Highly stable L-lysine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring: characterization, gene cloning and sequencing, and expression

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively. No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.     

Purification and characterization of ferulate and p-coumarate decarboxylase from Bacillus pumilus.

Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4-vinylphenol, respectively, by nonoxidative decarboxylation. The enzyme responsible for this activity has been purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extract from a culture induced by ferulic acid or p-coumaric acid shows three bands that are not present in the crude extract of an uninduced culture, while the purified enzyme shows a single band of 23 kDa; the molecular mass calculated by size exclusion chromatography is 45 kDa. Enzyme activity is optimal at 37 degrees C and pH 5.5 and is not enhanced by any cation. Kinetic studies indicated a Km of 1.03 mM and a Vmax of 0.19 mmol.min-1/mg.liter-1 for ferulic acid and a Km of 1.38 mM and a Vmax of 0.22 mmol.min-1/mg.liter-1 for p-coumaric acid.     

Purification and characterization of the sea urchin embryo hatching enzyme

The sea urchin hatching enzyme provides an interesting model for the control of gene expression during early development. In order to study its properties and developmental regulation, the hatching enzyme of the species Paracentrotus lividus has been purified. The fertilization envelopes of the embryos were digested before hatching by a crude culture supernatant previously made. The enzyme was then solubilized by 1 M NaCl and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and purified by hydrophobic chromatography on Procion-agarose. A 470-fold increase in specific activity was obtained. The kinetic parameters of the proteolytic activity using dimethylcasein as substrate are: Km = 120 micrograms x ml-1, Vm = 200 mumol x min-1 x mg-1, and kcat = 180 s-1 at 500 mM NaCl, 10 mM CaCl2, pH 8.0, at 35 degrees C. The purified enzyme is highly active on fertilization envelopes: at 20 degrees C and 500 mM NaCl, 10 mM CaCl2, pH 8.0, 100 ng of enzyme completely denudes embryos in about 20 min under standard conditions. The molecular mass of the enzyme was estimated as 57 kDa by gel filtration, 51 kDa by gel electrophoresis, and 52 kDa by amino acid analysis. The hatching enzyme was shown to be a glycoprotein which autolyzes to a 30-kDa inactive form. Antibodies raised against the 51- or 30-kDa forms reacted with both these forms. Immunoblotting experiments showed that the hatching supernatants contain important amounts of the autolyzed species.     

Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete <Emphasis Type="Italic">Fomitopsis palustris</Emphasis> grown on microcrystalline cellulose

An extracellular beta-glucosidase was purified 154-fold to electrophoretic homogeneity from the brown-rot basidiomycete Fomitopsis palustris grown on 2.0% microcrystalline cellulose. SDS-polyacrylamide gel electrophoresis gel gave a single protein band and the molecular mass of purified enzyme was estimated to be approximately 138 kDa. The amino acid sequences of the proteolytic fragments determined by nano-LC-MS/MS suggested that the protein has high homology with fungal beta-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-beta-D-glucoside (p-NPG) and cellobiose hydrolyses were 0.117 and 4.81 mM, and the Kcat values were 721 and 101.8 per sec, respectively. The enzyme was competitively inhibited by both glucose (Ki= 0.35 mM) and gluconolactone (Ki= 0.008 mM), when p-NPG was used as substrate. The optimal activity of the purified beta-glucosidase was observed at pH 4.5 and 70 degrees. The F. palustris protein exhibited half-lives of 97 h at 55 degrees and 15 h at 65 degrees, indicating some degree of thermostability. The enzyme has high activity against p-NPG and cellobiose but has very little or no activity against p-nitrophenyl-beta-lactoside, p-nitrophenyl-beta-xyloside, p-nitrophenyl-alpha-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the beta-glucosidase from F. palustris can be classified as an aryl-beta-glucosidase with cellobiase activity.     

A thermostable alpha-arabinofuranosidase from xylanolytic Bacillus pumilus: purification and characterisation

Bacillus pumilus PS213 secretes an alpha-L-arabinofuranosidase (AF) when grown in the presence of arabinogalactan or oat meal. The enzyme has been purified to homogeneity and characterised. Its molecular mass, as determined by gel filtration, is 220 kDa, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of approximately 60 kDa. According to the result of the mass spectrometry analysis showing a molecular mass of 56 kDa, the enzyme should be a homotetramer. The isoelectric point was found to be 5.2, the enzyme activity was optimal at 55 degrees C and pH 7.0. The enzyme retained 80% of its activity after 2 h at 65 degrees C and lost 50% of activity at 75 degrees C after 135 min. The Michaelis constant K(m) and V(max) for p-nitrophenylarabinofuranoside at 37 degrees C were 1.7 mM and 52.9 U mg(-1), respectively. N-terminal sequence analysis and internal peptide fragments showed homology with glycosyl hydrolases of family 51.     

