Matrix-bound Enzyme Research Articles

Enhancement of the activity, stability and reusability of an extracellular protease from Pseudomonas fluorescens 07A via three different strategies of immobilization

The bacterium Pseudomonas fluorescens 07A produces a protease with potential for industrial application. In order to remedy problems associated with the use of free enzymes and allow its reuse, the protease was immobilized on DEAE Sephacel® resin via three different strategies based on ionic interaction and covalent bonding. The matrix-bound enzymes were characterized in relation to their activity (pH, temperature and stability), reuse and storage. Immobilization raised the optimum temperature of activity from 37 °C to 50 °C, whereas the pH of highest activity changed from 7.5 to 7.0 or 8.0, depending on the immobilization strategy. Immobilization proved to be efficient for successive reuses, even leading to an increase in the enzymatic activity along the use. The immobilized enzyme also presented greater stability to high temperatures and storage conditions and has potential as a biocatalyst for industrial applications due to its high efficiency, stability and easy recovery.

Effect of calcium fluoride on the activity of dentin matrix-bound enzymes

ObjectiveMatrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) are two distinct enzymatic pathways responsible for the degradation of collagen fibrils in demineralized dentin. NaF and KF have been shown to inhibit salivary MMP-2, -9 and CCs. This study investigated the inhibitory effect of calcium fluoride (CaF2) on the dentin matrix-bound MMPs and CCs. DesignPhosphoric acid (10%)-demineralized dentin beams (1 × 2×6 mm) were incubated at 37 °C in an 1 ml of artificial saliva (AS, control), or AS with 6, 12, 24, 48, 120. 179 and 238 mM F containing CaF2 (n = 10/group) for 1, 7 and 21 days. All groups were further incubated in AS only for 6 months. Total MMP activity, dry mass loss, CTX and hydroxyproline (HYP) analyses were performed after each incubation. The beams were examined under scanning electron microscopy (SEM). MMP-2 and MMP-9 activities were screened with gelatin zymography. Data were analyzed by using ANOVA and Tukey HSD tests (p = .05). ResultsThe total MMP activity was similar for all groups after 21 days and 6 months. After 21 days, the cumulative mass loss and CTX levels were lower compared to control for the CaF2 ≥48 and CaF2≥120 mM, respectively (p < .05). After 6 months, no significant difference was detected in the dry mass loss and CTX compared to the control (p > .05), whereas HYP level was higher with F 24 and 238 mM groups. CaF2-like minerals were observed on the beams under SEM. There was no gelatinase inhibition in zymography. ConclusionCaF2 does not prevent the degradation of demineralized dentin matrices due to the catalytic activity of MMPs and CCs.

Dynamic Influence of pH on Metalloproteinase Activity in Human Coronal and Radicular Dentin

The aim of this study was to evaluate the effect of pH on the activation of matrix metalloproteinases (MMPs) of human coronal (CD) and radicular dentin (RD). CD and RD were pulverized to powder, and proteins were extracted with 1% phosphoric acid. The extracted proteins and the demineralized powder were separately incubated in the following solutions: 4-aminophenylmercuric acetate (control) or a buffer solution at different pHs (2.5, 4.5, 5.0, 6.0, and 7.0). After incubation, proteins were separated by electrophoresis to measure MMP activities by zymography. To assess the solubilized dentin collagen, the demineralized dentin powder was sustained in incubation buffer, and the amount of hydroxyproline (HYP) released was measured. Zymography revealed MMP-2 gelatinolytic activities for CD and RD in all experimental groups. For both substrates, the lowest pH solutions (2.5, 4.5, and 5.0) yielded higher gelatinolytic activity than those obtained by the highest pH solutions (6.0 and 7.0). For HYP analysis, no detectable absorbance values were observed for pHs of 2.5 and 4.5. The amount of HYP was higher for pH 7.0 than those of all other groups (p < 0.05), except for pH 6.0. No statistical differences were found between pHs 6.0 and 5.0 and control (p > 0.05). The MMP-2 enzyme from human CD and RD is dynamically influenced by pH: at low pH, the extracted enzyme activates this latent form, whereas collagen degradation by the matrix-bound enzyme is only observed when pHs are close to neutral.

Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: Biological/biochemical studies and chemical/biomimetical confirmation

Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]–poly(acetoxime methacrylate) [reactive ester polymer]– N ε-benzyloxycarbonyl l-lysine tert-butyl ester–Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.

Age-dependent increase of DNA topoisomerase II activity in quail oviduct; modulation of the nuclear matrix-associated enzyme activity by protein phosphorylation and poly(ADP-ribosyl)ation

Nuclear DNA topoisomerase II activity in quail oviduct tissue was found to increase by about 70% with age. This age-dependent increase was observed with both the enzyme in whole nuclear extract and nuclear matrix-associated topoisomerase II. Both purified topoisomerase II and the nuclear matrix-bound enzyme were found to be modifiable by phosphorylation and poly(ADP-ribosyl)ation. Phosphorylation of the purified enzyme by isolated nuclear protein kinase NII or protein kinase C resulted in a 2- to 3-fold increase in specific activity, while poly(ADP-ribosyl)ation by soluble poly(ADP-ribose) synthetase caused a 50% inhibition of the enzyme. Using immunoprecipitation and immunoblotting procedures, phosphorylation and poly(ADP-ribosyl)ation could also be demonstrated to occur with the nuclear matrix-associated enzyme. The nuclear matrix-associated NII-like protein kinase activity, assumed to be involved in post-translational modification of topoisomerase II, displayed a 1.4- to 1.6-fold increase in old animals compared to mature ones, while the matrix-bound poly(ADP-ribose) synthetase activity decreased by about 50%. It is suggested that age-correlated enhancement of DNA topoisomerase II activity, possibly due to age-dependent changes in activities of nuclear protein kinases and poly(ADP-ribose) synthetase, may result in alterations in the topological state of DNA, possibly affecting DNA replication, transcription and repair with age.

Pre-replicative association of multiple replicative enzyme activities with the nuclear matrix during rat liver regeneration.

As a step toward the molecular elucidation of the putative replicational apparatus associated with the nuclear matrix, we have investigated the possible matrix association of several replicational related enzymes. In addition to the previously identified DNA polymerase alpha, DNA primase, 3'-5' exonuclease, RNase H, and DNA methylase were all recovered at significant levels (20-30% of total nuclear activity) in nuclear matrix isolated from regenerating rat liver during maximal in vivo replication (22 h post-hepatectomy). In contrast, DNA ligase was not detected on the nuclear matrix even though significant activity was present in isolated nuclei. Examination of the replicative dependency of these enzyme activities following partial hepatectomy revealed pre-replicative elevations which were distinct for each matrix-bound enzyme. A second late-replicative peak in DNA methylase is consistent with a role of this matrix-bound enzyme in the maintenance of the inheritable methylation pattern. Mild sonication resulted in a significant release of all of these activities except RNase H. A major portion of the matrix-solubilized DNA polymerase alpha, DNA primase, 3'-5' exonuclease, and DNA methylase activities cosedimented on sucrose gradients between approximately 8-12 S. Our results are consistent with the organization of at least a portion of these replicative enzymes into nuclear matrix-bound replicational complexes. We also propose a novel pre-replicative assembly model of the matrix-bound replicational apparatus in which DNA primase plays an initial and critical role.

30] Snake venom NAD glycohydrolase: Purification, immobilization, and transglycosidation

This chapter presents purification, immobilization, and transglycosidation of snake venom nicotinamide adenine dinucleotide (NAD) glycohydrolase. The enzyme activity can be assayed titrimetrically by measuring the production of hydrogen ion, spectrophotometrically by converting NAD in samples of the reaction mixture to the NAD-cyano adduct absorbing at 327 nm, or spectrophotometricahy by monitoring the 275nm absorbance decrease accompanying the hydrolysis of the alternate substrate, nicotinamide l, N 6 -ethenoadenine dinucleotide (ɛ-NAD). The discontinuous cyanide addition assay is used predominantly for assay of crude preparations of enzyme. One unit of enzyme activity is the amount which catalyzes the hydrolysis of 1 μmol of NAD per minute. All purification steps are performed at 4°. The NADase from Bungarus fasciatus snake venom is adsorbed on Con A-Sepharose and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme is stable to repeated washings with buffer and storage at 4° and is used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide.

