许令侠,梁喆,孙建中,朱道辰

• 1:江苏大学环境与安全工程学院生物质能源研究院

摘要(Abstract)：

针对化学合成香草酸会造成环境污染、活性低，从植物中提取香草酸产量低、生产成本过高等问题，采用来源于Bacillus ligniniphilus L1的香草醛脱氢酶进行香草酸的生物转化。为了更好地提高酶促反应效率，在反应体系中添加适量的低共熔溶剂(DES,氯化胆碱∶1,4-丁二醇为1∶3)可以有效提升酶活并使其保持较高的稳定性。随即考察了反应温度、反应pH、反应时间和DES添加量对香草酸转化率的影响，并通过响应面实验进行工艺研究，得到最佳的转化工艺参数为反应温度36℃、反应pH 6.8、DES添加量17%,在此条件下香草酸的转化率达到84.57%,与未添加DES相比，转化率提升了37.98%。采用DES辅助酶法进行香草酸的生物转化，可以有效地提升香草醛脱氢酶的酶活和香草酸的转化率，制备过程绿色、安全、经济，符合香草酸绿色工业化生产的要求。

关键词(KeyWords)： 香草酸;低共熔溶剂;香草醛脱氢酶;生物转化;响应面优化

Abstract:

Keywords:

基金项目(Foundation): 江苏省研究生科研与实践创新计划项目(KYCX20＿3044)

