Kinetic behavior of penicillin acylase immobilized on acrylic carrier

The usefulness of Lilly's kinetic equation to describe penicillin G hydrolysis performed by immobilized penicillin acylase onto the acrylic carrier has been shown. Based on the experimental results characteristic kinetic constants have been estimated. The effect of noncompetitive inhibition of 6-amino penicillanic acid has not been found. Five components of reaction resistance have been defined. These components were also estimated for the reaction of the native enzyme as well as the Boehringer preparation.List of Symbols C _E g/m³ enzyme concentration - C _P,C _Q mol/m³ product concentrations - C _S mol/m³ substrate concentration - C _SO mol/m³ initial substrate concentration - K _A mol/m³ constant which defines the affinity of a substrate to the enzyme - K _iS mol/m³ substrate inhibitory constant - K _iP mol/m³ PhAA inhibitory constant - K _iQ mol/m³ 6-APA inhibitory constant - k ₃ mol/g/min constant rate of dissociation of the active complex - R(1) concentrational component of reaction resistance - R(2) resistance component derived from substrate affinity - R(3) resistance component due to the inhibition of the enzyme by substrate - R(4) resistance component due to the inhibition of the enzyme by PhAA - R(5) resistance component due to inhibition of the enzyme by 6-APA - r = dC_s/dt mol/m³ min rate of reaction - t min reaction time - (i) relative resistance of reaction     

Strategy for the simulation of batch reactors when the enzyme-catalyzed reaction is accompanied by enzyme deactivation

The balance equations pertaining to the modelling of batch reactors performing an enzyme-catalyzed reaction in the presence of enzyme deactivation are developed. The functional form of the solution for the general situation where both the rate of the enzyme-catalyzed reaction and the rate of enzyme deactivation are dependent on the substrate concentration is obtained, as well as the condition that applies if a maximum conversion of substrate is sought. Finally, two examples of practical interest are explored to emphasize the usefulness of the analysis presented.List of Symbols C _E mol/m³ concentration of active enzyme - C _E,O mol/m³ initial concentration of active enzyme - C _S mol/m³ concentration of substrate - C _S,O mol/m³ initial concentration of substrate - C _S,min mol/m³ minimum value for the concentration of substrate - k 1/s first order rate constant associated with conversion of enzyme/substrate complex into product - k ₁ 1/s first order deactivation constant of enzyme (or free enzyme) - k ₂ 1/s first order deactivation constant of enzyme in enzyme/substrate complex form - K _m mol/m³ Michaelis-Menten constant - p mol/(m³s) time derivative of C _S - q mol/m³ auxiliary variable - t s time elapsed after reactor startup Greek Symbols 1/s univariate function expressing the dependence of the rate of enzyme deactivation on C _S - mol/m³ dummy variable of integration - mol/m³ dummy variable of integration - 1/s univariate function expressing the dependence of the rate of substrate depletion on C _S - m³/(mol s) derivative of with respect to C _S     

Kinetic behaviour of penicillin acylase stabilized by poly (ethyleneimine)

The paper presents the kinetic evaluations of poly(ethyleneimine)-penicillin acylase preparations. The comparative studies show that the investigated system is much better than the native enzyme, and slightly worse than commercially available Boenringer preparation. Additionally, the high stability of PEI-enzyme system, very easy way of its preparation, high flexibility, and possibility to set the needed enzyme concentration are particularly favourable for use of the membrane bioreactor with PEI-enzyme system immobilized in its volume. Some advantages of the use of such bioreactor are also discussed.List of Symbols C _E IU/m³ activity concentration - C _S mol/m³ substrate concentration - C _P , C_Q mol/m³ products concentration - K _A mol/m³ constant which defines the affinity of a substrate to enzyme - K _iS mol/m³ substrate inhibitory constant - K _iP mol/m³ PhAA inhibitory constant - K _iQ mol/m³ 6-APA inhibitory constant - k ₃ , mol/IU min constant rate of dissociation of the active complex - mol/m³ min rate of reaction This work was supported by Government Committee of Science: Grant KBN # 3 0321 92 01     

