Variation In Enzyme Concentration Research Articles

The Effect of Enzyme Concentration and Hydrolysis Time on the Yield of Microcrystalline Cellulose from Rice Straw

The purpose of this study was to determine the effect of enzyme concentration and hydrolysis time on the yield of microcrystalline cellulose, and to determine whether the results of microcrystalline cellulose examination met the requirements of the British Pharmacopoeia and the United States Pharmacopoeia . The pretreatment process was carried out chemically using 96% alcohol, sodium hydroxide and hydrogen peroxide. The hydrolysis process was carried out enzymatically using the cellulase enzyme produced by the fungus Trichoderma viride , with variations in enzyme concentration of 5, 10 and 15% v/v for 0.5, 1 and 1.5 hours respectively. The highest yield of microcrystalline cellulose was produced from the hydrolysis process with a cellulase enzyme concentration of 5% v/v for 1.5 hours. Based on the two-way ANOVA test, it is known that the enzyme concentration and hydrolysis time have no significant effect on the yield of microcrystalline cellulose. Examination of organoleptic, identification, pH, water solubility, drying shrinkage, and starch absence test showed that the resulting microcrystalline cellulose complied with the requirements of the British Pharmacopoeia and the United States Pharmacopoeia.

KEBERADAAN BAKTERI ASAM LAKTAT PADA OLAHAN KECAP TETELAN TUNA (Thunnus sp) SELAMA PROSES FERMENTASI

Fish sauce is a food product made through a fermentation process from fish or fish waste. It has a distinctive taste and aroma as well as a long shelf life. Lactic acid bacteria activity is a crucial factor in the fermentation process. This study investigates the presence of lactic acid bacteria in processed tuna fish sauce (Thunnus sp) during the fermentation process. The research method used in this study is experimental, with a fermentation temperature of 50°C, various concentrations of papain enzyme treatment (A), including A1 (0%), A2 (9%), A3 (12%), and A4 (15%), different salt concentrations (B), including B1 (8%) and B2 (10%), and varying fermentation durations (C), including C0 (0 days), C1 (6 days), and C2 (12 days). The research results show that an increase in the total lactic acid bacteria (LAB) can lead to a more acidic fermentation condition, which inhibits the growth of other bacteria. Fish sauce with the addition of 15% papain enzyme hydrolyzes faster compared to the addition of 9% papain enzyme. This difference is due to variations in enzyme concentration, which can accelerate the hydrolysis process. Papain contains proteolytic enzymes that can break down proteins, softening the meat. The longer the fermentation process, the higher the growth of lactic acid bacteria, mainly due to the use of enzyme and salt concentrations.

Variations in Enzyme Concentrations During High-Temperature Composting of Livestock and Poultry Manure and Study on Enzymatic Reaction Mechanisms

Cow, pig, and chicken manure were collected from farms located in the proximity of Lanzhou University, Lanzhou City, Gansu Province, and were used as raw materials. Before composting, straws at a concentration of 10% were used as bulking agent. Different parameters were monitored during the composting process. Specifically, we analyzed changes in catalase, invertase, urease, and cellulase concentrations. In addition, the mechanisms of enzymatic reactions at high-temperatures were determined. The results showed that: (1) at the first stages of composting, the initial contents of catalase were 48.21±1.11, 45.04±0.85, and 44.29±0.65/0.002 mol·L−1·g−1, for cow, pig, and chicken manure, respectively. As composting proceeded, the content of catalase gradually increased. On the 6th day, catalase contents reached their maximum values, with numbers of 55.21±0.58, 57.28±0.14, and 62.18±0.45 mol·L−1·g−1, correspondingly. In addition, when composting ended, catalase content followed the order (chicken manure > cow manure > pig manure, with values of 34.33±0.58, 29.31±0.79, and 28.42±0.89/0.002 mol·L−1·g−1), in the same order; (2) According to the Michaelis-Menten expression, temperature greatly affects enzymatic reactions. Our data indicated that content of catalase, urease, sucrose, and cellulose, as well as number of microorganisms and compost temperature were positively correlated.

