Purification and characterisation of an unusually heat-stable and acid/base-stable class I fructose-1,6-bisphosphate aldolase from Staphylococcus aureus

The fructose-1,6-biphosphate aldolase (EC 4.1.2.13) from Staphylococcus aureus ATCC 12 600 was purified and biochemically investigated. It was found that this aldolase belongs to the class I type of aldolases since the fructose-1,6-bisphosphate cleavage activity was insensitivity to high levels of EDTA. Like class I aldolases of higher organisms, the S. aureus aldolase activity is inhibited on incubation with the substrate dihydroxyacetone-phosphate in the presence of NaBH4. Furthermore, the aldolase activity is not stimulated by monovalent or divalent cations. This enzyme exhibits an extreme stability to high temperature, acid and base. The purified enzyme is not activated after heating at 97 degrees C for 1.6 h. An incubation at 130 degrees C for 10 min is necessary to destroy irreversibly the activity of the aldolase. The optimal temperature for activity, however, is 37 degrees C. It is a monomer with a molecular weight of about 33,000 and exhibits a relatively broad pH optimum ranging over pH 7.5-9.0. Apart from fructose 1,6-bisphosphate as substrate (Km = 0.045 mM), this aldolase also revealed activity with fructose 1-phosphate (Km = 25 mM). The pH of the isoelectric point lies between 3.95 and 4.25.     

A novel alcohol resistant metalloproteinase, vimelysin, from vibrio sp. T1800: purification and characterization

We found a novel metalloproteinase, which has high activity at low temperatures and in the presence of organic solvents, in the culture supernatant of a marine bacterium, Vibrio sp. T1800. The metalloproteinase, named vimelysin, was purified from the culture supernatant by three column chromatographies. About 150 mg of purified vimelysin was obtained from 3.3 liters of the culture supernatant with a high yield of 57%. The purified vimelysin showed a single protein band on SDS-PAGE with molecular weight of 38,000. The isoelectric point of vimelysin was 4.3 by isoelectric focusing. The optimum pH of vimelysin was pH 8.0 or pH 6.5 using casein or furylacryloyl-glycyl-leucine amide (FAGLA) as substrates, respectively. The optimum temperature of vimelysin was 50 degrees C when casein was used as a substrate, but it was 15 degrees C when FAGLA was used as a substrate. Interestingly, vimelysin activity was completely retained after 48 h of incubation at 25 degrees C in the presence of 50% ethanol. Moreover, vimelysin showed 40% activity of the control even in the presence of 10% ethanol, while thermolysin showed only 5% activity under the same conditions.     

Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasite to groundnut rust, Puccinia arachidis

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Fusarium chlamydosporum and purified by ion-exchange chromatography and gel filtration. The molecular mass of purified chitinase was 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chitinase was optimally active at a pH of 5 and stable from pH 4 to 6 and up to 40 degrees C. Among the metals and inhibitors tested, mercuric chloride completely inhibited the enzyme activity. The activity of chitinase was high on colloidal and pure chitin. The purified chitinase inhibited the germination of uredospores of Puccinia arachidis and also lysed the walls of uredospores and germ tubes. The results from these experiments indicated that chitinase of F. chlamydosporum plays an important role in the biocontrol of groundnut rust.     

Purification and characterization of a cephalosporin esterase from Rhodosporidium toruloides

A novel cephalosporin esterase (EC 3.1.1.41) from Rhodosporidium toruloides was purified to gel electrophoretic homogeneity. The enzyme is a glycoprotein with a molecular mass of 80 kDa. Upon deglycosylation, several forms of the enzyme were observed with a molecular mass range between 60 and 66 kDa. The isoelectric point of the enzyme is approximately 5.6, with the pH optimum for activity occurring at 6.0. The optimal activity of the enzyme occurred at 25 degrees C, with the enzyme rapidly losing activity at temperatures above 25 degrees C. The enzyme deacetylated a variety of cephalosporin derivatives, including cephalosporin C; the Km for this substrate is 51.8 mM, and the Vmax is 7.9 mumol/min/mg. In addition to cephalosporins, the enzyme hydrolyzed short-chain p-nitrophenyl esters, with the activity decreasing with increasing ester chain length. The enzyme also has the ability to acetylate desacetyl cephalosporins in high yields under mild conditions in the presence of various acetyl donors. A comparison of the physical properties of the esterase with those of other well-characterized cephalosporin esterases indicates that the enzyme is unique in this class.     

Pattern of metabolism and composition of the fecal microflora in infants 10 to 18 months old from day care centers

A bacterial strain, Bacillus megaterium VUMB-109, has been isolated and identified which produces salt-tolerant, thermostable amylase (16 U/ml culture filtrate). Cultural conditions such as carbon and nitrogen sources, metal ions, temperature and pH have been optimized for enzyme production. The partially purified enzyme was active over a wide range of pH (4.5-10) and exhibited maximum activity at 95 degrees C, retaining 90% original activity at 100 degrees C. Partially purified enzyme was stable at 70 degrees C for 60 min. The enzyme was stable in NaCl up to 5m over 24 h without losing its original activity.     

