【作者】 张雪莹；

【导师】 宗敏华； 李宁；

【作者基本信息】 华南理工大学 ， 发酵工程， 2019， 博士

【摘要】 5-羟甲基糠醛(HMF)及糠醛是一类重要的生物基平台化合物,经碳水化合物脱水得到。5-羟甲基-2-糠酸(HMFCA)及2-糠酸(FCA)是上述呋喃醛选择性氧化后的产物,在高分子、医药及食品等领域具有广阔的应用前景。目前,呋喃羧酸主要是通过化学催化制备得到。尽管化学催化呋喃醛氧化合成呋喃羧酸已取得了重大进展,但该法存在反应条件苛刻、环境不友好、选择性不佳等缺点。生物催化能有效克服上述缺点,在合成化学中已成为了补充甚至替代化学催化的一种重要方法。尤其是对于HMF、糠醛等稳定性较差的生物基呋喃醛高值化转化,生物催化与化学催化相比更具应用潜力。与酶相比,全细胞生物催化剂更易制备、酶蛋白在细胞膜的保护下更稳定、且更易实现胞内辅酶再生,因而在氧化还原反应中备受关注。然而,HMF、糠醛对微生物和酶具有强烈的毒性及抑制作用,因此已报道的高底物耐受的、且能高效、高选择性催化呋喃醛氧化的生物催化剂仍有限。基于上述情况,本论文从土壤中分离到一株对HMF具有较高耐受能力、并且能够将HMF选择性氧化为HMFCA的Comamonas testosteroni SC1588。从该菌基因组中挖掘出能够催化HMF选择性氧化的关键醛脱氢酶基因,进行异源表达、分离纯化、并研究其酶学性质。之后,探讨了这些重组菌催化HMF选择性氧化的性能。最后,将上述醛脱氢酶分别与NADH氧化酶(NOX)在大肠杆菌(Escherichia coli)中进行共表达以促进NAD~+再生,提高反应选择性,建立高效、高选择性合成呋喃羧酸的生物催化途径。底物抑制和毒性研究表明,C.testosteroni SC1588细胞在催化HMF氧化反应中至少能耐受100 mmol/L的底物。目标产物HMFCA对该菌具有较强的抑制和毒性作用,这主要归因于其酸性,因为该菌的催化活性高度依赖于反应体系pH。通过添加弱碱控制反应体系pH及添加少量组氨酸均能显著提高该菌的催化性能。在最适反应条件下,当HMF浓度为160 mmol/L时,反应36 h后HMFCA产率为98%。该菌还可以催化糠醛、5-甲基糠醛和5-甲酰基-2-糠酸(FFCA)选择性氧化合成相应的羧酸,产率为90-93%。从该菌基因组中挖掘出5个能催化HMF选择性氧化的关键醛脱氢酶基因,包括2个香草醛脱氢酶(CtVDH1和CtVDH2)、2个松柏醛脱氢酶(CtCALDH1和CtCALDH2)和1个3-琥珀酰半醛吡啶脱氢酶(CtSAPDH),并在E.coli中克隆、表达。5个醛脱氢酶的亚基分子量均约为50 kDa。CtCALDH1为NAD~+依赖型脱氢酶,其它醛脱氢酶既可以NAD~+又可以NADP~+作为辅酶催化氧化反应。这些醛脱氢酶的最适pH均为7.0,最适反应温度为25-35oC,但是其在体外的稳定性均较差。各醛脱氢酶底物特异性迥异。CtCALDH1和CtCALDH2催化大多数芳基醛氧化的活性均较低,并且CtCALDH1不能接受FFCA、5-甲基糠醛和4-甲酰基苯甲酸作为底物;CtVDH1和CtVDH2对于大多数底物均表现出较高的氧化活性,而CtSAPDH催化各种芳基醛氧化的活性均适中。通常,这些醛脱氢酶催化苯甲醛衍生物氧化的活性高于其催化呋喃结构类似物氧化的活性。在所构建的5个过量表达醛脱氢酶的重组E.coli菌中,E.coli/pET-CtVDH1催化HMF选择性氧化效率最高。反应12 h后200 mmol/L HMF被全部转化,HMFCA产率和选择性分别为92%和89%。采用底物分批流加策略合成HMFCA,反应41.5 h后生成448 mmol/L目标产物。此外,E.coli/pET-CtVDH1细胞还能以HMF粗品(70%纯度)为底物合成HMFCA。糠醛、FFCA、5-甲氧甲基糠醛(MMF)、5-甲基糠醛和苯甲醛也可被E.coli/pET-CtVDH1细胞氧化生成相应的目标羧酸,产率为83-95%。将NOX分别与CtVDH1、CtVDH2、CtCALDH2和CtSAPDH在E.coli中进行共表达;系统地研究了各共表达重组菌催化HMF及其结构类似物(芳香醛)氧化特性。结果表明,E.coli-CtVDH1-NOX在HMF氧化中展现出最佳的催化性能,能在9 h内将250mmol/L HMF转化为HMFCA,其产率和选择性均为95%。E.coli-CtVDH2-NOX是催化糠醛和MMF选择性氧化的最适生物催化剂。当糠醛浓度为240 mmol/L时,反应24 h后FCA产率为90%。基于底物分批流加策略合成FCA,反应6 h后生成177 mmol/L目标产物,时空产率为3.3 g/L h。另外,当MMF浓度为240 mmol/L时,反应9 h后5-甲氧甲基-2-糠酸(MMFCA)产率为99%。采用底物分批流加策略合成MMFCA,反应8 h后生成287 mmol/L MMFCA,时空产率为5.6 g/L h。本研究不仅初步阐明了全细胞催化呋喃醛选择性氧化的反应特性、规律及相关醛脱氢酶的酶学性质,丰富了生物催化生物基呋喃醛高值化转化的理论知识,而且还建立了绿色、高效、高选择性制备呋喃羧酸的新工艺。生物催化将为清洁“生物炼制”的发展创造新的机遇。更多还原

【Abstract】 5-Hydroxymethylfurfural(HMF)and furfural are important biobased chemicals,which can be synthesized via carbohydrate dehydration.5-Hydroxymethyl-2-furancarboxylic acid(HMFCA)and 2-furoic acid(FCA)are the oxidation products of furan aldehydes above mentioned and have promising applications in polymer,pharmaceutical and food industries.Although significant advances in chemically catalytic oxidation of furan aldehydes into furan carboxylic acids have been achieved,these methods suffer from some problems,such as harsh reaction conditions,environmental infriendliness and unsatisfactory selectivities.Biocatalysis