Bacterial dextranase immobilised on zirconia coated alkylamine glass using glutaraldehyde

Bacterial dextranase has been immobilised on zirconia coated alkylamine glass through the process of glutaraldehyde coupling. The immobilised enzyme preparation exhibited 62% of the initial enzyme activity with a conjugation yield of 18 mg/g support. K_m of the immobilised enzyme exhibited a decline in its value as compared to the soluble enzyme while V_max remained unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme had its optimal pH at 5.4 while the alkylamine conjugated dextranase was optimally active in the pH range 5.2–6.2. The immobilised enzyme has also been characterised through its pI by a new method. The industrial importance of this work is discussed.     

The use of titanium(IV) oxide coated with diazotised 1,3-diaminobenzene for the immobilisation of carbohydrate-directed enzymes

The utility of porous titanium(IV) oxide particles as a matrix for the immobilisation of enzymes on column packings has been extended. On coating the particles with diazotised 1,3-diaminobenzene, their capacity for binding dextranase was increased two-fold. The stability of the enzyme-matrix bridge was enhanced by the covalent bond so formed. Excess diazonium groups were reacted with 2-naphthol. Investigations of the effects of change of dextran concentration, pH, temperature, and flow rate upon a continuously operated column of the immobilised dextranase permitted assessment of the kinetic aspects of the enzyme via Lineweaver-Burk plots. The change of reaction rate with temperature showed, according to Arrhenius plots, an abrupt change at 28°. Possible sources of the kinetic characteristics of the immobilised dextranase are discussed.     

Preparation and kinetic studies of immobilised yeast cytochrome c peroxidase.

Yeast cytochrome c peroxidase (CcP) was purified from baker's yeast and immobilised onto a nylon membrane. The kinetics of the soluble and immobilised forms of the enzyme were investigated for the catalysed oxidation of potassium ferrocyanide in the presence of H2O2 and m-chloroperoxybenzoic acid. The pH dependence of the two forms of the enzyme differed. Although both the soluble and the immobilised enzymes showed optimal activity at pH 6.2, a different kinetic behaviour was demonstrated. Both forms of the enzyme showed similar activity toward H2O2, although when m-chloroperoxybenzoic acid was replaced as the electron acceptor, the immobilised form of the enzyme had a reduced turnover number and an increased Km. The activation energy of immobilised CcP was greater in the presence of both H2O2 [16.6 kJ mol-1] and m-chloroperoxybenzoic acid [37.9 kJ mol-1] than for soluble CcP [11.4 and 23.4 kJ mol-1, respectively]. The activities of both soluble and immobilised CcP were greatly reduced above 45 degrees C, although at higher temperatures the immobilised enzyme retained a relatively greater percentage of its maximum activity.     

Characterisation of tyrosinase immobilised onto spacer-arm attached glycidyl methacrylate-based reactive microbeads

Immobilisation of tyrosinase onto modified poly(methyl methacrylate–glycidyl methacrylate–divinyl benzene), poly(MMA–GMA–DVB), microbeads was studied. The epoxy group containing poly(MMA–MMA–DVB) microbeads were prepared by suspension polymerisation. The epoxy groups of the poly(MMA–GMA–DVB) microbeads was converted into amino groups with either ammonia or 1,6-diaminohexane (i.e., spacer-arm). Tyrosinase was then covalently immobilised on aminated and the spacer-arm-attached poly(MMA–GMA–DVB) microbeads using glutaric dialdehyde as a coupling agent. Incorporation of the spacer-arm resulted an increase in the apparent activity of the immobilised tyrosinase with respect to the enzyme immobilised on the aminated microbeads. The activity yield of the immobilised tyrosinase on the spacer-arm-attached poly(MMA–GMA–DVB) microbeads was 68%, and this was 51% for the enzyme, which was immobilised on the aminated microbeads. Both immobilised tyrosinase preparation has resistance to temperature inactivation as compared to that of the free form. The temperature profiles were broader for both immobilised preparations than that of the free enzyme. Kinetic parameters were determined for immobilised tyrosinase preparations as well as for the free enzyme. The values of the Michaels constants (K_m) for all the immobilised tyrosinase preparations were significantly larger, indicating decreased affinity by the enzyme for its substrate, whereas V_max values were smaller for the both immobilised tyrosinase preparations. In a 40 h continuous operation with spacer-arm-attached poly(MMA–GMA–DVB) microbeads at 30 °C, only 3% of immobilised tyrosinase activity was lost. The operational inactivation rate constant (k_opi) of the immobilised tyrosinase was 1.25×10⁻⁵ min⁻¹.     

