The use of titanium (IV) oxide for the immobilisation of carbohydrate-directed enzymes.

The use of particles of porous titanium (IV) oxide as a suitable matrix for enzyme immobilisation has been investigated with dextranase. Treatment of the particles with enzyme in the presence and absence of ammonium ions showed that the presence of ammonia induced a greater coupling of protein, whereas a greater retention of enzyme specific activity was achieved in the absence of ammonia. Properties of the immobilised enzyme include a pH-dependence and reversibility of the coupling between enzyme and matrix. The immobilised dextranase was most stable at pH 5.0. Automated analytical techniques for measuring the activity of dextranase and other polysaccharidases in soluble and insoluble forms are also reported.     

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.     

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.     

Hydrolytic and chromatographic studies on the PEGylation of dextranase from Penicillium sp.

Dextranases catalyze the hydrolysis of the α-l,6-glucosidic bond of the polysaccharide dextran. Dextranases have been isolated from bacteria, yeast and fungi. Purified dextranase enzyme from Penicillium sp. was PEGylated (polyethylene glycol modification) with mPEG (5000 Da) and showed an increase in the dextranase protein molecular weight as estimated by Superose 12 (23 ml) column and this increment in the molecular weight is directly proportional to mPEG (5000 Da) concentration until a complete dextranase enzyme PEGylation (disappearance of dextranase peak). The residual activity of partially PEGylated dextranase (mPEG 5000 of 5.8 mg/ml) was 33.8% and for the completely PEGylated dextranase (mPEG 5000 of 29 mg/ml) it was 25.75%. Dextranase PEGylated with mPEG (30,000 Da) showed a little PEGylation at mPEG concentration of 5.8 mg/ml but at a concentration of 29 mg/ml several PEGylated peaks were produced with a difference in dextranase activity toward dextran T500, retardation in the activity with the increasing in the molecular weight was clearly appeared with Sephadex G75 but for Sephadex G200 a little retardation than Sephadex G75 has been appeared.     

Temperature and pH dependence of mold α-galactosidase

The effect of temperature and pH on kinetic behavior of α-galactosidase of Mortierella vinacea was investigated on the hydrolysis of p-nitrophenyl-α-D -galactopyranoside (PNPG). A very unusual kinetic behavior was observed for the soluble α-galactosidase i.e., substrate inhibition diminished gradually with increasing temperature or near the neutral pH range, and the kinetics approached the ordinary Michaelis-Menten (MM) type. On the other hand, with decreasing temperature or in acidic pH range, substrate inhibition was accelerated. Therefore, Arrhenius plots based on the initial reaction rate did not give straight lines. Furthermore, the slope in the Arrhenius plot changed with substrate concentration, which would make the determination of a characteristic value using conventional methods meaningless. However, the Arrhenius plots of individual kinetic parameters in the rate equation resulted in straight lines in the temperature range 15 to 50°C. From this, the drastic change in kinetic behavior could be explained in connection with the temperature and pH dependence of kinetic parameters in the model. For mold pellets (whole-cell enzyme), however, the influence of temperature and pH was less apparent than that of soluble enzyme because of the limitation in intraparticle diffusion. By using the rate equation that was determined for soluble enzyme and the theoretically derived effectiveness factor, the overall reaction rate for mold pellets at various temperature and pH could be predicted to some extent.     

Breaks in arrhenius plots of reactions involving membrane-bound and solubilized sialyltransferases,due to temperature dependence of kinetic parameters

Temperature dependence of asialomucin-sialyltransferase ($\text{CMP}\text{-N-}\text{acetylneuraminate:}\text{D}\text{-galactosyl-glyco-protein}$) N-acetylneuraminyltransferase, EC 2.4.99.1) activity is investigated. Discontinuities in Arrhenius plots are observed, whether the enzyme is membrane-associated or solubilized. These discontinuities cannot be firmly correlated with the phase-transition temperatures of either endogenous or exogenous phospholipids. Arrhenius plots of the kinetic parameters also exhibit sharp discontinuities, so that it is concluded that a significant change in K_m and V_max values occurs with varying temperature. Our results suggest that the biphasic behavior of Arrhenius plots may be attributed to the temperature dependence of the kinetic parameters for both membrane-associated and solubilized sialyltransferase activities.     

