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PubMed2002

Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents.

With the incorporation of lysozyme during the immobilization step, considerable enhancement of the operational stability of a biosensor has been demonstrated in the case of an immobilized single enzyme (glucose oxidase) system for glucose and multienzyme (invertase, mutarotase and glucose oxidase) system for sucrose. Thus an increased number of repeated analyses of 750 samples during 230 days for glucose and 400 samples during 40 days of operation for sucrose have been achieved. The increased operational stability of immobilized single and multienzyme system, will improve the operating cost effectiveness of the biosensor.

Animals↗

PubMed1979

Kinetic and spectroscopic evidence of cation-induced conformation changes in yeast K+ -activated aldehyde dehydrogenase.

The activity, stability and spectroscopic properties of yeast K+ -activated aldehyde dehydrogenase were measured at various times after removal from, and after returning to a solution containing K+. Enzyme activity is rapidly lost on removal of most of the K+ and rapidly regained if K+ is replaced immediately. These activity changes are slower than likely rates of K+ dissociation and association. These rapid changes in concentration result in altered enzyme stability with enzyme in K+ the more stable. U.v. difference spectra are produced whenever enzyme in an activating environment (K+ or Tl+) is compared with enzyme in a non-activating environment (Tris+ or Li+). These spectral changes occur within 10s. The saturation characteristics with K+ are hyperbolic for all three phenomena of activation, stabilization and spectral change, with estimated apparent dissociation constants (Ks) for K+ of 7.5 mM, 5.5 mM and 6 mM respectively. Continued incubation of enzyme in the absence of K+ results in the accumulation of an enzyme form that re-activates only slowly on replacing K+. Stability characteristics in various concentrations of K+ over equivalent time scales are consistent with the existence of additional conformations. Spectroscopic evidence also indicates such additional slow conformation changes. Results have been interpreted in terms of two separate conformation transitions induced or stabilized by K+.

Aldehyde Oxidoreductases↗

PubMed2003

Activity and stability of native and modified subtilisins in various media.

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.

Acrylic Resins↗

PubMed2006

A novel enzyme, lambda-carrageenase, isolated from a deep-sea bacterium.

A lambda-carrageenan-degrading Pseudoalteromonas bacterium, strain CL19, was isolated from a deep-sea sediment sample. A lambda-carrageenase from the isolate was purified to homogeneity from cultures containing lambda-carrageenan as a carbon source. This is the first report of the isolation of lambda-carrageenase together with the gene sequence for the enzyme. The molecular mass of the purified enzyme was approximately 100 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that the enzyme is a monomer. The optimal pH and temperature for activity were about 7 and 35 degrees C, respectively. The enzyme had specific activity of 253 U/mg protein. The enzyme required monovalent salts for the activity. Carbohydrates, such as sorbitol, sucrose, trehalose, improved the enzyme stability. The pattern of lambda-carrageenan hydrolysis showed that the enzyme is an endo-type lambda-carrageenase, and the final main product was a tetrasaccharide of the lambda-carrageenan ideal structure with galactose 2,6-disulfate at the reducing end, indicating the enzyme cleaves the beta-1,4 linkages of its backbone structure. Furthermore, the gene (cglA) encoding the enzyme was sequenced. It encoded a mature protein of 103 kDa (917 amino acids). Remarkably, the deduced amino acid sequence showed no similarity to any reported proteins.

Amino Acid Sequence↗

PubMed2003

Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11.

The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60 degrees C, pH 10, with casein as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60 degrees C even after 350 minutes of incubation. It was strongly activated by metal ions such as Ca+2, Mg+2, and Mn+2. Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate, p-chloromercuric benzoate (pCMB), and beta-mercaptoethanol (beta-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl2 and 1M glycine. The addition of 10mM CaCl2 and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10mM CaCl2 and 1M glycine.

Bacillus subtilis↗

PubMed2002

Stabilization of human pancreatic ribonuclease through mutation at its N-terminal edge.

Enzyme stability can be an important parameter in the design of recombinant toxins because unstable proteins are often degraded before they can reach their cellular target. There is great interest in the design of human pancreatic ribonuclease variants that could be cytotoxic against tumoral cells. To this end, some residues in the protein need to be substituted, but this may result in a loss of stability. Previous papers have reported the production of N- and C-terminal human pancreatic ribonuclease variants with increased thermal stability. Here, we investigated the contribution of the different amino acid changes at the N-terminus of the protein to its thermostability increase. We show that this increase correlates with the helical propensity of the first alpha-helix of the protein. On the other hand, deletion of the four last residues of the protein does not affect its thermal stability. These results set the basis for the design of a human pancreatic ribonuclease template on which amino acid substitutions can be made that could render the enzyme cytotoxic, without an important loss in its stability.

