Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.     

Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.     

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.     

Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive l-arabinose isomerase from the Bacillus stearothermophilus US100 strain

The araA gene encoding L-arabinose isomerase from Bacillus stearothermophilus US100 strain was cloned, sequenced and over-expressed in E. coli. This gene encodes a 496-amino acid protein with a calculated molecular weight of 56.161 kDa. Its amino acid sequence displays the highest identity with L-AI from Thermus sp. IM6501 (98%) and that of Geobacillus stearothermophilus T6 (97%). According to SDS-PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of nearly 225 kDa, composed of four identical 56-kDa subunits. The L-AI US100 was optimally active at pH 7.5 and 80 degrees C. It was distinguishable by its behavior towards divalent ions. Indeed, the L-AI US100 activity and thermostability were totally independent for metallic ions until 65 degrees C. At temperatures above 65 degrees C, the enzyme was also independent for metallic ions for its activity but its thermostability was obviously improved in presence of only 0.2 mM Co2+ and 1 mM Mn2+. The V(max) values were calculated to be 41.3 U/mg for L-arabinose and 8.9 U/mg for D-galactose. Their catalytic efficiencies (k(cat)/K(m)) for l-arabinose and D-galactose were, respectively, 71.4 and 8.46 mM(-1) min(-1). L-AI US100 converted the d-galactose into D-tagatose with a high conversion rate of 48% after 7 h at 70 degrees C.     

Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65 degrees C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (Vmax, 35.5 U/mg) and 129 mM (Vmax, 7.5 U/mg), respectively, at pH 6 and 65 degrees C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (kcat/Km) of each mutant at different pHs was significantly affected by an increase or decrease in Vmax. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.     

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.     

ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritimawas purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 degrees C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant ( S(0.5)) of 0.7 mM and a Hill coefficient of 2.2. The apparent K(m) for ATP was 0.2 mM and the apparent V(max) value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent K(m) for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent V(max) of about 13 U/mg. Divalent cations were required for maximal activity; Mg(2+), which was most effective, could partially be replaced by Mn(2+) and Fe(2+). Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 degrees C and showed a significant thermostability up to 100 degrees C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.     

ATP-dependent glucokinase from the hyperthermophilic bacterium Thermotoga maritima represents an extremely thermophilic ROK glucokinase with high substrate specificity

The gene (open reading frame (ORF) Tm1469, glk) encoding ATP-dependent ROK (repressors, ORFs, sugar kinases) glucokinase (ATP-GLK, EC 2.7.1.2) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 80 kDa composed of 36-kDa subunits. Rate dependence (at 80 degrees C) on glucose and ATP followed Michaelis-Menten kinetics with apparent Km values of 1.0 and 0.36 mM, respectively; apparent Vmax values were about 370 U mg(-1). The enzyme was highly specific for glucose as phosphoryl acceptor. Besides glucose only 2-deoxyglucose was phosphorylated to some extent, whereas mannose and fructose were not used. With a temperature optimum of 93 degrees C the enzyme is the most thermoactive bacterial ATP-GLK described.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.     

Characterization of a novel D-lyxose isomerase from Cohnella laevoribosii RI-39 sp. nov

A newly isolated bacterium, Cohnella laevoribosii RI-39, could grow in a defined medium with L-ribose as the sole carbon source. A 21-kDa protein isomerizing L-ribose to L-ribulose, as well as D-lyxose to D-xylulose, was purified to homogeneity from this bacterium. Based on the N-terminal and internal amino acid sequences of the purified enzyme obtained by N-terminal sequencing and quantitative time of flight mass spectrometry-mass spectrometry analyses, a 549-bp gene (lyxA) encoding D-lyxose (L-ribose) isomerase was cloned and expressed in Escherichia coli. The purified endogenous enzyme and the recombinant enzyme formed homodimers that were activated by Mn(2+). C. laevoribosii D-lyxose (L-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70 degrees C in the presence of Mn(2+) for D-lyxose and L-ribose, and its isoelectric point (pI) is 4.2 (calculated pI, 4.9). The enzyme is specific for D-lyxose, L-ribose, and D-mannose, with apparent K(m) values of 22.4 +/- 1.5 mM, 121.7 +/- 10.8 mM, and 34.0 +/- 1.1 mM, respectively. The catalytic efficiencies (k(cat)/K(m)) of CLLI were 84.9 +/- 5.8 mM(-1) s(-1) for D-lyxose (V(max), 5,434.8 U mg(-1)), 0.2 mM(-1) s(-1) for L-ribose (V(max), 75.5 +/- 6.0 U mg(-1)), and 1.4 +/- 0.1 mM(-1) s(-1) for D-mannose (V(max), 131.8 +/- 7.4 U mg(-1)). The ability of lyxA to permit E. coli cells to grow on D-lyxose and L-ribose and homology searches of other sugar-related enzymes, as well as previously described sugar isomerases, suggest that CLLI is a novel type of rare sugar isomerase.     