Bacterial Oxidation of Sulfide Minerals in Column Leaching Experiments at Suboptimal Temperatures

The purpose of the work was to quantitatively characterize temperature effects on the bacterial leaching of sulfide ore material containing several sulfide minerals. The leaching was tested at eight different temperatures in the range of 4 to 37°C. The experimental technique was based on column leaching of a coarsely ground (particle diameter, 0.59 to 5 mm) ore sample. The experimental data were used for kinetic analysis of chalcopyrite, sphalerite, and pyrrhotite oxidation. Chalcopyrite yielded the highest (73 kJ/mol) and pyrrhotite yielded the lowest (25 kJ/mol) activation energies. Especially with pyrrhotite, diffusion contributed to rate limitation. Arrhenius plots were also linear for the reciprocals of lag periods and for increases of redox potentials (dmV/dt). Mass balance analysis based on total S in leach residue was in agreement with the highest rate of leaching at 37 and 28°C. The presence of elemental S in leach residues was attributed to pyrrhotite oxidation.     

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.     

Characterization of a marine-derived dextranase and its application to the prevention of dental caries

The dextranase added in current commercial dextranase-containing mouthwashes is largely from fungi. However, fungal dextranase has shown much higher optimum temperature than bacterial dextranase and relatively low activity when used in human oral cavities. Bacterial dextranase has been considered to be more effective and suitable for dental caries prevention. In this study, a dextranase (Dex410) from marine Arthrobacter sp. was purified and characterized. Dex410 is a 64-kDa endoglycosidase. The specific activity of Dex410 was 11.9 U/mg at optimum pH 5.5 and 45 °C. The main end-product of Dex410 was isomaltotriose, isomaltoteraose, and isomaltopentaose by hydrolyzing dextran T2000. In vitro studies showed that Dex410 effectively inhibited the Streptococcus mutans biofilm growth in coverage, biomass, and water-soluble glucan (WSG) by more than 80, 90, and 95 %, respectively. The animal experiment revealed that for short-term use (1.5 months), both Dex410 and the commercial mouthwash Biotene (Laclede Professional Products, Gardena, CA, USA) had a significant inhibitory effect on caries (p = 0.0008 and 0.0001, respectively), while for long-term use (3 months), only Dex410 showed significant inhibitory effect on dental caries (p = 0.005). The dextranase Dex410 from a marine-derived Arthrobacter sp. strain possessed the enzyme properties suitable to human oral environment and applicable to oral hygiene products.     

Sensitive Detection of Organophosphorus Pesticides Using a Needle Type Amperometric Acetylcholinesterase-based Bioelectrode. Thiocholine Electrochemistry and Immobilised Enzyme Inhibition

An acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concentrations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of +0.410 V vs. Ag/AgCl, KCl sat was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthiocholine detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm ^?2 of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 μM for dipterex and 0.4 μM for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots.     

Investigation of kinetics of immobilized liver esterase by flow calorimetry

Flow calorimetry (FC) was shown to be a powerful tool for investigation of the kinetics of phenyl acetate hydrolysis catalyzed by pig liver carboxyl esterase. The enzyme was immobilized in alginate gel particles that were placed in a calorimetric flow column and the heat effect of enzyme reaction was followed in single flow and total recirculation conditions. It was shown that the registered temperature change was proportional to molar amount of substrate transformed in the column. A mathematical model describing the enzyme reaction, mass transfer, and heat effects in the calorimetric system was developed and used for the kinetic data evaluation. By combining data from single flow and recirculation modes true kinetic parameters were evaluated by the proposed mathematical procedure based on the model solution and successive approximations.

