Characterization of Trametes versicolor laccase for the transformation of aqueous phenol

Laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor was thoroughly characterized in terms of its catalytic stability and its effectiveness as a biocatalyst under various reaction conditions when using phenol as a model substrate. This enzyme demonstrated high or moderate degrees of stability at pHs from 5 to 8 at 25 degrees C and at temperatures from 10 to 30 degrees C at pH 6. Exponential decay expressions were successfully used to model laccase inactivation when incubated under various conditions of pH and temperature. Phenol transformation was optimum at pH 6, but significant transformation was observed over a pH range of 4-7, provided that sufficient laccase was present in the reacting solution. Partial inactivation of laccase was observed during the oxidation of phenol, even under conditions of optimal stability (pH 6 and 25 degrees C).     

Kinetic model of laccase-catalyzed oxidation of aqueous phenol

Laccase from Trametes versicolor (EC 1.10.3.2) catalyzes the oxidation of aqueous phenol by oxygen and has demonstrated good potential for applications in various industrial and environmental processes. A kinetic model of this system has been developed to facilitate a better understanding of the mechanisms and rate-limiting steps of enzyme-catalyzed transformation and to eventually assist in the choice and design of suitable reactor systems. A kinetic model was derived based on the differential and mass balance equations that describe the interactions of various forms of the enzyme with the aromatic substrate and oxygen. This model also incorporated an expression accounting for enzyme inactivation over time due to the reaction environment. The model was validated by comparing model predictions with experimental observations of phenol transformation and oxygen consumption over time at a variety of enzyme concentrations. Excellent agreement was found between experimental data and predictions of the kinetic model. Sensitivity analyses demonstrated that the reaction between oxidized-laccase and phenol was the rate-limiting step.     

Variable stoichiometry during the laccase-catalyzed oxidation of aqueous phenol

The oxidation of aqueous phenol through the catalytic action of laccase from Trametes versicolor was studied over a wide range of phenol concentrations and enzyme activities. The stoichiometric ratio, which is defined as the molar ratio of phenol transformed to oxygen consumed in the catalytic reaction, was found to increase with phenol concentration in the reaction mixture from a theoretical lower limit of 1 and to approach a theoretical upper limit of 4. A logistic equation was proposed to relate reaction stoichiometry to substrate concentration and was successfully used to relate these parameters over a range of phenol concentrations extending from approximately 0.15 to 8 mM. This expression was incorporated into two kinetic models in order to account for variations in reaction stoichiometry during the reaction and to extend the range over which the models may be accurately applied. The new models demonstrated an improved ability to predict concentrations of phenol and oxygen over time in a closed batch reaction system.     

Modeling of a Catalyzed Reaction Using Lipase Immobilized in a Poly(vinyl alcohol) Membrane

A mathematical model for the hydrolysis reaction of p‐nitro phenol laurate catalyzed by a lipase immobilized in a membrane was developed. In an earlier study this model reaction was found to show very different reaction rates when it was performed in aqueous micellar solution with free enzyme and with membrane immobilized enzyme. It was assumed that a local accumulation of substrate in the membrane is responsible for the observed rate enhancement. The conversion of p‐nitro phenol ester within the membrane was modeled by considering a combination of the convective flow through poly(vinyl alcohol) membrane pores, concentration polarization of substrate containing micelles at the membrane surface and the kinetics of the reaction with free enzymes. It was demonstrated that the model offered a comprehensive understanding of the interaction of the involved phenomena. The modeling results are in good agreement with the experimental data from 10 runs with different enzyme and substrate concentrations. The substrate concentration at the membrane surface increased by up to a factor of 3 compared to the feed concentration. This effect explains the observed rate enhancement. Moreover, the model was used to determine the unknown parameters, i.e., the intrinsic retention and the mass transfer coefficient, by fitting the model to the experimental data. The model may also be used to calculate the optimum operating conditions and design parameters of such a reactor.     