can overcome such drawbacks,which has been a good option to supplement and even replace chemical methods in synthetic chemistry.Especially for upgrading inherently unstable bio-based furans such as HMF and furfural,biocatalysis appears to be a preferred tool compared to chemical catalysis.Using of whole cells as biocatalysts in the redox reactions has attracted more interest than isolated enzymes,because they are more readily prepared,and the enzymes inside the cells are more stable under the protection of cell membranes.In addition,the regeneration of cofactors is easy in vivo.However,the substrates HMF and furfural are well-known potent inhibitors towards microorganisms and enzymes;thus,only limited biocatalysts were reported to show good substrate tolerance,and to enable efficient and selective oxidation of furan aldehydes.In this dissertation,a microbial strain Comamonas testosteroni SC1588 that is highly tolerant to HMF and is capable of selectively oxidizing HMF into HMFCA was isolated from soil samples.The genes of aldehyde dehydrogenases(ALDHs)from C.testosteroni SC1588 that are responsible for HMF oxidation were identified,heterologously expressed,purified and biochemically characterized.Subsequently,whole cells of recombinant E.coli harboring these ALDHs were used for catalytic oxidation of HMF.Moreover,NADH oxidase(NOX)and ALDHs were co-expressed in E.coli to promote the NAD~+regeneration,thus improving the selectivity of target products.An efficient and selective biocatalytic approach for the synthesis of furan carboxylic acids was developed.Based on the study on the substrate inhibition and toxicity against whole cells,it was shown that the HMF-tolerance level of C.testosteroni SC1588 was at least 100 mmol/L.The product HMFCA exerted significant inhibitory and toxic effects on this strain,which might be mainly attributed to the acidity of this compound,because its catalytic performances were highly dependent on pH.The catalytic performances of the strain were improved significantly by both adding histidine and tuning pH.Under the optimal reaction conditions,HMFCA was afforded in a yield of approximately 98%after 36 h at a substrate concentration of 160mmol/L.This strain was capable of selectively transforming furfural,5-methylfurfural and5-formyl-2-furancarboxylic acid(FFCA)to the target carboxylic acids with yields of 90-93%.The genes of aldehyde dehydrogenases including two vanillin dehydrogenases(CtVDH1and CtVDH2),two coniferyl aldehyde dehydrogenases(CtCALDH1 and CtCALDH2)and a3-succinoylsemialdehyde-pyridine dehydrogenase(CtSAPDH)that can catalyze oxidation of HMF were mined from the genome of C.testosteroni SC1588,cloned and heterologously expressed in E.coli.The subunit molecular masses of the five ALDHs were approximately 50kDa.CtCALDH1 was strictly dependent on NAD~+,while