作者(Author): 许令侠,梁喆,孙建中,朱道辰

参考文献(References)：

• [1]KAUR J,GULATI M,SINGH S K,et al.Discovering multifaceted role of vanillic acid beyond flavours:nutraceutical and therapeutic potential[J].Trends in Food Science & Technology,2022,122:187-200.
• [2]龚玉圆.香草酸抗耐甲氧西林金黄色葡萄球菌活性及生物被膜机制研究[D].宜昌：三峡大学，2022.
• [3]ALI W I,AL-ABBASY O Y,YOUNIS S A.Vanillic acid:an antioxidant used in preventing browning process in pear (Pyrus communis L.) juice[J].Annals of the Romanian Society for Cell Biology,2021,25(3):5272-5285.
• [4]SHARMA N,KHURANA N,MUTHURAMAN A,et al.Pharmacological evaluation of vanillic acid in rotenone-induced Parkinson's disease rat model[J].European Journal of Pharmacology,2021,903(12):174112.
• [5]JUNG Y,PARK J,KIM H L,et al.Vanillic acid attenuates testosterone-induced benign prostatic hyperplasia in rats and inhibits proliferation of prostatic epithelial cells[J].Oncotarget,2017,8(50):87194-87208.
• [6]钱伟.一种香草酸连续化生产设备及生产方法：中国，CN108129298A[P].2018-06-08.
• [7]LUDWIKOWSKI T M,WAGNER A O,MARGESIN R.Bioconversion of ferulic acid and vanillin to vanillic acid by cold-adapted Paraburkholderia aromaticivorans AR20-38:impact of culture conditions[J].Annals of Microbiology,2023,73(1):1-17.
• [8]KHOYRATTY S,KODJA H,VERPOORTE R.Vanilla flavor production methods:a review[J].Industrial Crops and Products,2018,125:433-442.
• [9]ASHENGROPH M,NAHVI I,ZARKESH-ESFAHANI H,et al.Novel strain of Bacillus licheniformis SHL1 with potential converting ferulic acid into vanillic acid[J].Annals of Microbiology,2012,62(2):553-558.
• [10]李金涛，张明祖，何金林，等.低共熔溶剂在高分子合成中的应用[J].化学进展，2022,34(10):2159-2172.
• [11]李凌娜，杨继威，张丽芬，等.响应面法优化低共熔溶剂提取迷迭香中迷迭香酸和鼠尾草酸的工艺[J].食品工业科技，2023,44(16):218-227.
• [12]SHANG X,DOU Y,ZHANG Y,et al.Tailor-made natural deep eutectic solvents for green extraction of isoflavones from chickpea (Cicer arietinum L.) sprouts[J].Industrial Crops and Products,2019,140:111724.
• [13]LIN J J,XIANG H,SUN-WATERHOUSE D X,et al.Deep eutectic solvents and alkaline extraction of protein from seabuckthorn seed meal:a comparison study[J].Food Science and Human Wellness,2022,11(4):1028-1035.
• [14]李利芬.基于氯化胆碱低共熔溶剂的木质素提取改性和降解研究[D].哈尔滨：东北林业大学，2015.
• [15]刘乾静，陈晓淼，王芷，等.低共熔溶剂预处理杨木水解渣拆解木质素[J].化工进展，2022,41(10):5612-5618.
• [16]仝争，王渝，何晓希，等.低共熔溶剂在生物催化中的应用研究进展[J].化学试剂，2022,44(12):1723-1730.
• [17]GORKE J,KAZLAUSKAS S R.Toward advanced ionic liquids.Polar,enzyme-friendly solvents for biocatalysis[J].Biotechnology & Bioprocess Engineering,2010,15(1):40-53.
• [18]DANESHJOU S,KHODAVERDIAN S,DABIRMANESH B,et al.Improvement of chondroitinases ABCI stability in natural deep eutectic solvents[J].Journal of Molecular Liquids,2017,227:21-25.
• [19]XU P,CHENG J,LOU W Y,et al.Using deep eutectic solvents to improve the resolution of racemic 1-(4-methoxyphenyl)ethanol through Acetobacter sp.CCTCC M209061 cell-mediated asymmetric oxidation[J].RSC Advances,2014,5(9):6357-6364.
• [20]徐万军.低共熔溶剂辅助糖苷酶转化黄酮苷的研究[D].重庆：重庆大学，2019.
• [21]LINDBERG D,REVENGA M D L F,WIDERSTEN M.Deep eutectic solvents (DESs) are viable cosolvents for enzyme-catalyzed epoxide hydrolysis[J].Journal of Biotechnology,2010,147(3-4):169-171.
• [22]ZHAO K H,CAI Y Z,LIN X S,et al.Enzymatic synthesis of glucose-based fatty acid esters in bisolvent systems containing ionic liquids or deep eutectic solvents[J].Molecules,2016,21(10):1294.
• [23]ZHU D,XU L,SETHUPATHY S,et al.Decoding lignin valorization pathways in the extremophilic Bacillus ligniniphilus L1 for vanillin biosynthesis[J].Green Chemistry,2021,23:9554-9570.
• [24]司海兵.嗜碱耐盐菌Bacillus ligniniphilus L1解聚木质素生成香草酸的代谢途径解析[D].镇江：江苏大学，2020.
• [25]张雪莹.睾丸酮丛毛单胞菌醛脱氢酶的挖掘、性质及其催化呋喃醛选择性氧化的研究[D].广州：华南理工大学，2019.
• [26]王艳玲，陈小燕，余强，等.低共熔溶剂在生物转化中的应用[J].新能源进展，2021,9(3):211-217.
• [27]YANG Z,PAN W.Ionic liquids:green solvents for nonaqueous biocatalysis[J].Enzyme & Microbial Technology,2005,37:19-28.
• [28]WAITE S L,LI H,PAGE A J.NO2 solvation structure in choline chloride deep eutectic solvents-the role of the hydrogen bond donor[J].Journal of Physical Chemistry B,2018,122(15):4336-4344.
• [29]XU W J,HUANG Y K,LI F,et al.Improving β-glucosidase biocatalysis with deep eutectic solvents based on choline chloride[J].Biochemical Engineering Journal,2018,138:37-46.
• [30]KOTOGáN A,KECSKEMéTI A,SZEKERES A,et al.Characterization of transesterification reactions by Mucoromycotina lipases in non-aqueous media[J].Journal of Molecular Catalysis B:Enzymatic,2016,127:47-55.
• [31]YANTA H,BELL E.The linkage between citrate,pH regulation and protein folding in glyoxysomal malate dehydrogenase[J].The FASEB Journal,2012,26(S1):581.
• [32]JUNEIDI I,HAYYAN M,HASHIM M A,et al.Pure and aqueous deep eutectic solvents for a lipase-catalysed hydrolysis reaction[J].Biochemical Engineering Journal,2017,117:129-138.