CN116622789A - 一种合成2,5-呋喃二甲醇或糠醇的方法 - Google Patents
一种合成2,5-呋喃二甲醇或糠醇的方法 Download PDFInfo
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Abstract
本发明属于生物化工及生物催化领域,公开了一种合成2,5‑呋喃二甲醇或糠醇的方法,包括如下步骤:(1)将醇脱氢酶YjgB基因与葡萄糖脱氢酶GDH Q252L基因共同连接到载体pETDuet‑1中,导入Escherichia coli BL21(DE3)中构建共表达体系,得到共表达重组菌E.coli‑YjgB‑GDH;(2)将上述重组菌加入含有呋喃醛、辅底物、碳酸钙的磷酸盐缓冲液中,反应后得到2,5‑呋喃二甲醇或糠醇;所述呋喃醛为5‑羟甲基糠醛或糠醛。本发明不仅具有高底物浓度、高时空收率和高选择性的优点,而且合成工艺条件温和、绿色环保。
Description
技术领域
本发明属于生物化工及生物催化领域,具体涉及利用共表达重组菌催化5-羟甲基糠醛和糠醛还原分别制备2,5-呋喃二甲醇和糠醇的方法。
背景技术
生物质是一种含碳可再生资源,可作为生产生物燃料和化学品的原料,以减少对化石资源的依赖。由戊糖和己糖制备的糠醛和5-羟甲基糠醛(HMF)可作为平台化合物生产大量高附加值中间体。在上述生物基呋喃中有一个高度活泼的醛基,通过选择性加氢还原其中的醛基可以合成糠醇和2,5-呋喃二甲醇(BHMF)。糠醇是高分子、食品和药物领域的一个重要的原料(Energy Environ.Sci.2016,9,1144)。BHMF是合成生物基聚酯、聚氨酯及聚醚、药物、生物燃料等的重要中间体(ChemSusChem.2013,6,630)。
基于金属包括贵金属(包括Pt,Pd,Au,Ir,Ru和Rh)以及非贵金属催化剂(Cu,Ni和Co)的化学催化还原呋喃醛合成对应的呋喃甲醇已取得了显著的进展(ACS Catal.2018,8,2959)。等利用担载在SiO2上Ir选择性催化HMF还原成BHMF,在333K和10bar H2下,HMF转化率为70%,BHMF选择性为83%(Appl.Catal.B:Environ.2019,241,270)。Wang等开发了一种高效、低成本负载Co的Zr基金属有机框架Co1.6/UiO-66-NH2,在100℃和异丙醇存在时,获得93%的HMF转化率和96%的BHMF选择性(Catal.Lett.2021,152,361)。Wang等以异丙醇为氢源,Ru/Co3O4作为催化剂,在190℃时BHMF产率可达82%(Catalysts 2017,7,92)。Ruan等利用高比表面积的SiO2纳米微球负载PtCuCo合金形成的催化剂,在3.0MPa H2、100℃下将糠醛转化为糠醇,12h后糠醛转化率达到98%,糠醇选择性为96%(Fuel 2023,335,126935)。化学方法仍存在反应条件激烈、环境不友好和能耗高等缺点。从经济和可持续性的角度,迫切需要开发经济、反应条件温和、高效且环境友好的呋喃甲醇合成方法。
生物催化具有效率高、反应条件温和、环境友好且选择性高等优点。目前,已报道的诸多生物催化剂能将呋喃醛还原为对应的呋喃甲醇(ACS Catal.2022,12,10080)。但由于HMF和糠醛对微生物细胞会产生高毒性和强抑制性,故生物催化高浓度底物还原仍然具有挑战性。
发明内容
针对现有技术存在的问题,本发明的目的在于提供一种高效、高选择性和高底物浓度的BHMF和糠醇合成方法。
本发明的目的通过以下技术方案实现:
一种合成2,5-呋喃二甲醇或糠醇的方法,包括如下步骤:
(1)将醇脱氢酶YjgB基因与葡萄糖脱氢酶GDH Q252L基因共同连接到载体pETDuet-1中,导入Escherichia coli BL21(DE3)中构建共表达体系,得到共表达重组菌E.coli-YjgB-GDH;
(2)将上述重组菌加入含有呋喃醛、辅底物、碳酸钙的磷酸盐缓冲液中,反应后得到2,5-呋喃二甲醇或糠醇;所述呋喃醛为5-羟甲基糠醛或糠醛。
优选地,步骤(1)中所述醇脱氢酶YjgB基因来源于Escherichia coli BL21(DE3);所述葡萄糖脱氢酶GDH基因来源于Bacillus megaterium IWG3,且是Q252L突变体。
优选地,步骤(1)中所述醇脱氢酶YjgB基因的核苷酸序列为SEQ ID.1;所述葡萄糖脱氢酶GDH Q252L基因的核苷酸序列为SEQ ID.2。
优选地,步骤(2)所述5-羟甲基糠醛浓度为0.02~1.0M;糠醛的浓度为0.02~0.5M。
优选地,步骤(2)中所述呋喃醛为商品化来源的高纯度5-羟甲基糠醛或糠醛或通过果糖、果葡糖浆脱水后制备的5-羟甲基糠醛粗品。
优选地,步骤(2)所述磷酸盐缓冲液中还含有乙酸乙酯或乙酸乙烯酯。
优选地,步骤(2)所述乙酸乙酯或乙酸乙烯酯的体积百分含量为5~20%。
优选地,步骤(2)中所述重组菌的浓度为20~100mg/mL。