The kinetic data for carboxyl esterase showed a slide substrate inhibition by phenyl acetate. The obtained kinetic parameters were as follows: Michaelis constant K_m=2 mmol dm⁻³ and substrate inhibition constant K_i=42 mmol dm⁻³. The method can be applied to kinetic study of immobilized enzymes directly in the flow calorimeter without any requirement of an independent analytical technique.     

Dextranase production from Penicillium funiculosum

Twenty-eight Penicillium cultures were screened for dextranase activity. Dextranase yield of about 2000 DU/ml was obtained with Penicillium funiculosum SH-5. Maximum dextranase concentration was attained only when cell mass decreased. The kinetics of the dextranase production was correlated with the cell mass by a two-parameter model. The optimum pH and temperature for dextranase were 5.0-5.5 and 55°C, respectively. Crude dextranase preparation was inhibitory to insoluble glucan formation by streptococcus mutans 6715 in vitro.     

Spectrophotometric assay for online measurement of the activity of lipase immobilised on micro-magnetic particles

A spectrophotometric assay has been adapted to directly measure the activity of enzymes immobilised on insoluble magnetic particles. Three different types of lipases (Candida antarctica lipase A and B and Thermocatenulatus lanuginosus lipase) were immobilised on two types of magnetic beads. The activity of the resulting immobilised lipase preparations was measured directly in the reaction solution by using a modified p-nitrophenol ester assay using a spectrophotometer. Removal of the solid particles was not necessary prior to spectrophotometric measurement, thus allowing reliable kinetic measurements to be made rapidly. The method was effective for a wide range of magnetic bead concentrations (0.01–0.2 mg ml⁻¹). In all cases the assay could determine the bead-related specific enzyme activity. The assay was validated by comparing with a pH-stat method using p-nitrophenol palmitate as the substrate with an excellent correlation between the two methods. The utility of the spectrophotometric assay was demonstrated by applying it to identify the best combination of lipase type, activation chemistry and magnetic particle. Epoxy activation of poly vinyl alcohol-coated magnetic particles prior to immobilisation of commercial C. antarctica lipase A gave the best preparation.     

Sensitive detection of organophosphorus pesticides using a needle type amperometric acetylcholinesterase-based bioelectrode. Thiocholine electrochemistry and immobilised enzyme inhibition

An acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concentrations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of +0.410 V vs. Ag/AgCl, KCl(sat) was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthiocholine detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm(-2) of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 microM for dipterex and 0.4 microM for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots.     

The use of polyaniline nanofibre as a support for lipase mediated reaction

Highly stable and recoverable polianiline nanofibres are developed for enzyme immobilisation and recovery. Candida rugosa lipase (LP) was immobilised onto a polyaniline nanofibre with cross-linking for enzyme aggregation. The optimal LP loading was 5 mg LP/1 mg polyaniline. The stability of the immobilised LP was measured and shown to be high under vigorous shaking at room temperature. This polyaniline nanofibre LP was easily separable with low-speed centrifugation and repeatedly usable. LP immobilised on polyaniline nanofibre demonstrated high stereoselectivity in the kinetic resolution of racemic (R,S)-ibuprofen and improved the long-term stability as compared to that by the free enzyme, allowing the supported enzyme to be repeatedly used for a series of chiral resolution reactions. The conversion from racemic ibuprofen to a chirally selective compound, a prophilic ester of ibuprofen, was approximately 30% with free LP and approximately 10% with immobilised LP. The enantiomeric excess using immobilised LP after 96 h reaction was 0.884.     

