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PubMed1994

Purification and characterization of a soluble lipolytic acylhydrolase from Cowpea (Vigna unguiculata L.) leaves.

With the use of [14C]monogalactosyl diacylglycerol as substrate for enzymatic test, a lipolytic acylhydrolase (EC 3.1.1.26) was purified 263-fold with a yield of 2.0% from soluble leaf extract of Vigna unguiculata L. cv. EPACE-1. The procedure involved ammonium sulfate precipitation, Q-Sepharose Fast Flow chromatography, gel filtration on Sephacryl 300 HR and chromatofocusing on Mono-P, followed by a semi-preparative electrophoresis on polyacrylamide gel. The purified enzyme had a molecular mass of about 80 kDa, as determined by gel filtration. On SDS-PAGE, it showed a single band corresponding to a molecular mass of 40 kDa. The isoelectric point of the enzyme was estimated to be 5.0-5.1 by isoelectric focusing and chromatofocusing. The Km value was 0.119 mM for monogalactosyl-diacylglycerol. The hydrolytic activity of the enzyme on different substrates was determined: the relative rates were digalactosyl-diacylglycerol > monogalactosyl-diacylglycerol > phosphatidylcholine > phosphatidylglycerol. For all substrates, the products of hydrolysis were free fatty acids. Triacylglycerols were not hydrolysed. The enzyme was activated by calcium but was not calcium-dependent. Experiments concerning the enzyme stability as affected by temperature and pH demonstrated that it was quite stable.

Carboxylic Ester Hydrolases↗
PubMed2004

Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups.

A method for immobilization of Candida rugosa lipase to two types of chitosan beads by activating the hydroxyl groups of chitosan using carbodiimide coupling agent has been successfully developed. The ability of carbodiimide to activate the hydroxyl groups of chitosan was confirmed using the electron spectroscopy for chemical analysis (ESCA) technique. The properties of lipase immobilized using dry and wet chitosan beads were also investigated and compared. Immobilization enhanced the enzyme stability against changes of pH and temperature. High storage stability of 30 days and an increased enzyme activity of 2,110% were observed in wet immobilized lipase. Immobilized lipase using dry and wet chitosan beads retained 78% and 85% of its initial activity after 10 batch hydrolytic cycles. The kinetic parameters Km and Vmax were determined for the free and immobilized lipase. The activation energy (Ea) was found to decrease for immobilization of lipase on chitosan beads.

Candida↗
PubMed1998

Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies.

The crystal structure of the thermostable xylanase from Thermomyces lanuginosus was determined by single-crystal X-ray diffraction. The protein crystallizes in space group P21, a = 40.96(4) A, b = 52. 57(5) A, c = 50.47 (5) A, beta = 100.43(5) degrees, Z = 2. Diffraction data were collected at room temperature for a resolution range of 25-1.55 A, and the structure was solved by molecular replacement with the coordinates of xylanase II from Trichoderma reesei as a search model and refined to a crystallographic R-factor of 0.155 for all observed reflections. The enzyme belongs to the family 11 of glycosyl hydrolases [Henrissat, B., and Bairoch, A. (1993) Biochem. J. 293, 781-788]. pKa calculations were performed to assess the protonation state of residues relevant for catalysis and enzyme stability, and a heptaxylan was fitted into the active-site groove by homology modeling, using the published crystal structure of a complex between the Bacillus circulans xylanase and a xylotetraose. Molecular dynamics indicated the central three sugar rings to be tightly bound, whereas the peripheral ones can assume different orientations and conformations, suggesting that the enzyme might also accept xylan chains which are branched at these positions. The reasons for the thermostability of the T. lanuginosus xylanase were analyzed by comparing its crystal structure with known structures of mesophilic family 11 xylanases. It appears that the thermostability is due to the presence of an extra disulfide bridge, as well as to an increase in the density of charged residues throughout the protein.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of brain-specific transglutaminases from rat.

