The artificial evolution of an enzyme by random mutagenesis: the development of formaldehyde dehydrogenase

A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction. This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase. The activity at 29 degrees C of the variant was 1.7-fold higher than that of the wild type. The K(m) values of the mutant at 37 degrees C were 0.40 mM for NAD(+) and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD(+) and 2.1 mM for formaldehyde. The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme. The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 degrees C, and 47 degrees C, respectively. The thermal stability of the mutant was lower than that of the wild type.     

The Sulfolobus tokodaii gene ST1704 codes highly thermostable glucose dehydrogenase

NAD(P)-dependent glucose-1-dehydrogenase (GDH) has been used for glucose determination and NAD(P)H production in bioreactors. Thermostable glucose dehydrogenase exhibits potential advantage for its application in biological processes. The function of the putative GDH gene (ST1704, 360-encoding amino acids) annotated from the total genome analysis of a thermoacidophilic archeaon Sulfolobus tokodaii strain 7 was investigated to develop more effective application of GDH. The gene encoding S. tokodaii GDH was cloned and the activity was expressed in Escherichia coli, which did not originally possess GDH. This shows that the gene (ST1704) codes the sequence of GDH. The enzyme was effectively purified from the recombinant E. coli with three steps containing a heat treatment and two successive chromatographies. The native enzyme (molecular mass: 160 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme utilized both NAD and NADP as the coenzyme. The maximum activity for glucose oxidation in the presence of NAD was observed around pH 9 and 75 °C in the presence of 20 mM Mg²⁺. The enzyme showed broad substrate specificity: several monosaccarides such as 6-deoxy- -glucose, 2-amino-2-deoxy- -glucose and -xylose were oxidized as well as -glucose as the electron donor. -Mannose, -ribose and glucose-6-phosphate were inert as the donor. The enzyme showed high thermostability: remarkable loss of activity was not observed up to 80 °C by incubation for 15 min at pH 8.0. In addition, the enzyme was stable in a wide pH range of 5.0–10.5 by incubation at 37 °C. From the steady-state kinetic analysis, the enzyme reaction of -glucose oxidation proceeds via a sequential ordered Bi–Bi mechanism: NAD and -glucose bind to the enzyme in this order and then -glucono-1,5-lactone and NADH are released from the enzyme in this order. The amino acid sequence alignment showed that S. tokodaii GDH exhibited high homology with the Sulfolobus solfataricus hypothetical glucose dehydrogenase and a Thermoplasma acidophilum one.     

Glutamate dehydrogenase activity profiles for type strains of ruminal Prevotella spp.

The glutamate dehydrogenase (GDH) activities for the type strains of Prevotella ruminicola (strain 23), Prevotella brevis (strain GA33), and Prevotella bryantii (strain B(1)4) were assessed by a combination of enzyme assays and analysis of migration patterns of GDH proteins following nondenaturing polyacrylamide gel electrophoresis. Unlike results with most other prokaryotes, but similar to results with other members of the family Bacteroidaceae, NADPH-utilizing specific activity was greatest in all species following ammonia-limited growth. Similar also to previous findings with P. bryantii, the NAD(P)H-utilizing GDH activity of P. ruminicola can be attributed to a single protein. However, P. brevis produces an additional GDH protein(s) in response to growth with peptides. These results conclusively demonstrate that all type strains of the ruminal Prevotella sp. grouping possess GDH activity.     

Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: evidence for two distinct enzyme forms.

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37 degrees C body temperature) and hibernating (torpid, 5 degrees C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5+/-2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335+/-5 kDa for euthermic and 320+/-5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and alpha-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5 degrees C). For example, the Km for alpha-ketoglutarate of euthermic GDH was higher at 5 degrees C (3.66+/-0.34 mM) than at 37 degrees C (0.10+/-0.01 mM), whereas hibernator GDH had a higher affinity for alpha-ketoglutarate at 5 degrees C (Km was 0.98+/-0.08 mM at 37 degrees C and 0.43+/-0.02 mM at 5 degrees C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37 degrees C and weakest with hibernator GDH at 5 degrees C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.     