Penicillium chrysogenum extracellular acid phosphatase: purification and biochemical characterization

An extracellular acid phosphatase (EC 3.1.3.2) from crude culture filtrate of Penicillium chrysogenum was purified to homogeneity using high-performance ion-exchange chromatography and size-exclusion chromatography. SDS-PAGE of the purified enzyme exhibited a single stained band at an Mr of approx. 57,000. The mobility of the native enzyme indicated the Mr to be 50,000, implying that the active form is a monomer. The isoelectric point of the enzyme was estimated to be 6.2 by isoelectric focusing. Like acid phosphatases from several yeasts and fungi the Penicillium enzyme was a glycoprotein. Removal of carbohydrate resulted in a protein band with an Mr of 50,000 as estimated by SDS-PAGE, suggesting that 12% of the mass of the enzyme was carbohydrate. The enzyme was catalytically active at temperatures ranging from 20 degrees C to 65 degrees C with a maximum activity at 60 degrees C and the pH optimum was at 5.5. The Michaelis constant of the enzyme for p-nitrophenyl phosphate was 0.11 mM and it was inhibited competitively by inorganic phosphate (ki = 0.42 mM).     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

Purification and characterization of mannose isomerase from Agrobacterium radiobacter M-1

A mannose isomerase from Agrobacterium radiobacter M-1 (formerly Pseudomonas sp. MI) was purified to electrophoretic homogeneity and characterized. A cell-free extract was separated by ammonium sulfate fractionation, Butyl-Toyopearl 650M, DEAE-Sepharose and hydroxylapatite column chromatography. Its molecular mass was estimated to be 44 kDa by SDS-PAGE and 90 kDa by gel filtration, in which the enzyme is most likely a dimer composed of two identical subunits. The purified enzyme had an optimum pH at 8.0, an optimum temperature at 60 degrees C, a pI of 5.2 and a Km of 20 mM, and specifically converted D-mannose and D-lyxose to ketose. The N-terminal amino acid sequence was identified.     

Characterization of a soluble inorganic pyrophosphatase from Microcystis aeruginosa and preparation of its antibody.

A soluble inorganic pyrophosphatase was isolated from a crude extract of Microcystis aeruginosa by adsorption chromatography. The enzyme was purified to homogeneity as judged by sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide gel electrophoresis and N-terminal amino acid analysis. The molecular mass was estimated to be 80 kDa by gel filtration chromatography, 87 kDa by nondenaturing polyacrylamide gel electrophoresis, and 28 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an isoelectric point of 4.5, which is similar to the pI values reported for other soluble inorganic pyrophosphatases. The sequence of 29 N-terminal amino acids was determined; only 4 of these amino acids are identical to those in the sequence of Saccharomyces cerevisiae inorganic pyrophosphatase. M. aeruginosa inorganic pyrophosphatase is a Mg(2+)-dependent enzyme exhibiting a pH optimum of around 7.5. Its KM value for inorganic pyrophosphate was estimated to be 1.30 mM. A specific antibody was raised in chicken to M. aeruginosa inorganic pyrophosphatase. No immunological cross-reactivity was seen when Western blots of partially purified S. cerevisiae or Escherichia coli inorganic pyrophosphatase were probed with the antibody.     

Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans

A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45 degrees C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both heparin and HS, similar to heparinase II from Flavobacterium heparinum.     

Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1

A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH(2)-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85 degrees C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co(2+) and Mn(2+). The k(cat)/K(m) for D-alaninamide was measured as 544.4 +/- 5.5 mM(-1) min(-1) at 50 degrees C with 1 mM Co(2+).     

Purification and thermal characterization of a novel peroxidase from a local chick pea cultivar

A novel peroxidase isolated from a local chick pea (Cicer arietinum L.) cultivar (Balksar 2000) was purified by means of ammonium sulfate precipitation, DEAE-cellulose chromatography and two runs on gel filtration. The purified enzyme has a specific activity of 2045 U/mg with 17 % activity recovery. The molecular mass of the enzyme was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. Optimum pH and temperature of the enzyme were 5.5 and 45 degrees C respectively. The thermal denaturation of local chick pea peroxidase was studied in aqueous solution at temperatures ranging from 45 degrees C to 65 degrees C. The temperature of 50% inactivation of the enzyme was found to be 68 degrees C. The enthalpy (DeltaH*) and free energy (DeltaG*) of thermal denaturation of chick pea peroxidase were 101.4 and 103.4 k J/mol respectively at 65 degrees C.Metals like Zn2+, Mn2+, Hg2+, Co2+ and Al3+ slightly inhibited the peroxidase activity while Ca2+, Mg2+ and Ba2+ have no effect on enzyme activity. The high specific activity and thermal stability make chick pea peroxidase an alternative to horseradish peroxidase (HRP) in various applications.     

Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1

Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn²⁺ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure. Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

Purification and characterization of a lactonase from Burkholderia sp. R-711, that hydrolyzes (R)-5-oxo-2-tetrahydrofurancarboxylic acid

A lactonase hydrolyzing (R)-5-oxo-2-tetrahydrofurancarboxylic acid to D-alpha-hydroxyglutaric acid was purified 170-fold with 2% recovery to near homogeneity from crude extracts of Burkholderia sp. R-711, which had been isolated as a bacterium able to assimilate (R)-5-oxo-2-tetrahydrofurancarboxylic acid. The molecular mass was estimated to be 33 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 38 kDa upon SDS-PAGE. The maximum activity was observed at pH 7.0-8.0 and 35-40 degrees C. The enzyme required no added cofactors or metal ions; the activity was inhibited to 60-100% by SH-blocking reagents, but was not affected by metal-chelating reagents. The enzyme showed lower activity and affinity toward (S)-5-oxo-2-tetrahydrofurancarboxylic acid, but did not act on other natural and synthetic lactones tested.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae

The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V(max)/K(M) ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1,10-phenanthroline having a K(i) value of 0.12 mM and cysteine with K(i) value of 0.31 mM, while EGTA stimulated LAP activity. Zn(2+), Mg(2+) and Mn(2+) all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases.     

Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae

Exopeptidases of Morimus funereus larvae were partially purified and characterized. Specific leucyl aminopeptidase (LAP) activity was increased eight-fold by gel filtration of the crude midgut extract. The partially purified LAP had a molecular mass greater than 100 kDa with pH optima from 7.0-9.0 and no strict substrate specificity. M. funereus LAP preferentially hydrolyzed p-nitroanilides with hydrophobic amino acids in the active site, with a K(m) for leucine-p-nitroanilide of 0.21 mM. Zymogram analysis of an electropherogram obtained by native polyacrylamide gel electrophoresis revealed four enzymatically active proteinases using leucine-p-nitroanilide and methionine-p-nitroanilide as substrates and two enzymatically active proteinases using lysine-p-nitroanilide as a substrate. Although the optimal temperature of LAP activity was 40 degrees C, the enzyme was active over a broad temperature range from 2 to 60 degrees C. Among a number of inhibitors tested, heavy metals and 1,10-phenanthroline completely inhibited the enzyme, while methanol, ethanol and EGTA stimulated somewhat LAP activity.     

Purification and partial characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase produced by<Emphasis Type="Italic">Thermomonospora fusca</Emphasis>

Thermomonospora fusca produced a relatively high level of alpha-L-arabinofuranosidase when growing on oat spelt xylan as the main carbon and energy source. The enzyme exhibited maximum relative activity (0.136 U/g protein) at pH 9.0 with 54 and 55% activity remaining at pH of 4.5 and 11.0, respectively. The apparent Km value for the crude alpha-L-arabinofuranosidase preparation was 180 mumol/L 4-nitrophenyl alpha-L-arabinofuranoside; the upsilon lim value was the release of 40 mumol/L 4-nitrophenol per min. Enzyme activity was eluted as a single peak (HPLC gel filtration chromatography) corresponding to molar mass of approximately 92 kDa. Native electrophoresis of crude cell lysate confirmed the presence of a single active intracellular alpha-L-arabinofuranosidase component. SDS-PAGE of this enzyme, developed as zymogram, did not demonstrate any activity; denaturing gel was stained and a protein band of relative molar mass of 46 kDa was revealed. Isoelectric focusing of a purified alpha-L-arabinofuranosidase yielded a single protein band for the corresponding activity zone with pI 7.9. The enzyme was purified approximately 21-fold the mean overall yield was about 16%.     

The purification and characterization of a dextranase from Lipomyces starkeyi

Dextranase produced by Lipomyces starkeyi was purified 43-fold, by carboxymethyl-Sepharose chromatography followed by agarose gel-filtration chromatography. The purified enzyme showed four bands by SDS/polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65 kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55 degrees C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40 degrees C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto-oligosaccharides from glucose to isomaltotetrose.