酿酒酵母孢子“微胶囊”表面展示乙醛脱氢酶以及检测乙醛的应用  

Displaying of acetaldehyde dehydrogenase on surface of“microcapsules”of Saccharomyces cerevisiae spores and applications for detection of acetaldehyde DU Xiangkun,FEI Kangqing,WANG Yasen,BAI Jiawen,

作　　者：杜祥坤 费康清 王亚森 白佳文 中西秀树[1] 李子杰[1] DU Xiangkun;FEI Kangqing;WANG Yasen;BAI Jiawen;NAKANISHI Hideki;LI Zijie(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2024年第22期318-326,共9页Food and Fermentation Industries

基　　金：国家自然科学基金(32171475)。

摘　　要：乙醛是对人体有毒的化合物,乙醛脱氢酶(acetaldehyde dehydrogenase,ALDH)能将乙醛氧化为无毒的乙酸,同时将NAD(P)+转化为NAD(P)H。目前检测乙醛的方法繁琐且耗时费力,该研究采用酿酒酵母孢子“微胶囊”表面展示热带假丝酵母(Candida tropicalis)LBBE-W1来源的乙醛脱氢酶,建立一种新颖快速检测乙醛的方法。首先,基于蛋白质免疫印迹与荧光结果证实ALDH能够在孢子中表达且定位在孢子壁上。然后,测定了孢子表面展示ALDH与游离ALDH的酶学性质。结果显示,孢子表面展示ALDH和游离酶的最适温度与pH值分别为40℃和9.0;与游离酶相比,孢子表面展示ALDH具有更高的活性,以及更佳的温度与pH稳定性。此外,孢子表面展示ALDH对十二烷基硫酸钠(sodium dodecyl sulfate,SDS)、蛋白酶K具有更强的耐受性;孢子表面展示ALDH与游离乙醛脱氢酶的最适底物都是乙醛,并且孢子表面展示ALDH具有良好的重复使用性能。最后,将孢子表面展示ALDH作为生物传感器检测乙醛,结果表明在0~500μmol/L的乙醛浓度范围内具有良好的线性关系,其R2值为0.9992。该研究成功构建一种新颖的生物传感器,为乙醛的检测提供一种新的方法。Acetaldehyde is a toxic compound to the human body.Acetaldehyde dehydrogenase(ALDH)can oxidize acetaldehyde to non-toxic acetic acid and convert NAD(P)+to NAD(P)H.At present,the methods for acetaldehyde detection are complicated and time-consuming.In this study,acetaldehyde dehydrogenase derived from Candida tropicalis LBBE-W1 was displayed on the surface of“microcapsules”of Saccharomyces cerevisiae spores to establish a novel and rapid detection method for acetaldehyde.Firstly,it was confirmed that ALDH could be expressed and localized on the spore wall based on western blotting and fluorescence results.Then,the enzymatic properties of ALDH on the spore surface and free ALDH were determined.Results showed that the optimum temperature and pH for ALDH displayed on spore surface and free enzyme were 40℃and 9.0,respectively.Compared with free enzyme,ALDH displayed on spore surface showed higher activity and better temperature and pH stability.In addition,ALDH on the surface of spores had stronger tolerance to SDS and protease K.The most suitable substrate for both ALDH on spore surface and free enzyme was acetaldehyde,and ALDH on spore surface showed good reuse performance.Finally,ALDH displayed on the spore surface was used as a biosensor to detect acetaldehyde.The results showed a good linear relationship in the concentration range of 0-500μmol/L with R 2 value of 0.9992.In this study,a novel biosensor was successfully constructed to provide a new method for acetaldehyde detection.

关 键 词：孢子表面展示 乙醛脱氢酶 生物传感器 酿酒酵母 乙醛检测 

分 类 号：O657.3[理学—分析化学]