Surface functionalization of wool via microbial-transglutaminase and bentonite as bio-nano-mordant to achieve multi objective wool and improve dyeability with madder

Recently, natural dyes have a widening scope in various traditional and advanced applications due to their eco-friendly environment. However, improved dyeability of natural dyes still remains a challenging task. This research was aimed to achieve multi-objective wool with improved dyeability using bio-nano-mordant composed of m-Trans-glutaminase, m-TGase, and bentonite nanoclay. Wool fiber was treated through sonochemical method using different concentrations of m-TGase and bentonite. The surface morphology of wool fabric samples was examined by field emission-scanning electron microscopy (FESEM), and Fourier transform Infrared Radiation (FTIR). Further, wool samples treated at different conditions were applied to madder for dyeability examination. The optimum conditions of color coordinates, color strength, K/S, and washing fastness of madder on treated wool fabric with m-TGase and bentonite, were also examined. The results revealed well-made interactions among m-TGase, bentonite, and wool fibers. In addition, surface morphology was strongly influenced by variations in enzyme concentrations so that extra addition of m-TGase lead to clear damage scales or less cuticle surface in SEM images. Moreover, the results showed that the value of K/S for treated wool samples was better than untreated samples. Indeed, amongst all, 5% concentrations of bio-nano-mordant for m-TGase and bentonite have the most constructive K/S values. Similarly, results of ΔE and antibacterial investigations also confirmed its superiority.

Enzymatic flux capacities in hummingbird flight muscles: a “one size fits all” hypothesis

Hummingbirds (family Trochilidae) are among the smallest endothermic vertebrates representing an extreme, among birds, in their physiological design. They are unique in their ability to sustain hovering flight, one of the most energetically demanding forms of locomotion. Given that hovering metabolic rate (HMR) in hummingbirds scales allometrically as M0.78(M is mass), we tested the hypothesis that variation in HMR may be correlated with variation in maximal enzyme activities (Vmaxvalues) of key enzymes in glucose and fatty acid oxidation pathways in the flight muscles of four species of hummingbirds ranging in body mass from 4 to 20 g. We also estimated metabolic flux rates from respirometric data obtained during hovering flight. The data are striking in the lack of correlation between Vmaxvalues and flux rates at most steps in energy metabolism, particularly at the hexokinase and carnitine palmitoyltransferase reactions. In the context of hierarchical regulation analysis, this finding suggests that metabolic regulation (resulting from variation in substrate, product, or allosteric regulator concentrations) dominates as the proximate explanation for the interspecific variation in flux. On the other hand, we found no evidence of hierarchical regulation of flux, which results from variation in Vmaxand is based on variation in enzyme concentration [E]. The evolutionary conservation of pathways of energy metabolism suggests that “one size fits all” among hummingbirds.

Kinetic modeling and exploratory numerical simulation of chloroplastic starch degradation

BackgroundHigher plants and algae are able to fix atmospheric carbon dioxide through photosynthesis and store this fixed carbon in large quantities as starch, which can be hydrolyzed into sugars serving as feedstock for fermentation to biofuels and precursors. Rational engineering of carbon flow in plant cells requires a greater understanding of how starch breakdown fluxes respond to variations in enzyme concentrations, kinetic parameters, and metabolite concentrations. We have therefore developed and simulated a detailed kinetic ordinary differential equation model of the degradation pathways for starch synthesized in plants and green algae, which to our knowledge is the most complete such model reported to date.ResultsSimulation with 9 internal metabolites and 8 external metabolites, the concentrations of the latter fixed at reasonable biochemical values, leads to a single reference solution showing β-amylase activity to be the rate-limiting step in carbon flow from starch degradation. Additionally, the response coefficients for stromal glucose to the glucose transporter kcat and KM are substantial, whereas those for cytosolic glucose are not, consistent with a kinetic bottleneck due to transport. Response coefficient norms show stromal maltopentaose and cytosolic glucosylated arabinogalactan to be the most and least globally sensitive metabolites, respectively, and β-amylase kcat and KM for starch to be the kinetic parameters with the largest aggregate effect on metabolite concentrations as a whole. The latter kinetic parameters, together with those for glucose transport, have the greatest effect on stromal glucose, which is a precursor for biofuel synthetic pathways. Exploration of the steady-state solution space with respect to concentrations of 6 external metabolites and 8 dynamic metabolite concentrations show that stromal metabolism is strongly coupled to starch levels, and that transport between compartments serves to lower coupling between metabolic subsystems in different compartments.ConclusionsWe find that in the reference steady state, starch cleavage is the most significant determinant of carbon flux, with turnover of oligosaccharides playing a secondary role. Independence of stationary point with respect to initial dynamic variable values confirms a unique stationary point in the phase space of dynamically varying concentrations of the model network. Stromal maltooligosaccharide metabolism was highly coupled to the available starch concentration. From the most highly converged trajectories, distances between unique fixed points of phase spaces show that cytosolic maltose levels depend on the total concentrations of arabinogalactan and glucose present in the cytosol. In addition, cellular compartmentalization serves to dampen much, but not all, of the effects of one subnetwork on another, such that kinetic modeling of single compartments would likely capture most dynamics that are fast on the timescale of the transport reactions.