Enzyme Stability↗

PubMed1976

Levansucrase of Bacillus subtilis. Characterization of a stabilized fructosyl-enzyme complex and identification of an aspartly residue as the binding site of the fructosyl group.

A covalently linked fructosyl-enzyme complex was isolated from a reaction mixture of enzyme and sucrose submitted to the quenching effect of a large decrease of the pH. The fructosyl-enzyme bond was shown to be stable under acidic and neutral conditions in the presence of high concentration of urea and of sodium dodecyl sulfate. This intermediate did not transfer at a measurable rate its fructosyl group to the usual fructosyl acceptors of the enzyme reaction under the usual conditions of enzyme activity. However stability measurements of the fructosyl-enzyme bond indicated a marked lability at pH values above 8.5. The apparent rate constant of the hydrolytic reaction of this bond evaluated under the standard state of molar concentration of hydroxide ion was of the same order of magnitude as the apparent rate constant of the hydrolytic reaction of the transient fructosyl-enzyme postulated from the kinetic analysis of levansucrase. Furthermore, nucleophilic agents like imidazole enhanced the hydrolytic reaction of the fructosyl-enzyme bond. Identification of the fructosyl binding site on the enzyme was accomplished by proteolytic hydrolysis of the trapped complex. Peptic digestion followed by pronase digestion released a fructosyl-aspartate compound that we have isolated in a high state of purity. The lability of the fructosyl-aspartate bond under mild alkaline conditions suggested that the fructosyl was linked through an ester bond involving the beta-carboxyl of the aspartate residue. Treatment of the trapped complex with cyanogen bromide released only one fructosylated peptide. The apparent molecular weight of this peptide was estimated to be lower than 10000.

Aspartic Acid↗

PubMed1997

Stability and stabilization of recombinant peroxidase in reversed micelles.

Stability of recombinant peroxidase lacking carbohydrate residues on the surface of the protein molecule has been characterized in reversed micelles of Aerosol OT in octane. The enzyme stability was found to depend on the surfactant hydration degree (w0 = [H2O]/[AOT]). Residual activity after 1 h incubation dropped to zero at w0 = 7 but was 54% at w0 = 25. However, the residual activity levels at all values of hydration degree were definitely low compared to that of glycosylated wild-type horseradish peroxidase. The stability of the enzyme apparently depends on the presence of carbohydrate residues. Stabilization of recombinant peroxidase in reversed micellar system involved sugar-containing co-surfactants such as Tweens and Spans is proposed. As an example, addition of 1 mM Span 80 (1% relative to AOT concentration) increased the recombinant peroxidase stability up to that of wild-type peroxidase.

Detergents↗

PubMed1978

Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase.

Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2M, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50 degrees or to 20 mM ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.

Adenosine Triphosphate↗

PubMed2001

Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution.

We attempted to apply the directed evolution approach to enhancing enzyme properties in the presence of organic solvents, in which enzyme stability and activity were often drastically reduced. Stability and catalytic activity of phospholipase A(1) in the presence of an organic solvent were enhanced by error-prone polymerase chain reaction (PCR) and DNA shuffling followed by a filter-based visual screening. Three mutants (SA8, SA17 and SA20) were isolated on indicator plates (i.e., 1% phosphatidylcholine gels containing 30% dimethyl sulfoxide (DMSO)) after a second mutant library was treated in 50% DMSO for 36 h. The half-life values of the three mutants exhibited an approximately 4-fold increase. The three mutants also exhibited increased stability in all organic solvents tested compared with the wild-type enzyme. Thus, an enzyme variant having superior catalytic efficiency in most of the organic solvents could be obtained by using any solvent suitable for designing the efficient screening system, regardless of the properties of the particular solvent.

Amino Acid Substitution↗

PubMed1988

Structure and stability of thermophilic enzymes. Studies on thermolysin.

The molecular mechanisms responsible for the unusual stability of enzymes isolated from thermophilic microorganisms are much more complex and subtle than was originally thought. In particular, a general mechanism cannot be proposed, since individual enzymes can be stabilized by specific molecular interactions and forces. The results of studies on thermophilic enzymes obtained in recent years in our laboratory will be summarized, with particular emphasis being placed on those obtained with thermolysin, a stable metalloendopeptidase isolated from Bacillus thermoproteolyticus. Fragmentation of thermolysin by limited proteolysis by added protease (subtilisin) or autolysis mediated by heat or the ion-chelating agent EDTA leads to quite selective peptide bond fissions, allowing isolation of 'nicked' thermolysin species. Correlation of the sites of proteolytic cleavage with the known three-dimensional structure of thermolysin allowed us to infer some of the key characteristics of the structure, folding, dynamics and stability of the thermolysin molecule. The potential utility of these and other studies on thermophilic enzymes in devising strategies for enhancing the stability of mesophilic enzymes using genetic engineering techniques is discussed.

