Enzyme deactivation during cellulose hydrolysis

Hydrolysis of cellulose by Trichoderma viride cellulase reached a plateau after some 25 hr. If the initial enzyme-to-substrate ratio was low, resuspension of substrate in fresh enzyme or addition of enzyme resulted in further high rate hydrolysis. This did not occur if the initial ratio was high. Over 75% hydrolysis might be achieved in the former case, while less than 60% in the latter. A model postulating inactivation of adsorbed enzyme–substrate complex which blocked further hydrolysis was proposed, and it was found to fit the data well. The proposed model had five parameters, four of which could be checked by graphical methods, and all of which had physical meanings. The parameters were estimated by a nonlinear least-squares minimization FORTRAN computer program, using numerical integration and optimization of the parameters. The model was used to predict the resuspension data, powdered enzyme addition data, cellobiose addition data, and cellulose addition data; the deviations from the model are discussed. It was found that average values could be used for four out of the five parameters, while the fifth (initial enzyme concentration) did not correlate with independent measurements such as the filter paper activity or protein concentration.     

Enzyme stabilization towards thermal,chemical and proteolytic deactivation

An enzyme stabilization technique which consists of entrapping protein within a polymeric network has been discussed. The high macromolecular concentration levels which lead to formation of the network are produced as a consequence of polarization phenomena which take place within an unstirred ultrafiltration membrane reactor. Increases in enzyme half-life were generally produced in connection with simple and complex deactivation phenomena of widely different natures (thermal, chemical and proteolytic). Experimental tests have been carried out on the following enzymes: β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21), β-d-fructofuranosidase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] and β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23).     

Enzyme deactivation in fixed bed reactors with michaelis-menten kinetics

Rate expression for enzyme poisoning which are consistent with a Michaelis-Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme. When enzyme deactivation results from the irreversible bonding of a product molecule to an existing substrate-enzyme complex, it is shown that minimum enzyme activity can occur in the interior of the bed, well away from the ends. This suggests that bed sectioning techniques may enable direct evaluation of fundamental poisoning mechanisms.     

Immobilization of tyrosinase for use in nonaqueous media: Enzyme deactivation phenomena

Summary We have investigated features for minimizing the inactivation of tyrosinase (E.C. 1.14.18.1) that is caused by immobilization on glass beads and Celite^®. The degree of inactivation is dependent on the enzyme loading and the carrier's surface area. Addition of a sacrificial protein during the immobilization procedure offers a protective effect with increased residual activity on the basis of comparable enzyme loading.     

Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt-beta-lactamase catalyzed hydrolysis of beta-lactams

Metallo-beta-lactamases are native zinc enzymes that catalyze the hydrolysis of beta-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted beta-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pK(a) = 8.9 +/- 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H(2)O/D(2)O) of 5.2 +/- 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt beta-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pK(a) 6.32 +/- 0.1 and 7.47 +/- 0.1 being in their deprotonated basic forms and one group of pK(a) 9.48 +/- 0.1 being in its protonated form.     

Airway mechanoreceptor deactivation.

Airway sensors play an important role in control of breathing. Recently, it was found that pulmonary slowly adapting stretch receptors (SARs) cease after a brief excitation following sodium pump blockade by ouabain. This deactivation can be explained by overexcitation. If this is true, mechanical stimulation of the SARs should also lead to a deactivation. In this study, we recorded unit activity of the SARs in anesthetized, open-chest, and mechanically ventilated rabbits and examined their responses to lung inflation at different constant pressures. Forty-seven of 137 units had a clear deactivation during the lung inflation. The deactivation threshold varied from unit to unit. For a given unit, the higher the inflation pressure, the sooner the deactivation occurs. For example, the SARs deactivated at 3.0 +/- 0.3 and 4.8 +/- 0.4 s when the lungs were inflated to constant pressures of 30 and 20 cmH(2)O, respectively (n = 25, P < 0.0001). The units usually ceased after a brief intense discharge. In some units, their activity shifted to a lower level, indicating a pacemaker switching. Our results support the notion that SARs deactivate due to overexcitation.     

pH-Dependent enzyme deactivation models

A pH-dependent "series-type" enzyme deactivation model using rapid protonation and deprotonation equilibria and the relatively slower inactivation rates is presented. From the enzyme activity-time trajectories at different pH the models presented permit the evaluation of some of the protonation and inactivation rate constants as well as the specific activities of the different enzyme forms. pH dependence of enzyme deactivations may also exhibit deactivation disguised kinetics. Three different examples of pH-dependent enzyme deactivations available in the literature are appropriately modeled to indicate the general applicability of the model. The model presented is consistent with the data and provides mechanistic insights into the pH-dependent deactivation of different enzymes.     