Efficient Elimination of Nonstoichiometric Enzyme Inhibitors from HTS Hit Lists

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

Sensitivity analysis and error structure of progress curves

Both the sensitivity of the monitored signal in progress curves to variations in enzyme concentration and the standard deviation of this signal were analyzed as a function of the proportion of transformed substrate. Three enzymes catalyzing essentially irreversible reactions were used as model systems: HIV-1 protease, glutathione reductase, and glutathione transferase. For all enzymes analyzed, the sensitivity was maximal when 60–80% of the substrate had been transformed. The standard deviation of reaction progress curve data replicates was also maximal at these substrate conversion levels, a result that was attributed to the influence of the sensitivity to random dispersion of the enzyme concentration. On this basis, we developed a model for the standard deviation of reaction progress curves that gave a good description of the experimental data and efficiently reduced the heteroscedasticity of residuals in a weighted fit of progress curves. This standard deviation model can be used for obtaining more efficient parameter estimates, to simulate noise in Monte Carlo procedures, and to delineate detection limits of enzyme inhibition. The transient increases in the sensitivity and in the standard deviation in progress curves are proposed to be features common to most enzymatic assays.

Roles of hierarchical and metabolic regulation in the allometric scaling of metabolism in Panamanian orchid bees

Assessment of the relative importance of variation in enzyme concentration [E] and metabolic regulation in accounting for interspecific variation in metabolic rates is an unrealized area of research. Towards this end, we used metabolic flux rates during hovering and enzymatic flux capacities (V(max) values, equal to [E]xk(cat), where k(cat) is catalytic efficiency) in flight muscles measured in vitro from 14 orchid bee species ranging in body mass from 47 to 1065 mg. Previous studies revealed that, across orchid bee species, wingbeat frequencies and metabolic rates decline in parallel with increasing body mass. V(max) values at some enzymatic steps in pathways of energy metabolism decline with increasing mass while, at most other steps, V(max) values are mass-independent. We quantified the relative importance of ;hierarchical regulation' (alteration in V(max), indicative of alteration in [E]) and ;metabolic regulation' (resulting from variation in substrate, product or modulator concentrations) in accounting for interspecific variation in flux across species. In addition, we applied the method of phylogenetically independent contrasts to remove the potentially confounding effects of phylogenetic relationships among species. In the evolution of orchid bees, hierarchical regulation completely accounts for allometric variation in flux rates at the hexokinase step while, at other reactions, variation in flux is completely accounted for by metabolic regulation. The predominant role played by metabolic regulation is examined at the phosphoglucoisomerase step using the Haldane relationship. We find that extremely small variation in the concentration ratio of [product]/[substrate] is enough to cause the observed interspecific variation in net flux at this reaction in glycolysis.

Is the regulation of galactose 1-phosphate tuned against gene expression noise?

The average number of mRNA molecules per active gene in yeast can be remarkably low. Consequently, the relative number of copies of each transcript per cell can vary greatly from moment to moment. When these transcripts are encoding metabolic enzymes, how do the resulting variations in enzyme concentrations affect the regulation of metabolic intermediates? Using a kinetic model of galactose utilization in yeast, we analysed the transmission of noise from transcription and translation on metabolic intermediate regulation. In particular, the effect of the kinetic properties of the galactose-1-phosphate uridylyltransferase reaction on the transmission of noise was analysed.

Fuel use in hawkmoth (Amphion floridensis) flight muscle: enzyme activities and flux rates.

