Glycerol Dehydrogenase from Cellulomonas sp. NT3060: Purification and Characterization

NAD⁺-dependent glycerol dehydrogenase from Cellulomonas sp. NT3060 was purified by a procedure of 10 steps involving crystallization. Dihydroxyacetone was identified as the oxidation product of glycerol with the enzyme. The purified enzyme did not lose activity on heating below 60°C. The enzyme oxidized other alcohols such as 1,2-propanediol, 2,3-butanediol and glycerol-α-monochlorohydrin, beside glycerol. The enzyme activity was inhibited by p-chloromercuribenzoate, Zn²⁺, Cu²⁺ and Cd²⁺. Oxidation of glyberol was activated by Na⁺ and reduction of dihydroxyacetone was activated by K⁺ at pH 7.5.     

Purification and Storage of Ribulose 1,5-bisphosphate Carboxylase from Rice Leaves

Ribulose 1,5-bisphosphate (RuBP) carboxylase was purified fromrice leaves. By using a buffer containing 12.5% (v/v) glycerolthroughout purification, the enzyme was protected from coldlability and was obtained at a high yield (5.5 mg/g fresh wt).The purified enzyme exhibited different rates of CO₂/Mg²⁺-activationby temperature pretreatment/storage. The purified enzyme was stable for at least one year in phosphatebuffer containing 12.5% (v/v) glycerol at 4°C or 50% (v/v)glycerol at –20°C. (Received March 1, 1983; Accepted June 27, 1983)     

Constituents of a Peptidal Antibiotic P168 Produced by Paecilomyces lilacinus (Thom) Samson

Two glycerol dehydrogenases, GD-I and GD-II, were purified from Geotrichum candidum by precipitation of ammonium sulfate, sequential column chromatography on Blue-Sepharose CL-4B and DEAE-Sepharose CL-6B and gel filtration on Cellulofine GC-700. The purified enzyme preparations were homogeneous upon disc gel electrophoresis. The molecular weights were estimated to be 135,000 for GD-I and 130,000 for GD-II, and their isoelectric points were 5.9 and 6.2, respectively. The specific activity of GD-I was twice that of GD-II. However, their other properties were very similar: Their optimum pHs for the oxidation of glycerol were 10.5, and those for the reduction of dihydroxyacetone were 5.5. The enzyme activities were highly activated by Cl-, but inhibited by PO₄^3?, BO₃^3? and 2-mercaptoethanol. The enzymes showed highest activity for glycerol, but also acted on several analogs of glycerol.     

Characterization of fructose-1,6-diphosphate-insensitive catabolic glycerol kinase ofPseudomonas aeruginosa

Glycerol kinase is induced in cells ofPseudomonas aeruginosa strain PAO when grown in the presence of glycerol or glycerol-3-phosphate. The enzyme was isolated from the soluble cytoplasmic fraction of cell extracts and purified 500-fold by ammonium sulfate precipitation and chromatography on columns of Sephadex G-25, DEAE-Sephadex, hydroxyapatite, and Sephadex G-200. A molecular weight of 120,000 was estimated by gel filtration of the catalytically active enzyme. In polyacrylamide gel electrophoresis the purified product contained one major band of Coomassie Blue staining material. The enzyme exhibited an apparent Km of 40 M for glycerol and 23 M for ATP. Of the nucleotide triphosphates tested, only ATP served as a phosphoryl group donor. Mg⁺⁺ or Mn⁺⁺ was required for activity, although a threefold greater concentration of Mn⁺⁺ was required when Mn⁺⁺ substituted for Mg⁺⁺. In contrast to most other catabolic glycerol kinases in bacteria, the enzyme was not inhibited by fructose-1,6-diphosphate nor by other tested metabolites.     

