Characterization of Peptostreptococcus asaccharolyticus Glutamate Dehydrogenase Purified by Dye-ligand Chromatography

Hornby, D.; Engel, Paul C.

doi:10.1099/00221287-130-9-2385

Cited by 13 publications

(16 citation statements)

References 24 publications

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“…The elution of the NAD-GDH from the anion-exchange column, followed by direct application onto the Blue-Sepharose CL-6B column, was crucial for the puri¢cation procedure. Triazine dyes have proved useful for the isolation of NADdependent enzymes [9]. The ¢nal preparation had a speci¢c activity of 70 U mg 3I and the enzyme was puri¢ed 350-fold.…”

Section: Resultsmentioning

confidence: 99%

NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8: purification and enzymatic properties

Ruiz

1998

FEMS Microbiology Letters

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An NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8 was purified 350-fold by a several-step procedure involving Blue-Sepharose chromatography. The native protein had a molecular mass of approximately 289 kDa, and consisted of six subunits with a molecular mass of 48 kDa each. The optimum pH for the deaminating reaction was 8.0. The optimum temperature was around 85^90³C. NAD-glutamate dehydrogenase showed a high coenzyme specificity, catalysed preferentially glutamate catabolism and presented K m values for NAD and L-glutamate of 0.27 þ 0.03 mM and 49 þ 10 mM, respectively. No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. Enzyme activity was found to be very stable at 72³C. Differential scanning calorimetry revealed a t m of 95³C for denaturation. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

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Section: Resultsmentioning

confidence: 99%

NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8: purification and enzymatic properties

Ruiz

1998

FEMS Microbiology Letters

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“…Although the P. laminosum enzyme was purified more than 4,400-fold, the preparation was not homogeneous when analyzed by polyacrylamide gel electrophoresis, indicating that this protein was present in a very low concentration even in ammonium-grown cells. However, the purified enzymes from other sources showed lower specific activities (between 3.5 and 8.5 U/mg of protein) (11,17) and were less highly purified (78-and 6-fold, respectively). Our enzyme recovery compares to that of the enzyme from the fungus Cenococcum graniforme (25) but is much lower than that of Chlorella spp.…”

Section: Discussionmentioning

confidence: 99%

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

et al. 1988

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The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-flxing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30°C it showed a specific activity of 32.9 gimol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (K,m = 1.25 mM) and NADPH (Km = 64 ,uM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [SJ0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60°C (Ea = 5.1 kcal/ mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.

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“…Moreover, this gene has already been successfully cloned with its own promoter in Escherichia coli, in which it was highly expressed (28). In P. asaccharolyticus GDH acts during hydroxyglutarate fermentation of glutamate, in which its role consists of degrading glutamate (11).…”

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confidence: 99%

Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

Rijnen

Courtin

Gripon

et al. 2000

Appl Environ Microbiol

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The first step of amino acid degradation in lactococci is a transamination, which requires an ␣-keto acid as the amino group acceptor. We have previously shown that the level of available ␣-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding ␣-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce ␣-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added ␣-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added ␣-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding ␣-ketoglutarate to improve aroma development in cheese.

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Characterization of Peptostreptococcus asaccharolyticus Glutamate Dehydrogenase Purified by Dye-ligand Chromatography

Cited by 13 publications

References 24 publications

NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8: purification and enzymatic properties

NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8: purification and enzymatic properties

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

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