优选地,步骤(2)中所述辅底物为葡萄糖;所述辅底物的摩尔浓度为呋喃醛摩尔浓度的2±0.5倍,所述碳酸钙的摩尔浓度为呋喃醛摩尔浓度的1.5±0.5倍。
优选地,步骤(2)中所述反应的pH为6~8;反应温度为20~35℃,反应时间为10min~25h。
所述HMF,BHMF,糠醛和糠醇的结构式如下:
本发明与现有技术相比,具有如下的优点:
1)本发明利用的生物催化剂(重组菌)在高底物浓度条件下能高效、高选择性催化呋喃醛还原,目标产物获得高时空收率。
2)本发明添加乙酸乙酯或乙酸乙烯酯能大大缩短反应时间,提高合成效率。
3)本发明反应过程简单、易控且条件温和,呋喃醛在反应过程中不易发生副反应,不仅提高产品品质、降低能耗,而且有利于简化后续目标产物的分离纯化工艺。
4)与游离酶催化相比,利用全细胞催化剂不仅能够降低催化剂的制备成本,而且由于酶蛋白在细胞膜的保护下具有更好的稳定性;此外,与酶催化相比,胞内的辅酶循环更容易实现,并且无需添加昂贵的辅酶。
附图说明
图1为YjgB和GDH Q252L基因扩增电泳图。图中标记:条带1,GDH Q252L纯酶蛋白条带;条带2,E.coli-YjgB-GDH共表达细胞上清液条带。
图2为HMF和BHMF样品分析的液相色谱图(HMF最大吸收波长283nm,保留时间9.0min;BHMF最大吸收波长224nm,保留时间7.5min)。
图3为糠醛和糠醇样品分析的液相色谱图(糠醛最大吸收波长277nm,保留时间14.4min;糠醇最大吸收波长215nm,保留时间12.9min)。
图4葡萄糖和果糖液相色谱图(葡萄糖保留时间11.9min,果糖保留时间19.3min)。
图5放大合成规模制备的BHMF核磁共振氢谱图(DMSO-d6,600MHz)。
具体实施方式
通过实施例进一步说明本发明,但不局限于实施例。实施例所用pET28a、pETDuet-1和E.coli BL21(DE3)均购自Novagen公司。
实施例1
共表达醇脱氢酶YjgB和GDH Q252L重组菌的构建方法
(1)以Escherichia coli BL21(DE3)基因组DNA为模板,通过设计特异性引物扩增YjgB
(GenBank accession number:CAQ34616.1)基因全长序列(核苷酸序列为SEQID.1);
(2)以GDH Q252L基因与pET28a载体构建的重组质粒pET28a-GDH为模板,通过设计特异性引物扩增GDH Q252L基因全长序列(核苷酸序列为SEQ ID.2);
(3)将所述醇脱氢酶基因YjgB和GDH Q252L基因通过同源重组分别连接到载体pETDuet-1限制性酶切位点EcoRI/SacI和NdeI/XhoI,得到共表达重组质粒,并测序验证;
(4)将上述共表达重组质粒转化至E.coli BL21(DE3),得到共表达YjgB和GDHQ252L的重组菌E.coli-YjgB-GDH;
表1引物信息
实施例2
E.coli-YjgB-GDH的诱导表达
将实施例1中获得的重组菌E.coli-YjgB-GDH接种至含有100μg/mL氨苄青霉素的LB液体培养基(胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,pH 7.2)中,在37℃、180rpm下培养12h。然后,按1%的接种量将上述菌悬液转种到含有100μg/mL氨苄青霉素的LB液体培养基中,于37℃、180rpm下培养,当菌液的OD600达到0.6~0.8时,加入0.3mM异丙基-β-D-硫代半乳糖苷,置于20℃、160rpm下诱导培养20h,培养结束后收集菌体细胞,并用50mM磷酸缓冲液(pH 7.0)清洗细胞2次,获得诱导表达的重组菌E.coli-YjgB-GDH。
实施例3
在2mL磷酸盐缓冲液(200mM,pH 7.0)中加入0.4mmol HMF(HMF初始浓度为200mM),0.8mmol葡萄糖,0.6mmol CaCO3,按50mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。利用液相色谱监控反应(图2)。8h后,HMF转化率为>99%,目标产物BHMF产率为97%。
实施例4
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入1.0mmol HMF(HMF初始浓度为500mM),2.0mmol葡萄糖,1.5mmol CaCO3,按50mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。8h后,HMF转化率为>99%,目标产物BHMF产率为96%。
实施例5
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入1.6mmol HMF(HMF初始浓度为800mM),3.2mmol葡萄糖,2.4mmol CaCO3,按50mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应,18h后,HMF转化率为>99%,目标产物BHMF产率为96%。
