒图谱；
图3是咖啡酸甲酯标准品和阿魏酸甲酯标准品以及反应产物的HPLC检测图。
具体实施方式
下面结合附图和具体实施例对本发明做进一步的说明：
如图1所示，本发明的酶法合成阿魏酸甲酯的方法，包括以下步骤：
S1、制备LcCOMT：
S11、构建生产转化菌株：
S111、用分别带有EcoR1和BamH1双酶切位点的引物(A1：5`-CGC GGA TCC ATG AATACG GAG CTG ATC CCA CC-3`；A2：5`-CGG AAT TCA CAT TAA GCA GAT GCC AGA CAC CC-3`)从川芎cDNA中通过PCR扩增出LcCOMT基因的全长阅读框。PCR体系为：Primer star mix(宝生物公司)25ul、cDNA2ul、引物A1 2ul、引物A2 2ul，双蒸水补足50ul。PCR体系为模板变性94℃5min，进入循环，模板变性94℃1min、退火66℃1min、延伸72℃2min，共35个循环，产物完整延伸72℃10min，4℃保存。
S112、将扩增出来的LcCOMT基因片段和pET28a分别用EcoR1和BamH1双酶切消化，获得两端带有粘性末端的片段；用T4DNA连接酶连接两个片段，体系为10*T4DNA ligasebuffer2.5μL、双酶切后的川芎咖啡酸-O-甲基转移酶DNA片段6μL、双酶切后的pET28a载体2μL、T4DNA ligase1μL，用灭菌的双蒸水补足25μL；16℃连接过夜；获得连接好的重组质粒(如图2所示)；
S113、取10μL重组质粒转化BL21感受态细胞，将转化成功的重组BL21基因工程菌涂布于含有50mg/L卡那霉素、10g/L蛋白胨、5g/L酵母提取物、10g/L NaCl、12g/L琼脂的LB平板中筛选阳性克隆；
S114、挑取筛选后的单克隆菌落于含10g/L蛋白胨、5g/L酵母提取物、10g/L NaCl、50mg/L卡那霉素的LB液体培养基中37℃，200rpm摇菌过夜。将初步筛选出来的重组菌株用菌落PCR和质粒双酶切鉴定后，经测序以确定阅读框的正确，得到预期的重组菌株；
S12、发酵扩大重组菌株：
S121、发酵培养基：蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L；溶解后，高温高压灭菌；
S122、发酵条件：37℃，200rpm将重组菌株培养至OD600＝0.6～0.8；加入IPTG，使其终浓度为1mM/L，25～30℃培养8～10h；
S13、制取LcCOMT的混合酶液
S131、将步骤S122获得的发酵液，4℃，5000g，离心10min离心收集菌体；
S132、用10mL 20mM的Tris-HCl pH7.9的缓冲液(20mM咪唑、10mM Tris-HCl pH7.9、500mM NaCl)重悬菌体，超声裂解菌体，超声程序为60W，工作3s、暂停3s，冰浴条件下超声30min；4℃，13000g离心15min去除细胞碎片，收集上清液；
S14、提纯LcCOMT溶液
S141、将上清液加入镍离子亲和层析柱，4℃下结合2h后，让上清液流出；
S142、用20mL的结合液洗脱杂蛋白，结合液为60mM咪唑、10mM Tris-HCl pH7.9、500mM NaCl，然后用5mL的洗脱液洗脱目的蛋白，洗脱液为200mM咪唑、10mM Tris-HCl pH7.9、500mM NaCl，得到LcCOMT蛋白溶液；
S143、将LcCOMT溶液在透析液中进行第一次透析，透析液成分是：2mM Tris-HCl(pH 7.4)、10％甘油、β-巯基乙醇0.5‰、250mM NACl，透析袋截留分子量为8000-14000；4-5h后更换透析液，进行第二次透析，第二次透析液成分：2mM Tris-HCl(pH 7.4)、10％甘油、β-巯基乙醇0.5‰、150mM NaCl；4-5h后，更换透析液，进行第三次透析，第三次透析液成分与第二次透析液成分相同，透析12-16h，反复透析三次，收集透析袋中的蛋白液；
S2、制备阿魏酸甲酯：
S21、配制10mL反应体系，其中：咖啡酸甲酯为200μM，SAM为1mM，二硫苏糖醇为1mM，Tris-HCl pH 7.0缓冲液为200mM，LcCOMT为40μg/mL，37℃，反应表达式如下：
反应1小时后，加1mL浓盐酸终止反应；
S22、向步骤S22的反应液中加5mL乙酸乙酯萃取，收集萃取液；再重复萃取两次，合并萃取液，共15mL，将萃取液转移到圆底烧瓶，通过旋蒸使其完全蒸发，溶质析出附着于圆底烧瓶内；向烧瓶内加入溶液(甲醇：甲酸＝95：5)洗涤烧瓶，使溶质溶解，收集洗涤液。再用溶液(甲醇：甲酸＝95：5)洗涤烧瓶数次，收集合并所有洗涤液，定容于5mL，作为样品待检测；