The relevance of transglutaminases with neural function and several disorders has been emphasized recently. Especially, many polypeptides associated with neurodegenerative diseases are suggested to be putative transglutaminase substrates such as beta amyloid protein of Alzheimer's disease, microtubule-associated proteins and neurofilaments, etc. In addition, the CAG repeated gene products with probable polyglutamine tract, putative transglutaminase substrates, were identified in several neurodegenerative disorders. However, the identity of the brain transglutaminase has not been confirmed, because of enzymic stability and low activity. In the present experiment, we have isolated brain-specific transglutaminases, designated as TGase NI and TGase NII, which are different from other types of transglutaminases in respects of molecular weights (mw. 45 kDa, 29 kDa respectively), substrate affinity, elution profile on ion-exchange chromatography, sensitivity to proteases and ethanol, and immunological properties. The enzymes were localized specifically in the brain tissues but not in the liver tissue. And neural cells such as pheochromocytoma cell, glioma cell, primary neuronal and glial cells were shown to be enriched with TGase NI and TGase NII. The possible biological roles of the enzymes were discussed not only on the aspect of crosslinking activity but also of signal transducing capacity of the enzyme in the brain.

Animals↗
PubMed2003

Impact of ionic liquid physical properties on lipase activity and stability.

Lipase activity and stability was investigated in dialkylimidazolium and pyrrolidinium-based ionic liquids with a variety of anions including hexafluorophosphate, acetate, nitrate, methanesulfonate, trifluoroacetate, and trifluoromethylsulfonate. The initial rate of lipase-catalyzed transesterification of methyl methacrylate in these ionic liquids and several organic solvents was examined as well as the polytransesterification of divinyl adipate and 1,4-butanediol. Free lipase (Candida rugosa) catalyzed the transesterification of methyl methacrylate in 1-butyl-3-methylimidazolium hexafluorophosphate at a rate 1.5 times greater than in hexane. However, no detectable activity was observed in all the "hydrophilic" ionic liquids studied. Methods of enzyme stabilization including adsorption, PEG-modification, and immobilization in polyurethane foam were ineffective in improving enzymatic activity in the hydrophilic ionic liquids. Polytransesterifications performed in 1-butyl-3-methylimidazolium hexafluorophosphate using Novozym 435 produced polyesters with weight average molecular weights limited to 2900 Da due to precipitation of the polymer. Solvatochromic studies and partition coefficient measurements suggest that ionic liquids are more polar and hydrophilic than organic solvents such as hexane, acetonitrile, and tetrahydrofuran. Stability studies indicate that lipases exhibit greater stability in ionic liquids than in organic solvents including hexane.

Anions↗
PubMed2004

Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors.

The activation of caspase-3 represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Upon induction of apoptosis, the large (p17) and small (p12) subunits, comprising active caspase-3, are generated via proteolytic processing of a latent proenzyme dimer. Two copies of each individual subunit are generated to form an active heterotetramer. The tetrameric form of caspase-3 cleaves specific protein substrates within the cell, thereby producing the apoptotic phenotype. In contrast to the proenzyme, once activated in HeLa cells, caspase-3 is difficult to detect due to its rapid degradation. Interestingly, however, enzyme stability and therefore detection of active caspase-3 by immunoblot analysis can be restored by treatment of cells with a peptide-based caspase-3 selective inhibitor, suggesting that the active form can be stabilized through protein-inhibitor interaction. The heteromeric active enzyme complex is necessary for its stabilization by inhibitors, as expression of the large subunit alone is not stabilized by the presence of inhibitors. Our results show for the first time, that synthetic caspase inhibitors not only block caspase activity, but may also increase the stability of otherwise rapidly degraded mature caspase complexes. Consistent with these findings, experiments with a catalytically inactive mutant of caspase-3 show that rapid turnover is dependent on the activity of the mature enzyme. Furthermore, turnover of otherwise stable active site mutants of capase-3 is rescued by the presence of the active enzyme suggesting that turnover can be mediated in trans.

Apoptosis↗
PubMed2006

Functional domains of human tryptophan hydroxylase 2 (hTPH2).

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.

Enzyme Induction↗
PubMed2004

Engineering a thermostable human prolyl endopeptidase for antibody-directed enzyme prodrug therapy.