Reactor design for the enzymatic isomerization of glucose to fructose

A comprehensive methodology is presented for the design of reactors using immobilized enzymes as catalysts. The design is based on material balances and rate equations for enzyme action and decay and considers the effect of mass transfer limitations on the expression of enzyme activity. The enzymatic isomerization of glucose into fructose with a commercial immobilized glucose isomerase was selected as a case study. Results obtained are consistent with data obtained from existing high-fructose syrup plants. The methodology may be extended to other cases, provided sound expressions for enzyme action and decay are available and a simple flow pattern within the reactor might be assumed.List of Symbols C kat/kg specific activity of the catalyst - D m²/s substrate diffusivity within the catalyst particle - Dr m reactor diameter - d d operating time of each reactor - E kat initial enzyme activity - E _i kat initial enzyme activity in each reactor - F m³/s process flowrate - F _i m³/s reactor feed flowrate at a given time - F ₀ m³/s initial feed flowrate to each reactor - H number of enzyme half-lives used in the reactors - K mole/m³ equilibrium constant - K _S mole/m³ Michaelis constant for substrate - K _P mole/m³ Michaelis constant for product - K _m mole/m³ apparent Michaelis constant f(K, K _s, K_p, s₀) - k mole/s · kat reaction rate constant - k _d d^–1 first-order thermal inactivation rate constant - L m reactor height - L _r m height of catalyst bed - N _R number of reactors - P _i kg catalyst weight in each reactor - p mole/m³ product concentration - R m particle radius - R _P ratio of minimum to maximum process flowrate - r m distance to the center of the spherical particle - s mole/m³ substrate concentration - s _0i mole/m³ substrate concentration at reactor inlet - s ₀ mole/m³ bulk substrate concentration - s mole/m³ apparent substrate concentration - T K temperature - t d time - t _i d operating time for reactor i - t _s d time elapsed between two successive charges of each reactor - V m³ reactor volumen - V _m mole/m³ s maximum apparent reaction rate - V _p mole/m³ s maximum reaction rate for product - V _R m³ actual volume of catalyst bed - V _r m³ calculated volume of catalyst bed - V _S mol/m³ s maximum reaction rate for substrate - v mol/m³ s initial reaction rate - v _i m/s linear velocity - v _m mol/m³ s apparent initial reaction rate f(K_m, s,V_m) - X substrate conversion - X _eq substrate conversion at equilibrium - =s/K dimensionless substrate concentration - ₀=s₀/K bulk dimensionless substrate concentration - _eq=s_eq/K dimensionless substrate concentration at equilibrium - local effectiveness factor - mean integrated effectiveness factor - Thiéle modulus - =r/R dimensionless radius - _s kg/m³ hydrated support density - substrate protection factor - s residence time     

Cost minimization in the predesign of an enzymatic CSTR: an overall approach

The balance equations pertaining to the modelling of a CSTR performing an enzyme-catalyzed reaction in the presence of enzyme deactivation are developed. Combination of heuristic correlations for the size-dependent cost of equipment and the purification-dependent cost of recovery of product with the mass balances was used as a basis for the development of expressions relating a (suitably defined) dimensionless economic parameter with the optimal outlet substrate concentration under the assumption that overall production costs per unit mass of product were to be minimized. The situation of Michaelis-Menten kinetics for the substrate depletion and first order kinetics for the deactivation of enzyme (considering that the free enzyme and the enzyme in the enzyme/substrate complex deactivate at different rates) was explored, and plots for several values of the parameters germane to the analysis are included.List of Symbols C _E mol m^–3 concentration of active enzyme - C _E,0 mol m^–3 initial concentration of active enzyme - C _p mol m^–3 concentration of product of interest - C _s mol m^–3 concentration of substrate - C _s,0 mol m^–3 initial concentration of substrate - I $ capital cost of equipment - k _d s^–1 deactivation constant of free enzyme - k _d s^–1 deactivation constant of enzyme in enzyme/substrate complex - K _m mol m^–3 Michaelis-Menten constant - K _m dimensionless counterpart of K _m - k _r s^–1 rate constant associated with conversion of enzyme/substrate complex into product - M _w kg mol^–1 molecular weight of product of interest - P $ kg^–1 cost of recovery of product of interest in pure form - Q m³s^–1 volumetric flow rate - V m³ volume of reactor - X $ kg^–1 global manufacture cost of product of interest in pure form - X dimensionless counterpart of X Greek Symbols ₁ $ m^–1.8 constant - ₂ $ m^–3 constant - t s useful life of CSTR - ₀ ratio of initial concentrations of enzyme and substrate - ratio of deactivation constant of free enzyme to rate constant of depletion of substrate - ratio of deactivation constants - univariate function expressing the dependence of the rate of enzyme deactivation on C _S - univariate function expressing the dependence of the rate of substrate depletion on C _S - dimensionless economic parameter     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