Lactate dehydrogenase from gastrocnemius muscle of turtle

Five bands of lactate dehydrogenase (LDH) isoenzymes were seen by polyacrylamide gel electrophoresis in gastrocnemius muscle of the turtle (Kachuga smithi). The major band was of M2H2 type and was partially purified by gel filtration and affinity chromatography. The specific activity of the enzyme was 2.6 units/mg protein. The half-life of the enzyme at 4 degrees C, was about 7 days. The optimum temperature for enzyme activity was 30 degrees C and the enzyme was irreversibly inactivated at 40 degrees C. The optimum pH for the forward reaction (pyruvate to lactate) was 5.5, while for reverse reaction it was between 8.0 to 9.5. The apparent Km values for pyruvate, NADH, lactate and NAD+ were 0.20, 0.013, 25 and 0.333 mM, respectively. Oxalate was found to be the inhibitor of LDH with Ki of about 4.2 mM.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Glutamate dehydrogenase and glutamine synthetase activities during the development of triticale grains

Glutamate dehydrogenase (GDH, EC 1.4.1.2–4) and glutamine synthetase (GS, EC 6.3.1.2) activities as well as protein content and dry matter in developing kernels of winter Triticale were determined. The relatively low level of GS activity compared to high level of NAD(P)H-dependent GDH activity during intensive filling of grains with storage compounds may indicate the participation of GDH in reductive amination of 2-oxoglutarate. The amination activity of this enzyme in all grain development phases exceeded the deaminating activity several fold. Moreover, the dynamics in the change of NAD(P)H-GDH and NAD(P)⁺-GDH activities were analysed in various tissues of the developing grains. The high amination activity of the enzyme in the seed coat, where the intensive protein synthesis occurs would also be an indication of the anabolic function of this enzyme.     

Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon that grows at temperatures up to 98 degrees C by fermenting peptides. Little is known about the primary metabolic pathways of this organism and, in particular, the role of enzymes that are dependent on thermolabile nicotinamide nucleotides. In this paper we show that the cytoplasmic fraction of cell extracts contained NADP-specific glutamate dehydrogenase (GDH) and NADP-specific alcohol dehydrogenase (ADH) activities, neither of which utilized NAD as a cofactor. The GDH is composed of identical subunits having an M(r) of 45,000 and had an optimal pH and optimal temperature for glutamate oxidation of 8.0 and > 95 degrees C, respectively. Potassium phosphate (60 mM), KCl (300 mM), and NaCl (300 mM) each stimulated the rate of glutamate oxidation activity between two- and threefold. For glutamate oxidation the apparent Km values at 80 degrees C for glutamate and NADP were 0.22 and 0.029 mM, respectively, and for 2-ketoglutarate reduction the apparent Km values for 2-ketoglutarate, NADPH, and NH4+ were 0.16, 0.14, and 0.63 mM, respectively. This enzyme is the first NADP-specific GDH purified form a hyperthermophilic organism. T. litoralis ADH is a tetrameric protein composed of identical subunits having an M(r) of 48,000; the optimal pH and optimal temperature for ethanol oxidation were 8.8 and 80 degrees C, respectively. In contrast to GDH activity, potassium phosphate (60 mM), KCl (0.1 M), and NaCl (0.3 M) inhibited ADH activity, whereas (NH4)2SO4 (0.1 M) had a slight stimulating effect. This enzyme exhibited broad substrate specificity for primary alcohols, but secondary alcohols were not oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold stress induces switchover of respiratory pathway to lactate glycolysis in psychrotrophicRhizobium strains