S23、高液检测，将上步待测样品过0.45μm滤头后，作为高液进样样品，流动相为甲醇：0.1％磷酸＝45：55，流速1mL/min，柱温30℃，检测波长330nm。出峰时间为7.4min，这与阿魏酸甲酯标准品(购于成都克洛玛生物科技有限公司)出峰时间相同，证明生成阿魏酸甲酯。如图3所示，根据咖啡酸甲酯和阿魏酸甲酯积分后的峰面积来计算咖啡酸甲酯的转化率，结果表明反应1h后，咖啡酸甲酯的转化率达到96.6％。
上述步骤S21中，在反应1小时后加浓盐酸阻止反应是为了检测1小时能反应的底物量，实际操作中可以等反应自然完成。萃取是为了将物质从水相转移到有机相，方便后续旋蒸后定容，便于用高液检测。
本领域的普通技术人员将会意识到，这里所述的实施例是为了帮助读者理解本发明的原理，应被理解为本发明的保护范围并不局限于这样的特别陈述和实施例。本领域的普通技术人员可以根据本发明公开的这些技术启示做出各种不脱离本发明实质的其它各种具体变形和组合，这些变形和组合仍然在本发明的保护范围内。
序列表
<110> 西南交通大学
<120> 一种酶法合成阿魏酸甲酯的方法
<141> 2019-03-29
<160> 2
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cttaatcttc tcgagtccat agctaaagct ggtcctggtg tttatgtttc gccttctcat 180
cttgcggctg ggcttccttc cagccaacct gacacgcctg ttatgcttga ccgcatcctc 240
cgcctcctgg ccagctactc tgtgctcaac tgtcaacttc gtgacctgcc tcaaggtcgg 300
gtcgagcgcc tttatggact ggctcctgtg tgcaagttct tgactaaaaa ctctgatggt 360
gtgtctatgg cgccactttt gctcatgaac caagacaaaa tccttatgga gagctggtat 420
cacctgaaag atgctgttct agatggtgga atacctttta acaaggcata tggaatgaca 480
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aatcattcta ctataactat gaagaaaatc ctccaaacat atgacggatt tggcggtctc 600
aaaactgtgg tggatgttgg cggaggcacc ggagccaccc ttaatatgat tatttctaaa 660
taccctaatc tcaaagggat taactttgat ctccctcatg ttgttgaaga tgctccatct 720
tatcctggtg tggagcatgt tggaggtgac atgtttgtta gcgtgcccaa aggggatgct 780
attttcatga agtggatatg tcacgattgg agcgatgcac attgtctgac attcttgaag 840
aactgctata aggcacttcc aaaggatgga aaggtgatac tagcagaatg cattcttcca 900
gaggctccag actccaaact tacaaccaag aatgtcattc atatagatgt tataatgctt 960
gcacataatc ctggcggaaa agaaagaact gagaaagatt ttgaggcctt gggaaaagag 1020
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Pro Gln Gly Arg Val Glu Arg Leu Tyr Gly Leu Ala Pro Val Cys Lys
100 105 110
Phe Leu Thr Lys Asn Ser Asp Gly Val Ser Met Ala Pro Leu Leu Leu
115 120 125
Met Asn Gln Asp Lys Ile Leu Met Glu Ser Trp Tyr His Leu Lys Asp
130 135 140