Immobilization of dextranase on bentonite

Dextranase (1,6-α- d-glucan 6-glucanohydrolase, EC 3.2.1.11) from Penicillium aculeatum culture has been immobilized on a bentonite support. The matrix-bound enzyme could be stored as acetone-dried powder or as a suspension in acetate buffer, pH 5.6, for about three weeks at 4°C without any loss of activity. There was no change in the specific activity of the enzyme on immobilization and the enzyme yield was 0.1–0.6 mg/g bentonite matrix. In the presence of sucrose, thermal stability of the immobilized enzyme was high and the bound enzyme could be used for about six cycles.

Properties and applications of immobilized snake venom NAD glycohydrolase

The NAD glycohydrolase (NADase) from Bungarus fasciatus snake venom was adsorbed on concanavalin A-Sepharose, and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme was stable to repeated washing with buffer and storage at 4°C. The bound enzyme exhibited the same K m value for hydrolysis of nicotinamide-1, N 6-ethenoadenine dinucleotide as previously measured with the soluble, purified form of the enzyme. The bound NADase was used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide. It was further demonstrated that the immobilized enzyme could be prepared directly from crude snake venom, thus avoiding the time required for purification. The application of the immobilized snake venom NADase for the preparation of pyridine nucleotide coenzyme analogs has many advantages over procedures used previously for analog synthesis.

Immobilization of glutamate dehydrogenase into proteic films. Stability and kinetic modulation by effectors

Bovine liver glutamate dehydrogenase ( l-glutamate:NAD(p) + oxidoreductase (deaminating), EC 1.4.1.3) was immobilised within a protein membrane using a co-crosslinking method. The retention of enzyme activity after immobilisation was dependent on the presence of NAD +, ADP or GTP during the insolubilisation process. The highest yield (40%) was obtained in the presence of 4 mM NAD + and 3.5 · 10 −2 M ADP and the immobilised enzyme was much more resistant to thermal denaturation. The optimal pH for matrix-bound glutamate dehydrogenase was shifted towards the alkaline by 0.85 units as compared to the free form. Deviations from Michaelis-Menten behaviour towards higher activity were observed with increasing concentrations of NAD + (for the enzyme immobilised in absence of ADP) and glutamate (for the enzyme immobilised with or without ADP). Due to the presence of effectors during immobilisation, apparent K m values for glutamate (at high NAD + concentration) measured in the presence of 3.5 mM ADP are not deeply modulated. At the opposite for glutamate dehydrogenase immobilised in presence of ADP, an important decrease in K m value is observed (when 4.6 · 10 −5 M GTP is added to the reaction milieu) as compared with immobilised enzyme in absence of ADP. This observation is in favor of a conformational state similar to that induced by ADP in the native enzyme case. We suggest that a conformational state could be frozen by a tight chemical immobilisation of glutamate dehydrogenase. The allosteric properties of immobilised glutamate dehydrogenase have been studied with ADP and GTP. Due to diffusion limitations, inhibition by GTP is slightly reduced, but ADP activation is dramatically reduced. An explanation could be a modification of the ADP-binding site. The reversibility of such modulations has been observed for all presented matrix-bound enzymes suggesting that the degree of association of the enzyme does directly affect the activity.

Affinity Chromatography of Glyceraldehyde-3-phosphate Dehydrogenase. A Comparative Study of the Enzymes from Yeast and Sturgeon Muscle