The fuels used by the hawkmoth Amphion floridensis to power flight are determined by nectar-feeding, with fed moths using primarily carbohydrate and unfed moths using primarily fat. To investigate the metabolic pathways underlying fuel-use flexibility in this species, we measured the maximal activities of several key metabolic enzymes in the flight muscle of fed and unfed individuals, for which metabolic rates and fuel utilization had been previously determined. Hexokinase (HK) and phosphofructokinase (PFK) occur at high activities and, during carbohydrate-fueled flight, are estimated to operate at fractional velocities comparable to those of exclusively carbohydrate-utilizing insects. Females exhibited higher glycolytic enzyme activities than did males, and males regulated PFK activity according to nectar feeding. Although beta-hydroxyacyl-CoA dehydrogenase (HOAD) was found at high activities, carnitine palmitoyl transferase (CPT) was not detectable, suggesting that fatty acids may be utilized via a carnitine-independent pathway during flight. Principal component analysis revealed a tendency for the activities of citrate synthase, HK, PFK, and HOAD to be positively correlated among individuals, as well as a lesser tendency for the activities of glycolytic vs. mitochondrial enzymes to be negatively correlated with each other. However, the principal components did not correlate with variation in either oxygen consumption rate or fuel use in vivo, suggesting that variation in enzyme concentration did not determine differences among individuals in metabolic performance during flight. J. Exp. Zool. 290:108-114, 2001.

Expression of cytochrome P4502E1 in human liver: assessment by mRNA, genotype and phenotype.

Cytochrome P4502E1 (CYP2E1) is constitutively expressed in human liver and is responsible for the metabolic bioactivation of a wide variety of xenobiotics, including a number of protoxins and procarcinogens. CYP2E1 expression is regulated at several levels including pre-transcriptional, transcriptional and post-transcriptional levels, and any variation in enzyme concentration and hence activity may represent increased risk of toxicity or carcinogenicity. We have investigated variability in the levels of CYP2E1 mRNA, protein and functional activity in a human liver bank, and attempted to relate these parameters to the RsaI restriction fragment length polymorphism in the 5'-flanking region. Variation in CYP2E1 mRNA (18-fold) was greater than the variation seen in CYP2E1 protein (twofold) and functional activity (fourfold) determined using two probe substrates, chlorzoxazone and p-nitrophenol. Although protein and functional activity showed a significant correlation (r = 0.93 and r = 0.83 for chlorzoxazone and p-nitrophenol, respectively), there was no correlation between any of these parameters and mRNA levels. Also, the variation in CYP2E1 activity could not be directly accounted for by the RsaI polymorphism in our samples. In conclusion, our results are consistent with a complex regulation of CYP2E1 and the fact that it is highly conserved in the human population. The absence of a relationship between the RsaI polymorphism and CYP2E1 activity is consistent with other studies performed in Caucasians, but does not exclude an effect of this polymorphism on inducibility of CYP2E1.

Annual and Diurnal Variations in, and Effects of Altered Photoperiod and Temperature, Ovariectomy, and Estradiol-17 β Replacement on Catechol-O-Methyltransferase Level in Brain Regions of the Catfish, Heteropneustes fossilis

The annual data show that catechol-O-methyltransferase (COMT) content increases with the progress of ovarian recrudescence in all the brain regions (telencephalon, hypothalamus, thalamus + tegmentum, and medulla oblongata) and declines after spawning to low values in quiescent phase. Diurnal variation in enzyme concentration with peak values at 24 hr was noticed in the hypothalamus throughout the reproductive cycle; in other brain regions, it was observed only in February and March. The stimulatory response of the enzyme to photoperiod and temperature alterations was differential and region-specific; an effect of photoperiod change was noticed only in the hypothalamus and was less in magnitude compared to the temperature effect. The response of the enzyme to ovariectomy (OVX) and E 2 supplementation was region-specific (hypothalamus only), season-specific (prespawning phase), and varied:inhibitory at 3 and 4 weeks, stimulatory at 6-week of OVX and after 0.05, 0.1, 5, and 10 μg/g doses of E 2, and none at week 2 and 5 of OVX and after 0.5 and 1 μg/g doses of E 2. The season-specific changes in hypothalamic COMT may be indicative of its involvement in catecholamine (and possibly catecholestrogen)-mediated neuroendocrine control of gonadotropin.