Two psychrotrophic strains of Rhizobium, DDSS69, a non-cold acclimated strain, and ATR1, a cold acclimated strain, were subjected to cold stress. A 4-fold increase in the specific activity of lactate dehydrogenase (LDH) was characteristic for cold stressed cells of DDSS69, whereas ATR1 showed a higher LDH activity in general, which increased 1.5-fold under cold stress. Cold sensitive mutants of DDSS69 which could not grow below 15 degrees C, in contrast to the wild type which could grow at 5 degrees C, were isolated using Tn5-tagged mutagenesis. These mutants showed a 40% lower LDH activity than the wild type grown at 5 degrees C that was comparable to the wild type grown at 15 degrees C. High specific activity of succinic dehydrogenase (SDH) at 28 degrees C in both strains and mutants indicated that aerobic respiration via the citrate cycle is the normal mode of saccharide utilization. Shifts to lower temperatures decreased the specific activity of SDH. However, alcohol dehydrogenase (ADH) activity remained very low in both the strains and the mutants at low temperatures indicating that a shift from aerobic saccharide metabolism to anaerobic one under cold stress involves lactate glycolysis rather than alcohol fermentation. There was an increase in membrane-bound ATPase activity under cold stress which is correlated to higher LDH activity. These data show that, in psychrotrophic Rhizobium strains, cold stress induces a switchover of respiratory metabolism from aerobic to anaerobic pathway, especially lactate glycolysis.     

Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion-exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0. 1% Triton X-100-treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K(m) value for NADP(+) than NAD(+), whereas the K(m) value for NAD(+) was higher than that for NADP(+) in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42 degrees C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N-terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH.     

Distribution and Solubilization of Particulate Gluconate Dehydrogenase and Particulate 2-Ketogluconate Dehydrogenase in Acetic Acid Bacteria

The distribution of two particulate enzymes, gluconate dehydrogenase (GDH) and 2-ketogluconate dehydrogenase (2KGDH), was investigated with cell free extract through 26 strains of genus Acetobacter and genus Gluconobacter. GDH activity was found in the cell free extracts from all strains of genus Gluconobacter and two species of genus Acetobacter, A. aceti and A. aurantium. High activity of 2KGDH was also found in the pigment-producing strains of genus Gluconobacter.

Best solubilization of particulate enzymes was attained with the highest recovery when 10 mg of Triton X–100 and 30 mg of protein of particulate fractions in 1 ml of 0.01 m phosphate buffer, pH 6.0, are incubated for 9 hr at 5°C with continuous stirring.

By comparison of the total enzyme activity of particulate enzymes with that of NAD(P)-linked enzymes in the cell free extract, it was obvious that the formation of ketogluconates by particulate enzymes was much more predominant, roughly over 100 times higher, as that of NAD(P)-linked enzymes.     

Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.

Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.     

Cold-adapted yeasts as producers of cold-active polygalacturonases

Eight cold-adapted, polygalacturonase-producing yeasts belonging to four species were isolated from frozen environmental samples in Iceland. They were identified as Cystofilobasidium lari-marini, Cystofilobasidium capitatum, Cryptococcus macerans and Cryptococcus aquaticus species by sequence analysis of rDNA regions. Growth behavior of the isolates was investigated. All strains could grow at 2 degrees C. Addition of glucose to pectin-containing culture medium had a repressive effect on enzyme production except for C. aquaticus, which showed increased polygalacturonase activity. Optimal temperature for enzyme production for the Cystofilobasidium strains was 14 degrees C, while that for the Cryptococcus strains was lower. Among the isolates, C. lari-marini S3B produced highest levels of enzyme activity at pH 3.2. Preliminary characterization of the polygalacturonases in the culture supernatant showed the enzyme from Cystofilobasidium strains to be optimally active at 40 degrees C and pH 5, and that from the Cryptococcus strains at 50 degrees C and pH 4. The polygalacturonase from C. macerans started to lose activity after 1 h of incubation at 40 degrees C, while that from the other strains had already lost activity at 30 degrees C. All the strains except C. aquaticus produced isoenzymes of polyglacturonase. In addition to polygalacturonase, the Cystofilobasidium strains produced pectin lyase, C. aquaticus pectin esterase, and C. macerans pectin lyase, pectate lyase and pectin esterase.     