We present a new antibody-directed enzyme prodrug therapy strategy (ADEPT) based on a post-proline cleaving endopeptidase and prodrugs, in which cytotoxic moieties are linked to a proline-containing peptide. Human prolyl endopeptidase was expressed in Escherichia coli and purified to homogeneity. The enzyme was active in buffer and in human serum but was rapidly thermally inactivated by incubation at 37 degrees C, thus preventing applications in vivo. While prolyl endopeptidase display on filamentous phage abolished viral infectivity and prevented directed evolution strategies based on phage display, we robotically screened 10752 individual colonies of mutant enzymes using a fluorogenic assay to improve enzyme stability. A single amino acid mutation (Glu289 --> Gly) improved protein stability, resulting in a half-life of 16 h at 37 degrees C in phosphate buffer. Two prodrugs were synthesized, in which an N-protected glycine-proline dipeptide was covalently coupled to doxorubicin and melphalan. (Benzyloxycarbonyl)glycylprolylmelphalan, but not the more sterically hindered doxorubicin prodrug, could be efficiently activated by prolyl endopeptidase [specific activity = 813.3 nmol min(-1) (mg of enzyme)(-1) at 25 degrees C]. The melphalan prodrug was essentially nontoxic to CHO, F9 teratocarcinoma, MCF7 breast adenocarcinoma, and p3U1 mouse myeloma cells up to millimolar concentrations, while prodrug incubation with the engineered prolyl endopeptidase mutant led to a cell killing profile superimposable to the one of melphalan. The prolyl endopeptidase mutant was then chemically coupled to the human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis. The resulting immunoconjugate retains antigen binding and enzymatic activity, thus opening the way to anticancer ADEPT applications.

Animals↗
PubMedDate not supplied

[Stabilization of glucoso-6-phosphate dehydrogenase by its substrate and cofactor in an ultrasonic field].

The inactivation kinetics of glucoso-6-phosphate dehydrogenase (GPDH) and its complexes with glucoso-6-phosphate and NADP+ was characterized in aqueous solutions at 36-47 degrees C under treatment with low frequency (27 kHz, 60 W/cm2) and high frequency ultrasound (880 kHz, 1 W/cm2). To this end, we measured three effective first-order inactivation rate constants: thermal k(in)* , total (thermal and ultrasonic) kin, and ultrasonic kin (US). The values of the constants were found to be higher for the free enzyme than for its complexes GP-DH-GP and GPDH-NADP+ at all temperatures, which confirms the enzyme stabilization by its substrate and cofactor under both thermal and ultrasonic inactivation. Effective values of the activation energies (Ea) were determined and the preexponential factors of the rate constants and thermodynamic activation parameters of inactivation processes (deltaH*, deltaS*, and deltaG*) were calculated from the temperature dependences of the inactivation rate constants of GPDH and its complexes. The sonication of aqueous solutions of free GPDH and its complexes was accompanied by a reduction of Ea and deltaH* values in comparison with the corresponding values for thermal inactivation. The Ea, deltaH*, and deltaS* inactivation values for GPDH are lower than the corresponding values for its complexes. A linear dependence between the growth of the deltaH* and deltaS* values was observed for all the inactivation processes for free GPDH and its complexes.

Enzyme Stability↗
PubMed1990

Production of L-tryptophan from D,L-5-indolylmethylhydantoin by resting cells of a mutant of Arthrobacter species (DSM 3747).

The reaction parameters and the stereospecificity of the enzymatic cleavage of D,L-5-indolylmethylhydantoin in producing L-tryptophan with resting cells of Arthrobacter sp. DSM 3747 were studied. When intact cells were tested, the optimal pH was between 8.5 and 9.0 and the optimal temperature was 50 degrees C. Both, L-N-carbamoylase and hydantoinase could be stabilized over 24 h at 30 and 40 degrees C by the addition of D,L-5-indolylmethylhydantoin. Furthermore, the hydantoinase was stable over 24 h at 50 degrees C by the addition of 0.5 mM Mn2+ ions. The treatment with sodium desoxycholate turned out to be successful in overcoming the poor availability of D,L-5-indolylmethylhydantoin for the cells. The optimal temperature with permeabilized cells decreased to 30 degrees C and therefore ensured a good enzyme stability. While the L-N-carbamoylase proved to be absolutely L-specific, the hydantoinase led to a mixture of enantiomers of N-carbamoyltryptophan. The produced D-N-carbamoyl-tryptophan caused an inhibition of the L-N-carbamoylase. The transformation yield from D,L-5-indolylmethylhydantoin always reached 100%.

Amidohydrolases↗
PubMed2001

Type I collagen stabilization of matrix metalloproteinase-2.