Purification and properties of a heat-stable and cold-labile NAD-specific glutamate dehydrogenase from Sporosarcina ureae

NAD-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.2) was purified to homogeneity from Sporosarcina ureae DSM 320; the native enzyme (M _r 250,000±25,000) is composed of subunits identical in molecular mass (M _r 42,000±3,000), suggesting a hexameric structure. In cell-free extracts and in its purified form, the enzyme was heat-stable, retaining 50% activity after 15 min incubation at temperatures up to 82°C. When exposed to low temperatures at pH values between 7.0 and 9.0. cell-free extracts and purified preparations lost enzyme activity rapidly and irreversibly. The addition of substrates, glycerol, or sodium chloride improved the stability of the enzyme with respect to cold lability and heat stability.Abbreviation NAD-GluDH nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase     

Production,Purification and Crystallization of 3α-Hydroxysteroid Dehydrogenase of Pseudomonas putida

The ability of formation of 3α-hydroxysteroid dehydrogenase was studied in bacteria and actinomycetes. The enzyme activity was found in several bacteria belonging to the genera Pseudomonas, Bacillus and Corynebacterium, when they were grown on cholic acid as a sole source of carbon. Of these bacteria, Pseudomonas putida NRRL B–11064 isolated from soil, showed the highest activity of 3α-hydroxysteroid dehydrogenase. The enzyme was purified from the cell-free extract by procedure including fractionation with ammonium sulfate and column chromatographies on DEAE-celluIose, Sephadex G–100 and hydroxylapatite. Crystals of the enzyme were obtained by the addition of ammonium sulfate to the purified enzyme in the presence of glycerol or polyethylene glycol. The overall purification was about 550-fold with an yield of 18.5%. The crystalline enzyme was homogeneous on polyacrylamide disc electrophoresis and analytical ultracentrifugation (s_20,w=3.2).     

A Novel Glycerol Kinase from Flavobacterium meningosepticum: Characterization,Gene Cloning and Primary Structure

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65°C for 10 min and at 37°C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.     

Purification, Characterization and Cloning of Phospholipase D from Peanut Seeds

We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the K _m value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl₂ and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2.     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Membrane-bound glycerol dehydrogenase catalyzes oxidation of D-pentonates to 4-keto-D-pentonates,D-fructose to 5-keto-D-fructose,and D-psicose to 5-keto-D-psicose

A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed with Gluconobacter thailandicus NBRC 3258. D-Pentonate 4-dehydrogenase activity in the membrane fraction was readily inactivated by EDTA and it was reactivated by the addition of PQQ and Ca²⁺. D-Pentonate 4-dehydrogenase was purified to two different subunits, 80 and 14 kDa. The absorption spectrum of the purified enzyme showed no typical absorbance over the visible regions. The enzyme oxidized D-pentonates to 4-keto-D-pentonates at the optimum pH of 4.0. In addition, the enzyme oxidized D-fructose to 5-keto-D-fructose, D-psicose to 5-keto-D-psicose, including the other polyols such as, glycerol, D-ribitol, D-arabitol, and D-sorbitol. Thus, D-pentonate 4-dehydrogenase was found to be identical with glycerol dehydrogenase (GLDH), a major polyol dehydrogenase in Gluconobacter species. The reaction versatility of quinoprotein GLDH was notified in this study.     

Cold Inactivation of Phosphoenolpyruvate Carboxylase and Pyruvate Orthophosphate Dikinase from the C4 Perennial Plant Atriplex halimus

Phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) cold inactivation was studied in leaf extracts from Atriplex halimus L. Both enzyme activities gradually reduced as the temperature and the total soluble protein decreased. Mg²⁺ at a concentration of 10 mM stabilized PEPC and PPDK activities against cold inactivation. At low Mg²⁺ concentration (4 mM), PEPC was strongly protected by phosphoenolpyruvate, glucose-6-phosphate, and, partially, byL-malate, while PPDK was protected by PEP, but not by its substrate, pyruvate. High concentrations of compatible solutes (glycerol, betaine, proline, sorbitol and trehalose) proved to be good protectants for both enzyme activities against cold inactivation. When illuminated leaves were exposed to low temperature, PPDK was partially inactivated, while the activity of PEPC was not altered.     

Purification and Stability during Storage of Phoshoenolpyruvate Carboxylase from Leaves of Amaranthus hypochondriacus,a NAD-ME Type C4 Plant

A traditional method is reported for purification of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) from leaves of Amaranthus hypochondriacus L. with a high yield of 50 %, 135-fold purification, and specific activity of 900 mmol kg^–1(protein) s^–1. PEPC was purified from light-adapted leaves of A. hypochondriacus, involving 40–60 % ammonium sulphate fractionation, followed by chromatography on columns of DEAE-Sepharose, hydroxylapatite (HAP), and Seralose 6-B. The enzyme appeared as a single band on 10 % SDS-PAGE, with a molecular mass of about 100 kDa. Kinetic studies with purified enzyme confirmed the PEPC to be the light-form of the enzyme. Glycerol generally increased the stability of PEPC. The stability and storage of the purified enzyme was studied at temperatures of 4 °C, –20 °C, and liquid nitrogen. PEPC maintained its activity for up to 3 months upon storage with 50 % (v/v) glycerol in liquid nitrogen.     

