摘要
谷氨酸脱氢酶 (GDH)是谷氨酸生物合成的关键酶 ,谷氨酸棒杆菌S91 1 4是目前我国味精工业应用最广泛的生产菌种 ,其谷氨酸脱氢酶的研究尚未见报道。分离纯化该菌中的谷氨酸脱氢酶 ,研究其辅酶组成 ,对揭示谷氨酸脱氢酶的分子结构和性质 ,提高谷氨酸产率很有必要。将培养至对数期中期的细胞离心收集并用含适量DTT、ED TA的Tris_HCl缓冲液 (pH 7 5 )洗涤 ,用Frenchpressurecellpress破碎 ,离心去除菌体碎片得无细胞抽提液。然后使用 KTA_10 0快速纯化系统经DEAE_纤维素柱、疏水柱 (HIC)、G_2 0 0凝胶过滤柱层析得到纯化大约 70倍的以NAD PH为辅酶的GDH和部分纯化的以NADH辅酶的GDH。这两个酶分别对NADPH、NADH高度专一 ,不能相互代替。经HPLC和SDS_PAGE测得前一种酶的分子量和亚基分子量分别为 188kD和 32kD ,表明该酶为具有相同亚基的六聚体。酶活性测定使用HITACHIU_30 0 0分光光度计利用NAD(P)H在 340nm氧化的初速度进行。蛋白质含量测定利用Bradford方法进行 ,并以牛血清白蛋白为标准蛋白。纯化结果表明S91 1 4中确实存在两种GDH ,其中以NADH为辅酶的GDH尚未见报道。和某些具有两种GDH的微生物一样 ,S91 1 4可能也是以NADPH为辅酶的GDH参与谷氨酸的合成代谢 ,以NADH为辅酶的GDH参与谷氨酸的分解代谢。
Glutamate dehydrogenase (GDH) is a key enzyme in the biosynthesis of glutamate. The GDHs from Corynebacterium glutamicum S 9114, the most commonly used strain in glutamate fermentation, were purified and their molecular structures and properties characterized. The coenzymes were also studied in the hope to increase glutamate production. Cells were harvested at mid_exponential phase by centrifugation and washed with Tris_HCl buffer containing DTT and EDTA (pH 7 5). The cells were then disrupted using a French pressure cell press and the supernatant was collected by centrifugation. The extract was concentrated by 70_fold using the AKTA_100 FPLC system employing a DEAE_cellulose ion exchange column, a hydrophobic interaction chromatography (HIC) and Sephadex G_200 gel filtration. The purified extracts contained NADPH_dependent GDH and NADH_dependent GDH. Both of the enzymes were highly specific for the coenzymes. The molecular masses of the NADPH_dependent GDH and its subunit were 188kD and 32kD respectively, suggesting the enzyme is a homo_hexamer. Our data reported for the first time the presence of NADH_ dependent GDH in Corynebacterium glutamicum S 9114, similar to other microorganisms containing both GDHs. The NADPH_dependent and NADH-dependent GDH in Corynebacterium glutamicum S 9114 may participate in the assimilation and dissimilation of ammonia respectively. The absorptions of NADPH_dependent GDH was very weak at 280nm but very high at 215nm, suggesting a low phenylalanine and tyrosine content in the enzyme.
出处
《生物工程学报》
CAS
CSCD
北大核心
2003年第6期725-729,共5页
Chinese Journal of Biotechnology
