A novel serine protease from pseuderanthemum latifolium B. Hansen: Characterization and fibrino(geno)lytic activities.

Protease (PPL) was isolated from Pseuderanthemum latifolium B. Hansen and had a molecular mass of 70 kDa. The N-terminal sequence of PPL showed 70-80% similarity with of subtilisin-like serine proteases from plants, but it did not show any sequence homology with known plant proteases. Serine protease inhibitors (PMSF, DFP) effectively blocked about 90% of PPL activity. PPL was highly activity at the pH range from 6 to 9 and temperatures from 50 °C to 80 °C, with an optimum at pH 7.0 and temperatures 70 °C. PPL had stability in a variety of pH, temperature, surfactant and oxidizing agents. PPL with concentration of 2.5 µg completely hydrolyzed the Aα-chain of fibrinogen within 5 min and hydrolyzed the Bß and the γ-chain after 10 h. Fibrin also was strong hydrolyzed by PPL with concentration of 0.3 µg. Thus, PPL is a unique serine protease, which it had strong fibrino(geno)lytic activities.

Molecular characterization and immunogenic function of ML1899 (LipG) of Mycobacterium leprae.

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.

Hanseniaspora thailandica BC9 ß-Glucosidase for the Production of ß-D-Hexyl Glucoside.

For biotechnological production of high-valued ß-D-hexyl glucoside, the catalytic properties of Hanseniaspora thailandica BC9 ß-glucosidase purified from the periplasmic fraction were studied, and the transglycosylation activity for the production of ß-D-hexyl glucoside was optimized. The constitutive BC9 ß-glucosidase exhibited maximum specific activity at pH 6.0 and 40ºC, and the activity of BC9 ß-glucosidase was not significantly inhibited by various metal ions. BC9 ß-glucosidase did not show a significant activity of cellobiose hydrolysis, but the activity was rather enhanced in the presence of sucrose and medium-chain alcohols. BC9 ß-glucosidase exhibited enhanced production of ß-D-hexyl glucoside in the presence of DMSO, and 62% of ß-D-hexyl glucoside conversion was recorded in 4 h in the presence of 5% 1-hexanol and 15% DMSO.

Hydrolysis of soybean isoflavones by Debaryomyces hansenii UFV-1 immobilised cells and free ß-glucosidase.

An intracellular ß-glucosidase from Debaryomyceshansenii UFV-1 was produced in an YP medium with cellobiose as the carbon source. This enzyme was purified, characterised and presented a Mr of 65.15kDa. Yeast cells containing the intracellular ß-glucosidase were immobilised in calcium alginate. The free ß-glucosidase and immobilised cells containing the enzyme presented optima values of pH and temperature of 6.0 and 45°C and 5.5 and 50°C, respectively. The free enzyme maintained 62% and 47% of its original activity after 90days at 4°C and after 15days at room temperature, respectively. The immobilisation process resulted in higher enzyme thermostability at 45 and 50°C. Soy molasses treatment with the free enzyme and the immobilised cells containing ß-glucosidase, for 2h at 40°C, promoted efficient hydrolysis of isoflavone glicosides to their aglycon forms. The results suggest that this enzyme could be used in the food industry, in the free or immobilised forms, for a safe and efficient process to hydrolyse isoflavone glycosides in soy molasses.

Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii.

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.

Debaryomyces hansenii UFV-1 intracellular alpha-galactosidase characterization and comparative studies with the extracellular enzyme.

Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s-1, respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The Km for hydrolysis of pNPalphaGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was a competitively inhibited by galactose (Ki = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.

Optimal activity and thermostability of xylose reductase from Debaryomyces hansenii UFV-170.