Sturgeon glyceraldehyde-3-phosphate dehydrogenase has been strongly adsorbed on an 8-azo-linked NAD+-Sepharose derivative. Specific elution of the matrix-bound enzyme was obtained with relatively low concentrations of NAD+ (20–100 μM) in elution buffer. Recovery of adsorbed enzyme amounted to 90–95% when an hydrophilic 1,3-diaminopropan-2-ol spacer arm was used instead of the more conventional diaminohexyl derivative. A capacity of 0.8 mg of sturgeon enzyme per g of derivative was estimated either under batchwise conditions or by frontal chromatography. Under similar conditions, purified apoglyceraldehyde-3-phosphate dehydrogenase from yeast did not bind significantly to the same NAD+ derivative. Crude extracts of sturgeon muscle can be purified more than 15-fold upon chromatography on this derivative. Unlike the sturgeon enzyme, yeast glyceraldehyde-3-phosphate dehydrogenase was significantly adsorbed to an N6-(6-aminohexyl)-AMP derivative under chromatographic or batchwise conditions. At least 40 units (i.e. 0.2–0.3 mg) of yeast glyceraldehyde-3-phosphate dehydrogenase can be bound per g of matrix. An 8-fold purification of the enzyme can be obtained upon specific elution with NAD+. Relatively large amounts of yeast enzyme (i.e. up to 100 mg) can be partially purified by this one-step procedure. The distinct adsorption properties of yeast and sturgeon glyceraldehyde-3-phosphate dehydrogenases are discussed in the light of the known cooperative coenzyme binding properties of these enzymes. The relevance of affinity chromatography to the preparation of sizeable amounts of glyceral-dehyde-3-phosphate dehydrogenase is discussed and compared with conventional procedures. Possible interference of glyceraldehyde-3-phosphate dehydrogenase with the ‘general ligand’ binding of other dehydrogenase and kinases in crude extracts is discussed.

Matrix-bound phosphoglucose isomerase. Formation and properties of monomers and hybrids.

Dimeric phosphoglucose isomerase from rabbit muscle was immobilized by reaction with cyanogen-bromide-activated Sephrose 4B. The catalytic parameters and stability properties of the free and matrix-bound isomerases were essentially identical. Total monomerisation of the matrix-bound enzyme was achieved with 8 M urea as determined by a study in which one subunit was labelled with iodo[14C]acetate and the other with the 3H-labelled reagent. Although matrix-bound monomers were devoid of isomerase activity, they were still capable of binding the substrate. Matrix-bound monomers exhibited the ability to redimerize with soluble isomerase subunits from either rabbit or human yielding catalytically active dimers. Yeast isomerase monomers, in contrast, did not yield active hybrids with the rabbit monomers. Furthermore, soluble subunits, which had been inactivated with pyridoxal 5'-phosphate were also capable of hybridizing with and inducing catalytic activity in the matrix-bound monomers. These studies indicate the prerequisite of dimer formation for catalytic activity but the independent action of the catalytic centers of the dimer.

Preparation and enzymatic properties of subtilisin Novo chemically attached to soluble DEAE-dextran and insoluble DEAE-Sephadex

Analogous soluble and insoluble derivatives of subtilisin Novo (EC 3.4.21.14) were prepared by coupling the enzyme to CNBr-activated DEAE-dextran and DEAE-Sephadex, respectively. The DEAE-dextran-subtilisin displayed pH optima and K m values for ester hydrolysis similar to subtilisin, whereas the pH versus activity profiles obtained with DEAE-Sephadex-subtilisin were shifted towards the alkaline pH region and the K m values were increased. Compared with subtilisin, DEAE-dextran-subtilisin showed a 40–65% reduction of k cat for hydrolysis of N- acetyl- l-tyrosine ethyl ester, p- tosyl- l-arginine methyl ester and benzyloxycarbonyl-glycyl- l-tyrosinamide and its maximum velocities for digestion of casein and clupein also amounted to 40–60% of the subtilisin values. With DEAE-Sephadex-subtilisin, in contrast, the maximum velocity of hydrolysis decreased to a greater extent for polypeptide substrates compared to ester substrates. The present results indicate that the chemical nature of a support can affect intrinsic properties of a matrix-bound enzyme in addition to the steric and diffusional effects usually observed with polymer-attached enzymes.

Immobilized glucuronosyltransferase for the synthesis of conjugates.

Partially purified rabbit liver UDPglucuronosyltransferase is immobilized on agarose by the cyanogen bromide activation method. Both soluble and matrix-bound enzyme preparations display very similar Km and pH optimum. The storage stability of the immobilized enzyme at 4 degrees is 5-10 times improved over the soluble preparations. The agarose-bound UDPglucuronosyltransferase is successfully used in the synthesis of p-nitrophenyl glucuronide in an overall yield of 50-70%. The matrix-bound enzyme is reusable over an extended period of time and offers an easy and convenient synthetic tool for various drug glucuronides.

