Product inhibition of the enzymatic hydrolysis of lactose

Kinetic experiments have been conducted with acetone-dried cells of Kluyveromyces fragilis to study product inhibition of the enzymatic hydrolysis of lactose. Both hydrolytic products, d-glucose and d-galactose, showed efficient inhibition effect on enzyme activity. The fact that d-glucose and d-galactose are mutually exclusive for the inhibition was verified by Dixon plots. The kinetic constants were also estimated using the experimental data. The rate equation was derived based on a multiple inhibition model of competitive inhibition of d-galactose and non-competitive inhibition of d-glucose. The good agreement between experiment and prediction indicated the validity of the established model.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.     

A calorimetric investigation of the binding of indole and phenylethane boronic acid to chymotrypsin

The heat of formation of the chymotrypsin-phenylethane boronic acid complex has been observed calorimetrically from pH 4 to 8 at 25 degrees C and is found to be pH-dependent, changing from near -6 kcal/mol at pH 4 to -13 kcal/mol at pH 8. The heat of formation of the chymotrypsin-indole complex is a nearly constant -6 kcal/mol over most of the same pH range. alpha-Chymotrypsin has been purified by pH gradient elution from an immobilized lima bean inhibitor column. Solutions of the enzyme up to 400 microM, prepared in this manner, have a zero heat of dilution from pH 5 to 8 in 0.1 M KCl, with or without added 0.05 M Tris, N-(tris[hydroxy-methyl]methyl-2-amino) ethanesulfonic acid, 4-morpholineethanesulfonic acid, or acetate buffers. Binding of phenylethane boronic acid causes a pH-dependent decrease in proton binding to chymotrypsin; the decrease in proton binding evoked by formation of the indole complex is much less, with a much smaller pH dependence. The calorimetric and proton-binding results are applied to a model for boronic acid binding (Hanai, K. (1976) J. Biochem. (Tokyo) 79, 107-116). We conclude that the thermodynamics of formation of the trigonal boronic acid complex are quite similar to those for the formation of the noncovalent complex formed by indole and related ligands. The trigonal-tetrahedral tautomerism in the boronic acid-chymotrypsin complex is characterized by thermodynamic changes similar to those accompanying the binding of virtual substrates to chymotrypsin.     

A calorimetric investigation of melting of tRNAAsp from brewer's yeast.

The thermodynamics of tRNAAsp unfolding was studied using a precision scanning microcalorimeter. The overall heat of melting was found to be about 55 J/g irrespective of the ionic strength and magnesium activity. The analysis of complex melting curves obtained in the absence of Mg2+ reveals four successive two-state transitions. The first was identified as the cooperative melting of the tertiary structure and the D region and the others as the melting of individual helical arms.     

AMP and IMP binding to glycogen phosphorylase b. A calorimetric and equilibrium dialysis study

Reaction microcalorimetry and equilibrium dialysis have been used to study the binding of AMP and IMP to glycogen phosphorylase b (EC 2.4.1.1) at 25 degrees C and pH 6.9. The combination of both techniques has enabled us to obtain some of the thermodynamic parameters for these binding processes. Four binding sites were found to be present in the dimeric active enzyme for both AMP and IMP. The binding to two high-affinity sites, which, in our opinion, correspond to the activator sites, seems to be cooperative. The two low-affinity sites, which would then correspond to the inhibitor sites, appear to be independent when the nucleotides bind to the enzyme. The negative delta G0 of binding/site at 25 degrees C is the result in all cases of a balance between negative enthalpy and entropy changes. The large differences in delta H and delta S0 for the binding of AMP to the activator sites (-27 and -70 kJ mol-1; -22 and -150 J X K-1 mol-1) suggest the existence of rather extensive conformational changes taking place in phosphorylase b on binding with the allosteric activator. Whereas the affinity of AMP for the activator sites is about 1 order of magnitude higher than that of IMP, the affinity of both nucleotides, including their delta H and delta S0 values, seems to be the same for the inhibitor sites.     

Anomerization and hydrolysis of lactose by beta-galactosidase from Saccharomyces lactis

Beta-Galactosidase from Saccharomyces lactis was found to be able to catalyze both the anomerization of alpha-lactose and the hydrolysis of beta-lactose; the rate of hydrolysis appeared to be four times higher with a 1:1 mixture of alpha and beta lactose than with a freshly prepared solution of alpha-lactose. The enzyme was also found to be unable to hydrolyze alpha-lactose. Thus, it appears that beta-galactosidase from S. lactis has its hydrolytic activity on lactose adapted only to the naturally more abundant beta-lactose.     

A calorimetric investigation of the growth of the luminescent bacteria Beneckea harveyi and Photobacterium leiognathi.