The activity of matrix metalloproteinase-2 (MMP-2) is regulated stringently on the posttranslational level. MMP-2 efficiently undergoes autolysis into inactive polypeptides in vitro, prompting the hypothesis that MMP-2 autolysis may function as an alternative mechanism for posttranslational control of MMP-2 in vivo. Moreover, MMP-2 binds to intact type I collagen fibrils; however, the functional consequences of this interaction have not been fully elucidated. To test the hypothesis that MMP-2 binding to type I collagen functions as a positive regulator of MMP-2 proteolytic potential, the effect of type I collagen on MMP-2 activity, inhibition by tissue inhibitor of metalloproteinase-2 (TIMP-2), and enzyme stability was examined. Here, we report that purified MMP-2 binds but does not cleave intact type I collagen. The presence of type I collagen affects neither enzymatic activity against a quenched fluorescent peptide substrate nor the kinetics of inhibition by TIMP-2. However, MMP-2 is stabilized from autolysis in the presence of type I collagen, but not by elastin, fibrinogen, or laminin. These data provide biochemical evidence that MMP-2 exosite interactions with type I collagen may function in the posttranslational control of MMP-2 activity by reducing the rate of autolytic inactivation.

Autolysis↗
PubMed2006

Isolation and partial characterization of trehalose 6-phosphate synthase aggregates from Selaginella lepidophylla plants.

Trehalose 6-phosphate synthase was purified from Selaginella lepidophylla plants and three aggregates of the enzyme were found by molecular exclusion chromatography, ion exchange chromatography and electrophoresis. Molecular exclusion chromatography showed four activity peaks with molecular weights of 624, 434, 224 and 115 kDa. Ion exchange chromatography allowed three fractions to be separated with TPS activity which eluted at 0.35, 0.7 and 1 M KCl. Native PAGE of each pool had three protein bands with apparent M(r) 660, 440 and 200 kDa. Western blot results showed that anti-TPS antibody interacted with 115 and 67 kDa polypeptides; these polypeptides share peptide sequences as indicated by internal sequence data. The effects of pH and temperature on enzyme stability and activity were studied. For fractions eluted at 0.35 and 1.0 M KCl, the optimum pH is 5.5, while an optimum pH of 7.5 for 0.7 M fraction was found. The three fractions eluted from ion exchange chromatography were stable in a pH 5-11 range. Optimal temperatures were 25, 45 and 55 degrees C for 0.7, 0.35 and 1.0 M fractions, respectively. The 0.7 M KCl fraction showed highest stability in a temperature range of 25-60 degrees C, whereas the 0.35 M KCl fraction had the lowest in the same temperature range.

Amino Acid Sequence↗
PubMed2002

Bacillus subtilis inorganic pyrophosphatase: the C-terminal signature sequence is essential for enzyme activity and conformational integrity.

Bacillus subtilis inorganic pyrophosphatase is the first member of a newly identified Family II of PPases. To examine the role of a signature sequence found near the C-terminus, two truncated variants and a series of site-specific mutants were produced. A truncation of 17 residues (17AATR) but also single alanine substitutions, R295A and K296A, produced inactive enzyme. Removal of 5 nonconserved terminal residues (5AATR) markedly affected enzyme stability. Replacing S294 with A, T, C, or V decreased activity, the latter two mutations showing the greatest effect. Substitutions V299I and V300I had no or minor effects, whereas V300W and V299G/V300W significantly reduced activity. The sizes of truncated proteins and the full-length PPase were indistinguishable by gel-filtration. We conclude that the C-terminus has no role in multimerization, while both its conserved and nonconserved regions are essential for full enzyme activity. The signature sequence is required for both the conformation and composition of the active site.

Bacillus subtilis↗
PubMed1996

Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus.

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40-55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5-5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatography. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.

Aflatoxins↗
PubMed1975

Irreversible stimulation of adenylate cyclase activity of fat cell membranes of phosphoramidate and phosphonate analogs of GTP.