Peculiarities of direct bioelectrocatalysis by laccase in aqueous-nonaqueous mixtures

The effect of concentration of ethanol and dimethyl sulfoxide on the catalytic activity of laccase is studied for the enzymatic reaction of catechol oxidation and bioelectrocatalytic reaction of oxygen reduction under the conditions of direct electron transfer. Laccase-Nafion composite is elaborated ensuring the enzyme stability in a wide potential range and a content of organic solvents. Based on the STM measurements, the structure of composite layer is proposed. It is shown that the mechanism of oxygen reduction reaction by laccase in organo-aqueous mixtures is similar to that earlier proposed for aqueous solutions. A decrease in the electrocatalytic activity of laccase in the oxygen reduction correlates with a decrease in the laccase enzymatic activity in the substrate oxidation. However, a decrease in the laccase activity in the composite is observed at a higher content of organic solvent in the mixture. The mechanism of laccase inactivation by organic solvents is proposed.     

Apparent zero-order kinetics of phenol biodegradation by substrate-inhibited microbes at low substrate concentrations

The reaction kinetics for phenol biodegradation at low substrate concentrations can be estimated based on the analysis of changes in the dissolved oxygen concentration in the bulk liquid during biodegradation. The measured oxygen concentration changes with an interesting behavior as biodegradation proceeds. The oxygen concentration in the bulk liquid decreases rapidly in the early stages of degradation and subsequently decreases linearly and then rapidly recovers to the initial saturated level. Taking into account the oxygen transfer rate between gas and liquid phases and oxygen consumption rate by microbes, the change in the dissolved oxygen concentration can be simulated with an unsteady state mass balance equation and three kinetic models for the rate of phenol metabolism: a substrate-inhibited model; a zero-order model; and a combined model. In the combined model, it is assumed that, at phenol concentrations above 10 mg/L, the degradation rate is expressed by a substrate-inhibited model; whereas at concentrations below 10 mg/L the zero-order model is applied. It was found that the characteristics of the change in the dissolved oxygen concentration, especially the rapid increase at the end of degradation, can only be described by the combined kinetic model. This result suggests that conventional Haldane-type kinetics would be unsuitable for estimating the phenol consumption rate at low phenol concentrations, in particular, at concentrations less than 10 mg/L. (c) 1996 John Wiley & Sons, Inc.     

Effect of ionic liquids activation on laccase from Trametes versicolor: Enzymatic stability and activity

The stability and activity of laccase from Trametes versicolor in two water‐soluble ionic liquids (ILs), namely 1‐butyl‐3‐methylimidazolium methyl sulfate, [bmim][MeSO₄] and 1,3‐dimethylimidazolium methyl sulfate, [mmim][MeSO₄], were investigated in this study. Thermal inactivation of laccase was characterized in the presence of these both ILs and as expected first‐order kinetics was followed. Inactivation rate constant (k), half‐life time (t_1/2), and energy of activation (Ea) were determined. Kinetics of 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) oxidation by laccase in the presence of these ILs was studied and Michaelis–Menten parameters were calculated. There is no enzymatic inactivation since the maximum reaction rate remained constant for IL concentrations up to 25%, and surprisingly, it was found that laccase was activated for concentrations of 35% of ILs, since the reaction rate increased 1.7 times.     

Microfabricated arrays of cylindrical wells facilitate single‐molecule enzymology of α‐chymotrypsin

Single‐molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single‐molecule enzymatic kinetics and provide comparisons to ensemble‐averaged kinetics. To isolate our model enzyme, α‐chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 μm in diameter and 1.35 μm in height. Inside the wells, a protease assay with a profluorescent substrate detects α‐chymotrypsin activity. We hold the concentration of α‐chymotrypsin at 0.39 nM in a given well with an enzyme‐to‐substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge‐coupled device. This method allows for the simultaneous real‐time characterization of hundreds of individual enzymes. We analyze single‐molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Growth kinetics of Pseudomonas putida in cometabolism of phenol and 4-chlorophenol in the presence of a conventional carbon source