Ala Val Leu Asp Gly Gly Ile Pro Phe Asn Lys Ala Tyr Gly Met Thr
145 150 155 160
Ala Phe Glu Tyr His Gly Lys Asp Pro Arg Phe Asn Lys Val Phe Asn
165 170 175
Gln Gly Met Ser Asn His Ser Thr Ile Thr Met Lys Lys Ile Leu Gln
180 185 190
Thr Tyr Asp Gly Phe Gly Gly Leu Lys Thr Val Val Asp Val Gly Gly
195 200 205
Gly Thr Gly Ala Thr Leu Asn Met Ile Ile Ser Lys Tyr Pro Asn Leu
210 215 220
Lys Gly Ile Asn Phe Asp Leu Pro His Val Val Glu Asp Ala Pro Ser
225 230 235 240
Tyr Pro Gly Val Glu His Val Gly Gly Asp Met Phe Val Ser Val Pro
245 250 255
Lys Gly Asp Ala Ile Phe Met Lys Trp Ile Cys His Asp Trp Ser Asp
260 265 270
Ala His Cys Leu Thr Phe Leu Lys Asn Cys Tyr Lys Ala Leu Pro Lys
275 280 285
Asp Gly Lys Val Ile Leu Ala Glu Cys Ile Leu Pro Glu Ala Pro Asp
290 295 300
Ser Lys Leu Thr Thr Lys Asn Val Ile His Ile Asp Val Ile Met Leu
305 310 315 320
Ala His Asn Pro Gly Gly Lys Glu Arg Thr Glu Lys Asp Phe Glu Ala
325 330 335
Leu Gly Lys Glu Ala Gly Phe Lys Ser Phe Asn Lys Ala Cys Cys Ala
340 345 350
Tyr Asn Thr Trp Val Ile Glu Tyr Tyr Lys
355 360
Claims (5)
1.一种酶法合成阿魏酸甲酯的方法，其特征在于：以咖啡酸-O-甲基转移酶为催化剂，以咖啡酸甲酯和S-腺苷甲硫氨酸为反应物，再加入缓冲液、还原剂配置成反应体系，制得阿魏酸甲酯；
上述过程反应式表示为：
所述咖啡酸-O-甲基转移酶为川芎咖啡酸-O-甲基转移酶。
2.根据权利要求1所述的酶法合成阿魏酸甲酯的方法，其特征在于：所述还原剂为二硫苏糖醇。
3.根据权利要求1所述的酶法合成阿魏酸甲酯的方法，其特征在于：所述缓冲液为三羟甲基氨基甲烷-盐酸缓冲液。
4.根据权利要求1所述的酶法合成阿魏酸甲酯的方法，其特征在于：配制10ml反应体系，其中：咖啡酸甲酯为200μM，S-腺苷甲硫氨酸为1mM，还原剂为1mM，pH 7.0的三羟甲基氨基甲烷-盐酸缓冲液为200mM，咖啡酸-O-甲基转移酶为40μg/mL。
5.根据权利要求4所述的酶法合成阿魏酸甲酯的方法，其特征在于：反应温度为37℃。
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| CN106906202A (zh) * | 2017-03-14 | 2017-06-30 | 西南交通大学 | 一种生产阿魏酸的固定化基因工程酶的制备方法 |
| CN109402087A (zh) * | 2018-12-07 | 2019-03-01 | 南京农业大学 | 一种新型阿魏酸酯酶及其制备方法和应用 |
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| CN106906202A (zh) * | 2017-03-14 | 2017-06-30 | 西南交通大学 | 一种生产阿魏酸的固定化基因工程酶的制备方法 |
| CN109402087A (zh) * | 2018-12-07 | 2019-03-01 | 南京农业大学 | 一种新型阿魏酸酯酶及其制备方法和应用 |
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