Xylose reductase (XR) is the enzyme that catalyzes the first step of xylose metabolism. Although XRs from various yeasts have been characterized, little is known about this enzyme in Debaryomyces hansenii. In the present study, response surface analysis was used to determine the optimal conditions for D. hansenii UFV-170 XR activity. The influence of pH and temperature, ranging from 4.0 to 8.0 and from 25 to 55 degrees C, respectively, was evaluated by a 2(2) central composite design face-centered. The F-test (ANOVA) and the Student's t test were performed to evaluate the statistical significance of the model and the regression coefficients, respectively. The NADPH-dependent XR activity varied from 0.502 to 2.53 U mL(-1), corresponding to 0.07-0.352 U mg(-1), whereas the NADH-dependent one was almost negligible. The model predicted with satisfactory correlation (R (2) = 0.940) maximum volumetric activity of 2.27 U mL(-1) and specific activity of 0.300 U mg(-1) at pH 5.3 and 39 degrees C, which were fairly confirmed by additional tests performed under these conditions. The enzyme proved very stable at low temperature (4 degrees C), keeping its activity almost entirely after 360 min, which corresponded to the half-time at 39 degrees C. On the other hand, at temperatures >or=50 degrees C it was lost almost completely after only 20 min.

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids.

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

Cloning and structural analysis of Mycobacterium leprae serine hydroxymethyltransferase.

Serine hydroxymethyltransferase (SHMT) plays a key role in cell physiology as it participates in the different interconversion pathway of folate coenzymes, provides almost exclusively folate one carbon fragments for the biosynthesis of a variety of end products. For the first time, Mycobacterium leprae glyA gene, encodes the enzyme serine hydroxymethyltransferase, has been cloned in Escherichia coli, over-expressed and purified the protein product (mlSHMT) for folding and stability studies under various denaturating conditions. The recombinant mlSHMT exists as homo-dimer of molecular mass about 90 kDa under physiological conditions . The studies on catalytic properties of mlSHMT show that the enzyme catalyzes the H(4)-folate dependent retro-aldol cleavage of L-serine, however, D-alanine dependent transaminase activity was absent in the enzyme. Further analysis of the enzyme kinetics for hydroxymethyltransferase reaction for mlSHMT demonstrates a comparable K(m) value for L-serine to SHMTs from other sources but significantly lower catalytic efficiency (k(cat)/K(m)). The mlSHMT is resistant to alkaline denaturation and exist as apo-dimer up to pH 10.5. Urea and guanidinium chloride induces dissociation of mlSHMT dimer into monomer at low denaturant concentrations, and leads to loss of enzymatic activity.

Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources.

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45 degrees C, respectively. The enzyme was activated by Cu(2+) (at a concentration of 1.0 mM) and Mn(2+) and inhibited by Hg(2+), Fe(2+), Fe(3+), Zn(2+), and Co(2+). The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1-10-phenanthroline, and iodoacetic acid. The K(m) and V(max) values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 micromol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.

Expression, purification, and characterization of human acetyl-CoA carboxylase 2.

The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could be more similar to the native enzyme.

Pyruvate carboxylase from Mycobacterium smegmatis: stabilization, rapid purification, molecular and biochemical characterization and regulation of the cellular level.

This is the first report on the purification and characterization of an anaplerotic enzyme from a Mycobacterium. The anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages. We have purified and characterized a pyruvate carboxylase (PYC) from Mycobacterium smegmatis and cloned and sequenced its gene. We have developed a very rapid and efficient purification protocol that provided PYC with very high specific activities (up to 150 U/mg) that remained essentially unchanged over a month. The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs for activity and inhibited by ADP, by excess Mg(2+), Co(2+), and Mn(2+), by aspartate, but not by glutamate and alpha-ketoglutarate. Supplementation of minimal growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged. These observations would not fit the idea that the M. smegmatis enzyme fulfills a straightforward anaplerotic function; in a closely related organism, Corynebacterium glutamicum, PYC is the major anaplerotic enzyme. Growth on glucose provided 2-fold higher cellular PYC level than that observed with glycerol. The PYCs of M. smegmatis and Mycobacterium tuberculosis were highly homologous to each other. In M. smegmatis, M. tuberculosis and M. lepra, pyc was flanked by a putative methylase and a putative integral membrane protein genes in an identical operon-like arrangement. Thus, M. smegmatis could serve as a model for studying PYC-related physiological aspects of mycobacteria. Also, the ease of purification and the extraordinary stability could make the M. smegmatis enzyme a model for studying the structure-function relationships of PYCs in general. It should be noted that no crystal structure is available for this enzyme of paramount importance in all three domains of life, archaea, bacteria, and eukarya.

Purification and characterization of the NAD-preferring glucose 6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum).

