文献类型：期刊文献

中文题名：没食子酸脱羧酶的分离与纯化

英文题名：Separation and Purification of Gallic Acid Decarboxylase

作者：李文君[1] 王成章[1]

第一作者：李文君

机构：[1]中国林业科学研究院林产化学工业研究所生物质化学利用国家工程实验室国家林业局林产化学工程重点开放性实验室江苏省生物质能源与材料重点实验室

年份：2016

卷号：28

期号：7

起止页码：1109-1115

中文期刊名：天然产物研究与开发

外文期刊名：Natural Product Research and Development

收录：CSTPCD;;北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；

基金：国家863项目(2014AA021802);国际合作项目(中俄项目2014DFR31300)

语种：中文

中文关键词：没食子酸脱羧酶;微生物降解;焦性没食子酸;结构表征

外文关键词：gallic acid decarboxylase; microbial degradation; pyrogallic acid; structural characterization

分类号：Q814.1

摘要：本实验研究了没食子酸脱羧酶（Gallicaciddecarboxylase，GAD）的提取、分离、分析及结构表征。建立了考马斯亮蓝法（Y=4．8748X-0．0255，帮=0．9922）和双缩脲法（Y=0．2476X＋0．0003，R2=0．9993）测定GAD蛋白含量的线性方程。对GAD粗酶液，先采用pH为6，60％／70％的（NH。）2S0。盐沉淀32h；然后，利用35kD透析膜处理料液12—16h；再将透析液经二乙氨基乙基（DiethylAminoethanol，DEAE）纤维素树脂，在pH为6．5时饱和吸附量可达2．838mg／g，采用0．4mol／LNaCl溶液进行洗脱的洗脱液经过G-100葡聚糖凝胶纯化。采用SDS．PAGE聚丙烯酰胺电泳分离得到蛋白样品条带，MALDI-TOF-MS进行结构表征，得到的蛋白分子质量45．62kD，等电点5．19，与已知蛋白质gi／334732950匹配度为53，说明两者具有相似性，但匹配到的序列组仅占蛋白全序列的3％，初步推测GAD可能是一种新的酶。
This paper mainly studied the extraction,separation,analysis and structural characterization of gallic acid de- earboxylase（GAD）. The linear regression equation was established to determine GAD content by Coomassie brilliant blue method （ Y = 4. 8748X-0. 0225, R2 = 0. 9922 ） and biuret colorimetry method （ Y = 0. 2476X ＋ 0. 0003,R2 = 0. 9993 ）. 60% and 70% Ammonium sulfate was used to purify crude enzyme solution twice for 32 h with pH 6;then ,35 kD dialysis membrane was used to filter the solution for 12-16 h;DEAE cellulose resin was applied to sequentially purify the solution and found the saturated adsorption amount of resin was 2. 838 mg/g with the buffer of pH 6.5 ; G-100 Sepha- dex gel was selected to further purify enzyme extract in the present of 0.4 mol/L NaC1 as the eluent. SDS-PAGE polyac- rylamide gel electrophoresis was used to separate the protein and MALDI-TOF-MS was applied to analyze and identify its structure：the molecular weight was 45.62 kD, isoelectric point 5.19, matching to protein gi/334732950, it showed they had similar properties, but because matching rate was only 3%, hence it was supposed that GAD could be a new protein.