Cloning, expression and characterization of trehalose-6-phosphate phosphatase from a psychrotrophic bacterium, Arthrobacter strain A3

A trehalose-6-phosphate phosphatase (TPP) gene, otsB, from a psychrotrophic bacterium, Arthrobacter strain A3, was identified. The product of this otsB gene is 266 amino acids in length with a calculated molecular weight of 27,873 Da. The protein was expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant TPP catalyzed the dephosphorylation of trehalose-6-phosphate to form trehalose and showed a broad optimum pH range from 5.0 to 7.5. This enzyme also showed an absolute requirement for Mg(2+) or Co(2+) for catalytic activity. The recombinant TPP had a maximum activity at 30 °C and maintained activity over a temperature range of 4-30 °C. TPP was generally heat-labile, losing 70 % of its activity when subjected to heat treatment at 50 °C for 6 min. Kinetic analysis of the Arthrobacter strain A3 TPP showed ~tenfold lower K (m) values when compared with values derived from other bacterial TPP enzymes. The highest k (cat)/K (m) value was 37.5 mM(-1) s(-1) (repeated three times), which is much higher than values published for mesophilic E. coli TPP, indicating that the Arthrobacter strain A3 TPP possessed excellent catalytic activity at low temperatures. Accordingly, these characteristics suggest that the TPP from the Arthrobacter strain A3 is a new cold-adapted enzyme. In addition, this is the first report characterizing the enzymatic properties of a TPP from a psychrotrophic organism.     

Purification and characterization of alpha-L-arabinofuranosidase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produced an alpha-L-arabinofuranosidase when grown in the presence of L-arabinose, sugar beet arabinan, or oat spelt xylan. At the end of a fermentation, about 40% of the activity was extracellular, and enzyme activity in the cell-free supernatant could reach 25 U/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 20000, and the enzyme was purified eightfold by anion-exchange and hydrophobic interaction chromatographies. The molecular weight of T-6 alpha-L-arabinofuranosidase was 256,000, and it consisted of four identical subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The native enzyme had a pI of 6.5 and was most active at 70 degrees C and at pH 5.5 to 6.0. Its thermostability at pH 7.0 was characterized by half-lives of 53, 15, and 1 h at 60, 65, and 70 degrees C, respectively. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax, a Km, and an activation energy of 749 U/mg, 0.42 mM, and 16.6 kcal/mol, (ca. 69.5 kJ/mol), respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by 1 mM Hg2+. T-6 alpha-L-arabinofuranosidase released L-arabinose from arabinan and had low activity on oat spelt xylan. The enzyme acted cooperatively with T-6 xylanase in hydrolyzing oat spelt xylan, and L-arabinose, xylose, and xylobiose were detected as the end reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and functional characterization of a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667

The gene encoding a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667, abfB, was cloned and sequenced. The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with alpha-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism. A histidine tag was introduced at the C terminus of AbfB, and the recombinant protein was overexpressed in Lactococcus lactis under control of the tightly regulated, nisin-inducible nisA promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure. AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70 degrees C), and it had an optimum pH of 6.0 and an optimum temperature of 45 degrees C. The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55 degrees C. Chelating and reducing agents did not have any effect on its activity, but the presence of Cu(2+), Hg(2+), and Zn(2+) markedly reduced enzymatic activity. The protein exhibited a high level of activity with p-nitrophenyl alpha-L-arabinofuranoside, with apparent K(m) and V(max) values of 0.295 mM and 417 U/mg, respectively. AbfB released L-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. No endoarabinanase activity was detected. These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of L-arabinose-containing polysaccharides.     