The ability of 5'-guanylylimidodiphosphate (Gpp(NH)p) to stimulate irreversibly the adenylate cyclease activity of fat cell membranes has been studied by preincubating the membranes with this or related analogs followed by assaying after thoroughly washing the membranes. Activation can occur in a simple Tris-HCl buffer, in the absence of added divalent cations and in the presence of EDTA. Dithiothreitol enhances the apparent degree of activation, perhaps by stabilization. The importance of utilizing optimal conditions for stabilizing enzyme activity, and of measuring the simultaneous changes in the control enzyme, is illustrated. The organomercurial, p-aminophenylmercuric acetate, inhibits profoundly the activity of the native as well as the Gpp(NH)p-stimulated adenylate cyclase, but in both cases subsequent exposure to dithiothreitol restores fully the original enzyme activity. However, the mercurial-inactivated enzyme does not react with Gpp(NP)p, as evidenced by the subsequent restoration of only the control enzyme activity upon exposure to dithiothreitol. Thus, reaction with Gpp(NH)p requires intact sulfhydryl groups, but the activated state is not irreversibly destroyed by the inactivation caused by sulfhydryl blockade. GTP and, less effectively, GDP and ATP inhibit activation by Gpp(NH)p, but interpretations are complicated by the facts that this inhibition is overcome with time and that GTP and ATP can protect potently from spontaneous inactivation. These two nucleotides can be used in the Gpp(NH)p preincubation to stabilize the enzyme. The Gpp(NH)p-activated enzyme cannot be reversed spontaneously during prolonged incubation at 30 degrees C in the absence or presence of GTP, ATP, MgCl2, glycine, dithiothreitol, NaF or EDTA. The strong nucleophile, neutral hydroxylamine, decreases the Gpp(NH)p-activated enzyme activity and no subsequent activation is detected upon re-exposure to the nucleotide.

Adenosine Triphosphate↗
PubMed2003

Hydroperoxidase II of Escherichia coli exhibits enhanced resistance to proteolytic cleavage compared to other catalases.

Catalase (hydroperoxidase) HPII of Escherichia coli is the largest catalase so far characterized, existing as a homotetramer of 84 kDa subunits. Each subunit has a core structure that closely resembles small subunit catalases, supplemented with an extended N-terminal sequence and compact flavodoxin-like C-terminal domain. Treatment of HPII with trypsin, chymotrypsin, or proteinase K, under conditions of limited digestion, resulted in cleavage of 72-74 residues from the N-terminus of each subunit that created a homotetramer of 76 kDa subunits with 80% of wild-type activity. Longer treatment with proteinase K removed the C-terminal domain, producing a transient 59 kDa subunit which was subsequently cleaved into two fragments, 26 and 32 kDa. The tetrameric structure was retained despite this fragmentation, with four intermediates being observed between the 336 kDa native form and the 236 kDa fully truncated form corresponding to tetramers with a decreasing complement of C-termini (4, 3, 2, and 1). The truncated tetramers retained 80% of wild-type activity. The T(m) for loss of activity during heating was decreased from 85 to 77 degrees C by removal of the N-terminal sequence and to 59 degrees C by removal of the C-terminal domain, revealing the importance of the C-terminal domain in enzyme stability. The sites of cleavage were determined by N- and C-terminal sequencing, and two were located on the surface of the tetramer with a third being exposed by removal of the C-terminal domain.

Amino Acid Sequence↗
PubMed2004

Mannan-penicillin G acylase neoglycoproteins and their potential applications in biotechnology.

Mannan-penicillin G acylase neoglycoproteins were prepared by the conjugation of Saccharomyces cerevisiae mannan with enzyme penicillin G acylase using the reductive amination method. Eight neoglycoproteins preparations were obtained after gel chromatography. The preparations contained from 42 to 67% (w/w) saccharides and their molar masses varied from 283 to over 1000 kDa. Significant biospecific interaction of separated fractions with the lectin concanavalin A was evaluated by the precipitation and sorption method (equilibrium constants) and further characterized using surface plasmon resonance to determine kinetic association and dissociation constants. K (D) was determined over the range 10(-7) M. High-molar-mass preparations appeared to be more suitable for preparation of stable and active complexes with concanavalin A for prospective use as a penicillin G acylase biocatalyst in enzyme reactors. The enzyme stability of such complexes was significantly increased compared with the original neoglycoprotein. Lower-molar-mass preparations were more suitable for applications such as biocatalysts in bioanalytical devices.

Adsorption↗
PubMed2003

Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane.

Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane. The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells. The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity. An increase in both the alginate and CaCl2 concentrations used for bead preparation led to a decrease in enzyme stability. An increase in the alginate concentration led to an increase in bead diameter. The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl2 concentrations higher than 0.4 M were utilised for bead preparation.

Alginates↗