Kinetic study on thermal stability of immobilized invertase

The kinetic study of the thermal stability of three kinds of invertases: native, immobilized on porous glass covalently, and on ion-exchange resin ionically, has been carried out, measuring their enzymatic activity for sucrose hydrolysis. Thermal deactivations of all invertases obeyed first-order kinetics, being independent of substrate concentration, with k_d and ΔE_d, ΔS_d* as shown in Tables I and II, respectively. Based on these parameter values, the effects of immobilization and pH at deactivation on the stability have been considered, and it was suggested that the ionic bond gives a more loosely deformed enzyme than the covalent bond.     

Isolation, Purification, and Characterization of Catalase from the Methylotrophic Yeast Pichia pastoris

Catalase (CAT_pp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CAT_pp solutions and of its membrane-concentrated form (CAT_pp-conc) were studied. Rates of H₂O₂ decomposition and kinetic characteristics K _m and k _cat of CAT_pp and CAT_pp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30°C, as well as the effective constant k _in of the enzyme inactivation rate during the catalysis and the constant k ₂ of the interaction rate of the Complex I catalases with H₂O₂. Thermal inactivation of CAT_pp in solutions at 45°C was characterized by the effective rate constant k _in ^*, and the low-frequency (27 kHz) ultrasonic inactivation of CAT_pp at 20°C was characterized by the firstorder rate constant k _in (US). All spectral and kinetic characteristics of CAT_pp and CAT_pp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CAT_cb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CAT_pp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of k _cat/K _m, thermal stability comparable with the thermal stability of CAT in terms of k _in ^*, the minimal k _in, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm².     

Adaptation of Drosophila enzymes to temperature IV. Natural selection at the alcohol-dehydrogenase locus

Studies were carried out on the temperature-dependent kinetic properties (K_m, Q₁₀, E_a, thermostability) of alcohol-dehydrogenase allozymes from D. melanogaster. It was shown that there is a parallelism between the biochemical properties of the enzymes and the behaviour of the genes in natural and cage populations. Furthermore, the relationship between the temperature-dependent kinetic properties of alcohol dehydrogenase and assay temperature was examined in three tropical and two temperate Drosophila species. K_m patterns were similar among species from the same habitat and different between habitats. No such parallelism was seen with respect to thermal inactivation. The Q₁₀ values in general reflected temperature-dependent changes in K_m. It is discussed that the mechanistic connection observed between the biochemical and population levels (intra-and interspecifically) strongly suggests that temperature acts as a selective factor on the structural Adh locus in the genus Drosophila.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

Expression,purification and immobilization of the intracellular invertase INVA,from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on crystalline cellulose and Nylon-6

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD _Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K _m and V _max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V _max increased up to 5.7-fold, following immobilization, whereas K _m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

Adaptation of malate dehydrogenase to environmental temperature variability in two populations of Potentilla glandulosa Lindl.

Summary The responses of the kinetic properties of malate dehydrogenase to environmental temperature variability were compared for two populations of Potentilla glandulosa (Rosaceae). The two populations are native to regions of contrasting climates, with the inland population experiencing a high level of temperature variability during growth and the coastal populaton a low level of temperature variability. The substrate binding ability, as measured by apparent K _m of both populations was relatively insensitive to assay temperature (Q ₁₀<2.0) over the range of temperatures likely to be encountered during growth. The breadth of this thermal optimum was different for the two populations with the K _m of the inland plants exhibiting relative temperature insensitivity over a much wider range of temperatures than the K _m of the coastal plants. There was no difference between the two populations in the thermal stability of MDH activity.     

Enhanced activity and stability of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase by immobilization on aminopropyl glass

Immobilization of Bacillus licheniformis l-arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q _m) and affinity (k _a). The pH and temperature for immobilization were optimized to be pH 7.1 and 33°C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k _cat/K _m) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t _1/2 increased from 2 to 275 h) at 50°C following immobilization.     

