Identification of a novel esterase from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2

Extremophiles - In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the...     

Proteomics analysis of aromatic catabolic pathways in thermophilic Geobacillus thermodenitrificans NG80-2

Geobacillus thermodenitrificans NG80-2 is a crude oil-degrading thermophilic bacterium isolated from an oil reservoir in China. In this study, the gene clusters and pathways for the degradation of benzoate (via benzoyl-CoA), phenylacetate (via phenylacetyl-CoA), 4-hydroxyphenylacetate (via 3,4-dihydroxyphenylacetate) and anthranilate (via 3-hydroxyanthranilate) were confirmed using combined in silico analysis and proteomics approaches. It was observed that synthesis of the enzymes catalyzing initial activation, ring oxidation and ring cleavage reactions were generally induced specifically by their respective substrates, while many of the enzymes catalyzing downstream reactions exhibited broader substrate specificities. Novel genes encoding benzoyl-CoA epoxidase and 3,4-dihydroxyphenylacetate 2,3-dioxygenase, and a paaX homologue that serves as a positive regulator of benzoate degradation were proposed. Downregulation of the glycolysis pathway, along with upregulation of the gluconeogenesis pathway and the glyoxylate bypass (phenylacetate) were detected in association with the utilization of the aromatics. This novel proteomics approach confirmed the presence of multiple metabolic pathways for aromatic compounds in NG80-2, which is highly advantageous to the survival of this thermophilic bacterium under reservoir conditions.     

Characterization of two long-chain fatty acid CoA ligases in the Gram-positive bacterium Geobacillus thermodenitrificans NG80-2

The functions of two long-chain fatty acid CoA ligase genes (facl) in crude oil-degrading Geobacillus thermodenitrificans NG80-2 were characterized. Facl1 and Facl2 encoded by GTNG_0892 and GTNG_1447 were expressed in Escherichia coli and purified as His-tagged fusion proteins. Both enzymes utilized a broad range of fatty acids ranging from acetic acid (C₂) to melissic acid (C₃₀). The most preferred substrates were capric acid (C₁₀) for Facl1 and palmitic acid (C₁₆) for Facl2, respectively. Both enzymes had an optimal temperature of 60 °C, an optimal pH of 7.5, and required ATP as a cofactor. Thermostability of the enzymes and effects of metal ions, EDTA, SDS and Triton X-100 on the enzyme activity were also investigated. When NG80-2 was cultured with crude oil rather than sucrose as the sole carbon source, upregulation of facl1 and facl2 mRNA was observed by real time RT-PCR. This is the first time that the activity of fatty acid CoA ligases toward long-chain fatty acids up to at least C₃₀ has been demonstrated in bacteria.     

Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90°C for up to 1 h. Its optimal pH and temperature were observed to be 7.6–7.8 and 60°C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K_m and V_max values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 mol min^–1 mg^–1 protein and 1,149 mol min^–1 mg^–1 protein, respectively. The turnover rate (k_cat) and catalytic efficiency (k_cat/ K_m) for Leu-p-NA and LeuGlyGly were 10,179 s^–1 and 49,543 s^–1 and 15,470 mM^–1 s^–1 and 1981.7 mM^–1 s^–1, respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, -mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co²⁺ .     

嗜热脱氮土壤芽孢杆菌β-葡萄糖苷酶的克隆与重组表达及其酶学性质研究

【目的】克隆嗜热脱氮土壤芽孢杆菌中的β-葡萄糖苷酶基因bglB,在E.coli中异源表达,纯化并研究其酶学性质。【方法】利用PCR技术从嗜热脱氮土壤芽孢杆菌的基因组DNA中克隆得到bglB基因,将该基因克隆到表达载体pGEX-2TL上并在大肠杆菌BL21(DE3)中表达,对纯化后的β-葡萄糖苷酶的酶学性质及寡聚状态进行分析。【结果】重组表达的β-葡萄糖苷酶最适温度为65°C,最适pH为7.0,能在pH 5-10、60°C下稳定存在4 h,并能在较高的离子强度(880 mmol/L K+)下发挥其功能。Al3+离子对其有强烈的激活作用,Co2+有一定的抑制作用。最适反应条件下该酶比活力为0.043 IU/mg。该酶具有多种寡聚体形式,这些寡聚体均有β-葡萄糖苷酶活性。【结论】获得一个耐热耐盐的中性β-葡萄糖苷酶,为进一步研究β-葡萄糖苷酶的催化作用机理,提高其热稳定性提供一定的帮助。     

Purification and characterization of an extremely stable glucose isomerase from Geobacillus thermodenitrificans TH2