Direct calorimetric determinations of the rate of heat production along with simultaneous determinations of the rate of photon emission and the number of viable cells have provided insight into the growth of Beneckea harveyi and Photobacterium leiognathi. These experiments were performed with a Tronac isothermal microcalorimeter modified with a fiber optic light guide to allow in situ detection of light. Escherichia coli and a dark variant of P. leiognathi were also examined to provide points of reference. It is demonstrated that B. harveyi seems to pause in the rate of metabolic heat production at the same point in time that the enzyme luciferase begins to be synthesized. This effect is not removed if B. harveyi is grown in conditioned medium. The thermograms for all species are correlated with cell generation time. The heat production per cell indicates that uncrowded cultures produce more heat than older, more crowded cultures, supporting the original observation of Bayne-Jones and Rhees (1929). These observations reopen for examination the suggestion that living systems tend toward a state of minimum metabolism per unit mass.     

The kinetic of lactose hydrolysis for the beta-galactosidase from Aspergillus niger

The kinetics of glucose liberation from lactose by means of the beta-glactosidase from Aspergillus niger has been studied in a wide range of the main variables. The analysis shows that the kinetic models proposed so far are not adequate. The main finding is that the reaction rate is not linearly correlated to the enzyme concentration-it increase more than proportionally. This nonlinear relationship results because this lactase can distinguish between alpha-and beta-galactose alpha-Galactose acts as competitive and anticompetitive inhibitor while beta-galactose is a competitive one. The competitive inhibition of the alpha-anomer is approximately 12 times more sever than that of the beta-anomer. The kinetics, including a simplified model for the mutarotation of galactose is given for a temperature of 50 degrees C at a pH of 3.5-the most likely conditions for the application of this lactase in acid whey treatment.     

Glycoprotein-surfactant interactions: a calorimetric and spectroscopic investigation of the phytase-SDS system

The interactions of sodium dodecyl sulfate (SDS) and two glyco-variants of the enzyme phytase from Peniophora lycii were investigated. One variant (Phy) was heavily glycosylated while the other (dgPhy) was enzymatically deglycosylated. Effects at 24 degrees C of titrating SDS to Phy and dgPhy were studied by Isothermal Titration Calorimetry (ITC) and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. Comparisons of results for the two variants were used to elucidate glycan-surfactant interrelationships. The CD spectra suggested that both the native and the SDS-denatured states of the two variants were mutually similar, and hence that the denaturation process was structurally equivalent for the two glyco-variants. The denatured state was far from fully unfolded and probably retained a substantial content of native-like structure. Furthermore, it was found that the glycans brought about only a small increase in the resistance towards SDS induced denaturation. The SDS concentration required to denature half of the protein molecules differed less than 1 mM for the two variants. The affinity for SDS of both variants was unusually low. The amount of bound SDS (w/w) at different stages of the binding isotherm was 3-10 times lower than that reported for the most previously investigated globular proteins. Analysis of the relative affinity of the glycan and peptide moieties suggested that the carbohydrates bind much less surfactant. At saturation, glycans adsorbed about half as much SDS (in g/g) as the peptide moiety of Phy and about five times less than average proteins.     

A cytochemical kinetic investigation of the feulgen hydrolysis of fixed chromatin

Summary Measurements of the initial slope of the Feulgen hydrolysis curve under various conditions of preparation, temperature, and acid concentration demonstrate that this slope has the kinetic properties of the reaction rate of a simple hydrolytic reaction. This result allows a considerable refinement in both the kinetic analysis of chromatin fractions and the cytophotometric measurement of DNA amounts.     

Selection of yeast strains for lactose hydrolysis in dairy effluents

The ability of eight selected yeast strains growing in unsupplemented whey permeate to abate its lactose concentration has been studied. All strains reached 1 × 10⁸ colony forming units (cfu) ml⁻¹ by 20h of culture. Rates of lactose disappearance in permeate were poorly correlated with intracellular lactase levels. Maximal lactase activity levels were registered by Candida kefir NCYC 143 and Kluyveromyces marxianus NCYC 1548 cells, which reached 200 nmol ONP min⁻¹ 10⁻⁷ cells. Large fluctuations in enzymatic activity were observed during incubation, presumably as a result of the reciprocal effects between lactase activity and both galactose (0–1.9 mM) and lactose (0–143 mM) concentrations in the permeate.     