Amino Acid Sequence↗

PubMed2002

Structural significance of the plasma membrane calcium pump oligomerization.

The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles was evaluated using a combined spectroscopic and kinetic approach and related to the enzyme stability. Energy transfer between fluorescein-5'-isothiocyanate and eosin-5'-isothiocyanate attached to different PMCA molecules was used to determine the dissociation constant of dimeric PMCA (140 +/- 50 nM at 25 degrees C) and characterize the time course of dimerization. The enzyme thermal stability at different dimer/monomer ratios was evaluated, quantifying the kinetic coefficient of thermal inactivation. This coefficient decreases with PMCA concentration, becoming approximately constant beyond 300 nM. Thermal treatment leads to the formation of inactive monomers that associate only with native monomers. These mixed dimers are formed with a kinetic coefficient that is half that determined for the native dimers. We proposed a model for PMCA thermal inactivation that considers the equilibria among dimers, monomers, and mixed dimers, and the inactivation of the last two species through irreversible steps. The numerical resolution of the differential equations describing this model fitted to the experimental data allowed the determination of the model coefficients. This analysis shows that thermal inactivation occurs through the denaturation of the monomer, which lifetime is 25 min at 44 degrees C. The obtained results suggest that PMCA dimerization constitutes a mechanism of self protection against spontaneous denaturation.

Calcium-Transporting ATPases↗

PubMed2000

Identification of specific histidine residues and the carboxyl terminus are essential for serotonin N-acetyltransferase enzymatic activity.

Melatonin is synthesized in pinealocytes of the pineal gland and in photoreceptors of the retina. Synthesis rate from serotonin to melatonin is controlled by the rapid and dramatic enzymatic increase in darkness of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, EC 2.3.1.87) and hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The primary structure of these critical indoleamine enzymes is now known and the regulation of the enzyme catalysis can be examined. As a first step, the conserved cysteine (C) and histidine (H) residues were targeted for site-directed mutagenesis as potential amino acid residues involved in the N-acetylation reaction of AA-NAT. Our studies concluded that among 6 histidine (H) to alanine (A) mutations, three residues (H110A, H118A, H120A) within the AA-NAT protein showed little or no enzymatic activity, whereas the others (H28A, H70A, H125A) retained enzymatic activity, compared to the unaltered AA-NAT protein. Cysteine to alanine mutations, C37A and C177A, had no significant effect on the AA-NAT enzymatic activity; however, C61A had a four-fold increase in K(m) for acetyl CoA and an altered sensitivity to the thiol modification chemical, N-ethylmaleimide (NEM), implying that C61 may participate in the acetyl CoA binding. Further studies examined the AA-NAT enzyme regulation of the highly conserved carboxyl terminus. When 12 terminal amino acid residues were deleted systematically from the carboxyl terminus of the 205 amino acid residue AA-NAT protein, enzyme activity was retained. However, further residue deletion resulted in enzyme activity plummeting, implicating that the essential information either for the correct structural folding into an active enzyme form or for enzyme stability is in the 193 residues. To test the relative importance of the AA-NAT carboxyl terminal region, a single leucine (L) was altered to alanine (A) or proline (P). Both mutants, either L193A or L193P, had a marked decrease in AA-NAT enzymatic activity and a decrease in thermal stability, suggesting the leucine, in addition to the cysteine and histidine residues, is involved in either enzyme catalysis or stability. In light of the recently reported three-dimensional structure of AA-NAT (17,18), the site-directed mutagenesis data demonstrate experimentally the importance of essential amino acid residues for acetyl CoA binding and AA-NAT activation.

Acetyl Coenzyme A↗

PubMed1993

Purification and properties of a lipase from Penicillium expansum.

Penicillum expansum DSM 1994 produces a new, inducible extracellular lipase when grown in medium containing 0.1% olive oil. Maximum activity was obtained after 4 days of incubation at 20 degrees C. The enzyme was purified 219-fold by cross-flow filtration, ammonium sulfate precipitation and hydrophobic interaction chromatography to a final specific activity of 558 U/mg. The molecular weight of the homogeneous lipase was (25 kDa) determined by gel filtration and SDS-PAGE, however, it forms active dimers and higher aggregates as observed after native PAGE. The enzyme was identified as a glycoprotein with a pI of 5.5. The N-terminal sequence shows a homology to sequences of other lipase just behind their consensus sequence. Enzyme stability was enhanced by the addition of Tween 20 and Lubrol PX. The enzyme showed a maximum activity at pH 9 at 45 degrees C and was stable at a broad pH range of 6-10. Lipase of P. expansum showed a preference for triacylglycerols, but no positional specificity.

Amino Acid Sequence↗

PubMed1980

Influence of lecithin liposomes on chlorophyllase-catalyzed chlorophyll hydrolysis: comparison of intramembraneous and solubilized Phaeodactylum chlorophyllase.

