Isolation and properties of malate dehydrogenase from meso- and thermophilic bacteria

A scheme of purification of malate dehydrogenase from Macromonas bipunctata strain D-405 and Vulcanithermus medioatlanticus DSM 14978T was developed. This scheme was used to obtain electrophoretically homogeneous enzyme preparations of the mesophilic bacterium M. bipunctata (specific activity, 26.9 +/- 0.8 U/mg protein; yield, 10.9%) and the thermophilic bacterium V. medioatlanticus (specific activity, 5.0 +/- 0.2 U/mg protein; yield, 19.2%). Using these high-purity enzymatic preparations, the physicochemical and regulatory properties of malate dehydrogenase were studied and the differences in kinetic characteristics and thermal stability of the preparations were determined.     

Simple efficient methods for the isolation of malate dehydrogenase from thermophilic and mesophilic bacteria.

Malate dehydrogenase from a number of bacteria drawn from several genera and representing the mesophilic, moderately thermophilic and extremely thermophilic classes was isolated by procedures which involve only a small number of steps (in most cases only two), of which the key one is affinity chromatography on 5'-AMP--Sepharose and/or on NAD+--hexane--agarose. Electrophoretic analysis of the native enzymes in polyacrylamide gel and of the denaturated enzymes in sodium dodecyl sulphate/polyacrylamide gel revealed no significant protein impurity in the purified preparations. The yields ranged from about 40% to over 80%. The malate dehydrogenases from the extreme thermophiles and from some of the moderate thermophiles are appreciably less efficient catalytically than their mesophilic homologues.     

Isocitrate dehydrogenase from thermophilic and mesophilic bacteria. Isolation and some characteristics

1. Simple methods incorporating the principle of selective enzyme elution from a triazinyl dye adsorbent with a mixture of NADP+ and isocitrate are described for isolating NADP+-linked isocitrate dehydrogenase in pure state from several mesophilic and thermophilic bacteria. 2. Several characteristics of the isocitrate dehydrogenases have been examined, viz. molecular size, amino acid composition including the content of sulphydryl groups, thermostability and structural homology by the criterion of immunological cross-section.     

NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm. Isolation and properties

The NADP-dependent decarboxylating malate dehydrogenase was isolated from the cytoplasmic fraction of bovine adrenal cortex and purified 3530-fold by 3-fold ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 M and DEAE-Sephadex A-50 with subsequent two-fold gel filtration through Toyopearl HW-55. The specific activity of homogeneous enzyme preparations was equal to 60 U/mg protein with a 30% yield. The enzyme molecular weight as determined by gel filtration on Sephadex G-20 was 155000. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate malate dehydrogenase dissociated into two subunits with Mr 77000. The Arrhenius plot for the reaction rate showed a break at 30 degrees C. The values of activation energy and temperature coefficient above and below the breakpoint were equal to 45049 and 147188 J X mol-1; 1.68 and 2.63, respectively. Within the temperature range of 26-40 degrees C, malate dehydrogenase exhibited hyperbolic kinetics with respect to the substrate. At 30 degrees C, Km for malate was equal to 250 microM, whereas at 40 degrees C it was 130 microM. The curve for the dependence of the initial reaction velocity versus NADP concentration was S-shaped. The Hill coefficient was 1.4, which testifies to positive cooperativity of NADP interaction with malate dehydrogenase.     

Purification and physicochemical properties of malate dehydrogenase from bacteria of the genus Beggiatoa

Homogeneous malate dehydrogenase (MDH) with a specific activity of 20-24 units per mg protein was purified from the sulfur bacterium Beggiatoa leptomitiformis strain D-402 grown organotrophically and lithotrophically and from the organotrophic bacterium Beggiatoa alba. MDHs from the B. leptomitiformis strain D-402 grown under organotrophic conditions and from B. alba are homodimers with the subunit molecular weight of 40 kD. Tetrameric MDH is formed in B. leptomitiformis strain D-402 grown under lithotrophic conditions. The dimeric and tetrameric forms of MDH from B. leptomitiformis D-402 display some differences in kinetic properties.     

Isolation and purification of malate dehydrogenase isoforms from phototrophic purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris

A five-step procedure was used to obtain electrophoretically pure preparations of malate dehydrogenase (EC 1.1.1.37) from Rhodobacter sphaeroides and Rhodopseudomonas palustris. The procedure included extraction, ammonium sulfate fractionation, gel filtration, and ion exchange and gel permeation chromatography. The enzyme was found to exist in two isoforms, dimeric and tetrameric, formed by the oligomerization of identical subunits. The isoforms are assumed to be involved in different metabolic processes.     