Phylogenetic Analysis of Glycolytic Enzyme Expression

Although differences among species in enzyme maximal activity or concentration are often interpreted as adaptive and important for regulating metabolism, these differences may simply reflect phylogenetic divergence. Phylogenetic analysis of the expression of the glycolytic enzymes among 15 taxa of a North American fish genus (Fundulus) indicated that most variation in enzyme concentration is due to evolutionary distance and may be nonadaptive. However, three enzymes' maximal activities covary with environmental temperature and have adaptive value. Additionally, two pairs of enzymes covary, indicating coevolution. Thus, metabolic flux may be modulated by many different enzymes rather than by a single rate-limiting enzyme.

Detection of cathepsin B- and L-, elastase-, tryptase-, trypsin-, and dipeptidyl peptidase IV-like activities in crevicular fluid from gingivitis and periodontitis patients with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin.

Crevicular fluid samples were collected from 20 gingivitis and periodontitis patients using filter paper strips; these were then eluted into buffer. Portions of each sample were combined and the activities of this pooled eluate against different peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin (AFC) were examined with respect to their pH profiles and effector responses. Ca-thepsin B- and L-like activity was detected with Bz-Val-Lys-Lys-Arg-AFC; elastase-like activity with MeOSuc-Ala-Ala-Pro-Val-AFC; tryptase-like activity with Z-Ala-Ala-Lys-AFC; trypsin-like activity with Z-Gly-Gly-Arg-AFC; and dipeptidyl peptidase (DPP) IV-like activity with Ala-Pro-AFC. The selectivity and sensitivity of these assays were improved by choice of appropriate conditions. The cathepsin B- and L-, elastase-, tryptase-, and trypsin-like activities all had properties consistent with those from host sources, whilst partial inactivation of the DPP IV-like activity by heat treatment (60 degrees C for 30 min) suggested that it may have represented a mixture of human and Bacteroides gingivalis enzymes. Individual patient eluates showed wide variations in enzyme concentrations, but generally elastase-like activity was by far the highest. The sensitivity of the assays with AFC-linked substrates was such that it should prove possible to measure all five different types of activity in crevicular fluid samples from local periodontal disease sites.

The matrix method of metabolic control analysis: its validity for complex pathway structures

The sensitivities of the variables of a metabolic system (such as fluxes and concentrations) to variations in enzyme concentration are expressed in metabolic control analysis as control coefficients. The matrix method is a system of writing matrix equations that generate expressions for the control coefficients in terms of the characteristics of the components (principally the enzymes). Previously, the matrix method has been considered in terms of simple pathway structures; here we justify its applicability to complex pathways, such as those with multiple branches. It is shown that this requires modification of the branch point relationship to take account of changes of flux along the limbs of the branch and of stoichiometric factors. The method of deriving the flux control coefficients with respect to different fluxes in the system is extended to cope with these circumstances.

Regulation of enzyme levels by proteolysis: The role of pest regions

Enzymes can be regulated in a variety of ways. Readily reversible mechanisms, such as phosphorylation, are frequently used by cells to control metabolic pathways. Less often, enzyme levels are regulated by changing the rate at which the protein is destroyed. Although these changes, too, are reversible through protein synthesis, large variations in enzyme concentration can be produced in very short periods of time by combinations of transcriptional control, translational control and rapid degradation. We recently examined the primary sequences of proteins whose intracellular half-lives are less than two hours. With a single exception, each short-lived protein contains one or more regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). These PEST regions range in length from 12 to 60 residues, and they are often flanked by possibly charged amino acids. Similar inspection of 35 more stable, structurally characterized proteins revealed only three weak PEST regions. All PEST proteins appear to be important regulatory molecules, and their fast turnover surely reflects a metabolic requirement for rapid changes in their concentrations. Known PEST proteins include oncogene products, key enzymes and components of signal pathways. In addition, there are a number of PEST-containing proteins that are suspected of being rapidly degraded. These proteins include Drosophila homeotic proteins (e.g., notch, snake, caudal, ftz and even-skipped) and a host of yeast cdc mutants. PEST regions, which target the molecules containing them for destruction, thus appear to be widely distributed among metabolically unstable proteins.

The development and application of a radioimmunoassay for rat phosphoenolpyruvate carboxykinase.