other four ALDHs were both NAD~+-and NADP~+-dependent.Optical pH and temperature of these ALDHs were 7.0 and25-35oC,respectively.Nonetheless,all the ALDHs exhibited unsatisfactory stability in vitro.The recombination ALDHs had different substrate specificity.CtCALDH1 and CtCALDH2exhibited low activities towards most aromatic aldehydes,and CtCALDH1 could not accept FFCA,5-methylfurfural and 4-formylbenzoic acid as substrates.CtVDH1 and CtVDH2exhibited satisfactory activities towards most substrates,while CtSAPDH displayed moderate activities.In general,benzylaldehydes appeared to be preferable substrates of these ALDHs compared to their structural analogs(furan aldehydes).Of recombinant E.coli strains expressing ALDHs tested,E.coli/pET-CtVDH1 displayed the highest catalytic efficiency in biocatalytic oxidation of HMF.Upon the reaction of 12 h,HMF of 200 mmol/L was completely converted,and HMFCA was affored in the yield and selectivity of 92%and 89%,respectively.HMFCA of 448 mmol/L was produced in 41.5 h by a fed-batch strategy.E.coli/pET-CtVDH1 could also biotransform crude HMF(70%of the purity)into HMFCA.In addition,various aldehydes such as furfural,FFCA,5-methoxymethylfurfural(MMF),5-methylfurfural and benzaldehyde were selectively transformed into the corresponding acids with yields of 83-95%by this biocatalyst.NOX was co-expressed with CtVDH1,CtVDH2,CtCALDH2 and CtSAPDH in E.coli,respectively.The catalytic performances of these recombinant cells in the oxidation of HMF and its structural analogs(aromatic aldehydes)were investigated.It was found that E.coli-CtVDH1-NOX showed the best catalytic performance in the oxidation of HMF,among these biocatalysts examined.HMF was converted into HMFCA with the yield and selectivity of both 95%within 9 h when the substrate concentration was 250 mmol/L.Of whole-cell biocatalysts tested,E.coli-CtVDH2-NOX gave the highest catalytic efficiency toward furfural and MMF.FCA was obtained in a yield of 90%within 24 h when the furfural concentration was 240 mmol/L.FCA of 177 mmol/L was produced in 6 h by a fed-batch strategy,and its productivity was 3.3 g/L h.Moreover,MMF was converted into5-methoxymethyl-2-furancarboxylic acid(MMFCA)in a yield of 99%within 9 h when its concentration was 240 mmol/L.MMFCA of 287 mmol/L was produced in 8 h by a fed-batch strategy,and its productivity was 5.6 g/L h.In this study,the catalytic performances of whole cells in selective oxidation of furan aldehydes as well as the enzyme properties of ALDHs were uncovered,which enriches the theoretical knowledge for biocatalytic valorization of biobased furan aldehyde.In addition,a green,efficient and highly selective biocatalytic approach for the synthesis of furancarboxylic acids was developed.Biocatalysis will creat new opportunities for the development of clean"biorefinery".更多还原