类产碱假单胞菌谷氨酸脱氢酶的提纯、鉴定及某些特性的初步研究

从类产碱假单胞菌纯化出电泳纯的谷氨酸脱氢酶，用聚丙烯酰胺梯度凝胶电泳和ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为２９０ ｋＤ，亚基分子量为４７ ｋＤ，提示该酶为六聚体．该酶对ＮＡＤＰ（Ｈ）和底物均具有高度专一性，对谷氨酸、α－酮戊二酸及ＮＡＤＰ＋ 的Ｋｍ 值分别为：２８ ｍ ｍ ｏｌ／Ｌ、１．２ｍ ｍ ｏｌ／Ｌ及０．０６３ ｍ ｍ ｏｌ／Ｌ．用Ｈｉｌｌ作图法求得酶对ＮＨ＋４ 和ＮＡＤＰＨ 的［Ｓ］０．５分别为２４ ｍ ｍ ｏｌ／Ｌ和０．０３７ ｍ ｍ ｏｌ／Ｌ．最适反应温度为５０℃，催化氨化反应和脱氨反应的最适ｐＨ 分别为８．０和８．８，在热稳定性方面不及嗜热细菌的谷氨酸脱氢酶稳定．提纯的谷氨酸脱氢酶在低温（４℃）条件下，可在Ｔｒｉｓ－ＨＣｌ缓冲液中贮存半年以上，活力无明显下降，冷冻则可导致纯酶液迅速失活．氮源对菌体谷氨酸脱氢酶水平有显著影响．

Purification, Identification and Some Properties of Glutamate Dehydrogenase from Pseudomonas Pseudoalcaligenes

Glutamate dehydrogenase (GDH) from Pseudomonas pseudoalcaligenes was purified to homogeneity. The molecular size and the subunit size estimated by native gradient PAGE and SDS PAGE were 290 kD and 47 kD respectively, indicating that the GDH was a hexamer with identical subunits. The enzyme was highly specific for NADP(H) and the substrates, and showed K m values as follows: glutamate, 28 mmol/L, α ketoglutarate, 1 2 mmol/L and NADP +, 0 063 mmol/L. Hill plots gave [S] 0 5 of 24 mmol/L for ammonia and 0 037 mmol/L for NADPH. The maximal activity was obtained at 50℃ and the optimal pH values were 8 0 and 8 8 for amination and deamination, respectively. The enzyme was relatively unstable to heat as compared with the GDHs from thermophilic bacteria. Experiments also revealed that the purified GDH could be stored at 4℃ in Tris HCl buffer for more than six months and had no obvious loss of activity, but freezing would result in rapid inactivation of it. Nitrogen source had a significant effect on the intracellular level of GDH.