1.Chlorophyllase-catalyzed chlorophyll hydrolysis is greatly enhanced by the addition of divalent cations (Mg2+) combined with a reducing agent (dithiothreitol, ascorbate). A similar effect is obtained by the addition of lecithin. In the presence of lecithin, dithiothreitol has only slight or no influence on chlorophyll hydrolysis. Mg2+ eliminates the activating effect of lecithin. 2. In the absence of Mg2+ + dithiothreitol or of lecithin, Triton X-100 has a slight activating effect on chlorophyllase-catalyzed chlorophyll hydrolysis, but only at low concentrations (0.01--0.02%). In the presence of Mg2+ and dithiothreitol or of lecithin, Triton X-100 (greater than or equal to 0.02%) inhibits this reaction. 3. Whereas chlorophyllase combines with chlorophyll, no combination of chlorophyllase and lecithin could be detected. 4. Solubilized chlorophyllase is stabilized by its substrate, chlorophyll. Enzyme stabilization is eliminated by lecithin, whereas in the absence of chlorophyll, denaturation is somewhat increased by dithiothreitol. 5. No clear difference was found between the actions of intramembraneous and solubilized chlorophyllase. The results suggest that chlorophyllase is situated within membranes in such a way that the active group protrudes into the aqueous medium surrounding the membrane. 6. A hypothesis explaining the activating effects which Mg2+ combined with a reducing agent and lecithin have upon chlorophyllase-catalyzed chlorophyll hydrolysis is presented.

Chlorophyll↗

PubMed2001

Assimilatory nitrate reductase from the haloarchaeon Haloferax mediterranei: purification and characterisation.

Haloferax mediterranei can use nitrate as sole nitrogen source during aerobic growth. We report here the purification and biochemical characterisation of the assimilatory nitrate reductase (EC 1.6.6.2) from H. mediterranei. The enzyme, as isolated, was composed of two subunits (105+/-1.3 kDa and 50+/-1.3 kDa) and behaved as a dimer during gel filtration (132+/-6 kDa). A pH of 9 and elevated temperatures up to 80 degrees C (at 3.1 M NaCl) are necessary for optimum activity. The enzyme stability and activity of the enzyme depend upon the salt concentration. Reduced methyl viologen was as effective as the natural electron donor ferredoxin in the catalytic process. In contrast, NADPH and NADH, which are electron donors in nitrate reductases from different non-photosynthetic bacteria, were ineffective.

Ferredoxins↗

PubMed1978

De novo purine synthesis in avian liver. Co-purification of the enzymes and properties of the pathway.

The enzymes of the de novo purine biosynthetic pathway have been partially co-purified from pigeon liver by a method dependent upon the use of the nonionic polymer polyethylene glycol for enzyme stabilization and cofractionation. Although the enzymes did not appear to constitute a large macromolecular complex it was evident that some particular inter-relationship between them was preserved during the purification procedure. Analysis of the end products and pathway intermediates was carried out primarily by sensitive high pressure liquid chromatographic techniques. Substrate and cofactor requirements were confirmed and optimal conditions of pH, temperature, and K+ ion activation established. At phosphoribosyl pyrophosphate (PP-ribose-P) concentrations below 0.3 mM the activity of the first pathway enzyme amidophosphoribosyltransferase was rate-limiting, and the inhibition of this enzyme by AMP regulated the rate of purine ring synthesis. At higher concentrations of PP-ribose-P, aminoimidazole ribonucleotide synthetase, the fifth enzyme of the pathway became rate limiting and was subject to inhibition by added AMP. It was evident that the regulation of purine synthesis was quite complex and that AMP inhibition (perhaps reflected in a low adenylate energy charge) can be effected at different points on the purine pathway.

Amidophosphoribosyltransferase↗

PubMed1997

Purification and some properties of a novel dsRNA degrading nuclease bound to rye germ ribosomes.

A two-step procedure including affinity chromatography for purification of rye germ ribosomal nuclease that degrades double-stranded RNA from a virus of Penicillium chrysogenum and the poly(I).poly(C) complex was developed. The specific activity towards poly(I).poly(C) of the obtained nuclease preparations was 30 times as high as that of ribosomes. The recovery of activity was 3.4% when the Octyl-Sepharose column was used, and 2.0% in the case of the Phenyl-Sepharose column. On polyacrylamide/SDS gel electrophoresis the nuclease was resolved into two proteins of molecular mass 62 kDa and 57 kDa, respectively. 2-Mercaptoehanol and Mn2+ stimulated the activity of the purified enzyme. Glycerol (20%-50% concentration) stabilized enzyme. In addition to activity towards dsRNA and ssRNA the enzyme cleaves native and denatured DNA. It is suggested that this type of a nuclease takes part in regulation of the mRNA level in cytoplasm.

Penicillium chrysogenum↗