Characteristics of glutamate dehydrogenase, a new diagnostic marker for the genus Fusobacterium

Enzymes representative of carbohydrate and nitrogen metabolism were screened for their presence and activity amongst species of the genus Fusobacterium. Glutamate dehydrogenase (GDH) was reliably detected in all 25 strains studied. The pH profile of this enzyme and the DNA base composition of selected strains were also determined. DNA base composition of selected strains ranged between 28-32.9 mol% G + C. GDH was active between pH 7.5-11.5 but two pH profiles of activity, with optima at 9.5 and 10.5, were discernible among species. Apart from Fusobacterium nucleatum, which had a heterogeneous enzyme pattern, the GDH electrophoretic mobility was constant within a species but in a few cases the enzyme bands overlapped. A combination of the pH profile, the GDH electrophoretic pattern and the DNA base composition provided clear separation of the test organisms into discrete groups; however, a larger number of strains must be examined before the full potential of these tests can be evaluated.     

Inhibition of lactate dehydrogenase C4 (LDH-C4) blocks capacitation of mouse sperm in vitro

Lactate dehydrogenase C4 (LDH-C4) is a tissue-specific enzyme in the mammalian testis and the only lactate dehydrogenase isozyme of sperm. Inhibitors of LDH activity were used to determine whether this enzyme plays a role in sperm capacitation, the acrosome reaction and/or fertilization. Oxamate or its derivative was used to inhibit sperm LDH activity in a medium promoting capacitation. Complete inhibition of LDH activity blocked capacitation. This effect could be reversed partially by the addition of dbcAMP or pentoxifylline to the culture medium. Western blotting showed that oxamate and N-isopropyl oxamate inhibited the tyrosine phosphorylation of proteins during the sperm capacitation process. Presumably, glycolysis is the primary energy pathway for sperm metabolism. The oxidation of reduced NAD with the conversion of pyruvate to lactate by LDH provides ATP necessary for protein kinase A (PKA) activity. Our data indicate that LDH-C4 plays an important metabolic role in sperm capacitation.     

Proposed Enzymes of Auxin Biosynthesis and Their Regulation II. Tryptophan Dehydrogenase Activity in Plants.

In pea, maize and tomato plants a hitherto undescribed L-tryptophan dehydrogenase activity (TDH) has been detected. This enzyme catalyzes the reversible formation of indolepyruvic acid (IPyA) from L-tryptophan (L-trp). TDH and L-glutamate dehydrogenase (GDH), related enzymes in their mode of action, could be separated by gel chromatography. Enzymatic activity of TDH was sustained by both pyridine coenzymes NAD/NADP. With pea TDH the coenzyme NAD displays, at optimum pH 8.5 and at room temperature, only about 40-70 % of the activity of NADP. The amination of IPyA is catalysed more actively than the deamination of L-trp. L-trp/IPyA, L-glu/ketoglutarate, L-ala/pyruvate reacted as dehydrogenase substrates; L-phe/ phenylpyruvate, D-trp and D-phe did not react with pea enzyme extracts. A considerable similarity between the active centres of TDH and GDH has been found using inhibitors: absence of heavy metals, presence of a carbonyl group, indispensibility of bivalent ions for the enzyme activity. Pea TDH and GDH were distinctly inhibited by sodium azide. For the activity of TDH the presence of SH groups is less important than for GDH. The TDH activity in the investigated plants was lower than the GDH activity. The possible role of TDH in the regulation of the IPyA pool is discussed.Doc. RNDr. PhMr. M. Kutáček died on 28 November, 1989. The final form for print was prepared by dr. Ivana Machdckovd of the same Institute, who will also answer the reprint requests. Received June 6, 1990; accepted October 10, 1990     

The Antarctic Psychrobacter sp. TAD1 has two cold-active glutamate dehydrogenases with different cofactor specificities. Characterisation of the NAD+-dependent enzyme