Dihydroxyacetone kinase from a methylotrophic yeast,Hansenula polymorpha CBS 4732

Dihydroxyacetone (DHA) kinase was purified to electrophoretic homogeneity from methanol-grown Hansenula polymorpha CBS 4732. The enzyme was a dimer with a molecular weight of 150,000, and had an isoelectric point of 4.9. The enzyme was active toward DHA, and D- and L-glyceraldehydes as phosphorylation acceptors, and only ATP served as a donor. ADP inhibited the enzyme at a physiological concentration. Magnesium ion was essential for the activity and stability. Some other divalent cations can substitute in part the magnesium ion. The DHA kinases found in cells grown on methanol and glycerol were immunologically identical, but were different from those of other methylotrophic yeasts as shown by immunotitration. A mutant (204D) derived from the yeast, which could not grow on methanol or DHA but could so on glycerol, was deficient in DHA kinase. Glycerol kinase activity was found in glycerol-grown 204D cells as well as the parent strain.Abbreviation DHA dihydroxyacetone     

Production and Purification of the Heavy-Chain Fragment C of Botulinum Neurotoxin, Serotype B, Expressed in the Methylotrophic YeastPichia pastoris

A recombinant H_cfragment of botulinum neurotoxin, serotype B (rBoNTB(H_c)), has been successfully expressed in a Mut⁺strain of the methylotrophic yeastPichia pastorisfor use as an antigen in a proposed human vaccine. The fermentation process consisted of batch phase on glycerol, followed by glycerol and methanol fed-batch phases yielding a final cell mass of 60 g/L (dcw) and was easily scaled-up to 60 L. A multistep ion-exchange chromatographic purification process was employed to produce 99% pure H_cfragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified H_cfragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin.     

Towards a phylogeny of phototrophic purple sulfur bacteria — the genus Ectothiorhodospira

While most strains of heterofermentative lactobacilli and strains of Leuconostoc species contained only traces of a dehydratase reacting with glycerol or propanediol-1,2, three strains of Lactobacillus brevis and one strain of L. buchneri that metabolized glycerol readily in the presence of glucose, contained propanediol-1,2 dehydratase (EC 4.2.1.28). This cobamide requiring enzyme from L. brevis B 18 was partially purified. It reacts with the substrates propanediol-1,2, glycerol and ethanediol-1,2 with the relative activities of about 3:2:1. This ratio remained unchanged throughout the purification procedure. The substrate affinities were measured: propanediol-1,2 K _m=0.6 mM, glycerol K _m=4 mM, ethanediol-1,2 K _m=5.3 mM coenzyme B₁₂ (substrate glycerol) K _m=0.007 mM. The activity of the dehydratase was promoted by potassium or ammonium ions and inhibited by sodium, lithium, magnesium or specially manganese. The apparent molecular weight of propanediol-1,2 dehydratase was determined as M_r=180,000.Dedicated to Prof. Dr. H. G. Schlegel on behalf of his 60th birthday     

Purification and characterization of an extracellular lipase from Clostridium tetanomorphum

Summary The strictly anaerobic bacterium Clostridium tetanomorphum formed an extracellular lipase when the growth medium contained glycerol in addition to fermentable substrates such as l-glutamate or glucose. The lipase was purified from the concentrated culture supernatant and exhibited a final specific activity of 900 U/mg. The purified lipase had a Stokes’ radius of 5.0 nm and a sedimentation coefficient of 5.7S. The native molecular mass calculated from these values was 118,000 Da, which is considerably higher than the molecular mass calculated by PAGE (70,000 Da). With p-nitrophenyl esters of different fatty acids as substrates enzyme activity was highest when the acyl chain was short (C₂). The purified lipase showed no protease or thioesterase activity.     

Synthesis of l-Tryptophan from Indole and dl-Serine by Tryptophan Synthetase Entrapped in Fibres

Optimal culture conditions for microbial production of tryptophan synthetase were studied. It was found that on cultivation of Escherichia coli 476, a tryptophan auxotroph, in a medium containing 5g/liter glycerol as C source, supplemented with 1 g/liter of acid-treated peptone, cells with high tryptophan synthetase activity could be obtained.