An optical biosensor employing tiron-immobilised polypyrrole films for estimating monophenolase activity in apple juice

A method is described for the incorporation of tiron as a substrate for tyrosinase enzyme into a polypyrrole film deposited on indium titanium oxide (ITO) glass. The presence of tiron in the polypyrrole film is verified by cyclic voltammetry (CV). The enzyme activity using the polypyrrole-tiron film is confirmed by the catalytic conversion of immobilised substrate to quinones by the enzyme. The use of both potentiometric and optical methods for the detection of the catalytic activity of the polypyrrole-tiron film and their potential use for the determination of monophenolase activity of apple polyphenol oxidase is described. This is the first report of this kind whereby tiron has been immobilised in a polypyrrole matrix for the enzyme activity determination.     

Characterization of an Extracellular Dextranase from Fusarium moniliforme

An extracellular dextranase (EC 3.2.1.11) was purified approximately 75-fold from cell-free culture filtrates of Fusarium moniliforme. The purified dextranase was of the endo type, and isomaltose was identified as the primary end product of dextran hydrolysis. The molecular weight of the dextranase was determined to be 39,000 by gel permeation chromatography. The enzyme was most active at pH 5.5, and the temperature optimum was near 55 C. Activity was not inhibited by either ethylenediaminetetraacetic acid or iodoacetate. The Km for dextran with an average molecular weight of 10,000 was estimated to be 1.1 X 10(-4) M. The electrophoretic mobility of the dextranase was distinctly different from that of a Penicillium-derived commercial dextranase. The F. moniliforme dextranase was also found to differ from the commercial preparation by its greater relative activity against glucans isolated from Streptococcus mutans.     

Immobilization of dextranase from Chaetomium erraticum

In order to facilitate the Co-Immobilization of dextransucrase and dextranase, various techniques for the immobilization of industrial endo-dextranase from Chaetomium erraticum (Novozymes A/S) were researched. Adsorption isotherms at various pH-values have been determined for bentonite (Montmorillonite), hydroxyapatite and Streamline DEAE. Using bentonite and hydroxyapatite, highest activity loads (12,000 Ug(-1); 2900 Ug(-1), respectively) can be achieved without a significant change of the apparent Michaelis-Menten constant K(M). For successful adsorption, enzyme to bentonite ratios greater than 0.4 (w/w) have to be used as lower ratios lead to 90% enzyme inactivation due to bentonite contact. In addition, covalent linkage using the activated oxiran carriers Eupergit C and Eupergit C250L as well as linkage with aminopropyl silica via metaperiodate activation of glycosyl moiety of dextranase are discussed. This is also the first report probing the structure of a matrix containing dextranase by use of substrate species with different molecular weights. From this we can observe a relationship between the porosity of Eupergit and dextran dependent activity. For the reactor concept using Co-Immobilisates, hydroxyapatite will be preferred to Eupergit because of its higher specific activity and dispersity.     

Expression, purification and characterization of a recombinant Lipomyces starkey dextranase in Pichia pastoris

The DEX gene encoding an extracellular dextranase from Lipomyces starkeyi was cloned into vector pPIC9k-His6 and was expressed in Pichia pastoris GS115 strain under the control of AOX1 promoter. After 107 h of the 5L-scaled fermentation, wet cells weight of the recombinant P. pastoris Mut(+) strain reached to 588.6g/L, and the concentration of dextranase and enzyme activity in the supernatant were 0.46 g/L and 83900 U/L, respectively. The activity of dextranase was improved 17.56-fold by cation-exchange chromatography only with a final yield of 71.61% and the specific activity of the purified enzyme was 181.96 U/mg. The purified dextranase, analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. Then the factors affecting the dextranase activity were evaluated. The optimal temperature and pH was 30 degrees C and pH 4.5, respectively. Metal ions Al(3+), Cu(2+), Fe(3+), and SDS could completely inhibit the enzyme activity, whereas Mg(2+) enhanced 145% of the enzyme activity. These characters are much different from what was previously reported for the L. starkeyi dextranase that was either expressed in S. cerevisiae or purified from natural L. starkeyi.     