The extremely thermophilic eubacterium, Thermotoga maritima, contains a novel iron-hydrogenase whose cellular activity is dependent upon tungsten.

Thermotoga maritima is the most thermophilic eubacterium currently known and grows up to 90 degrees C by a fermentative metabolism in which H2, CO2, and organic acids are end products. It was shown that the production of H2 is catalyzed by a single hydrogenase located in the cytoplasm. The addition of tungsten to the growth medium was found to increase both the cellular concentration of the hydrogenase and its in vitro catalytic activity by up to 10-fold, but the purified enzyme did not contain tungsten. It is a homotetramer of Mr 280,000 and contains approximately 20 atoms of Fe and 18 atoms of acid-labile sulfide/monomer. Other transition metals, including nickel (and also selenium), were present in only trace amounts (less than 0.1 atoms/monomer). The hydrogenase was unstable at both 4 and 23 degrees C, even under anaerobic conditions, but no activity was lost in anaerobic buffer containing glycerol and dithiothreitol. Under these conditions the enzyme was also quite thermostable (t50% approximately 1 h at 90 degrees C) but extremely sensitive to irreversible inactivation by O2 (t50% approximately 10 s in air). The optimum pH ranges for H2 evolution and H2 oxidation were 8.6-9.5 and greater than or equal to 10.4, respectively, and the optimum temperature for catalytic activity was above 95 degrees C. In contrast to mesophilic Fe hydrogenases, the T. maritima enzyme had very low H2 evolution activity, did not use T. maritima ferredoxin as an electron donor for H2 evolution, was inhibited by acetylene but not by nitrite, and exhibited EPR signals typical of [2Fe-2S]1+ clusters. Moreover, the oxidized enzyme did not exhibit the rhombic EPR signal that is characteristic of the catalytic iron-sulfur cluster of mesophilic Fe hydrogenases. These data suggest that T. maritima hydrogenase has a different FeS site and/or mechanism for catalyzing H2 production. The potential role of tungsten in regulating the activity of this enzyme is discussed.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Gene Cloning,purification, and characterization of a phosphodiesterase from Delftia acidovorans

A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans, the gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 65 degrees C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(p-nitrophenyl)phosphate with a K(m) of 2.9 +/- 0.1 mM and a k(cat) of 879 +/- 73 min(-1). The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E. coli. The IS1071 transposon insertion sequence was found downstream of pdeA.     

Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase

The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli. The preparation yields 470 UL(-1) of enzyme at a specific activity of 9.4 U mg(-1). During retroaldol cleavage of KDPG, the enzyme shows a k(cat) that decreases with decreasing temperature. A more than offsetting decrease in K(m) yields an enzyme that is more efficient at 40 degrees C than at 70 degrees C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.     

An L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization

The araA gene encoding an L-arabinose isomerase (L-AI) from the acido-thermophilic bacterium Acidothermus cellulolytics ATCC 43068 was cloned and overexpressed in Escherichia coli. The open reading frame of the L-AI consisted of 1,503 nucleotides encoding 501 amino acid residues. The recombinant L-AI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be approximately 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was optimally active at 75°C and pH 7.5. It required divalent metal ions, either Mn²⁺ or Co²⁺, for both enzymatic activity and thermostability improvement at higher temperatures. The enzyme showed relatively high activity and stability at acidic pH. It exhibited over 90% of its maximal activity at pH 6.0 and retained 80% of activity after 12 h incubation at pH 6.0. Catalytic property study showed that the enzyme had an interesting catalytic efficiency. Its apparent K _m, V _max, and catalytic efficiency (k _cat/K _m) for D-galactose was 28.9 mM, 4.9 U/mg, and 9.3 mM⁻¹ min⁻¹, respectively. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield over 50% after 12 h under optimal conditions, suggesting its potential in D-tagatose production.     

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.