Psychrobacter sp. TAD1 is a psychrotolerant bacterium from Antarctic frozen continental water that grows from 2 to 25 degrees C with optimal growth rate at 20 degrees C. The new isolate contains two glutamate dehydrogenases (GDH), differing in their cofactor specificities, subunit sizes and arrangements, and thermal properties. NADP+-dependent GDH is a hexamer of 47 kDa subunits and it is comparable to other hexameric GDHs of family-I from bacteria and lower eukaria. The NAD+-dependent enzyme, described in this communication, has a subunit weight of 160 kDa and belongs to the novel class of GDHs with large size subunits. The enzyme is a dimer; this oligomeric arrangement has not been reported previously for GDH. Both enzymes have an apparent optimum temperature for activity of approximately 20 degrees C, but their cold activities and thermal labilities are different. The NAD+-dependent enzyme is more cold active: at 10 C it retains 50% of its maximal activity, compared with 10% for the NADP+-dependent enzyme. The NADP+-dependent enzyme is more heat stable, losing only 10% activity after heating for 30 min, compared with 95% for the NAD+-dependent enzyme. It is concluded that in Psychrobacter sp. TAD1 not only does NAD+-dependent GDH have a novel subunit molecular weight and arrangement, but that its polypeptide chains are folded differently from those of NADP+-dependent GDH, providing different cold-active properties to the two enzymes.     

The Effect of Red and Far-red Light on Subsequent Enzyme Activity in Arena Coleoptiles

The effect of red light (660 nm), far-red light (730 nm) and dark treatment on the subsequent enzyme activity in homogenates of Avena coleoptiles was investigated. The activities of succinic dehydrogenase (SDH), lactic dehydro-genase (LDH) and glucose-6-P dehydrogenase (GDH) were investigated. The activity of SDH was greatest in material receiving continuous darkness. LDH and GDH activity was stimulated by both light treatments compared with the dark values. Little or no difference in enzyme activity was found using either a single 15 min flash of light or continuous light for 24 h. Admixtures of extracts from dark treated and light treated material in a 1:1 ratio gave unexpected levels of enzyme activity. In all cases such admixtures gave much less than the anticipated enzyme activity.     

Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria

In amino acid production by coryneform bacteria, study on relationship between change in enzyme activities and production of a target amino acid is important. In glutamate production, Kawahara et al. discovered that the effect of decrease in 2-oxoglutamate dehydrogenase complex (ODHC) on glutamate production is essential (Kawahara et al., Biosci. Biotechnol. Biochem. 61(7) (1997) 1109). Significant reduction of the ODHC activity was observed in the cells under the several glutamate-productive conditions in Corynebacterium glutamicum. Recent progress in metabolic engineering enables us to quantitatively compare the flux redistribution of the different strains after change in enzyme activity precisely. In this paper, relationship between flux redistribution and change in enzyme activities after biotin deletion and addition of detergent (Tween 40) was studied in two coryneform bacteria, C. glutamicum and a newly isolated strain, Corynebacterium efficiens (Fudou et al., Int. J. Syst. Evol. Microbiol. 52(Part 4) 1127), based on metabolic flux analysis (MFA). It was observed that in both species the specific activities of isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) did not significantly change throughout the fermentation, while that of the ODHC significantly decreased after biotin depletion and Tween 40 addition. Flux redistribution clearly occurred after the decrease in ODHC specific activity. The difference in glutamate production between C. glutamicum and C. efficiens was caused by the difference in the degree of decrease in ODHC specific activity. The difference in Michaelis-Menten constants or K(m) value between ICDH, GDH, and ODHC explained the mechanism of flux redistribution at the branch point of 2-oxoglutarate. It was found that the K(m) values of ICDH and ODHC were much lower than that of GDH for both strains. It was quantitatively proved that the ODHC plays the most important role in controlling flux distribution at the key branch point of 2-oxoglutarate in both coryneform bacteria. Flux redistribution mechanism was well simulated by a Michaelis-Menten-based model with kinetic parameters. The knowledge of the mechanism of flux redistribution will contribute to improvement of glutamate production in coryneform bacteria.