NAD+-Dependent (S)-Specific Secondary Alcohol Dehydrogenase Involved in Stereoinversion of 3-Pentyn-2-ol Catalyzed by Nocardia fusca AKU 2123

An NAD⁺-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K _m and V _max for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 μmol/min/mg, and 0.33 mM and 130 μmol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K _m and V _max for 2-hexanone reduction were 2.5 mM and 260 μmol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH₂-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.     

Optimal temperature and concentration profiles in a cascade of CSTR's performing Michaelis-Menten reactions with first order enzyme deactivation

A necessary condition is found for the intermediate temperatures and substrate concentrations in a series of CSTR's performing an enzyme-catalyzed reaction which leads to the minimum overall volume of the cascade for given initial and final temperatures and substrate concentrations. The reaction is assumed to occur in a single phase under steady state conditions. The common case of Michaelis-Menten kinetics coupled with first order deactivation of the enzyme is considered. This analysis shows that intermediate stream temperatures play as important a role as intermediate substrate concentrations when optimizing in the presence of nonisothermal conditions. The general procedure is applied to a practical example involving a series of two reactors with reasonable values for the relevant five operating parameters. These parameters are defined as dimensionless ratios involving activation energies (or enthalpy changes of reaction), preexponential factors, and initial temperature and substrate concentration. For negligible rate of deactivation, the qptimality condition corresponds to having the ratio of any two consecutive concentrations as a single-parameter increasing function of the previous ratio of consecutive concentrations.List of Symbols C _E,0 mol.m^–3 Initial concentration of active enzyme - C _E,i mol.m^–3 Concentration of active enzyme at the outlet of the i-th reactor - C _S,0 mol.m^–3 Initial concentration of substrate - C _S,i mol.m^–3 Concentration of substrate at the outlet of the i-th reactor - Da _i Damköhler number associated with the i-th reactor ((V _i.k_v,0.C_E,0)/(Q.C_S,0)) - Da _min Minimum value of the overall Damköhler number - Da _tot Overall Damköhler number - E _d J.mol^–1 Activation energy of the step of deactivation of the enzyme - E _m J.mol^–1 Standard enthalpy change of the step of binding of substrate to the enzyme - E _v J.mol^–1 Activation energy of the step of enzymatic transformation of substrate - i Integer variable - j Dummy integer variable - k Dummy integer variable - k _d,i s^–1 Kinetic constant associated with the deactivation of enzyme in the i-th reactor (k _d,o·exp{–E _d/(R.T _i}) - k _d,0 s^–1 Preexponential factor of the kinetic constant associated with the deactivation of the enzyme - K _m,i mol.m^–3 Equilibrium constant associated with the binding of substrate to the enzyme in the i-th reactor, (k _m,o·exp{–E _m}(R.T _i}) - K _m,0 mol.m^–3 Preexponential factor of the Michaelis-Menten constant associated with the binding of substrate to the enzyme - k _v,i s^–1 Kinetic constant associated with the transformation of the substrate by the enzyme in the i-th reactor (k _v,o·exp{–E _v/(R.T _i})) - k _v,0 s^–1 Preexponential factor of the kinetic constant associated with the transformation of the substrate by the enzyme - N Number of reactors in the series - Q m³.s^–1 Volumetric flow rate of reacting liquid through the reactor network - R J.K^–1.mol^–1 Ideal gas constant - T _i K Absolute temperature at the outlet of the i-th reactor - T ₀ K Initial absolute temperature - V _i m³ Volume of the i-th reactor - v _max mol.m^–3.s^–1 Maximum rate of reaction under saturation conditions of substrate - x _i Normalized concentration of substrate (C_S,i/C_{S, 0}) - x _i,opt Optimum value of the normalized concentration of substrate - y _i Dimensionless temperature (exp{–T ₀/T _i}) - y _i,opt Optimum value of the dimensionless temperature Greek Symbols Dimensionless preexponential factor associated with the Michaelis-Menten constant (K _m,0/C_s,0) - Dimensionless activation energy of the step of enzymatic transformation of substrate (E _v/R.T₀)) - Dimensionless standard enthalpy change of the step of binding of substrate to the enzyme (E _m/(R.T₀)) - Dimensionless activation energy of the step of deactivation of the enzyme (E _d/(R.T₀)) - Dimensionless deactivation preexponential factor ((k _d,0.C_S,0)/(k_v,0.C_E,0)     