Purification and characterization of a prolidase from Aureobacterium esteraromaticum

An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440,000 by gel permeation chromatography, and about 40,000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro. Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45 degrees C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60 degrees C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn2+ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.     

Expression and characterization of the recombinant gene encoding chitinase from Aeromonas caviae

The gene encoding a chitinase from Aeromonas caviae was cloned by PCR techniques. Its recombinant gene expression was performed using pET20b(+) in Escherichia coli BL21 (DE3). The recombinant chitinase with the extra 33 and 13 amino acids in its N- and C-termini, respectively, was purified to near homogeneity using His-Tag affinity chromatography. The recombinant chitinase was found to be present in both the culture medium and the cytoplasm. A single protein band on the native polyacrylamide gel was confirmed by both the activity staining and protein staining. The optimum pH and temperature of the recombinant chitinase were determined to be 6.25-6.5 and 42.5 degrees C, respectively. It was stable within the pH range of 5-7. Significant activity stimulation by Cu2+ and inhibition by Fe3+ and Hg2+ were observed. Detergents such as SDS and Triton X-100 strongly inhibited the enzyme activity. Substrates such as 4-methylumbelliferyl-N,N'-diacetylchitobioside and 4-methylumbelliferyl-N,N',N"-triacetylchitotriose were hydrolyzed by the recombinant chitinase; however, 4-methylumbelliferyl-N-acetylglucosaminide was not cleaved during the activity assay periods. When chitin power was suspended in buffer with the chitinase (pH 6.5 and 42.5 degrees C), N-acetylchitooligosaccharides [(GlcNAc)n, n = 1-4] were detected at 24 h.     

Purification and some characteristics of a Ca2+-activated proteolytic enzyme from beef cardiac muscle

A calcium-dependent neutral proteinase was purified from beef cardiac muscle. The crude extract prepared from cardiac muscle was subjected to acid precipitation and salt fractionation and then further purified by column chromatography on Sepharose 6B, DE-52, and Sephadex G-200 columns in succession. The final preparation showed an 11 300 fold increase in specific activity of the Ca2+-activated enzyme. Average enzyme protein yield was 2.4 microgram/g fresh tissue. The enzyme was maximally active at pH 7.6 in the presence of 4 mM calcium. Proportionality of enzyme activity in partially purified preparations was retained when activity was measured at 25 degrees C using casein as the substrate. The rate of proteolysis by the purified enzyme was linear for 60 min under similar assay conditions. Fractionation of muscle homogenates showed that 70 to 73% of the total enzyme activity was present in the 24 000 X g and 30 000 X g supernatants. The enzyme was labile in aqueous solutions and storage at 4 degrees C and --20 degrees C resulted in considerable loss of activity, unless glycerol (50% v/v) was added to the solution.     

Purification and characterization of ovine pancreatic elastase

Elastase was isolated from ovine pancreas and purified to homogeneity by two different procedures. One involved precipitation with ammonium sulphate, p-aminobenzamidine-Sepharose chromatography, CM-Sepharose ion exchange chromatography and S-300 Sephacryl chromatography. The other involved the direct adsorption of elastase by tri-L-alanyl-Sepharose chromatography and a CM-Sepharose step. The enzyme, which was produced in an inactive form in the pancreas, was activated with a trace of trypsin prior to chromatography. Ovine pancreatic elastase has an isoelectric point above pI 9.3 and its molecular mass is estimated at approximately 25 kDa. The optimal pH range for activity is between 8.0 and 8.4 and the enzyme is unstable at pH below 4.0 and above 10.0 and at temperatures above 65 degrees C. The kinetic properties of the enzyme were determined with succinyl-Ala-Ala-Ala-p-nitroanilide as the substrate. Km and kcat Km-1 proved to be similar to the kinetic parameters of porcine elastase determined simultaneously.     

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by beta-adrenergic signals, leading to a rise in intracellular 3',5'-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 +/- 0.08. A change in assay temperature from a euthermic value (37 degrees C) to one typical of hibernating body temperature (5 degrees C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 degrees C to 8.7 at 5 degrees C, (b) K(m) values at 37 degrees C for Mg.ATP (49.2 +/- 3.4 microM) and for two phosphate acceptors, Kemptide (50.0 +/- 5.5 microM) and Histone IIA (0.41 +/- 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 degrees C, and (c) inhibition by KCl, NaCl and NH4Cl was reduced. However, temperature change had little or no effect on K(m) values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 degrees C and activation energies that were 29.1 +/- 1.0 kJ/mol for temperatures > 9 degrees C and 2.3-fold higher at 68.1 +/- 2.1 kJ/mol for temperatures < 9 degrees C, whereas the rabbit enzyme showed a breakpoint at 17 degrees C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 +/- 0.18 at 37 degrees C to 2.19 +/- 0.07 at 5 degrees C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with 32P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 degrees C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor.     