Ion exchange immobilization of changed beta-galactosidase fusions for lactose hydrolysis

The use of charged peptides fused to enzymes for immobilization onto ion-exchange membranes was explored for the enzyme x-galactosidase. The additional charged peptides, containing 1, 5, 11, and 16 aspartates, fused to x-galactosidase, for the most part did not interfere with the kinetic behavior for lactose hydrolysis. There was a 2-fold decline in V(m) for the 16-aspartate fusion, but the others were quite similar to the wild type enzyme (BGWT). BGWT and the fusions all retained approximately 50% of their activities when adsorbed onto ion-exchange membranes. In contrast to BGWT, the enhanced binding strength of the 11 aspartate fusion provided the ability to hydrolyze whey permeate at 0.3 M ionic strength without enzyme leakage, and to immobilize the enzyme directly from diluted cell extract with 83% purity. (c) 1994 John Wiley & Sons, Inc.     

A calorimetric investigation of the nutritive value of formic acid- and formaldehyde—Formic acid-treated grass silages

Experiments were conducted to study the utilisation of a ryegrass mixture preserved chemically by formic acid (silage 1) of mean gross energy (GE) 18.6 MJ/kg dry matter (DM), and formaldehyde—formic acid (silage 2) of mean GE 18.3 MJ/kg DM. The parent grass and resulting silages were analysed chemically and calorimetric balance trials were performed with four sheep fed at maintenance and 1.5 times maintenance.Silages 1 and 2 had similar, high digestibility coefficients for DM (0.732 and 0.718), organic matter (OM) (0.755 and 0.742), and energy (E) (0.730 and 0.710). The digestible energy (DE) contents of silages 1 and 2 were 13.3 and 12.7 MJ/kg DM, respectively, at the lower level of feeding and 13.8 and 12.8 MJ/kg DM, respectively, at the higher level. The metabolisable energy (ME) contents of silages 1 and 2 were 11.6 and 10.8 MJ/kg DM, respectively, at the lower level of feeding and 12.0 and 11.0 MJ/kg DM, respectively, at the higher level.The mean efficiency of utilisation of ME for growth (K_g) for silages 1 and 2 was 0.43 and 0.39, respectively; these results are in broad agreement with those predicted by other workers.     

A comparative spectroscopic and calorimetric investigation of the interaction of amsacrine with heme proteins,hemoglobin and myoglobin

The binding of the anilido aminoacridine derivative amsacrine with the heme proteins, hemoglobin, and myoglobin, was characterized by various spectroscopic and calorimetric methods. The binding affinity to hemoglobin was (1.21?±?.05) × 10⁵ M^?1, while that to myoglobin was three times higher (3.59?±?.15) × 10⁵ M^?1. The temperature-dependent fluorescence study confirmed the formation of ground-state complexes with both the proteins. The stronger binding to myoglobin was confirmed from both spectroscopic and calorimetric studies. The binding was exothermic in both cases at the three temperatures studied, and was favored by both enthalpy and entropy changes. Circular dichroism results, three-dimensional (3D) and synchronous fluorescence studies confirmed that the binding of amsacrine significantly changed the secondary structure of hemoglobin, while the change in the secondary structure of myoglobin was much less. New insights, in terms of structural and energetic aspects of the interaction of amsacrine with the heme proteins, presented here may help in understanding the structure-activity relationship, therapeutic efficacy, and drug design aspects of acridines.     

A new graphical method for determining parameters in Michaelis-Menten-type kinetics for enzymatic lactose hydrolysis

A new graphical method was developed to determine the kinetic parameters in the Michaelis-Menten-type equation. This method was then applied to studying the kinetics of lactose hydrolysis by Aspergillus niger beta-galactosidase. In this study, the reaction temperature ranged between 8 and 60 degrees C, and the initial lactose concentration ranged between 2.5 and 20%. A kinetic model similar to the conventional Michaelis-Menten equation with competitive product inhibition by galactose was tested using this graphical method as well as a nonlinear computer regression method. The experimental data and the model fit together fairly well at 50 degrees C. However, a relative large disparity was found for reactions at 30 degrees C. A three-parameter integrated model derived from the reversible reaction mechanism simulates the experimental data very well at all temperatures studied. However, this reversible reaction model does not follow the Arrhenius temperature dependence. Nevertheless, reaction rate constants for the proposed model involving the enzyme-galactose complex (in addition to the Michaelis complex) as an intermediate in lactose hydrolysis follow the Arrhenius temperature dependence fairly well, suggesting that this model can be best used for describing the enzymatic lactose hydrolysis. The lack of fit between the model predictions and data may be largely attributed to the effects of galactose mutarotation and oligosaccharide formation during lactose hydrolysis.     

Preparation of immobilized lactase. Continued studies on the enzymatic hydrolysis of lactose

A soluble fungal lactase (lactase-W) of greater activity that the previously available fungal lactase (lactase-M) has been covalently coupled to ZrO₂-coated porous glass particles and 1 mm diameter porous TiO₂ particles. The immobilized lactase-W appears to give results similar to the lactase-M except for the operational half-life. At 30°C the half-life of the lactase-M appears to exceed that of the lactase-W by approximately 100 days under operational conditions.