Na/K-ATPase under Oxidative Stress: Molecular Mechanisms of Injury

1. The authors compare oxidative injury to brain and kidney Na/K-ATPase using in vitro and in vivo approaches. The substrate dependence of dog kidney Na/K-ATPase was examined both before and after partial hydrogen peroxide modification. A computer simulation model was used for calculating kinetic parameters.2. The substrate dependence curve for the unmodified endogenous enzyme displayed a typical curve with an intermediate plateau, adequately described by the sum of hyperbolic and sigmoidal components.3. The modified enzyme demonstrated a dependent curve that closely approximates normal hyperbola. The estimated ATP K _m value for the endogenous enzyme was about 85 M; the K _h was equal to 800 M. The maximal number of protomers interacting was 8. Following oxidative modification, the enzyme substrate dependence curve did not show a significant change in the maximal protomer rate V _m, while the K _m was increased slightly and interprotomer interaction was abolished.4. Na/K-ATPase from an ischemic gerbil brain showed a 22% decrease in specific activity. The maximal rate of ATP hydrolysis by an enzyme protomer changed slightly, but the sigmoidal component, characterizing the enzyme's ability to form oligomers was abolished completely. The K _m value was almost unchanged, but the Hill coefficient fell to 1. These data show that Na/K-ATPase molecules isolated from the ischemic brain have lost the ability to interact with one another.5. We suggest that the most important consequence of oxidative modification is Na/K-ATPase oligomeric structure formation and subsequent hydrolysis rate suppression.     

Optimum temperature policy for batch reactors performing biochemical reactions in the presence of enzyme deactivation

