酵母3-脱氧葡糖醛酮代谢酶的分离纯化及部分性质

3-脱氧葡糖醛酮 ( 3- deoxyglucosone)是美拉德反应的主要中间产物 ,对生物体具有毒性作用 .用硫酸铵分部沉淀、DEAE- cellulose52、Hydroxyapatite、DEAE- Sepharose CL- 6B柱层析从酿酒酵母 YBr-M( S.cerevisiae YBr-M)抽提液中分离纯化了 3-脱氧葡糖醛酮代谢酶 (以 NADPH为辅酶 ) .该酶是单一的分子 ,分子量为 44k D,反应最适 p H为 7.0 ,p H6.0～ 8.0之间酶活性相对稳定 ,以 3-脱氧葡糖醛酮为底物的米氏常数 Km 为 2 .2 5mmol/ L.在 35℃以下保温 30 min酶活不变 ,50℃保温 30 min后酶活损失 50 % .该酶对二羰基化合物的活性较高 ,对单羰基化合物则较低 ,其催化作用受碘乙酸、N-乙基顺丁烯二酰亚胺的抑制 ,而被β-巯基乙醇、二硫苏糖醇激活 ,催化作用必须以 NADPH为专一辅酶 ,当用 NADH代替 NADPH时 ,活力只有 5.3% .

Purification and Properties of 3-Deoxyglucosone Metabolizing Enzyme from S. cerevisiae Y_(Br-M)

Deoxyglucosone, a highly reactive and toxic compound, is a major intermediate in the Maillard reaction. An NADPH dependent 3 deoxyglucosone metabolizing enzyme from S. cerevisiae Y Br M was isolated and purified by ammoniun sulfate fractionation, DEAE cellulose 52 column chromatography, Hydroxyapatite column chromatography and DEAE sepharose CL 6B column chromatography. The molecular weight of the enzyme was estimated to be 44 000 dalton and the enzyme was a monomer. The optimum pH of the enzyme activity was 7 0. The K m for 3 deoxyglucosone was 2 25 mmol/L. The enzyme was stable at pH6 0-8 0. The enzyme was stable at 35℃. It lost 50% of its activity when incubated at 50℃ for 30 min. 2 Oxoaldehyde compounds were found to be good substrates and monocarbonyl compounds were poor substrates for this reductase. The enzyme activity was inhibited by iodoacetic acid and N ethylmaleimide; β Mercaptoethanol and dithiothreitol activated the activity of enzyme. The enzyme that specifically required NADPH was a coenzyme for activity and was inactive when NADH was substituted for NADPH.