The enzyme was extracted from cells and 3-fold purified by heat treatment and ammonium sulfate precipitation. The overall yield of the isolation procedure was 60%.

The partially purified tryptophan synthetase was entrapped in cellulose triacetate fibres. Under storage conditions, in refrigerator, the entrapped enzyme was stable at least for 6 months. The activity of the entrapped enzyme was about 75% with respect to the free enzyme.

Similar behaviour for the free and entrapped enzyme was observed as to the effect of temperature and pH on the enzymic activity. The operational stability of the entrapped tryptophan synthetase was very good (activity unchanged after 50 days) provided the accumulation of indole on the fibres was avoided.     

Effects of Organic Solvents on Streptomyces griseus Protease I with Transfer Action

The protease I from Streptomyces griseus var. alcalophilus strongly catalyzed the transfer reaction forming hydroxamic acids of various amino acids, especially aromatic and hydrophobic amino acids. Dimethyl sulfoxide (DMSO), 1,4-butane diol, and glycerol protected this enzyme from heat denaturation. Moreover, only DMSO strongly activated the transfer action of protease I and the velocity of reaction was accelerated three times in the presence of 50% DMSO. The yield of transfer product strongly depended on reaction time and the concentrations of enzyme and substrates in the presence of DMSO. The velocity of the transfer reaction was faster than that of the hydrolysis reaction under the conditions of 0.1 M Phe-OEt, 0.75 M NH₂OH, and 50% DMSO.     

Fructose-1,6-bisphosphatase from a cold-hardy insect: Control of cryoprotectant glycerol catabolism

Fructose 1,6-bisphosphatase (FBPase) from the larvae of the gall moth, Epiblema scudderiana, was purified to homogeneity with a final specific activity of 1.6 U/mg protein. The enzyme had a native molecular weight of 74.0 ± 6.5 kD and a subunit molecular weight of 37.6 ± 3.0 kD; the dimeric structure of the enzyme in this species is unusual. The pH optimum was 7.00 in imidazole buffer at 22°C and rose to 7.31 at 5°C. An Arrhenius plot of enzyme activity vs. temperature was linear with an activation energy of 91 ± 4.1 kJ/mol^?1. K_m values for FBPase decreased from 4.7 ± 0.34 μM at 22°C to 1.3 ± 0.05 μM at 5°C. No allosteric activators were identified, but the enzyme was inhibited by fructose 2,6-bisphosphate (F2,6P₂), AMP, ADP, dihydroxyacetonephosphate, glycerol, and KCI. Inhibition by AMP and F2,6P₂ increased at low temperature, and effects of these compounds may be key to preventing futile cycling of carbon at the FBPase/phosphofructokinase loci during the biosynthesis of glycerol cryoprotectant. Oppositely, glycerol clearance in the spring and reconversion into glycogen is promoted by interactions of temperature, inhibitors, and glycerol that promote FBPase activity: I₅₀ values for AMP and F2,6P₂ increase at 22°C (compared with 5°C), high glycerol levels override F2,6P₂ inhibition of the enzyme, and deinhibitors (ATP, citrate) partially reverse AMP inhibition of the enzyme. © 1995 Wiley-Liss, Inc.     

Purification and Properties of Pyranose Oxidase from Coriolus versicolor

Coriolus versicolor KY2912 grown on a medium containing glucose, sucrose or glycerol produced pyranose oxidase. Pyranose oxidase (glucose-2-oxidase) was purified by HPA-75 chromatography, Sepharose 4B and Sephadex G-100 gel filtration, and hydroxyapatite chromatography. The purified enzyme preparation showed a single protein band on acrylamide gel electrophoresis. The highest activity was obtained when D-glucose was employed as substrate and molecular oxygen as electron acceptor. The enzyme was most active at pH 6.2 and 50°C, stable in the pH region between 5.0 and 7.4, and the activity was completely lost above 70°C. The activity was inhibited by Ag⁺ , Cu²⁺ and PCMB. The enzyme contained FAD covalently bound to the polypeptide chain. The enzyme consisted of identical subunits with a molecular weight of 68,000, and showed a total molecular weight of 220,000.