Fluorocitrate inhibition of aconitase. Reversibility of the inactivation

Fluoride ion is released nearly stoichiometrically when (?)-erythro-fluorocitrate is incubated with aconitase. The release of F^? parallels the loss in activity and could arise from direct displacement of F^? by a base on the enzyme or from dehydration to fluoro-cis-aconitate and attack of an enzymic base to release F^?. Aconitase inactivated by ¹⁴C-fluorocitrate does not retain radioactivity when passed through G-50 Sephadex or precipitated by ammonium sulfate. Fullenzymicactivity can be regained after either of these treatments by activation by cysteine and ferrous salts. These data are consistent with the report of fluorocitrate being a competitive (and non-competitive) inhibitor of aconitase (Villafranca, J.J. (1972) Intra-Science Chem. Rept. 6 (4), 1–11) which rapidly inactivates the enzyme. This inactivated enzyme may be a very labile covalent complex, a very tight complex between enzyme and fluoro-cis-aconitate or a tight complex between a defluorinated deravitive of fluorocitrate.In the course of Peters (1957) extensive work on the toxic effects of fluoroacetate, he determined that fluoroacetate was metabolically converted to fluorocitrate. This finding and the fact that citrate levels rise soon after ingestion of fluoroacetate led to the suggestion that fluorocitrate inactivates aconitase (E.C. 4.2.1.2).Recently, conflicting reports concerning the site of inactivation in mitochondria by the inhibiting isomer of fluorocitrate, (?)-erythro-fluorocitrate (1R:2R, 1-fluoro-2-hydroxy-1,2,3-propanetricarboxylate) have appeared (Eanes et al. 1972; Brand et al, 1973). Eanes et al. (1972) contends that the tricarboxylate carrier is the site of inhibition, while Brand et al. (1973) has compelling evidence that aconitase is the site of inhibition. This controversy is a matter of intrepretation of the results and a greater knowledge of the inactivation of aconitase by fluorocitrate may be useful in these interpretations. The results reported herein are concerned with the mechanism of inactivation of purified mitochondrial aconitase by fluorocitrate and demonstrate that this reaction is readily reversible.     

Reversibility of the ampicillin-and nitrite-induced inactivation of beta-lactamase I.

beta-Lactamase I was isolated from Bacillus cereus 569/H. Treatment with ampicillin in the presence of sodium nitrite at pH 4 or 5 resulted in the inactivation of the enzyme presumably by modification of a carboxyl group in the active site. However, this inactivation was rapidly, reversible at neutral pH and the available evidence points to the participation of a second carboxyl group which is involved in the reactivation process.     

Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

Preparation of immobilized alpha-amylase covalently attached to granular polyacrylonitrile

In this report, alpha-Amylase originating from Bacillus subtilis (liquefying type) was immobilized on partially imidoesterized polyacrylonitrile (PAN) by covalent bonding. For the preparation of immobilized alpha-amylase, which has a high activity and high stability to repeated use, the optimum conditions for the preparation reaction were investigated. The optimum conditions for the preparation reaction were quantified on the basis of the enzymatic activity, the preservation of the activity during repeated use in batch process and the protein content on the support. Further-more, enzymatic properties of immobilized alpha-amylase prepared at optimum conditions were compared with the native enzyme. The optimum temperature and reaction time for the imidoes-terification reaction were 30 degrees c and 6 h, respectively, whereas those of the amidinatin reaction were 30-40 degrees C and more than 3 h, respectively; the optimum pH range was 9-10. Immobilized alpha-amylase prepared at the optimum conditions was very stable against the repeated use and had more than 90% of relative to activity of the first use after the tenth procedure. The initial reaction rate of immobilized alpha-amylase was lower than native alpha-amylase, but same amount of reducing sugars were produced after the reaction passed for more than 90 min. The immobilized alpha-amylase was less stabel at the high temperature and the more basic media. However, after long incubation time, immobilized alpha-amylase was more stable than the native enzyme in exposure to heat and a storng base.     

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.     