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.     

NAD-dependent formate dehydrogenase from methylotrophic bacteria. Isolation and properties]

NAD-Dependent formate dehydrogenase (FDH) has been isolated from methylotrophyc strain Bacterium sp 1 by (NH4)2SO4 fractionation of cell extract, ion-exchange chromatography and preparative isotachophoresis. Preparation of FDH is homogeneous in analytical polyacrylamide gel electrophoresis and under ultracentrifugation. Sedimentation coefficient of FDH is 4.9S. Mikhaelis constants are 1.1-10(-4) M for NAD and 1.5-10(-2) M for formate. In the absence of sulfhydril compounds FDH is unstable, but it is stable in the presence of mercaptoethanol or ditiotreitol.     

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.     

Characterization of an essential histidine residue in thermophilic malate dehydrogenase

Heat-stable malate dehydrogenase isolated from Thermus flavus AT62 was completely inactivated by treatment with diethylpyrocarbonate. The inactivation was accompanied by the loss of 1.2 histidine residues per subunit of the enzyme. The enzyme was protected from inactivation by NADH. The enzyme was also inactivated by dye-sensitized photooxidation. Methionine residues, in addition to histidine residues, were destroyed in the inactivated enzyme. Kinetic analyses of the inactivation indicated that the pK value of the residue involved in the inactivation was 8.20 at 25.0 degrees C and 7.52 at 60.0 degrees C. From the pK values and the heat of ionization calculated from the van't Hoff plot of pKs, a histidine residue was identified to be primarily involved in the inactivation. The effect of temperature on the pK value of the essential group in this enzyme from a thermophilic organism is discussed.     

Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost

Abstract During the first stage of the preparation of mushroom compost oxygen is believed to be readily available. However we measured methane in the evoking air above the compost piles and were able to isolate thermophilic methanogenic bacteria from this compost. The isolates grow only on H₂ and CO₂ as energy and carbon source and do not require complex factors for growth. On the basis of nutritional and morphological characteristics these methanogens were identified as strains of Methanobacterium thermoautotrophicum .     

Isolation of extrachromosomal deoxyribonucleic acids from extremely thermophilic bacteria

Eight strains of thermophilic bacteria were examined for the presence of covalently closed circular deoxyribonucleic acid molecules by caesium chloride-ethidium bromide density gradient centrifugation. Four of the eight strains tested, Thermus flavus BS1, AT61, AT62 and Thermus thermophilus HB8 carried covalently closed circular DNA molecules. Thermus flavus BS1 haboured two species of plasmids with molecular weights of 6.1 X 10(6) and 17.0 X 10(6) as determined by electron microscopy. Thermus thermophilus HB8, T. flavus AT61 and T. flavus AT62 carried plasmids with molecular weights of 6.2 X 10(6), 6.6 X 10(6) and 6.6 X 10(6), respectively. Plasmids from T. flavus AT61 and AT62 were indistinguishable in their electrophoretic patterns in agarose or acrylamide gel after digestion with various restriction endonucleases. This is the first evidence for the presence of plasmids in extremely thermophilic bacteria, though their functions are unknown.     

Isolation of thermophilic iron-oxidising bacteria from sulfidic waste rock

Summary A thermophilic, rod-shaped, iron-oxidising bacterium was isolated by enrichment culture of rock samples from an overburden dump at the Rum Jungle mine site in Australia's Northern Territory. Oxidation of ferrous iron and sulfur occurred at 50–55°C, with a temperature maximum of 60°C. The isolate required yeast extract for growth. The pH optimum for iron oxidation at 50°C was 1.4. Rapid iron-oxidation occurred at a pH as low as 0.35, but little or no oxidation occurred at or above pH 2.2.     

Purification and properties of malate dehydrogenase from Pseudomonas testosteroni.

