Thermal inactivation of free and immobilized inulinase

Thermal inactivation of the Kluyveromyces marxianus inulinase in a free form and immobilized on VION KN-1 cation exchange fiber was studied. Atomic force microscopy demonstrated an oligomeric structure of this enzyme, composed of two subunits differing in their size. It was assumed that the intersubunit contacts were destroyed at 60°C, and the inulinase molecule dissociated into two monomers located separately.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

Flow kinetics of immobilized beta-glucosidase

The enzyme beta-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 degrees C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 degrees C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.     

Thermal inactivation of immobilized enzymes in ideal continuous reactors

The limitation of thermal inactivation on catalytic activity in continuous enzymatic reactions is considered. Where an enzyme is retained in a reaction environment which is open to mass transfer of reaction components, the effect of enzyme inactivation on reactant conversion depends on the order of the chemical reaction and the pattern of fluid flow through the reaction volume. Equations expressing conversion as a function of time for first-order inactivation are presented for Michaelis-Menten kinetics and the limiting fluid flow conditions of plug flow and complete back-mixing. Substrate protection or destruction of an enzyme is also considered and it is shown theoretically that the catalytic life of an enzyme may be optimized by the proper choice of fluid flow pattern.     

Gluco-oligosaccharide synthesis by free and immobilized beta-glucosidase

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. (c) 1993 John Wiley & Sons, Inc.     

Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products

Inactivation of immobilized penicillin acylase has been studied in the presence of substrate (penicillin G) and products (phenylacetic acid and 6-aminopenicillanic acid), under the hypothesis that substances which interact with the enzyme molecule during catalysis will have an effect on enzyme stability. The kinetics of immobilized penicillin acylase inactivation was a multistage process, decay constants being evaluated for the free-enzyme and enzyme complexes, from whose values modulation factors were determined for the effectors in each enzyme complex at each stage. 6-Aminopenicillanic acid and penicillin G stabilized the enzyme in the first stage of decay. Modulation factors in that stage were 0.96 for penicillin G and 0.98 for 6-aminopenicillanic acid. Phenylacetic acid increased the rate of inactivation in both stages, modulating factors being -2.31 and -2.23, respectively. Modulation factors influence enzyme performance in a reactor and are useful parameters for a proper evaluation. (c) 1996 John Wiley & Sons, Inc.     

Stability and reactivity of acid phosphatase immobilized on composite beads of chitosan and ZrO2 powders

Equal weights of chitosan and ZrO2 powders were mixed in acetic acid solution to prepare the composite beads. They were then cross-linked with glutaraldehyde and stored with and without freeze-drying before use. The physicochemical properties of acid phosphatase immobilized on four types of the supports (wet/dried pure chitosan beads, wet/dried chitosan-ZrO2 composite beads) were compared. Various parameters including glutaraldehyde concentration, cross-linking time, enzyme concentration, temperature, and pH on enzyme activity were studied. It was shown that the activity yield of enzyme immobilized on the dried chitosan-ZrO2 beads was the highest, and the relative activity remained above 83.2% within pH 2.9-5.8. Regardless of wet or dried beads, the Michaelis constant KM and maximum rate of reaction Vmax of acid phosphatase immobilized on chitosan-ZrO2 composite beads were 1.8 times larger than those on pure chitosan beads. Of the four immobilized enzymes, the use of wet chitosan-ZrO2 bead as the support showed the lowest thermal deactivation energy (78 kJ mol(-1)).     

Studies on the thermal inactivation of immobilized enzymes

The thermal inactivation of a great number of immobilized enzymes shows a biphasic kinetics, which distinctly differs from the first-order inactivation kinetics of the corresponding soluble enzymes. As shown for alpha-amylase, chymotrypsin, and trypsin covalently bound to silica, polystyrene, or polyacrylamide, the dependence of the remaining activities on the heating time can be well described by the sum of two exponential terms. To interpret this mathematical model function, the catalytic properties of immobilized enzymes (number of active sites in silica-bound trypsin, K(M) and E(a) values in silica-bound alpha-amylase and chymotrypsin) at different stages of inactivation and the influence of various factors (coupling conditions, addition of denaturants or stabilizers, etc.) on the thermal inactivation of silica-bound alpha-amylase were studied. Furthermore, conformational alterations in the thermal denaturation of spin-labeled soluble and silica-bound beta-amylase were compared by electron spin resonance (ESR) studies. The results suggest that the biphasic inactivation kinetics reflects two different pathways according to which catalytically identical enzyme molecules are predominantly inactivated.     

Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-life of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.     

Unfolding and inactivation of Ampullarium crossean beta-glucosidase during denaturation by guanidine hydrochloride

Changes of activity and conformation of Ampullarium crossean beta-glucosidase in different concentrations of guanidine hydrochloride (GuHCl) have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreased distinctly with increasing guanidine concentrations, the emission peaks appeared red shifted (from 338.4 to 350.8 nm), whereas a new fluorescence emission peak appeared near 310 nm. Changes in the conformation and catalytic activity of the enzyme were compared. A corresponding rapid decrease in catalytic activity of the enzyme was also observed. The extent of inactivation was greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. k(+0)>k(+0)' also showed that the enzyme was protected by substrate to a certain extent during guanidine denaturation.     