实施例6
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入2.0mmol HMF(HMF初始浓度为1M),4.0mmol葡萄糖,3.0mmol CaCO3,按100mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。25h后,HMF转化率为>99%,目标产物BHMF产率为98%。
实施例7
在50mL磷酸盐缓冲液(200mM,pH7.0)中加入50.0mmol HMF(HMF初始浓度为1M),100.0mmol葡萄糖,75.0mmol CaCO3,按100mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,室温下于磁力搅拌器250rpm搅拌,反应过程中用NaOH调控反应液pH维持6.5-7.0。通过液相色谱监测,反应10h时,HMF完全转化,产物只有BHMF。目标产物产率达到92%,时空产率为11g/L·h。反应后,反应液用乙酸乙酯萃取3次,收集有机相,除去有机溶剂后得到目标产物。反应初始时加入HMF 6.3g,反应结束得到BHMF 5.0g,BHMF得率为79%。
实施例8
在2mL磷酸盐缓冲液(200mM,pH 7.0)中加入0.4mmol糠醛(糠醛初始浓度为200mM),0.8mmol葡萄糖,0.6mmol CaCO3,按50mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。利用液相色谱监控反应(图3)。2h后,糠醛转化率为>99%,目标产物糠醇产率为98%。
实施例9
在2mL磷酸盐缓冲液(200mM,pH 7.0)中加入1.0mmol糠醛(糠醛初始浓度为500mM),2.0mmol葡萄糖,1.5mmol CaCO3,按50mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。8h后,糠醛转化率为96%,目标产物糠醇产率为95%。
实施例10
在2mL磷酸盐缓冲液(200mM,pH 7.0)中加入1.2mmol糠醛(糠醛初始浓度为600mM),2.4mmol葡萄糖,1.8mmol CaCO3,按50mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。12h后,糠醛转化率为16%,目标产物糠醇产率为8%。
实施例11
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入0.2mmol HMF(HMF初始浓度为100mM),0.4mmol葡萄糖,0.3mmol CaCO3,按20mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。6h后,HMF转化率为>99%,目标产物BHMF产率为95%。
实施例12
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入0.2mmol HMF(HMF初始浓度为100mM),0.4mmol葡萄糖,0.3mmol CaCO3,加入5%(v/v)的乙酸乙酯,按20mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。1h后,HMF转化率为>99%,目标产物BHMF产率为97%。
实施例13
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入0.2mmol HMF(HMF初始浓度为100mM),0.4mmol葡萄糖,0.3mmol CaCO3,加入20%(v/v)的乙酸乙酯,按20mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。1h后,HMF转化率为>99%,目标产物BHMF产率为91%。
实施例14
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入0.2mmol HMF(HMF初始浓度为100mM),0.4mmol葡萄糖,0.3mmol CaCO3,加入5%(v/v)的乙酸乙烯酯,按20mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。1h后,HMF转化率为>99%,目标产物BHMF产率为94%。
实施例15
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入0.2mmol HMF(HMF初始浓度为100mM),0.4mmol葡萄糖,0.3mmol CaCO3,加入20%(v/v)的乙酸乙烯酯,按20mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。1h后,HMF转化率为>99%,目标产物BHMF产率为99%。
实施例16