Growth kinetics of Pseudomonas putida (ATCC 49451) in cometabolism of phenol and 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) were studied. In the ternary substrate mixture, phenol and SG are growth substrates while 4-cp is a nongrowth substrate. Cell growth on phenol was found to follow Andrews kinetics and cells displayed substrate inhibition pattern on sodium glutamate in the range of 0-4 g L(-1) as well. A cell growth model for the ternary substrate system was established based on a simplified cell growth mechanism and subsequently modified by experimental results. Model analysis over a wide range of substrate concentrations shows that the inhibition of SG is much larger than phenol at low phenol concentrations (/=600 mg L(-1)). The nongrowth substrate, 4-cp, inhibits cell growth mainly through inactivation of cells (cell decay) and competitive inhibition to cell growth on phenol. In the absence of SG, 4-cp retards cell growth severely and cells cannot grow at 250 mg L(-1) 4-cp. Addition of sodium glutamate, however, greatly attenuates the toxicity of 4-cp and supports cell growth at 4-cp concentration higher than 250 mg L(-1). By using the proposed cell growth model, we were able to optimize the amount of SG needed to enhance cell growth rate and validate model predictions against experimental data.     

Kinetics of the hydrolysis of lean meat protein by alcalase: Derivation of two alternative rate equations and their fit to experimental data

Two rate equations have been developed to model the hydrolysis of ground lean meat protein by Alcalase. The first equation was based on classical Michaelis-Menten kinetics and the second on the adsorption of enzyme to the protein prior to reaction. It was assumed that this adsorption could be modelled by a Langmuir-type adsorption isotherm. Each equation considered the enzyme to be competitively inhibited by reaction product, and considered enzyme inactivation to be first order. Both rate equations have been fitted to experimental data obtained from the hydrolysis of meat protein by Alcalase. Initial rate data indicated that the adsorption model was more appropriate. However, both equations gave satisfactory fits to 11 reaction progress curves determined over a wide range of enzyme and substrate concentrations.     

Inhibitory kinetics of phenol on the enzyme activity of beta-N-acetyl-D-glucosaminidase from green crab (Scylla serrata)

Chemical pollution such as chromium and phenol in the sea water has been increasing in recent years in China sea. At the same time, marine shellfish such as prawn and crab are sensitive to this pollution. beta-N-acetyl-D-glucosaminidase (NAGase, EC.3.2.1.52) catalyzes the cleavage the oligomers of N-acetylglucosamine (NAG) into the monomer. In this paper, the effects of phenol on the enzyme activity from green crab (Scylla serrata) for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) have been studied. The results showed that appropriate concentrations of phenol could lead to reversible inhibition on the enzyme and the inhibitor concentration leading to 50% activity lost, IC(50), was estimated to be 75.0+/-2.0 mM. The inhibitory kinetics of phenol on the enzyme in the appropriate concentrations of phenol has been studied using the kinetic method of substrate reaction. The time course of the enzyme for the hydrolysis of pNP-NAG in the presence of different concentrations of phenol showed that at each phenol concentration, the rate decreased with increasing time until a straight line was approached. The results show that the inhibition of the enzyme by phenol is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction on phenol with the enzyme.     

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water‐soluble IL 1‐ethyl‐3‐methylimidazolium ethylsulfate ([emim][EtSO₄]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl–agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis‐Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO₄]. When concentration of the IL was augmented, the values of V_max for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase–glyoxyl–agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO₄]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first‐order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO₄]. Finally, thermal stability was scarcely affected by the presence of the IL. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:790–796, 2014     

Kinetics of inactivation of Ulva pertusa Kjellm alkaline phosphatase by ethylenediaminetetraacetic acid disodium

Ulva pertusa Kjellm alkaline phosphatase (EC 3.3.3.1) is a metalloenzyme, the active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory described by Tsou of the substrate reaction during irreversible inhibition of enzyme activity has been employed to study the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction at different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA indicated a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-EDTA complex is a relative rapid reaction, following by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity.     