Purification and immobilization of dextranase

An enzymic characteristic of Novo dextranase was presented. In addition to a high dextranolytic activity (7,200 U/ml), the crude enzyme also contained small amounts of protease, glucoamylase, polygalacturonase, carboxymethylcellulase, laminarinase and chitinase. A highly purified dextranase was then simply separated from a commercial preparation by column chromatographies on DEAE-Sepharose, CM-Sepharose, and by chromatofocussing on Polybuffer Exchanger PBE-94. The enzyme was recovered with an over 200-fold increase in specific activity and a yield of 84%. The final preparation was homogeneous, as observed during high performance liquid chromatography (HPLC). Size-exclusion HPLC indicated that dextranase had a molecular mass of 35 kDa and its isoelectric point, established by chromatofocussing, was 4.85. Analysis of the dextran break-down products indicated that purified dextranase represents an endolytic mode of action, and isomaltose and isomaltotriose were identified as the main reducing sugars of dextran hydrolysis. The enzyme was then covalently coupled to the silanized porous glass beads modified by glutaraldehyde (Carrier I) or carbodiimide (Carrier II). It was shown that immobilization of dextranase gave optimum pH and temperature ranges from 5.4 to 5.7 and from 50°C to 60°C, respectively. The affinity of the enzyme to the substrate decreased by a factor of more than 13 for dextranase immobilized on Carrier I and increased slightly (about 1.4-times) for the enzyme bound to Carrier II.     

Stability of free and immobilised peroxidase in aqueous–organic solvents mixtures

The activity and stability of horseradish (Amoracia rusticana) peroxidase (HRP) free in solution and immobilised onto silica microparticles was studied in the presence of organic co-solvents.

The effect of several hydrophilic organic solvents, namely dimethyl sulfoxide, dimethylformamide, dioxan, acetonitrile and tetrahydrofuran, in the activity and stability of free HRP was studied. From the solvents tested, DMSO led to the highest activities and stabilities. After 2 h of incubation at 35°C, the remaining activity of the enzyme in the presence of 30% of each solvent was less than 30%, with exception of DMSO for which the enzyme remained fully active.

In order to increase stability, HRP was covalently immobilised onto silica microparticles. The half-life of the enzyme in buffer at 50°C increased from 2 to 52 h when the enzyme was immobilised. The stability of both free and immobilised HRP was also studied at 50°C in aqueous mixtures of 3.5, 20, 35 and 50% (v/v) DMSO. Free HRP stability was not affected by the presence of 3.5 and 20% DMSO, but higher contents lead to a more pronounced deactivation. Immobilised HRP stability increased with DMSO content up to 20%, decreasing for higher contents. The enzyme half-life increased more than 300% when changing from buffer to 20% DMSO.

The deactivation of free HRP was modelled using the simple exponential decay, and the deactivation of immobilised HRP was described by a two-step inactivation model.     

A comparison of some activated matrices for preparation of immunosorbents

The adsorption characteristics of monoclonal anti-(β-galactosidase) immobilised to a number of commercially available pre-activated matrices have been investigated in a series of small scale experiments. Binding characteristics were determined by batch isotherm techniques and estimates were obtained of the rate constants governing adsorption to the immobilised antibodies. The capacity of the different matrices for binding antibody and the specific activity of immunosorbents were measured.There was little effect of support matrix on the dissociation constant, K_d, for the interaction between β-galactosidase and immobilised anti-(β-galactosidase). However, the maximum amounts of antibody that could be immobilised, rates of adsorption and desorption of the enzyme to the immobilised antibody and the specific activity of immunosorbents were affected by the choice of support matrix. The importance of the relative sizes of the antigen and immobilised antibody and the influence of the nature of the support matrix on the properties of the resulting immunosorbent when used in large scale applications are discussed.     

Penicillium notatum 1 a new source of dextranase

Summary Two hundred and fifteen fungal strains were screened for extracellular dextranase production with a diffusion plate method. The best enzymatic activity (12–19 DU ml^–1) was achieved byPenicillium notatum 1, a species for which the dextranase productivity has not yet been published. Some of the parameters affecting enzyme production have been standardized. The enzyme in crude state was relatively stable, its maximal activity was at 50°C and at pH 5.0. Conidia of the selected strain were mutagenized, and isolated mutants were tested for production of dextranase in submerged culture. The most active mutant,P. notatum 1-I-77, showed over two-times higher dextranase activity than the parentP. notatum 1     

Some properties of an endodextranase inhibitor from continuous cultures of Streptococcus sobrinus.

Cell-free filtrates of Streptococcus sobrinus, cultured at low growth rate in the chemostat, contain a dextranase inhibitor that can completely inhibit the activity of S. sobrinus endodextranase. The range of conditions under which inhibition occurs, and the situations in which enzyme activity can reappear, have been examined in continuous cultures of strain 6715-13WT and the dextranase-deficient mutant 6715-13-201. A purified preparation of the inhibitor was specific for S. sobrinus dextranase, having no action on dextranases from other oral streptococci. The percentage inhibition of S. sobrinus dextranase varied with the enzyme concentration, and the complete inhibition of low amounts of enzyme indicated a very tight bond between the inhibitor and the enzyme.     