We employed a newly developed radioimmunoassay to quantitate P-enolpyruvate carboxykinase protein directly in liver and kidney of intact rats. The radioimmunoassay was dependent upon the presence of sodium dodecyl sulfate in the competitive binding reaction mixture. In conjunction with assayable activity measurements, the radioimmunoassay results also made it possible to assess the average specific activity of the enzyme. In the fed state, liver P-enolpyruvate carboxykinase was 0.89 microM (micromoles/kg of tissue) and the kidney enzyme was 1.90 microM. Following a 48-h fast, the enzyme in liver increased to 2.83 microM and that in kidney to 6.83 microM. Although liver enzyme concentration increased 3-fold, total liver enzymic activity was increased only about 30%. The discrepancy was due to the decrease in liver weight which occurred during fasting. The kidneys, which do not lose weight during fasting, had a 65% elevation of total organ activity. Total organ enzyme activity was predominantly dependent on changes in enzyme mass. In the fed state, the kidney/liver enzyme mass ratio was 0.44, in the fasted state it was 0.68. Variation in enzyme concentration, enzyme half-life, and response to tryptophan and chronic triamcinolone administration all gave evidence for organ-specific regulation.

Denaturation and renaturation of bovine liver glutamic dehydrogenase after dissociation in various denaturants.

Abstract Oligomeric glutamic dehydrogenase from bovine liver is dissociated to inactive monomers (Mr = 56 000) under a wide variety of conditions: 3 ≥ pH ≥ 12, 6 ᴍ guanidine · HCl, 6 ᴍ urea, 0.2% sodium dodecylsulfate. High hydrostatic pressure (< 1 kbar) only affects the association equilibrium of the native hexamer to higher polymers. The respective reaction volume (ΔV=28 ± 5 ml · mol-1 at 298 K, 1 bar) is linearly dependent on temperature and pressure. At p > 1.5 kbar dissociation of the hexamer occurs; this reaction is accompanied by irreversible deactivation. Depending on the denaturant applied for the monomerization, the final conformational stale of the polypeptide chain differs widely regarding its residual structure. As taken from laser light scattering measurements the rate of dissociation at pH 1.8 follows first order kinetics with a rate constant k1 = 0.42 ± 0.06 s-1. In the range of the oligomer ⇌ monomer transition, dissociation is accompanied by irreversible aggregation leading to inactive high molecular weight material. At low concentration (c < 5 µg/ml) this side reaction can be slowed down, so that the reconstitution of the enzyme can be monitored using spectroscopic techniques. Concentration dependent stopped-flow experiments prove the regain of fluorescence to be a rapid first order process; the respective half-times at pH 7.4 are τ1/2 = 2.0 ± 0.5 ms and 0.7 ± 0.2 ms for the “renaturation” from 6 ᴍ guanidine · HCl, pH 6, and pH ~ 2, respectively. The product of reconstitution shows the fluorescence and circular dichroism pattern characteristic for the native enzyme. However, no reactivation can be achieved under any of the following conditions: optimum protection against chemical modification; variation of enzyme concentration, temperature, and hydrostatic pressure; addition of specific ligands such as coenzymes, substrates, ADP, membrane constituents (cardiolipin, electron transfer particles ETPH). Obviously, the “renaturation” (D → N) of glutamic dehydrogenase is governed by a side reaction according to N → D ⇌ R → A which causes aggregation of intermediates R instead of reconstitution of the native enzyme.

Reactivation of Alkaline Phosphatase in Ultra High-Temperature, Short-Time Processed Liquid Milk Products

Alkaline phosphatase (enzyme) reactivation was studied in liquid milk products heated to 87.8 to 121.1 C for less than 1 s in a continuous, two-phase, slug-flow heat exchanger. The effects of magnesium concentration, pH, incubation temperature, homogenization pressure, processing temperature, fat content, and initial enzyme concentration were investigated. Jersey milk from one farm showed seasonal variations in enzyme concentration and its reactivation behavior. Increased reactivation in products with high fat content was due to high initial enzyme concentration in the product. Homogenization of products before heating decreased reactivation. Maximum reactivation occurred in products heated to 104.4 C, incubated at 34 C, and adjusted to pH 6.5. Maximum velocity of reactivation and reactivation constant varied with milk samples. Activation energy for the control and samples with magnesium was 22.646 +/- .118 and 24.100 +/- .210 kJ mole-1, respectively. The enzyme from raw and reactivated cream contained two major isozymes, and the reactivated isozyme differed from the control. The official method for differentiating residual and reactivated enzymes was modified in terms of magnesium concentration and extent of reactivation of the enzyme in the reactivated product.