Shear-induced inactivation of alpha-amylase in a plain shear field

A newly developed shearing device was used to study shear-induced inactivation of thermostable alpha-amylase in a plain shear field, under conditions comparable to extrusion. The results show that the inactivation can be described well with a first-order process, in which the inactivation energy largely depends on the shear stress, instead of specific mechanical energy or strain history. The resulting dependency of the rate of inactivation on the shear stress is very strong and nonlinear, which leads to the conclusion that in many cases the maximally applied shear stress determines the inactivation. Quantification of the inactivation rates gives design criteria for the application of enzymes in more viscous systems than conventionally used, provided that the reactor is designed such that no peak shear stresses occur.     

Reversibility of oxygen induced inactivation of nitrogenase in some enterobacteria

Aerobic and microaerobic diazotrophs possess numerous oxygen restriction strategies to protect nitrogenase from inactivation by oxygen without interfering with energy generation through oxidative phosphorylation. Protection by conformational change in nitrogenase was first detected and described in Azotobacter. This strategy once considerd unique for Azotobacter has been shown in this study to occur in Citrobacterfreundii (Braak) Werkman and Gillen and Klebsiella pneumoniae subspecies rhinoscleromatis (Trevisan) Migula also. However, in these enteric bacteria the entire enzyme is not protected probably due to the absence of any respiratory protection similar to that found in the aerobe, Azotobacter.     

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.     

Reversibility of acid denaturation of recombinant interferon-gamma

It has been shown that interferon-gamma (IFN-gamma) loses activity after acid treatment and this property can be used to distinguish it from other types of interferons. Therefore, reversibility of acid denaturation of IFN-gamma was examined using the recombinant human protein. The fluorescence spectra showed that conformation of the protein is similar before and after acid treatment, suggesting reversibility of the acid denaturation. The antiviral activity of the protein was also identical in the same treatment. However, the antiviral activity was significantly reduced when it was determined by directly diluting the acidic samples into the assay medium containing high salts and serum proteins. Similar results were obtained with the recombinant murine IFN-gamma. This observation demonstrates that acid denaturation of the IFN-gamma is dependent on the way the protein is renatured, and hence that the difference in response to acid treatment between IFN-gamma and other interferons is quantitative rather than qualitative.     

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.     

Hydrolysis of soluble starch using Bacillus licheniformis alpha-amylase immobilized on superporous CELBEADS

In the present work, indigenously prepared rigid superporous (pore size of approximately 3 microm) cross-linked cellulose matrix (CELBEADS) has been used as a support for the immobilization of Bacillus licheniformis alpha-amylase (BLA). Optimum pH and temperature, and Michaelis-Menten constants were determined for both free and immobilized BLA. Immobilized BLA was observed to produce a different saccharide profile than free BLA at any value of dextrose equivalent. It was observed that pH, temperature, and initial starch concentration has a significant effect on the saccharide profile of starch hydrolysate produced using immobilized BLA in the batch mode, whereas the ratio of concentration of enzyme units to initial starch concentration has no influence on the same. Hence immobilized BLA can be used as an additional tool for production of maltodextrins with different saccharide profiles. Immobilized BLA has better thermostability than free BLA. Immobilized BLA was found to retain full activity even after eight batches of hydrolysis, each of 8h duration at 55 degrees C and 90 mg/mL initial starch concentration. A semiempirical model has been used for the prediction of saccharide composition of starch hydrolysate with respect to time.     

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.     

Simultaneous inactivation of the wprA and dltB genes of Bacillus subtilis reduces the yield of alpha-amylase

AIMS: In Gram-positive bacteria, signal peptide-bearing secretory proteins are translocated through the cytoplasmic membrane and fold into their native conformation on the outside of the cell. The products of the Bacillus subtilis wprA and dltB genes separately influence post-translocational stages of the secretion process by mediating proteolytic degradation and folding of secretory proteins. Inactivation of either wprA or dltB in B. subtilis increases the yield of secretory proteins released into the culture medium in an intact and biologically active conformation. The aim of this work was to study the combined influence of these genes. METHODS AND RESULTS: A wprA/dltB double mutant was constructed, but did not have an additive effect on secretion and caused a significant reduction in the yield of alpha-amylase. CONCLUSIONS AND SIGNIFICANCE: The activities of the wprA gene and the dlt operon interact in a negative way to influence the growth cycle and protein secretion. The mechanism by which this may occur, and its potential significance for the secretion of native and non-native proteins from B. subtilis and related bacteria, is discussed.     