Rapid determination of an amino acid: tRNA ligase · aminoacyl adenylate complex on DEAE-cellulose filter disks

A rapid and convenient method for the detection and quantitative analysis of an amino acid:tRNA ligase·aminoacyl adenylate complex on DEAE-cellulose filter disks at pH 7.5 is presented. Results for L -phenylalanine: tRNA ligase (Escherichia coli K 10) demonstrate that 2 mol of L-phenylalanyl adenylate bind to 1 mol of enzyme. At 4°C, the decomposition of the ligase · aminoacyl adenylate complex was shown to follow first-order kinetics with a half-life of 16 min. With respect to the carboxylate-activating reaction catalyzed by L -phenylalanine:tRNA ligase the DEAE-cellulose matrix-bound enzyme appeared to be active.

Acid Phosphatase Heterogeneity in Horse Neutrophil and Eosinophil Leukocytes1

Two distinct groups of acid phosphatase containing granules were characterized in neutrophils, each group displaying different multiple forms of the enzyme. The heavy granule acid phosphatase showed a lysosomal location. A second lighter group of particles contained a thermolabile, thiol-dependent acid p-nitrophenyl and alpha-naphtylphosphatase, an enzyme clearly different from lysosomal acid phosphatase. Acid phosphatase activity from eosinophil leukocytes appeared to be totally associated with the typical eosinophil granules. On mechanical disruption of these particles, an acid phosphatase was released which differed in substrate and inhibitor specificity, in electrophoretic pattern, and in thermosensitivity, from the remaining matrix-bound enzyme.

The preparation of matrix-bound proteases and their use in the hydrolysis of proteins

Four proteases, crude acid protease from Aspergillus, pronase, amino-peptidase M, and prolidase, have been covalently attached to activated agarose and to amino propyl glass beads. The matrix-bound enzymes have been tested as catalysts for the complete hydrolysis of protein substrates, with the primary goal to isolate unstable amino acid derivatives present in the substrate protein. Under conditions used in the present work, the total amino acid release from the protease-catalyzed hydrolysis of four substrate proteins (pancreatic ribonuclease, egg white lysozyme, yeast enolase, and bovine insulin) was 95–103% of that observed in standard acid hydrolysis. Recovery of individual amino acids showed greater deviation from the theoretical values, but cystine was the only amino acid recovered in low yields (42–77%) from all four proteins. Derivatized amino acids, such as methionine sulfoxide, O-(butylcarbamoyl)-serine, and N-glycosyl asparagine have been obtained from chemically modified proteins or from unmodified glycoprotein in good yield, and normal amino acid constituents of proteins which cannot be quantified after acid hydrolysis (tryptophan, asparagine, and glutamine) have also been determined either directly after proteolysis or after proteolysis in conjunction with acid hydrolysis.

An enzyme thermistor

A simple device comprising a heat sensor placed in close proximity to a matrix-bound enzyme preparation and connected to a temperature measuring unit has been developed to measure directly the temperature changes that occur in the environment of enzyme activities. The sensitivity of the system was established by pumping pulses of substrate past trypsion or apyrase immobilised to glass-beads. Injection of a pulse containing only one μmole trypsin substrate (benzoyl- l-arginine ethyl ester, BAEE) in Tris-HCl buffer gave ΔT of 0.01 °C, corresponding to 10% full scale deflection, and a linear relationship was found within the range tested of upto 8 μmoles BAEE.

An apparatus for the determination of kinetic parameters associated with immobilized enzymes

The advantages associated with the described immobilized enzyme reactor include: (1) Use of the common rate equations and kinetic parameters. (2) Detection of significant lag periods. (3) Quantitative measure for non-covalently attached enzyme. (4) The means for washing the immobilized enzyme allows for the repeated use of the same matrix-bound enzyme. (5) Constant temperature control. (6) Both unbound native and matrix-bound enzyme may be reacted under identical conditions. (7) No grinding of the glass-bound enzyme or other matrix fragmentation occurs because no abrasive forces are required for stirring.

Matrix-bound enzymes. I. The use of different acrylic copolymers as matrices.

Matrix-bound enzymes. I. The use of different acrylic copolymers as matrices.