Screening of porous and cellular materials for covalent immobilisation of Agaricus bisporus tyrosinase

We conducted a systematic study of covalent immobilisation of Agaricus bisporus tyrosinase onto typical enzyme carriers. Acrylic beads, two commercial silica gels with different pore structures and mesoporous silica foam (MCFs) beads functionalised using different organosilanes showed that only aminated MCFs offer active preparations with immobilisation efficiencies greater than 100% and a similar ratio of diphenolase (L-DOPA) to monophenolase (L-tyrosine) activities as the free enzyme. The native enzyme was entirely inactivated during incubation at 55°C for 30 min, whereas the enzyme immobilised on acrylic carrier or MCF retained 46 and 35%, respectively, of the initial activity after similar treatment. Susceptibility of native and immobilised tyrosinase to suicide inactivation in the presence of L-tyrosine and L-DOPA was tested in repeated batch tests. However, none of the preparations obtained in the L-DOPA solution was operationally stable enough to be used for practical applications.     

Production and Properties of Alkaline Dextranase from a Newly Isolated Brevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.

Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.     

New and effective entrapment of polyelectrolyte-enzyme-complexes in LentiKats

Amyloglucosidase EC 3.2.1.3 was used as model for the immobilisation of enzymes in poly(vinylalcohol) hydrogel (LentiKats) lenses. The entrapment of the enzyme in PVA-hydrogel based on a two-step procedure, firstly its coupling to polyelectrolytes increased the structure level of the enzyme, and subsequently, the resulting complex was entrapped in LentiKats. The immobilised enzyme retained 45% of its original activity and lost no activity over five repeated batch runs.     

Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis

A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.     

Immobilisation and kinetics of Penicillium notatum dextranase on controlled porous glass

Two highly purified enzymic fractions of dextranase (1,6-- -glucan 6-glucanohydrolase, EC 3.2.1.11) from Penicillium notatum have been immobilised on silanised porous glass modified by glutaraldehyde, carbodiimide and bis-oxirane binding. The marked shifts in the pH and temperature optima as well as the changes in the kinetics (K_m, V_max, E_a) of the solid-phase dextranases were observed and discussed. The immobilisation of enzymes on alkylamine glass through the process of glutaraldehyde coupling proved to be the best of the methods studied. The dextranase preparations obtained in this way showed a catalytic activity at wider pH and temperature ranges than those of the free enzymes. They were also characterized by a relatively high affinity to the substrate and good storage stability. The usefulness of the bound dextranase in batch and column hydrolysis of dextran was also established.     

Immobilised monomers of human liver arginase.

Human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was immobilised by attachment to nylon with glutaraldehyde as a crosslinking agent. Incubation of the immobilised tetrameric enzyme with EDTA followed by dialysis resulted in the dissociation of the enzyme into inactive matrix-bound and solubilised subunits. Both species recovered enzymatic activity after incubation with Mn2+, and the activity of the reactivated matrix-bound subunits was nearly 25% of that shown by the enzyme initially attached to the support in the tetrameric form. When the reactivated bound subunits were incubated with soluble subunits in the presence of Mn2+, they 'picked-up' from the solution an amount of protein and enzymatic activity almost identical to that initially lost by the immobilised tetramer after the dissociating treatment with EDTA. This occurred only in the presence of Mn2+. It is suggested that the reactivation of the subunits of arginase involves the initial formation of an active monomer, which then acquires a conformation that favours a reassociation to the tetrameric state.     

Biological activity of the vegetalizing factor: Decrease after coupling to polysaccharide matrix and enzymatic recovery of active factor

Summary The inducing activity of the vegetalizing factor decreases after covalent coupling to CNBr-Sepharose with reduced binding capacity. The residual inducing activity is probably due to the release of a small amount of the factor from Sepharose beads. Covalent coupling to activated CH-Sepharose completely inactivated the vegetalizing factor, whereas the neuralizing factor retained its full activity. The biological activity was also very much reduced when the vegetalizing factor was bound to Sephadex beads, a derivative of dextran. Fully active factor was recovered after enzymatic degradation of the dextran matrix with dextranase. The experiments suggest that the neuralizing factor acts on the cell surface of ectoderm cells, whereas the vegetalizing factor must probably be internalized to become biologically active.