Time-dependent inactivation of immobilized glucose oxidase and catalase

Homogeneous membranes containing immobilized glucose oxidase and catalase were stored in buffered solutions at 37 degrees C to determine the mechanisms and rates of catalyst inactivation. The experiments were designed so that inactivation occurred homogeneously throughout the membrane, thereby simplifying the analysis. The mechanism of inactivation is consistent with the reaction of hydrogen peroxide and certain catalytic intermediates of both enzymes. Based on this information, numerical simulations were developed that incorporate spatially heterogeneous catalytic and inactivation processes.     

Purification of cytosolic beta-glucosidase from pig liver and its reactivity towards flavonoid glycosides

Flavonoid glycosides are common dietary components which may have health-promoting activities. The metabolism of these compounds is thought to influence their bioactivity and uptake from the small intestine. It has been suggested that the enzyme cytosolic beta-glucosidase could deglycosylate certain flavonoid glycosides. To test this hypothesis, the enzyme was purified to homogeneity from pig liver for the first time. It was found to have a molecular weight (55 kDa) and specific activity (with p-nitrophenol glucoside) consistent with other mammalian cytosolic beta-glucosidases. The pure enzyme was indeed found to deglycosylate various flavonoid glycosides. Genistein 7-glucoside, daidzein 7-glucoside, apigenin 7-glucoside and naringenin 7-glucoside all acted as substrates, but we were unable to detect activity with naringenin 7-rhamnoglucoside. Quercetin 4'-glucoside was a substrate, but neither quercetin 3, 4'-diglucoside, quercetin 3-glucoside nor quercetin 3-rhamnoglucoside were deglycosylated. Estimates of K(m) ranged from 25 to 90 microM while those for V(max) were about 10% of that found with the standard artificial substrate p-nitrophenol glucoside. The non-substrate quercetin 3-glucoside was found to partially inhibit deglycosylation of quercetin 4'-glucoside, but it had no effect upon activity with p-nitrophenol glucoside. This study confirms that mammalian cytosolic beta-glucosidase can deglycosylate some, but not all, common dietary flavonoid glycosides. This enzyme may, therefore, be important in the metabolism of these compounds.     

Thermal inactivation of butyrylcholinesterase and acetylcholinesterase

Differences were observed in the extent of thermal inactivation of human butyrylcholinesterase (BuChE) and eel acetylcholinesterase (AChE). BuChE was more resistant to 57°C inactivation than was AChE. Thermal inactivation of BuChE was reversible and followed first-order kinetics. AChE thermal inactivation was irreversible and did not follow first-order kinetics. AChE was marginally protected from thermal inactivation by the nonspecific salts ammonium sulfate and sodium chloride and to a greater extent by the active site-specific salts choline chloride, sodium acetate, and acetylcholine iodide. This protection was accompanied by a loss of absorbance at 280 nm. This data supports the hypothesis that thermal inactivation of AChE occurs by conformational scrambling and that aromatic amino acid residue(s) are involved in this process.Recipient of a research fellowship from the UNCW graduate school.     

Reversibility of the acid inactivation of immobilized alpha-amylase

The process of reactivation of acid-inactivated alpha-amylase of Bacillus subtilis in weakly alkaline media was examined. The reactivation of alpha-amylase immobilized on carboxyl polyelectrolytes developed in a larger measure than that of the native enzyme. The stabilizing effect of the cross polymer decreased as its porosity increased.     

Molecular orbital theory on cellulolytic reactivity between pNP-cellooligosccharides and beta-glucosidase from Cellulomonas uda CS1-1

A beta-glucosidase with the molecular mass of 160,000 Da was purified to homogeneity from cell extract of a cellulolytic bacterium, Cellulomonas uda CS1-1. The kinetic parameters (Km and Vmax) of the enzyme were determined with pNP-cellooligosccharides (DP 1-5) and cellobiose. The molecular orbital theoretical studies on the cellulolytic reactivity between the pNP-cellooligosaccharides as substrate (S) molecules and the purified beta-glucosidase (E) were conducted by applying the frontier molecular orbital (FMO) interaction theory. The results of the FMO interaction between E and S molecules verified that the first stage of the reaction was induced by exocyclic cleavage, which occurred in an electrophilic reaction based on a strong charge-controlled reaction between the highest occupied molecular orbital (HOMO) energy of the S molecule and the lowest occupied molecular orbital (LUMO) energy of the hydronium ion (H3O+), more than endocyclic cleavage, whereas a nucleophilic substitution reaction was induced by an orbital-controlled reaction between the LUMO energy of the oxonium ion (SH+) protonated to the S molecule and the HOMO energy of the H2O2 molecule. A hypothetic reaction route was proposed with the experimental results in which the enzymatic acid-catalyst hydrolysis reaction of E and S molecules would be progressed via SN1 and SN2 reactions. In addition, the quantitative structure-activity relationships (QSARs) between these kinetic parameters showed that Km has a significant correlation with hydrophobicity (logP), and specific activity has with dipole moment, respectively.