Immobilization of thermostable exo-inulinase from mutant thermophilic Aspergillus tamarii-U4 using kaolin clay and its application in inulin hydrolysis

In this study, attempts were made to immobilize purified exo-inulinase from mutant thermophic Aspergillus tamarii-U4 onto Kaolinite clay by covalent bonding cross-linked with glutaraldehyde with an immobilization yield of 66% achieved. The free and immobilized inulinases were then characterized and characterization of the enzymes revealed that temperature and pH optima for the activity of the free and immobilized enzymes were both 65?°C and pH 4.5 respectively. The free inulinase completely lost its activity after incubation at 65?°C for 6 h while the immobilized inulinase retained 16.4% of its activity under the same condition of temperature and incubation time. The estimated kinetic parameters K_m and V_max for the free inulinase as estimated from Lineweaver-Burk plots were 0.39?mM and 4.21?µmol/min for the free inulinase and 0.37?mM and 4.01?µmol/min for the immobilized inulinase respectively. Inulin at 2.5% (w/v) and a flow rate of 0.1?mL was completely hydrolysed for 10?days at 60?°C in a continuous packed bed column and the operational stability of the system revealed that the half-life of the immobilized inulinase was 51?days. These properties make the immobilized exo-inulinase from Aspergillus tamarii-U4 a potential candidate for the production of fructose from inulin hydrolysis.     

Cephalexin synthesis by immobilised penicillin G acylase under non-isothermal conditions: reduction of diffusion limitation

The effect of thermodialysis on the enzymatic kinetic synthesis of the antibiotic cephalexin was investigated. As reference points, two existing models for an immobilised enzyme (Assemblase^®) and for the free enzyme were used. For Assemblase^®, it is known that diffusion limitation occurs and that therefore considerably more of the undesired side-product phenylglycine is formed.

The enzyme was immobilised on a membrane, and under isothermal conditions (293 K) the course of the reaction resembled that of the Assemblase^® enzyme. However, if a temperature gradient was applied across the membrane, with an average temperature of 293 K for the enzyme, than the course of the reaction changed. For large temperature gradients (30° and more), the course of the reaction resembled that of free enzyme. Thermodialysis enhances mass transfer across the membrane and therewith reduces diffusion limitations in the immobilised enzyme on the membrane.

The stability of the immobilised enzyme is such that the reactor can be re-used repeatedly. This, together with the positive effect of the temperature gradient on the course of the reaction, makes thermodialysis an interesting new technique that has potential to be applied on a larger scale if the membrane surface area per volume of reactor can be improved.     

一株产右旋糖酐酶青霉的分离及酶的纯化和性质

[目的]从土壤中筛选到一株新的产右旋糖酐酶的真菌F1001,为酶法制备药用级右旋糖酐提供新的右旋糖酐酶产生菌株.[方法]通过形态特征和ITS rDNA序列分析方法鉴定菌株.利用硫酸铵盐析、Sepharose 6B凝胶柱纯化,得到纯度较高的酶蛋白.以右旋糖酐70 kDa为底物,对右旋糖酐酶酶学性质及催化机理进行研究.[结...     

Production,characterization and (co-)immobilization of dextranase from <Emphasis Type="Italic">Penicillium aculeatum</Emphasis>

Fermentation kinetics of Penicillium aculeatum ATCC 10409 demonstrated that fungal growth and dextranase release are decoupled. Inoculation by conidia or mycelia resulted in identical kinetics. Two new isoenzymes of the dextranase were characterized regarding their kinetic constants, pI, MW, activation energy and stabilities. The larger enzyme was 3-fold more active (turnover number: 2,230 ± 97 s⁻¹). Pre-treatment of bentonite with H₂O₂ did not affect adsorption characteristics of dextranase. Enzyme to bentonite ratios above 0.5:1 (w/w) resulted in a high conservation of activity upon adsorption. Furthermore, dextranase could be used in co-immobilizates for the direct conversion of sucrose into isomalto-oligosaccharides (e.g. isomaltose). Yields of co-immobilizates were 2–20 times that of basic immobilizates, which consist of dextransucrase without dextranase.