A necessary condition is found for the optimum temperature policy which leads to the minimum reaction time for a given final conversion of substrate in a well stirred, enzymatic batch reactor performing an enzyme-catalyzed reaction following Michaelis-Menten kinetics in the presence of first order enzyme decay. The reasoning, which is based on Euler's classical approach to variational calculus, is relevant for the predesign steps because it indicates in a simple fashion which temperature program should be followed in order to obtain the maximum advantage of existing enzyme using the type of reactor usually elected by technologists in the fine biochemistry field. In order to highlight the relevance and applicability of the work reported here, the case of optimality under isothermal operating conditions is considered and a practical example is worked out.List of Symbols C _E mol.m^–3 concentration of active enzyme - C _E ^* dimensionless counterpart of C_E - C _E,0 mol.m^–3 initial concentration of active enzyme - C _E,b mol.m^–3 final concentration of active enzyme - C _E,opt ^* optimal dimensionless counterpart of C_E - C _smol.m^–3 concentration of substrate - C _S ^Emphasis>/* dimensionless counterpart of C_S - C _S,0mol.m^–3 initial concentration of substrate - C _S,bmol.m^–3 final concentration of substrate - E enzyme in active form - E ₃ ^* dimensionless counterpart of E_a,3 - E _a,1J.mol^–1 activation energy associated with k₁ - E _a,3J.mol^–1 activation energy associated with k₃ - E _d enzyme in deactivated form - ES enzyme/substrate complex - k ₁ s^–1 kinetic constant associated with the enzyme-catalyzed transformation of substrate - k _1,0 s^–1 preexponential factor associated with k₁ - k ₂ mol^–1.m³s^–1 kinetic constant associated with the binding of substrate to the enzyme - k _–2 s^–1 kinetic constant associated with the dissociation of the enzyme/substrate complex - K _2,0 mol.m^–3 constant value of K₂ - K _2,0 ^* dimensionless counterpart of K_2,0 - k ₃ s^–1 kinetic constant associated with the deactivation of enzyme - k _3,0 s^–1 preexponential factor associated with k₃ - k _3,0 ^* dimensionless counterpart of k_3,0 - P product - R J.K^–1.mol^–1 ideal gas constant - S substrate - t s time since start-up of reaction - T K absolute temperature - T ^* dimensionless absolute temperature - T _i,opt ^* optimal dimensionless isothermal temperature of operation - T _opt ^* optimal dimensionless temperature of operation - t _b s time of a batch - t _b ^* dimensionless counterpart of t_b - t _b,min ^* minimum value of the dimensionless counterpart of t_b Greek Symbols dimensionless counterpart of C_E,0 - dimensionless counterpart of C_E,b - dummmy variable of integration - dummy variable of integration - auxiliary dimensionless variable - ^* dimensionless variation of k₁ with temperature - _i ^* dimensionless value of k₁ under isothermal conditions - _opt ^* optimal dimensionless variation of k₁ with temperature     

Purification and Characterization of Extracellular Chitin Deacetylase from Colletotrichum lindemuthianum

Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mm sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with the blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS–PAGE and gel filtration, respectively, suggesting that the enzyme is a single polypeptide. The optimum temperature was 60°C and the optimum pH was 11.5–12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer and dimer, and inactive with N-acetylglucosamine. The K_m and k_cat for glycol chitin were 2.55 mm and 27.1s^?1, respectively, and those for chitin pentamer were 414 μm and 83.2s^?1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis–Menten kinetics.     

Purification and characterization of a NAD+-dependent secondary alcohol dehydrogenase from propane-grown Rhodococcus rhodochrous PNKb1

NAD⁺-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD⁺-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C₂–C₈) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K _m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC high performance liquid chromatography - DEAE diethyl amino ethyl - IEF isoelectrofocusing - NTG nitrosoguanidine - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - pI isoelectric point     

Purification and characterization of glutamine synthetase from the commerical mushroom Agaricus bisporus

Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30°C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20°C. The enzyme exhibits a K _m for glutamate, ammonium, and ATP of 22mm, 0.16mm and 1.25mm respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10mm concentrations of l-alanine, l-histidine, l-tryptophan, anthranilic acid, and 5-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K _i-values were 890 m and 240 m for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K _i was 17 m for both methionine sulfoximine and phosphinothricine.     

Intraparticle mass-transfer resistance and apparent time stability of immobilized yeast alcohol dehydrogenase