A kinetic approach to the thermal inactivation of an immobilized triosephosphate isomerase

The process of thermal inactivation of triosephosphate isomerase covalently attached to a silica-based support activated with p-benzoquinone was found to be a complex one. At 50 degrees C, a characteristic activation preceding the thermal inactivation was observed. Following the intramolecular changes caused by heat, the values of K(M) and V(max) were determined during the activation. It was presumed that the complex thermal inactivation kinetics reflects the microheterogeneity of the immobilized enzyme molecules. The phosphate ion proved to be a better stabilizer than the substrate. (c) 1992 John Wiley & Sons, Inc.     

Production of ethanol from starch by free and immobilized Candida tropicalis in the presence of alpha-amylase

Candida tropicalis is a potentially useful organism for the commercial production of ethanol as it is capable of fermenting starch at a low rate. To enhance this carbon source utilization and increase the rate of alcohol production, we pretreated corn soluble starch with alpha-amylase. Starch liquefaction was sufficient to drive the fermentation and to convert 96% substrate to ethanol. Indeed, in the presence of exogenous alpha-amylase, 9% (w/v) soluble starch was converted to 43.1g ethanol/l in 65 h with a productivity of 0.65 g/l h. Thus, bio-ethanol production using free and calcium alginate-immobilized C. tropicalis does not require the saccharification step. Furthermore, fed-batch fermentation by free C. tropicalis cells increased the final concentration to 56 g ethanol/l, reaching published values for Saccharomyces cerevisiae recombinant strains expressing both alpha-amylase and glucoamylase.     

Thermal inactivation and reactivity of beta-glucosidase immobilized on chitosan-clay composite

The dried and wet chitosan-clay composite beads were prepared by mixing equal weights of cuttlebone chitosan and activated clay and then spraying drop-wise through a syringe, with and without freeze-drying, respectively. These beads were then immersed in 5 g/L of glutaraldehyde solution at a dosage of 0.5 g/L and were cross-linked, which were finally used as supports for beta-glucosidase immobilization. The properties of the enzyme immobilized on wet- and dried-composite beads were compared. Kinetic modeling of thermal inactivation of free and immobilized enzymes was also investigated. For a given enzymatic reaction, the rate constant related to the decomposition of the enzyme-substrate complex to final product and the uncomplexed enzyme using dried-composite immobilized enzyme was larger than those using both free and wet-composite immobilized enzymes.     

Some properties of free and immobilized alpha-amylase from Penicillium griseofulvum by solid state fermentation

Alpha-amylase was produced from Penicillium griseofulvum by an SSF technique. Alpha-amylase was immobilized on Celite by an adsorption method. Various parameters, such as effect of pH and temperature, substrate concentration, operational and storage stability, ability to hydrolyze starch and products of hydrolysis were investigated; these findings were compared with the free enzyme. The activity yield of immobilization was 87.6%. The optimum pH and temperature for both enzymes were 5.5 degrees C and 40 degrees C, respectively. The thermal, and the operational and storage stabilities of immobilized enzyme were better than that of the free enzyme. Km and Vmax were calculated from Lineweaver-Burk plots for both enzymes. Km values were 9.1 mg mL(-1) for free enzyme, and 7.1 mg mL(-1) for immobilized enzyme. The Vmax of the immobilized enzyme was approximately 40% smaller than that of the free enzyme. The hydrolysis ability of the free and immobilized enzyme were determined as 99.3% and 97.9%, respectively. Hydrolysis products of the a-amylase from P. griseofulvum were maltose, unidentified oligosaccharides, and glucose.