[Purification and characterization of glutamate dehydrogenase. from Corynebacterium glutamicum S9114].
Glutamate dehydrogenase (GDH) is a key enzyme in the biosynthesis of glutamate. The GDHs from Corynebacterium glutamicum S9114 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 S9114, similar to other microorganisms containing both GDHs. The NADPH-dependent and NADH-dependent GDH in Corynebacterium glutamicum S9114 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.
Wang
Yan
Y
School of Biotechnology, Southern Yangtze University, Wuxi 214036, China.
Song
Xiang
X
Yang
Ping-Ping
PP
Duan
Zuo-Ying
ZY
Mao
Zhong-Gui
ZG
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity
D009249:NADP; D005969:Glutamate Dehydrogenase