The stability and, consequently, the lifetime of immobilized enzymes (IME) are important factors in practical applications of IME, especially so far as design and operation of the enzyme reactors are concerned. In this paper a model is presented which describes the effect of intraparticle diffusion on time stability behaviour of IME, and which has been verified experimentally by the two-substrate enzymic reaction. As a model reaction the ethanol oxidation catalysed by immobilized yeast alcohol dehydrogenase was chosen. The reaction was performed in the batch-recycle reactor at 303 K and pH-value 8.9, under the conditions of high ethanol concentration and low coenzyme (NAD⁺) concentration, so that NAD⁺ was the limiting substrate. The values of the apparent and intrinsic deactivation constant as well as the apparent relative lifetime of the enzyme were calculated.The results show that the diffusional resistance influences the time stability of the IME catalyst and that IME appears to be more stabilized under the larger diffusion resistance.List of Symbols C _A, C_B, C_E mol · m^–3 concentration of coenzyme NAD⁺, ethanol and enzyme, respectively - C _p mol · m³ concentration of reaction product NADH - d _p mm particle diameter - D _eff m² · s^–1 effective volume diffusivity of NAD⁺ within porous matrix - k _d s^–1 intrinsic deactivation constant - K _A, K_A, K_B mol · m^–3 kinetic constant defined by Eq. (1) - K _A ^x mol · m^–3 kinetic constant defined by Eq. (5) - r _A mol · m^–3 · s^–1 intrinsic reaction rate - R m particle radius - R _v mol · m^–3 · s^–1 observed reaction rate per unit volume of immobilized enzyme - t _E s enzyme deactivation time - t _r s reaction time - V mol · m^–3 · s^–1 maximum reaction rate in Eq. (1) - V ^x mol · m^–3 · s^–1 parameter defined by Eq. (4) - V _f m³ total volume of fluid in reactor - w _s kg mass of immobilized enzyme bed - factor defined by Eqs. (19) and (20) - kg · m^–3 density of immobilized enzyme bed - unstableness factor - effectiveness factor - Thiele modulus - relative half-lifetime of immobilized enzyme Index o values obtained with fresh immobilized enzyme     

Purification,characterization and thermostability of lipase from a thermophilic Bacillus sp. J33

A thermostable lipase produced by a thermophilic Bacillus sp. J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography. The enzyme is a monomeric protein having molecular weight of 45 kDa. It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C12 and C4. The K_m and V_max for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 M min^-1 ml^-1 respectively. The immobilized enzyme was stable for 12 h at 60°C. Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers. Lipase acquired a remarkable stability, since no deactivation occurred at 70°C for 150 min in the presence of additives.     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 M phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K _m and k _cat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K _m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K _m and k _cat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.     

Developmental changes in the activity and substrate specificities of mouse brain monoamine oxidase

Developmental changes in monoamine oxidase (MAO) activity in the mouse brain were investigated with the substrates -phenylethylamine (PEA), tryptamine, and 5-hydroxytryptamine (5-HT). In the newborn brain, MAO activity towards PEA was found to be much lower than the adult and to be inhibited by clorgyline in a double-sigmoidal fashion. The inhibition curve shifted to a single-sigmoidal pattern with age. MAO activity towards 5-HT as substrate was inhibited by 90% and in a single-sigmoidal manner by clorgyline throughout the postnatal life. Lineweaver-Burk plots with PEA as substrate presented two linear lines (apparentK _m: 28.6 and 4.1 M) for the newborn and one line (apparentK _m: 11.4 M) for the adult, respectively. The plot with highK _m value for the newborn brain disappeared in a clorgyline-treated preparation. These findings suggest that age-dependent alterations in the ratio of MAO-A/MAO-B activity affect the substrate specificity of the enzyme.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

Process of penicillin G hydrolysis catalyzed by penicillin acylase immobilized on acylic carrier

The usefulness of penicillin acylase immobilized onto butyl acrylate — ethyl glycol dimethacrylate (called in this paper acrylic carrier) in penicillin G hydrolysis performed in a stirred tank reactor is shown. The enzyme-acrylic carrier preparation does not deteriorate its own properties in the mixing condition of slurry reactor. The experiments were carried out in a batch and a continuous stirred tank reactor as well as continuous stirred tank reactors in series. It was found to be a satisfactory agreement between experimental and predicted results. It also indicated the optimal substrate concentration range which provides the most effective enzyme operation. A superiority of the three reactors in series over the batch reactor is shown.List of Symbols C_E g/m³ equivalent enzyme concentration - C_SO mol/m³ initial penicillin G concentration - K_A mol/m³ substrate affinity constant - K_iS mol/m³ substrate inhibitory constant - K_iP mol/m³ PhAA inhibitory constant - K_iQ mol/m³ 6-APA inhibitory constant - k₃ mol/g min constant rate of dissotiation of the active complex - r mol/m³ rate of reaction - t min. reaction time - t_j min. maintenance time - degree of conversion - _B, _F dimensionless time - min. residence time - PA penicillin acylase - PG penicillin G - PhAA phenylacetic acid - 6-APA 6-aminopenicillanic acid