Free vs chitosan-immobilized urease: Microenvironmental effects on enzyme inhibitions

Chitosan gel membranes were prepared by a solvent-evaporation method and used as a support for covalent immobilization of jack bean urease. The effects of the local microenvironment created by both the electrostatic potential of the polycationic support and the enzyme reaction on the inhibition of urease by phosphate buffer were investigated as a function of pH and compared with other urease competitive inhibitions. It was found that the kinetic behaviour of chitosan-immobilized urease in the inhibition is resultant of structural, diffusion limitation-related and microenvironmental factors. More importantly, it was shown that this behaviour is microenvironment-dependent when either a pH-dependence of enzyme inhibition (phosphate and F^?) or electrostatic inhibitor-support repulsion (Ni^2?+?) prevailed in the system. Other inhibitors uncharged that are do not show pH-dependented inhibition (boric and acetohydroxamic acids), the inhibition only depends on factors other than microenvironmental. Knowledge of such microenvironmental effects is of practical and theoretical importance in designing applications of immobilized enzymes and in clarifying the mode of action of enzymes in biological membranes in their native milieu.     

Inhibition of chitosan-immobilized urease by slow-binding inhibitors: Ni, F and acetohydroxamic acid

The inhibitions by Ni²⁺ and F⁻ ions and by acetohydroxamic acid of jack bean urease covalently immobilized on chitosan membrane was studied (pH 7.0, 25°C) and compared with those of the native enzyme. The reaction progress curves of the immobilized urease-catalyzed hydrolysis of urea were recorded in the absence and presence of the inhibitors. They revealed that the inhibitions are of the competitive slow-binding type similar to those of native urease. The immobilization weakened the inhibitory effect of the inhibitors on urease as measured by the inhibition constants K_i^*. The increase in their values: 17.9-fold for Ni²⁺, 26.5-fold for F⁻ and 1.7-fold for acetohydroxamic acid, was accounted for by environmental effects generated by heterogeneity of the urease–chitosan system: (1) mass transfer limitations imposed on substrate and reaction product in the external solution, and (2) the increase in local pH on the membrane produced by both the enzymatic reaction and the electric charge of the support. By relating the K_M/K_i^* ratio to the electrostatic potential of chitosan it was found that while the reduced Ni²⁺ inhibition is mainly brought about by the potential, inhibition by acetohydroxamic acid is independent of the potential, and the acid inhibits urease in its non-ionic form. The reduction in F⁻ inhibition was ascribed to the increased pH in the local environment of the immobilized enzyme.     

Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ相似文献   

Poly(methylene co-guanidine) coated alginate as an encapsulation matrix for urease

Urease was encapsulated within alginate beads, coated with poly(methylene co-guanidine) membranes via polyelectrolyte complexation. Membrane thickness increased with reaction time to 53 μm after 80 min, and to 59 μm with an increase in co-guanidine concentration from 2.5 to 20 mg ml⁻¹. A 70% mass and 31% activity yield of urease resulted following encapsulation. Although co-guanidine strongly inhibited freely soluble urease (I_0.5=5.8 μg ml⁻¹ co-guanidine), immobilization stabilized the enzyme against inactivation. Encapsulated activity declined as the polycation concentration used for membrane formation increased; however an activity loss of only 35% was observed when the co-guanidine concentration was as high as 5 mg ml⁻¹. Glucose protected against inactivation, with 0.5 increasing to 28.5 μg ml⁻¹ for the freely soluble enzyme. When the beads were coated with co-guanidine in the presence of glucose, encapsulated urease activity was fully retained.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Development of potentiometric urea biosensor based on urease immobilized in PVA-PAA composite matrix for estimation of blood urea nitrogen (BUN)

A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1–1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinity™ BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, β-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity.     