The D-glucose/D-xylose isomerase was purified from a thermophilic bacterium, Geobacillus thermodenitrificans TH2, by precipitating with heat shock and using Q-Sepharose ion exchange column chromatography, and then characterized. The purified enzyme had a single band having molecular weight of 49 kDa on SDS-PAGE. In the presence of D-glucose as a substrate, the optimum temperature and pH of the enzyme were found to be 80°C and 7.5, respectively. The purified xylose isomerase of G. thermodenitrificans TH2 was extremely stable at pH 7.5 after 96 h incubation at 4°C and 50°C. When the thermal stability profile was analyzed, it was determined that the purified enzyme was extremely stable during incubation periods of 4 months and 4 days at 4°C and 50°C, respectively. The K _m and V _max values of the purified xylose isomerase from G. thermodenitrificans TH2 were calculated as 32 mM and 4.68 μmol/min per mg of protein, respectively. Additionally, it was detected that some metal ions affected the enzyme activity at different ratios. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.     

Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans

Cytochromes bd are terminal oxidases in the respiratory chains of many prokaryotic organisms. They reduce O? to 2H?O at the expense of electrons extracted from quinol. The oxidases can be divided into two subfamilies, L and S, based on the presence of either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I designated as 'Q-loop'. The L-subfamily members, e.g. the enzyme from Escherichia coli, are relatively well-studied and were shown to generate proton-motive force. The S-subfamily comprises the majority of cytochromes bd including the enzyme from Geobacillus thermodenitrificans but is very poor studied. We compared the properties of cytochromes bd from G. thermodenitrificans and E. coli at room temperature using a combination of absorption, CD and MCD spectroscopy. The G. thermodenitrificans enzyme does contain the high-spin heme b(HS) ("b(595)") despite the fact that its characteristic Q(00)-band ("α"-band) at 595nm is not seen in the absorption spectra; stoichiometry of hemes b(LS), b(HS) and d per the enzyme complex is suggested to be 1:1:1. At 1mM CO, 20-25% of ferrous heme b(HS) in the G. thermodenitrificans oxidase binds the ligand, while in case of the E. coli enzyme such a reaction is minor. In the G. thermodenitrificans oxidase, the excitonic interaction between ferrous hemes b(HS) and d decreased as compared to that in the E. coli bd. The latter may suggest that the two enzymes differ in the distance between heme d and heme b(HS) and/or in the angle between their porphyrin planes.     

Purification and characterization of leucine aminopeptidase from kidney bean cotyledons

A leucine aminopeptidase (EC 3,4,11.1) was purified from cotyledons of resting kidney beans ( Phaseolus vulgaris L. cv. Processor) by acidic extraction, ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, Mono Q HPLC and Superose HPLC columns. The yield of the 317-fold purified enzyme was 9%. On gel filtrations on Sephacryl S-300 and Superose HPLC the elution volumes of the enzyme corresponded to an M, of 360 000. The enzyme gave one band on native gel electrophoresis and an electrophoretic titration in an immobilized pH gradient gave a single curve with a pI of 4.8. Two bands were observed in an SDS-gel electrophoresis with M_r values of 58 000 and 60 000 both with and without reduction by 2-mercaptoethanol, indicating that subunits of the enzyme are not linked by disulphide bridges. The purified enzyme most rapidly liberated Leu and Ala of the N-termini of di-and oligopeptides, optimally at pH 9.0 ± 0.5. The enzyme was stable in the presence of glycerol, dithiothreitol and Mg²⁺, while the latter also had an activating effect. Bestatin inhibited the enzyme competitively with Leu-Gly-Gly with a Kⁱ-value of 1.5 nM . These observations indicate that the purified aminopeptidase from the cotyledons of resting kidney beans corresponds to the cytosolic leucine aminopeptidase of mammalian tissues (EC 3.4, 11.1). The high enzyme activity observed suggests that this aminopeptidase has an important role in the production of free amino acids during germination.     

Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V

The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E. coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20 degrees C and 50 degrees C. At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed.     

