聚乙烯醇脱氢酶的提取及检测  

Extraction and detection of polyvinyl alcohol dehydrogenase

作　　者：李敏[1,2] 廖鲜艳[1] 堵国成[1] 陈坚[1] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,无锡214122 [2]内蒙古师范大学生命科学与技术学院,呼和浩特010022

出　　处：《生物加工过程》2009年第6期35-39,共5页Chinese Journal of Bioprocess Engineering

基　　金：教育部新世纪优秀人才支持计划资助项目(NCET-05-0488;NCET-07-0378);国家重点基础研究发展计划(973计划)资助项目(2007CB714036);国家杰出青年基金资助项目(20625619);教育部长江学者和创新团队计划资助项目(IRT0532)

摘　　要：为解决结合在细胞上的可溶性蛋白聚乙烯醇脱氢酶(PVADH)的检测困难问题,从提取及检测两方面对该酶进行研究,并对检测方法进行改进。结果表明,非离子型表面活性剂Triton X-100对可溶性蛋白PVADH的提取效果优于离子型表面活性剂烷基苯磺酸钠(LAS)和溴化十六烷基吡啶(CPB),酶活力比LAS和CPB提取后所得酶活力分别提高246.5%和831.3%。而非离子型表面活性剂中,Triton X-100与Tween 80相比,所得最高酶活提高了101.4%。Triton X-100浓度和提取时间对测定有明显影响,以1%Triton X-100提取18 h为宜,最高比酶活达14.9U/g。在PVADH检测体系中,加入电子受体启动反应比加入酶液与底物启动反应可使酶活性分别提高60.6%和126.5%;酶液与吡咯喹啉醌(PQQ)预先保温对检测该酶活性是十分重要的,可使酶活性提高59.1%。在检测系统中加入的KCN、CaC l2和PQQ的适宜浓度分别为1.0 mmol/L、0.5 mmol/L和2μmol/L,可使测定酶活分别提高37.1%、38.7%和214.0%。The extraction and the detection of cell-associated polyvinyl alcohol dehydrogenase （PVADH） were studied. Results showed that PVADH activity reached the maximum as extracted by non ionic surfactant Triton X-100, which were 246. 5% and 831.3% higher than those of ionic surfactant sodium alkyl benzene sulfonate （LAS） and cetylpyridinium bromide （CPB）. As for non ionic suffactants, PVADH ac- tivity was enhanced by 101.4% compared with that of Tween 80. The concentration and the extraction time obviously affected PVADH activities and the appropriate values were 1% and 18 h, respectively. Under the condition, the maximum PVADH activity of 14. 9 U/g was obtained. In PVADH detection system, PVADH activity determined by using the electron acceptors as the starter was enhanced by 60. 6% and 126. 5% compared with that of enzyme solutions and the substrate. It was very important for the detection of PVADH activity keeping enzyme solutions and pyrroloquinoline quinone （PQQ） at 30 ℃ for 10 min before determination and 59. 1% of the activity was increased. The suitable eoneentrations of KCN, CaCl2 and PQQ were 1.0 mmol/L, 0. 5 mmol/L and 2 μmol/L, and 37. 1%, 38.7% and 214. 0% of PVADH activity were enhanced with addition of them in the detection system.

关 键 词：脱氢酶 提取 检测 聚乙烯醇 表面活性剂 

分 类 号：TQ92[轻工技术与工程—发酵工程]