Purification and characterization of the D-hydantoinase from Bacillus circulans

A D-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from Bacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0-10.0 and a temperature of 75 degrees C. The enzyme is the most stable in a pH range of 8.5-9.5 and up to 60 degrees C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.     

Maltose phosphorylase from Lactobacillus brevis: purification, characterization, and application in a biosensor for ortho-phosphate

With the goal to obtain maltose phosphorylase as a tool to determine ortho-phosphate, the enzyme from Lactobacillus brevis was purified to 98% by an expeditious FPLC-aided procedure which included anion exchange chromatography, gel filtration, and hydroxyapatite chromatography. The native maltose phosphorylase had a molecular mass of 196 kDa and consisted of two 88 kDa subunits. In isoelectric focusing two isoforms with pI values of 4.2 and 4.6 were observed. Maximum enzyme activity was obtained at 36 degrees C and pH 6.5 and was independent of pyridoxal 5'-phosphate. The apparent K(m) values with maltose and phosphate as substrates were 0.9 mmol l-1 and 1.8 mmol l-1, respectively. Maltose phosphorylase could be stored in 10 mM phosphate buffer pH 6.5 at 4 degrees C with a loss of activity of only 7% up to 6 months. The stability of the enzyme at high temperatures was enhanced significantly using additives like phosphate, citrate, and imidazole. The purified maltose phosphorylase was used as key enzyme in a phosphate sensor consisting of maltose phosphorylase and glucose oxidase. A detection limit of 0.1 microM phosphate was observed and the sensor response was linear in the range between 0.5 and 10 microM.     

Purification and some properties of human erythrocyte hexokinase

1. Human erythrocyte hexokinase (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified 50 000--100 000-fold with a final specific activity of about 25--50 units/mg protein using gel-filtration, ion-exchange chromatography and affinity chromagraphy. 2. After isoelectrofocusing ofthe preparation one major protein band could be detected besides a minor band. THe isoelectric point of the major protein band was found to be 4.7. 3. After purification the enzyme could be stabilized in a medium containing inorganic phosphate, glucose, glycerol and mercaptoethanol. 4. The molecular weight was determined by gel-filtration and was found to be 132 000+/-8000. 5. The enzyme shows a broad pH optimum ranging from 7.0 to 8.4. 6. The kinetic behavior of the purified enzyme at 37 degrees C was somewhat different from the normal Michaelis-Menten kinetics due to its instability. The affinity constants were 0.048--0.080 mM for glucose and 0.57--1.0 mM for Mg-ATP. 7. The enzyme was specific for Mg- ATP as the nucleotide substrate. Mg-UTP, Mg-ITP,Mg-GTP and Mg-CTP were not converted to corresponding diphosphates. Several hexoses could be phosphorylated by the enzyme. Mannose could be phosphorylated at the same rate as glucose, although the affinity for the enzyme was lower (5m=0.60mM). Much lower rates and lower affinities were found with 2-deoxy-D-glucose (5m=1.0mM), D(+)-glucosamine (5m=4.5 mM) and fructose (5m=10 mM). N-acetyl-D-glucosamine , galactose andsorbose were not phosphorylated at all.     

Practical considerations for the use of the optical disector in estimating neuronal number

A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to amino-acid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced amino-acid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.     

Purification and characterization of a novel thermostable alpha-L-arabinofuranosidase from a color-variant strain of Aureobasidium pullulans

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced alpha-L-arabinofuranosidase (alpha-L-AFase) when grown in liquid culture on oat spelt xylan. An extracellular alpha-L-AFase was purified 215-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chromatography, and SP-Sephadex C-50 column chromatography. The purified enzyme had a native molecular weight of 210,000 and was composed of two equal subunits. It had a half-life of 8 h at 75 degrees C, displayed optimal activity at 75 degrees C and pH 4.0 to 4.5, and had a specific activity of 21.48 mumol min-1. mg-1 of protein against p-nitrophenyl-alpha-L-arabinofuranoside (pNP alpha AF). The purified alpha-L-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. The K(m) values of the enzyme for the hydrolysis of pNP alpha AF, arabinan, and debranched arabinan at 75 degrees C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The alpha-L-AFase activity was not inhibited at all by L-arabinose (1.2 M). The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM).     