Direct electron transfer catalysed by enzymes: application for biosensor development

The ability to catalyse an electrode reaction via direct (mediatorless) electron transfer has been demonstrated for a number of redox enzymes. In the case of mediatorless electron transfer, the electron is transferred directly from the electrode to the substrate molecule via the active site of the enzyme, or vice versa. The electron itself is the second substrate for the reaction. An important point characterizing bioelectrocatalysis is the catalytic removal of the reaction over-voltage. Therefore the enzyme attached to the electrode is able to catalyse electrode reaction and forms a 'molecular transducer'. The substrate can be detected by potentiometric measurement of the removal of reaction over-voltage. The enzyme laccase is able to catalyse the reaction of oxygen electroreduction. Therefore a laccase molecular layer attached to the electrode surface forms an oxygen transducer. The formation of the layer results in a change of the electrocatalytic feature of the electrode. Laccase label coupled with either ligand or receptor allows the detection of ligand-receptor complex formation/dissociation on the electrode surface. The detection is virtually reagentless. The substrates for the reaction are molecular oxygen and the electron itself. Numerous reagentless immunosensors of different formats (competitive, displacement and sandwich) have been developed, as well as the reagentless detection system for immunofiltration/immunochromatography.     

Suicide-peroxide inactivation of microperoxidase-11: a kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H2O2. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H2O2] > [AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, alphai, and the apparent inactivation rate constant, ki, were obtained as 0.282 +/- 0.006 min(-1) and 0.497 +/- 0.013(-1) min at [H2O2] = 1.0 mM, 27 degrees C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).     

Biosensor for the determination of phenols based on cross-linked enzyme crystals (CLEC) of laccase

Cross-linked enzyme crystals (CLECs) are a versatile form of biocatalyst that can also be used for biosensor application. Laccase from Trametes versicolor (E.C.1.10.3.2) was crystallized, cross-linked and lyophilized with beta-cyclodextrin. The CLEC laccase was found to be highly active towards phenols like 2-amino phenol, guaiacol, catechol, pyrogallol, catechin and ABTS (non-phenolic). The CLEC laccase was embedded in 30% polyvinylpropylidone (PVP) gel and mounted into an electrode to make the sensor. The biosensor was used to detect the phenols in 50-1000 micromol concentration level. Phenols with lower molecular weight such as 2-amino phenol, catechol and pyrogallol gave a short response time where as the higher molecular weight substrates like catechin and ABTS had comparatively a long response time. The optimum pH of the analyte was 5.5-6.0 when catechol was used as substrate. The CLEC laccase retained good activity for over 3 months.     

PAR对氨基酰化酶不可逆抑制动力学研究

本文将邹氏的在酶的活性修饰剂存在下的底物反应动力学理论应用于氨基酰化酶被金属螯合剂PAR脱锌而失活的动力学研究。通过对不同浓度的PAR存在下底物反应过程和含有PAR的不同浓度的底物中酶促反应的分析,讨论了PAR对氨基酰化酶的脱锌机制。这一过程很可能按如下机制进行:首先,PAR与酶分子活性部位的锌结合,形成一复合物,这一步是较快的反应,然后发生一个可逆的构象变化,最后是不可逆的去锌步骤。锌的存在显然稳定了酶活性部位的构象,而这正是酶活性所必需的。     

Affinity labeling of catalytic subunit of bovine heart muscle cyclic AMP-dependent protein kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.     

Ceramic honeycomb as support for covalent immobilization of laccase from Trametes versicolor and transformation of nuclear fast red

The covalent immobilization of laccase on an inorganic ceramic support was investigated. The intention was to find a system of enzyme and reactor for a universal immobilization procedure. Laccase from Trametes versicolor as model enzyme was chosen. The special honeycomb structure of the monolith can be applied for intensive mixing of the reaction compounds. An appropriate reactor with ceramic material was constructed allowing different setup for enzyme immobilization and its application. To test the success of the immobilization, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was used. The immobilized laccase was found to be stable over a time period of over 3 months. As an example for possible application for treatment of wastewater containing dyes, the conversion of nuclear fast red as model substrate was tested.