Nicotinamide adenine dinucleotide-linked malate dehydrogenase has been purified from Pseudomonas testosteroni (ATCC 11996). The purification represents over 450-fold increase in specific activity. The amino acid composition of the enzyme was determined and found to be quite different from the composition of the malate dehydrogenases from animal sources as well as from Escherichia coli. Despite this difference, however, the data show that the enzymatic properties of the purified enzyme are remarkably similar to those of other malate dehydrogenases that have been previously studied. The Pseudomonas enzyme has a molecular weight of 74,000 and consists of two subunits of identical size. In addition to L-malate, the enzyme slowly oxidizes other four-carbon dicarboylates having an alpha-hydroxyl group of S configuration such as meso- and (-) tartrate. Rate-determining steps, which differ from that of the reaction involving L-malate, are discussed for the reaction involving these alternative substrates. Oxidation of hydroxymalonate, a process previously undetected with other malate dehydrogenases, is demonstrated fluorometrically. Hydroxymalonate and D-malate strongly enhance the fluorescence of the reduced nicotinamide adenine dinucleotide bound to the enzyme. The enzyme is A-stereospecific with respect to the coenzyme. Malate dehydrogenase is present in a single form in the Pseudomonas. The susceptibility of the enzyme to activation or inhibition by its substrates-particularly the favoring of the oxidation of malate at elevated concentrations-strongly resembles the properties of the mitochondrial enzymes. The present study reveals that whereas profound variations in chemical composition have occurred between the prokaryotic and eukaryotic enzymes, the physical and catalytic properties of malate dehydrogenase, unlike lactate dehydrogenase, are well conserved during the evolutionary process.     

Isolation and characterization of two forms of malate dehydrogenase from human placenta

• 1.1. The purification and characterization of the cytoplasmic and mitochondrial forms of malate dehydrogenase from human placenta are described.
• 2.2. Both enzymes are composed of two subunits and have similar molecular weights and similar pH optima.
• 3.3. However, they differ with respect to thermal stability, excess substrate inhibition and electrophoretic mobility.

     

嗜热菌的耐热L—乳酸脱氢酶的研究

About 200 strains of extreme thermophilic bacteria were isolated from hot springs in Guandong province. A strain, HG25, was found to produce thermostable intracellular L-lactate dehydrogenase (EC. 1.1.1.27). It has the characteristic of Thermus sp. The cells were gram-negative, non-sporulating, nonmotile, aerobic rods containing yellow pigment. The optimum temperature for growth was between 65 degrees C to 75 degrees C, the maximum 85 degrees C, and minimum 40 degrees C. The generation time at the optimum was about 80 min. Starch was not hydrolyzed. Acid was not produced from glucose. The G+C content in DNA was 62-65 mol% (Tm). As the properties of strain HG25 is similar to those of Thermus aquaticus and T. thermophilus HB 8 belonging to the genus Thermus. The thermostable L-lactate dehydrogenase was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. For pyruvate reduction, the optimum temperature of the enzyme was 60 degrees C and pH 8.0. After incubation in 0.1 mol/L phosphate buffer pH 7.4 at 70 degrees C for 10 min, the enzyme retained about 85% of its original activity. The half-live time (t1/2) at 85 degrees C was 10 min.     

Isolation and characterization of thermophilic bacteria from natural and artificial habitats

Several thermophilic Bacillus Strains were isolated from natural as well as artificial habitats. They grow optimally on a carbohydrate-containing medium at a temperature of 65 to 68°C and a pH value of 6 to 7 under aerobic conditions. They utilize glucose, sucrose and sodium acetate as carbon and energy sources. They can be differentiated by acid formation and composition of intracellular fatty acid fraction as well as growth on xylose, lactose, starch, cellobiose, ribose and galactose.     

Purification and properties of malate dehydrogenase from the extreme thermophileBacillus caldolyticus

The enzyme malate dehydrogenase (EC 1.1.1.37) from an extreme thermophileB. Caldolyticus was purified to about 91% homogeneity. The molar mass of the enzyme was determined as 73 000 daltons and it is composed of two subunits, each with a molar mass of 37 000. Initial velocity studies with oxaloacetic acid and NADH as substrates at pH 8.1, over a range of temperatures, indicate that the enzyme operates via a sequential type mechanism. Van't Hoff plots of the kinetic parameters displayed sharp changes in slope at characteristic temperatures, whereas the Arrhenius plot exhibited no such breaks over the temperature interval investigated. The enzyme was found to be stable at 41°C and lower temperatures. At 51°C and 59°C an almost immediate 20% reduction in activity was obtained, but no further inactivation occurred during the 60 min of incubation. At 59°C the enzyme lost 50% of its initial activity in about 38 s. High concentration of NADH was observed to greatly stabilize the enzyme at that temperature.It is suggested that the slope changes in the Van't Hoff plots and the stability profies at 51°C and 59°C are representative of a temperature induced conformational change in the enzyme.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.