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入1.0mmol HMF(HMF初始浓度为500mM),2.0mmol葡萄糖,1.5mmol CaCO3,加入5%(v/v)的乙酸乙酯,按50mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。4h后,HMF转化率为>99%,目标产物BHMF产率为94%,BHMF的时空收率为15g/L h。
实施例17
在2mL磷酸盐缓冲液(200mM,pH7.0)中加入1.0mmol HMF(HMF初始浓度为500mM),2.0mmol葡萄糖,1.5mmol CaCO3,加入5%(v/v)的乙酸乙烯酯,按50mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。6h后,HMF转化率为>99%,目标产物BHMF产率为89%。
实施例18
将2g D-果糖溶于6mL去离子水,取2mL该果糖溶液于反应釜中,加入8mL丙酮和50mM HCl,于油浴锅中120℃,磁力搅拌反应1h。反应结束加入1mL NaOH将反应液调到pH 7。利用液相色谱监控反应(图4),果糖转化率为85%,HMF产率和选择性分别为57%和67%。溶液中果糖含量为44.5mM,HMF浓度约为170mM。反应液于40℃减压旋蒸5min以除去丙酮,获得HMF粗品。
在2mL磷酸盐缓冲液(200mM,pH8.0)中加入0.28mL上述HMF粗品(HMF初始浓度为50mM),0.2mmol葡萄糖,0.15mmol CaCO3,按20mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。20min后,HMF转化率为99%,目标产物BHMF产率为>99%。
实施例19
用去离子水将2.27mL果葡糖浆溶液(含56%果糖和44%葡萄糖)稀释到6mL,取2mL该溶液于反应釜中,加入8mL丙酮和50mM HCl,于油浴锅中120℃,磁力搅拌反应1h。反应结束加入1mL NaOH将反应液调到pH 7。果糖转化率为79%,葡萄糖转化率为30%,目标产物HMF产率和选择性分别为58%和74%。溶液中剩余葡萄糖浓度108mM,果糖浓度41.4mM,HMF浓度116.8mM。将反应液于40℃减压旋蒸5min以除去丙酮,获得HMF粗品。
在2mL磷酸盐缓冲液(200mM,pH8.0)中加入0.11mL上述HMF粗品(HMF初始浓度为20mM,葡萄糖初始浓度为23.5mM),0.06mmol CaCO3,按20mg/mL(按细胞湿重计)的浓度加入经实施例2获得的重组菌E.coli-YjgB-GDH,混合均匀后,于30℃及150rpm下反应。10min后,HMF转化率为98%,目标产物BHMF产率为93%。
Claims (10)
1.一种合成2,5-呋喃二甲醇或糠醇的方法,其特征在于,包括如下步骤:
(1)将醇脱氢酶YjgB基因与葡萄糖脱氢酶GDH Q252L基因共同连接到载体pETDuet-1中,导入Escherichia coli BL21(DE3)中构建共表达体系,得到共表达重组菌E.coli-YjgB-GDH;
(2)将上述重组菌加入含有呋喃醛、辅底物、碳酸钙的磷酸盐缓冲液中,反应后得到2,5-呋喃二甲醇或糠醇;所述呋喃醛为5-羟甲基糠醛或糠醛。
2.根据权利要求1所述的方法,其特征在于,步骤(1)中所述醇脱氢酶YjgB基因来源于Escherichia coli BL21(DE3);所述葡萄糖脱氢酶GDH基因来源于Bacillus megateriumIWG3,且是Q252L突变体。
3.根据权利要求1所述的方法,其特征在于,步骤(1)中所述醇脱氢酶YjgB基因的核苷酸序列为SEQ ID.1;所述葡萄糖脱氢酶GDH Q252L基因的核苷酸序列为SEQ ID.2。
4.根据权利要求1所述的方法,其特征在于,步骤(2)所述5-羟甲基糠醛浓度为0.02~1.0M;糠醛的浓度为0.02~0.5M。
5.根据权利要求4所述的方法,其特征在于,步骤(2)中所述呋喃醛为商品化来源的高纯度5-羟甲基糠醛或糠醛或通过果糖、果葡糖浆脱水后制备的5-羟甲基糠醛粗品。
6.根据权利要求1所述的方法,其特征在于,步骤(2)所述磷酸盐缓冲液中还含有乙酸乙酯或乙酸乙烯酯。
7.根据权利要求5所述的方法,其特征在于,步骤(2)所述乙酸乙酯或乙酸乙烯酯的体积百分含量为5~20%。
8.根据权利要求1-7任意一项所述的方法,其特征在于,步骤(2)中所述重组菌的浓度为20~100mg/mL。
9.根据权利要求8所述的方法,其特征在于,步骤(2)中所述辅底物为葡萄糖;所述辅底物的摩尔浓度为呋喃醛摩尔浓度的2±0.5倍,所述碳酸钙的摩尔浓度为呋喃醛摩尔浓度的1.5±0.5倍。
10.根据权利要求9所述的方法,其特征在于,步骤(2)中所述反应的pH为6~8;反应温度为20~35℃,反应时间为10min~25h。
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