Immunoaffinity purification and characterization of leucine aminopeptidase from human liver

Leucine aminopeptidase was purified from human liver cytosol to homogeneity, 1538-fold, with a yield of 84.4% by immunoaffinity chromatography. Increases in the activity and the stability of the enzyme were simultaneously observed during the purification procedure, suggesting the presence of some endogenous inhibitor in cytosol. The specific activity and Km value of the enzyme for L-leucine amide were found to be 58.00 mumol/min/mg of protein and 4.02 mM, respectively, at pH 8.0. The molecular weight of the enzyme was determined to be 360,000 by both polyacrylamide gradient gel electrophoresis and Sephadex G-200 gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of native and dimethyl suberimidate cross-linked enzyme indicate that the native enzyme has two subunits of Mr 53,000 (a) and 65,000 (b) and is a hexamer arranged as a trimer of dimers (3 X (a X b)). The optimum pH was 10.5, and the enzyme was stable in the pH range from 7.5-8.5. The enzyme was activated by divalent metal ions, especially by Mg2+ and Mn2+, with no change in Km value. The enzyme was inhibited by metal-chelating agents, indicating it to be a metalloenzyme. Amastatin and bestatin strongly inhibited the enzyme, but leupeptin did not. The enzyme had a broad substrate specificity toward oligopeptides and amino acid amides but had little or no activity toward chromogenic substrates. The enzyme also could hydrolyze natural substrates contained in liver cytosol and accordingly produce many kinds of amino acids commonly found in proteins.     

Production and characterization of exopolysaccharides by Geobacillus thermodenitrificans ArzA-6 and Geobacillus toebii ArzA-8 strains isolated from an Armenian geothermal spring

The thermal ecosystems, including geothermal springs, are proving to be source of thermophiles able to produce extracellular polysaccharides (EPSs). Among the sixteen thermophilic bacilli isolated from sediment sampled from Arzakan geothermal spring, Armenia, two best EPSs producer strains were identified based on 16S rRNA gene sequence analysis and phenotypic characteristics, and designated as Geobacillus thermodenitrificans ArzA-6 and Geobacillus toebii ArzA-8 strains. EPSs production was investigated under different time, temperature and culture media’s composition. The highest specific EPSs production yield (0.27 g g⁻¹ dry cells and 0.22 g g⁻¹ dry cells for strains G. thermodenitrificans ArzA-6 and G. toebii ArzA-8, respectively) was observed after 24 h when fructose was used as sole carbon source at 65 °C and pH 7.0. Purified EPSs displayed a high molecular mass: 5 × 10⁵ Da for G. thermodenitrificans ArzA-6 and 6 × 10⁵ Da for G. toebii ArzA-8. Chemical composition and structure of the biopolymers, determined by GC–MS, HPAE-PAD and NMR, showed that both the two EPSs are heteropolymers composed by mannose as major monomer unit. Optical rotation values [α] ^25 °C_D of the two EPSs (2 mg ml⁻¹ H₂O) were − 142,135 and − 128,645 for G. thermodenitrificans ArzA-6 and G. toebii ArzA-8, respectively.

     

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.     

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.     

Purification and partial characterization of barley leucine aminopeptidase

A peptidase acting on Leu-Gly-Gly and Leu-Tyr at pH 8 to 10 was purified about 670-fold from germinated grains of barley (Hordeum vulgare L.). Gel electrophoretic analyses indicated a purity of about 90%. The purified enzyme is remarkably similar to mammalian leucine aminopeptidases (EC 3.4.1.1) both in chemical and in enzymatic properties. It has a sedimentation constant of 12.7S and a molecular weight of about 260,000. The enzyme has a high activity on leucine amide and di- and tripeptides with N-terminal leucine or methionine; leucyl-β-naphthylamide, in contrast, is hydrolyzed very slowly. The enzyme also liberates N-terminal amino acids from the insulin B chain. The pH optima for the hydrolysis of different substrates depend on the buffers used; highest reaction rates are generally obtained at pH 8.5 to 10.5. Mg²⁺ and Mn²⁺ ions stabilize (and probably activate) the enzyme. In contrast to mammalian leucine aminopeptidases, the barley enzyme is inactivated in the absence of reducing sulfydryl compounds.     

Identification and characterization of Paragonimus westermani leucine aminopeptidase

Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.     

Purification,characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.     

Schistosoma mansoni: purification and characterization of a membrane-associated leucine aminopeptidase

Membrane-associated leucine aminopeptidase (EC 3.4.11.1, LAP) has been purified to homogeneity from Schistosoma mansoni egg homogenates by a combination of ultracentrifugation, chromatofocusing, and molecular sieve chromatography. A 260-fold increase in specific activity was observed after purification. This is a metalloenzyme, containing carbohydrate moieties. Optimal enzyme activity was found at neutral pH. Enzyme activity was measured using L-leucine-7-amino-4-trifluoromethylcoumarin (L-Leu-AFC); in addition, schistosome egg LAP hydrolyzed a variety of other aminopeptidase substrates. Hydrolysis of L-Leu-AFC was inhibited by a number of aminopeptidase inhibitors, including 1,10-phenanthroline, bestatin, and amastatin.