Prevalence of Toxoplasma gondii in Canadian market-age pigs

Triacylglycerol lipase (L3) was purified from Aspergillus oryzae RIB128 by ammonium sulfate fractionation, acetone precipitation, anion-exchange chromatography, and gel filtration. The purified enzyme was formed from a glycoprotein and a monomeric protein with molecular masses of 25 and 29 kDa, by SDS-PAGE and gel filtration, respectively. The optimum pH at 40 degrees C was 5.5 and the optimum temperature at pH 5.5 was 40 degrees C. The enzyme was stable between a pH range of 4.0-7.5 at 30 degrees C for 24 h, and at up to 30 degrees C at pH 5.5 for 1 h. Heavy metal ions, detergents, DFP, and DEP strongly inhibited the enzyme activity. The lipase hydrolyzed not only triacylglycerols but also monoacylglycerols and diacylglycerols. The enzyme had higher specificity toward triacylglycerols of middle-chain saturated fatty acids than short-chain or long-chain fatty acids. The enzyme had 1,3-positional specificity. The N-terminal amino acid sequence of the enzyme was not significantly similar to that of other lipases with published sequences.     

Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk

Ovine milk that had been standardized to 6% fat was inoculated with Escherichia coli 405 CECT and Pseudomonas fluorescens 378 CECT at a rate of 10(6) and 10(7) cfu/ml, respectively, and treated with high hydrostatic pressure. Treatments consisted of combinations of pressure (300, 400, 450, and 500 MPa), temperature (2, 10, 25, and 50 degrees C), and time (5, 10, and 15 min). Inactivation (> 6 log cfu/ml) of both strains was observed at 50 degrees C for all pressures and treatment times. A similar level of inactivation occurred at > or = 450 MPa and 25 degrees C for E. coli and at > or = 400 MPa and 10 degrees C for P. fluorescens. Destruction was lowest at 10 degrees C for E. coli and at 25 degrees C for P. fluorescens. The test strain of E. coli was more baroresistance than was the P. fluorescens strain.     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50,900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42 degrees C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 micrograms/ml) and not inhibited by antipain dihydrochloride (120 micrograms/ml), aprotinin (4 micrograms/ml), bestatin (80 micrograms/ml), chymostatin (50 micrograms/ml), E-64 (20 micrograms/ml), leupeptin (4 micrograms/ml), Pefabloc SC (2000 micrograms/ml), pepstatin (4 micrograms/ml), phosphoramidon (660 micrograms/ml), or phenylmethylsulfonyl fluoride (400 micrograms/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.     

Purification and properties of dipeptidyl amino-peptidase I from chicken liver (author's transl)

Dipeptidyl aminopeptidase I (E.S. 3.4.14.1) from chicken liver was purified by the following steps: homogenization at pH 5, thermic precipitation, acetone fractionation and Sephadex G-100, DEAE-cellulose and organomercurial-Sepharose column fractionations. The purified enzyme appears to be homogeneous by polyacrylamide gel electrophoresis at both pH 4.5 and 8.3 and has an isoelectric point of 5.7 +/- 0.05. The molecular weight of the enzyme reale 167,000 +/- 17,000 on the Sephadex G-150 column chromatography. The optimum pH for hydrolysis of Gly-Phe-p-nitroanilide (GPNA) and Gly-Phe-B-naphthylamide was 5.8. The value of Km for the hydrolysis of GPNA was estimated at 3.3 mM. The enzyme required halide ions for activity and was activated by thiol reagents (dithiothreitol, cysteine and 2-mercaptoethanol). Accordingly, DAP I was inhibited by thiol-blocking reagents (PCMB, IAA, Hg2+). The enzyme oxidation with oxygen current was fostered by chloride anion (50 nM); nevertheless the activity was recovered when cysteine was present in the incubation mixture; the latter, besides, seems to perform as enzyme protector.     

Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum

A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) from Clostridium hydroxybenzoicum JW/Z-1T was purified and partially characterized. The estimated molecular mass of the enzyme was 270 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single band of 57 kDa, suggesting that the enzyme consists of five identical subunits. The temperature and pH optima were 50 degrees C and pH 7.0, respectively. The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ . mol(-1) for the temperature range from 22 to 50 degrees C. The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 10(3) min(-1), respectively, at pH 7.0 and 25 degrees C. The enzyme optimally catalyzed the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0. The enzyme did not decarboxylate 2-hydroxybenzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2,3,4-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 3-F-4-hydroxybenzoate, or vanillate. The decarboxylase activity was inhibited by 25 and 20%, respectively, by 2,3,4- and 3,4,5-trihydroxybenzoate. Thiamine PPi and pyridoxal 5'-phosphate did not stimulate and hydroxylamine and sodium borohydride did not inhibit the enzyme activity, indicating that the 3,4-dihydroxybenzoate decarboxylase is not a thiamine PPi-, pyridoxal 5'-phosphate-, or pyruvoyl-dependent enzyme.