The Inositol Lipids of Paramecium tetraurelia and Preliminary Characterizations of Phosphoinositide Kinase Activity in the Ciliary Membrane

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphati-dylinositol-bis-phosphate (PI-P₂) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [γ-³²P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P₂. Kinase activity was activated by millimolar [Mg²⁺] and inhibited by millimolar [Ca²⁺]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated     

Supported bilayer lipid membranes modified with a phosphate ionophore

This article reports the electrical responses of a phosphate ionophore, the cyclic polyamine 3-decyl-1,5,8-triazacyclodecane-2,4-dione (N₃-cyclic amine) incorporated into metal supported bilayer lipid membranes (s-BLM). Teflon coated silver wire was used as a support. In a potentiometric mode, the ionophore had a response that was linearly related to the logarithm of HPO₄²⁻ concentration and was also dependant on pH. Selectivity coefficients for other anions compared to HPO₄²⁻ ions, determined by the separate solution method, fell within the range 1.73 × 10⁻⁴ to 6.38 × 10⁻².     

Purification and properties of two aromatic aminotransferases in Bacillus subtilis.

Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized. One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient. Previous studies have shown that mutants lacking this enzyme require histidine for growth. Mutants in the other enzyme termed aromatic aminotransferase are prototrophs. Neither enzyme is active on any other substrate involved in amino acid synthesis. The two enzymes can be distinguished by a number of criteria. Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively. Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate. Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction. The two enzymes have similar pH optima and employ a ping-pong mechanism of action. The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine. This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase. Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase. The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.     

Interactions of barley α-amylase isozymes with Ca2 , substrates and proteinaceous inhibitors

-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2 +, different substrates, and proteinaceous inhibitors for -amylases. Isozyme specific effects of Ca^2 + on the 80% sequence identical barley -amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2 + with identical ligands. A fully hydrated fourth Ca^2 + at the interface of the AMY2/barley -amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2 + occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2 +. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered 'sugar tongs' site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley -amylases are reported in this review.     

Several compartments of Saccharomyces cerevisiae are equipped with Ca2+-ATPase(s)

Abstract Sucrose density fractionation of yeast membranes revealed two major and two minor peaks of ⁴⁵Ca²⁺ transport activity which all co-migrate with marker enzymes of the endoplasmic reticulum, Golgi and membranes associated with these compartments as well as with ATPase activity measured when all other known ATPase are inhibited. Co-migration of ⁴⁵Ca²⁺ transport and ATPase activities was also found after removal of plasma membranes by concanavalin A treatment. SDS-PAGE at pH 6.3 shows the Ca²⁺-dependent formation of acyl phosphate polypeptides of about 110 and 200 kDa. It is concluded that several compartments or sub-compartments of yeast are equipped with Ca²⁺-ATPase(s). It is proposed that these compartments are derived from the protein secretory apparatus of yeast.     

Interactions between human blood serum inhibitors and native and modified dextran proteinases--terrilytin and trypsin]

Interactions between native terrylytin and trypsin and their derivatives modified by water-soluble dextrans on one hand and human blood serum inhibitors on the other, were studied. It was shown that modification of the enzymes results in changes in the type of their inhibition by blood serum due to a decrease of affinity of polymeric enzyme forms for alpha 2-macroglobulin and alpha 1-antitrypsin. The inhibition constants for native and modified forms of terrylytin and trypsin were calculated. The effects of steric and electrostatic factors on the interaction between inhibitors of blood and polymeric forms of proteinases are discussed.     

Peroxisomal dihydroxyacetone phosphate acyltransferase. Effect of acetaldehyde on the intact and solubilized activity

The peroxisomal enzyme dihydroxyacetone phosphate (DHAP) acyltransferase shows a differential response to acetaldehyde. Employing whole peroxisomes, the enzyme displays a 130-400% stimulation of activity when assayed in the presence of 10-250 mM acetaldehyde. Following taurocholate solubilization of the enzyme the response to 0.25 M acetaldehyde is one of almost total inhibition. This inhibition of the taurocholate-solubilized enzyme is not observed at acetaldehyde concentrations below 200 mM. The stimulation of DHAP acyltransferase by acetaldehyde is solely a response of the peroxisomal enzyme as evidenced by its insensitivity to N-ethylmaleimide and 5 mM glycerol 3-phosphate. Furthermore, microsomal dihydroxyacetone phosphate acyltransferase activity is inhibited at all acetaldehyde concentrations. The activation of membrane-bound DHAP acyltransferase by acetaldehyde appears to be specific for this enzyme in comparison to several other peroxisomal and microsomal enzymes. The specificity of activation and differential response of the peroxisomal enzyme to acetaldehyde indicates that the microenvironment of the peroxisomal membrane is important for normal enzymatic function of this enzyme.     

Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate

The initial rate and enantioselectivity of enzymatic asymmetric hydrolysis of amino acid esters were examined in methylimidazolium-based ionic liquids with anions including tetrafluoroborate, chloride, bromide and bisulfate and in typical organic solvents. Papain displayed much higher enantioselectivity but lower activity in phosphate buffer solution of 1-butyl-3-methylimidazolium tetrafluoroborate BMIM·BF₄ than in other media tested (i.e. E=100, V ₀=0.21 mM min^-1 in BMIM·BF₄, E=2, V ₀=0.43 mM min^-1 in phosphate buffer, E=14-92, V ₀=0.22-0.25 mM min^-1 in organic solvents for D,L-phenylglycine methyl ester). The influence of BMIM·BF₄ on enzyme activity and enantioselectivity also varied with the substrate and the enzyme used. All of the enzymes assayed showed no activity or low enantioselectivity in the ILs with anions including chloride, bromide and bisulfate.     

Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an -amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for - and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe³⁺, Cd²⁺, Pb²⁺, Hg²⁺, Ni²⁺ and Ag¹⁺ were potent inhibitors whereas Zn²⁺, Mg²⁺, Mn²⁺ and Al³⁺ were mild inhibitors. Ca²⁺, Ba²⁺, Sr²⁺ and K⁺ stimulated amylase activity in the order of Ca²⁺ > Ba²⁺ > Sr²⁺ > K⁺. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both - as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca²⁺ and PCMB inhibition by the addition of glutathione (reduced). The K_m for - and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.     

Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity.

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid beta-glycerophosphatase, alpha-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.     

Electrostatic effects on the kinetics of bound enzymes.

1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.     

Immobilization of fuculose-1-phosphate aldolase from E. coli to glyoxal-agarose gels by multipoint covalent attachment

Recombinant fuculose 1-phosphate aldolase (FucA) from E. coli has been immobilized by multipoint covalent attachment to glyoxal-agarose gels. Experiments, varying the main parameters that control the immobilization process (surface density of aldehyde groups, temperature, pH), were carried out. An immobilization yield of 80-90% and FucA retained activity on immobilized derivative of 10-20% can be achieved when pH 10, 20°C and 200 µmoles cm^-3 of aldehyde groups was used. The observed activity loss in the immobilization process might be related to the fact that the complex quaternary structure of the enzyme could not be maintained. A short contact-time enzyme support is required to obtain high ratio of active to total immobilized enzyme.


A highly loaded derivative of immobilized FucA (65 AU cm^-3 of support) has been prepared to use in aldol condensation reactions. Reactions catalyzed by these aldolases involve the use of non-conventional media because of substrate solubility. For instance, the condensation of dihydroxyacetone phosphate (DHAP) and Z-amino-propanal, Z-(R)-alaninal and Z-(S)- alaninal in highly concentrated water-in-oil emulsions gave synthetic yields of 40, 25 and 29% respectively.     

Localization in the Cell and Extraction of Alkaline Phosphatase from Bacillus subtilis

Study of protoplasts, lysed protoplasts, and cells treated with lysozyme in the absence of osmotic stabilizer suggested that the alkaline phosphatase (EC 3.1.3.1.) of Bacillus subtilis is located in the protoplasmic membrane. Cytochemical evidence in support of this view is presented. The enzyme protein was strongly bound to the membrane structure and could not be solubilized by a number of treatments known to release enzymes from membranes and other lipoprotein structures. Alkaline phosphatase was, however, solubilized by treatment of intact B. subtilis cells or isolated protoplasmic membranes with strong salt solutions at pH 7.2, suggesting that electrostatic forces are responsible for the association between membrane and enzyme protein. Dialysis of alkaline phosphatase solutions against buffer of low ionic strength resulted in precipitation of the enzyme.