木糖异构酶酿酒酵母孢子“微胶囊”的构建及酶学性质分析  

Yeast spores function as microcapsules for enzyme immobilization

作　　者：李毅[1] 李子杰[1] 中西秀树[1] 高晓冬[1] 

机构地区：[1]江南大学生物工程学院糖化学与生物技术教育部重点实验室

出　　处：《食品与发酵工业》2015年第4期6-11,共6页Food and Fermentation Industries

基　　金：国家自然科学基金(21302069);中国博士后科学基金(2014M551500)

摘　　要：将来源于嗜热细菌Thermus thermophilus HB8的木糖异构酶(XI)基因xyl A与SPR1信号肽序列(ss)融合后连接到p RS424-TEFpr表达载体上,将重组质粒p RS424-TEFpr-ss-xyl A转化酿酒酵母AN120野生型菌株、osw2Δ缺陷型菌株以及dit1Δ缺陷型菌株并进行产孢。通过荧光定位、蛋白免疫印迹分析以及酶活测定,表明酿酒酵母孢子"微胶囊"固定化酶系统构建成功。由于该固定化酶的最佳反应温度为85℃,避免了酿酒酵母孢子在D-葡萄糖为底物条件下萌发的弊端。同时,根据蛋白免疫印迹分析和重复利用性结果,表明二酪氨酸层的存在对于酶的包埋,对高温的耐受性都表现出了较好的特性。此外对野生型和osw2Δ孢子固定的XI的酶学性质进行了研究,相对于游离酶,孢子固定化酶更加稳定。在p H为6的酸性条件或p H为9的碱性条件下,固定化酶相对酶活能达到60%以上,同时在温度95℃时,固定化的酶相对酶活也能达到60%以上。与野生型孢子相比,osw2Δ孢子展现出了潜在的优势,既可以有效阻止酶的释放,又提高了底物分子的通透性。The gene xyl A encoding xylose isomerase from Thermus thermophilus HB8 was fused with SPR1 signal sequence and inserted into p RS424-TEFpr vector. The recombinant plasmid p RS424-TEFpr-ss-xyl A was transformed into yeast AN120 wild type strain,osw2Δ and dit1Δ strain and sporulate,respectively. The results of GFP localization,western blot analysis and enzyme assay demonstrated that the system of yeast spores as microcapsules for enzyme immobilization has been successfully established. As the optimal temperature of immobilized XI was 85 ℃,which avoided the disadvantage of spore germination in the presence of D-glucose as the substrate. According to the analysis of western bolt and reusability,it demonstrated that the dityrosine layer showed excellent properties for enzyme entrappment and thermostability. In addition,the enzymatic properties of XI immobilized on wild type and osw2Δ spores were investigated. Compared with free enzyme,the stability of immobilized enzyme on spores was obviously improved.At the acidic condition（ p H = 6. 0） or alkaline condition（ p H = 9. 0）,the relative activity of immobilized enzyme on yeast spores was above 60 % of the highest activity. Moreover,immobilized XI still had 60% of the highest activity at a high temperature of 95 ℃. Compared with wild type spores,osw2Δ spores showed a potential advantage that the dityrosine layer could not only prevent the release of enzyme,but also improved the interaction between the substrate and enzyme.

关 键 词：酿酒酵母 孢子 木糖异构酶 酶固定化 

分 类 号：TS261.11[轻工技术与工程—发酵工程]