Product deactivation in recombinant Streptomyces

The production of tyrosinase by Streptomyces antibioticus (p1J7O2) was investigated as a model system for recombinant protein production by Streptomyces. Product deactivation was found to have a severe effect on the levels of tyrosinase obtained. Tyrosinase deactivation was detected during all phases of batch cultures, with higher specific deactivation rates observed during the stationary phase. The specific deactivation rate exhibited an Arrhenius dependence on temperature, with approximately a twofold increase in the deactivation rate between 25 degrees C and 30 degrees C. The effect of deactivation on the determination of tyrosinase production kinetics is discussed. A strategy was implemented to increase tyrosinase productivity by enriching the growth medium and reducing the culture temperature during the period of maximum tyrosinase production. This strategy resulted in a shorter culture time and a 2.5-fold increase in tyrosinase activity compared to a culture grown at 25 degrees C using a standard growth medium.     

Microheterogeneity of enzymes and deactivation

The influence of microheterogeneity on enzyme inactivation kinetics is presented. Examples of different enzymes are given where microheterogeneity has been detected by different techniques. The different statistical models are presented which include the influence of microheterogeneity on enzyme inactivation kinetics and stability. As the microheterogeneity of the enzyme increases, there is a sharper decline in the normalized activity during the initial stages of the deactivation but a greater stability and activity, compared to similar homogeneous enzyme, as the deactivation proceeds. Microheterogeneity makes the deactivation reaction have a higher apparent order of reaction. The implications of microheterogeneity on enzyme inactivations are high lighted by different examples. The analysis provides fresh physical insights into the chemistry, subpopulations, structure, and function of enzymes.     

Resistance to deactivation of enzyme-collagen membranes

Amongst the attractive properties of immobilized enzymes, an enhanced stability is very often underlined. In our case, the covalent attachment of numerous enzymes from different classes to water-insoluble collagen films allowed us to study their resistance to inactivation or denaturation after coupling. The influence of heat, denaturing reagents like concentrated urea or guanidinium chloride, the incubation in the presence of glutaraldehyde, have been tested on aspartate amino-transferase either in soluble form or bound on collagen films. The fact that diffusional effects can lead to an apparent enhancement of stability after immobilization has been taken into account and their influence studied for both thermal and storage stability : diffusional limitations are partly responsible for the enhanced stability of the bound enzyme but the binding to the collagen membrane itself increases its storage stability. The resistance of proteolytic enzymes to autolysis has also been checked.     

Single-step unimolecular non-first-order enzyme deactivation kinetics

A two-parameter deactivation model is proposed to describe the kinetics of activity stabilization for some enzymes. The single-step unimolecular mechanism exhibits non-first-order deactivation kinetics since the final enzyme state, E(1) is not completely inactivated. The usefulness of the model is demonstrated by applying it to the inactivation of different enzymes. The influence of the concentration of active ester, ionic strength, and pH on the model parameters is examined during the inactivation of electric eel acetylcholinesterase.(25) In general, inactivators would decrease the level of activity stabilization, alpha(1), and increase the first-order inactivation rate constant, k(1). The effect of protecting agents would be to increase alpha(1) and to decrease k(1).     

Peroxidase-dependent deactivation of prostacyclin synthetase.

A study of the enzymes of the arachidonic acid cascade revealed a high sensitivity of prostacyclin synthetase and a complete resistance of thromboxane A2 synthetase to time-dependent destruction by an oxidant [Ox] released during the peroxidase-catalyzed reduction of hydroperoxy fatty acids. The destructive action of [Ox] derived from prostaglandin G1 (PGG1), 15-hydroperoxy-PGE1, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid upon prostacyclin synthetase was prevented by 2-aminomethyl-4-t-butyl-6-iodophenol. On the other hand, deactivation resulting from PGG2 metabolism was neither time-dependent nor sensitive to 2-aminomethyl-4-t-butyl-6-iodophenol. The possibility that the action of [Ox] may alter the arachidonic acid cascade in favor of thromboxane A2 is discussed in view of its possible implications in inflammatory and other pathological processes.     

Cellulase deactivation in a stirred reactor

During the production or downstream processing of an enzyme it is always subjected to shear stress, which may deactivate the enzyme. This susceptibility of enzymes to shear stress is a major concern as it leads to the loss of enzyme activity and is, therefore, a major consideration in the design of the processes involving enzyme production and its application.In the present work the cellulase enzyme was subjected to shear stress in a stirred reactor with an objective of investigating its deactivation under various conditions such as different agitation speeds, concentrations of enzyme, concentrations of buffer, pH ranges, buffer systems and the presence of gas–liquid interface. It was found that the extent of deactivation depends upon the conditions under which the enzyme was subjected to shear.     

Cas9 deactivation with photocleavable guide RNAs

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Studies on the deactivation of immobilized urease

The results of experiments in a fixed-bed reactor and a CSTR containing urease immobilized on a nonporous support and conducted in the absence of diffusional limitations are reported. Kinetic parameters were established by separate batch experiments. The key observation was that the product ammonia attacked the free form of the enzyme and thereby illustrates the importance of mechanism in determining deactivation kinetics.