摘要
设计并优化了从猪心中提取高纯度苹果酸脱氢酶的技术路线,采用组织破碎、二度硫酸铵盐析、DEAE Sepharose F.F.离子交换层析、Phenyl Sepharose 6 F.F.疏水层析及Sephadex G-75Fine凝胶过滤等方法进行分离纯化,苹果酸脱氢酶比酶活达1 212.97 U/mg,纯化倍数达122.64倍,酶活力收率为53.64%。Sephadex G-75 Fine凝胶过滤和SDS-PAGE电泳结果显示纯化的苹果酸脱氢酶相对分子质量约为69 000,含有2个亚基,每个亚基的相对分子质量约为34 000。以制备的苹果酸脱氢酶和谷草转氨酶配成双酶反应体系,对葡萄酒中L-苹果酸的含量进行了测定;通过标准样品和葡萄酒样品精密度及回收率（93.2%～104%）实验。
The extraction of Malate dehydrogenase（MDH） from Swine heart was designed and optimized, by tissue crushing, ammonium sulfate duel-precipitation and purification through DEAE Sepharose F. F, Phenyl Sepharose 6 F. F. hydrophobie chromatography and Sephadex G- 75 Fine. The final specific activity of MDH was up to 1 212.97 U/mg and 122.64-fold purification was obtained with enzymatic activity recovery of 53.64%. The molecular weight of malate dehydrogenase was 69 000, consists of two subunits , and the moleeular weight of single subunits was about 34 000 by analysis from Sephadex G-75 Fine gel and SDS-PAGE. A new method was developed for assaying I.-malie acid in wine by MDH and GOT dual-enzymatic system. The assay was linear over L malic acid of 0. 005 to 0.3 g/L at the temperature of 25 ℃ and detective wavelength at 340 nm. It was proved to be simply and accurately applicable to L- malic acid determination in wine based on the precision and recovery（93.2%-104%）.
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2008年第5期57-61,共5页
Journal of Food Science and Biotechnology
基金
国家863计划项目(2006AA10Z305)
关键词
苹果酸脱氢酶
柱层析
纯化
L-苹果酸测定
Malate dehydrogenase
chromatography
purification
L-malic acid determination