An NAD-preferring glucose 6-phosphate dehydrogenase of Acetobacter hansenii (formerly known as Acetobacter xylinum) has been purified to apparent homogeneity and kinetically characterized. The purified enzyme was stabilized by the use of glycerol, MgSO4, and 2-mercaptoethanol at pH 5.4. The molecular weight of the enzyme, determined by nondenaturing gel filtration, is 243,000. The subunit molecular weight is 60,140 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. At pH 5.4 the enzyme has Kms of 0.104 and 0.34 mM for NAD+ and NADP+, respectively; the Kms for glucose 6-phosphate are 0.071 and 0.089 mM, using NAD+ and NADP+, respectively; and the kcat values are 128,000 and 77,300 min-1 with NAD+ and NADP+, respectively. The Kms for NADP+ and glucose 6-phosphate are approximately 10 times higher than the corresponding Kms for the NADP-specific glucose 6-phosphate dehydrogenase in the same organism, but the kcat is also approximately 10-fold higher, so that the kcat/Km values for these two activities are nearly identical at pH 5.4. Both the NAD- and NADP-linked activities of the NAD-preferring enzyme are inhibited by ATP. The NADP-specific glucose 6-phosphate dehydrogenase is insensitive to ATP at pH 6.7 and 9.5, but at pH 5.4 ATP inhibits this enzyme. The possible roles of these two glucose 6-phosphate dehydrogenases in the metabolism of A. hansenii are discussed.

Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residues (C-less permease), Asp237 and Lys358 were replaced with Cys or other amino acids to pursue the proposal that the two residues form a charge pair [King, S. C., Hansen, C. L., & Wilson, T.H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Individual replacement of Asp237 with Cys, Ala, or Lys or replacement of Lys358 with Cys, Ala, or Asp virtually abolishes active lactose transport. However, simultaneous replacement of both residues with Cys and/or Ala yields permease with high activity. Therefore, neutral amino acid substitutions at either position are detrimental only because they leave the opposing charge unpaired. Strikingly, moreover, when Asp237 is interchanged with Lys358, high activity is observed. The results indicate strongly that Asp237 and Lys358 interact to form a salt bridge and that neither residue nor the salt bridge per se is important for activity. Immunoblots reveal low membrane levels of the active mutants lacking the putative salt bridge, suggesting a role for the salt bridge in either permease folding or stability and raising the possibility that the salt bridge may exist in a folding intermediate but not in the mature protein. Remarkably, however, a mutant with Cys in place of Asp237 is restored to full activity by carboxymethylation which recreates a negative charge at position 237. Pulse-chase analysis and heat-inactivation studies indicate that the stability of the double mutant with Cys at positions 237 and 358 is comparable to C-less. Therefore, the interaction between Asp237 and Lys358 is likely to be important for permease folding and is maintained in the mature protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of NADP-linked glucose-6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum).

The NADP-linked glucose-6-phosphate dehydrogenase from Acetobacter hansenii (formerly known as Acetobacter xylinum) has been purified to apparent homogeneity. The sequence of the 10 N-terminal amino acids was determined. The subunit molecular weight of the enzyme is 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gel filtration studies under nondenaturing conditions revealed that the molecular weight of the enzyme is 200,000 to 220,000 at pH 6.5 and 9.5, suggesting that the native enzyme is a tetramer. Specificity studies at both pH 6.5 and 9.5 demonstrated that the enzyme is a typical NADP-preferring glucose-6-phosphate dehydrogenase. The enzyme's catalytic activity increases with increasing pH, kcat being approximately 4 times greater at pH 9.5 than at pH 6.7 and the Km for NADP+ being 3 times lower at the higher pH; but the Km for glucose 6-phosphate is nearly 20 times higher at pH 9.5 than at pH 6.7, suggesting that the enzyme is catalytically more efficient at the lower pH. At pH 6.7, initial velocity measurements, product inhibition by NADPH, and inhibition by glucosamine 6-phosphate yielded results that were consistent with a steady-state random mechanism. At pH 9.5, steady-state kinetic analyses suggested that the mechanism is ordered, with coenzyme binding first, but nonlinear double-reciprocal plots were observed in the presence of NADPH when glucose 6-phosphate was varied and a complete kinetic analysis was not undertaken. Among several nucleotides and potential inhibitory ligands examined, only 2',5'-ADP inhibited the enzyme significantly.