Purification and characterization of a thermostable pullulanase fromThermoactinomyces thalpophilus

Summary Thermoactinomyces thalpophilus No. 15 produced an extracellular pullulanase in an aerobic fermentation with soluble starch, salts, and complex nitrogen sources. Acetone fractionation, ion-exchange chromatography, and gel filtration purified the enzyme from cell-free broth 16-fold to an electrophoretically homogeneous state (specific activity, 1352 U/mg protein; yield, 4%). The purified enzyme (estimated MW 79 000) was optimally active at pH 7.0 and 70°C and retained 90% relative activity at 80°C (30 min) in the absence of substrate. The enzyme was activated by Co²⁺, inhibited by Hg²⁺, and exhibited enhanced stability in the presence of Ca²⁺. The enzyme hydrolyzed pullulan (K _m 0.32%, w/v) forming maltotriose, and hydrolyzed amylopectin (K _m 0.36%, w/v), amylopectin beta-limit dextrin (K _m 0.45%, w/v) and glycogen beta-limit dextrin (K _m 1.11%, w/v) forming maltotriose and maltose.     

Protein adsorption on Ion exchange resin: Estimation of equilibrium isotherm parameters from batch kinetic data

The simple Langmuir isotherm is frequently employed to describe the equilibrium behavior of protein adsorption on a wide variety of adsorbents. The two adjustable parameters of the Langmuir isotherm—the saturation capacity, orq _m, and the dissociation constant,K _d—are usually estimated by fitting the isotherm equation to the equilibrium data acquired from batch equilibration experiments. In this study, we have evaluated the possibility of estimatingq _m andK _d for the adsorption of bovine serum albumin to a cation exchanger using batch kinetic data. A rate model predicated on the kinetic form of the Langmuir isotherm, with three adjustable parameters (q _m,K _d, and a rate constant), was fitted to a single kinetic profile. The value ofq _m determined as the result of this approach was quantitatively consistent with theq _m value derived from the traditional batch equilibrium data. However, theK _d value could not be retrieved from the kinetic profile, as the model fit proved insensitive to this parameter. Sensitivity analysis provided significant insight into the identifiability of the three model parameters.     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Amino acid modified chitosan beads: Improved polymer supports for immobilization of lipase from Candida rugosa

Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (k_cat/K_m) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads.     

An investigation of the properties of glucoamylase immobilized on glass beads involving 5-diazosalicylic acid bonded to a titanium (IV) oxide film

Optimum conditions have been determined for the immobilization of glucoamylase on glass involving diazotized 5-aminosalicylic acid bonded to a deposited imperfectly crystallized film of TiO₂. The changes in the kinetic and thermodynamic characteristics of the enzyme on immobilization have been determined. There are significant differences in the behaviour of the immobilized enzyme towards its substrates, maltose and starch. The apparent K_m for starch increased on immobilization whereas that for maltose decreased. The pH optimum for the immobilized preparation showed a shift to acid pH relative to that of the free enzyme.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

Investigation of deactivation thermodynamics of lipase immobilized on polymeric carrier

In the present work, we have investigated biochemical thermo-kinetic stability of lipases immobilized on a biocompatible polymeric material. Immobilization of lipase Candida rugosa (CRL) was carried out on biocompatible blend of poly vinyl alcohol (PVA) and chitosan (CHY) support via entrapment and glutardehyde (Glu) cross-linking method to produce PVA:CHY:CRL and PVA:CHY:Glu:CRL as robust biocatalyst. These immobilized lipases were characterized by various physico-biochemical characterization techniques. Later on, thermal and solvent stability of polymer immobilized lipase was determined in term of half-life time (t _0.5), D values, enthalpy (ΔH°), entropy (ΔS°), and free energy (ΔG°) of deactivation at different temperatures and in various solvents. The thermodynamic deactivation stability trend was found as: cross-linked lipase CRL > entrapped lipase CRL > free lipase CRL. Moreover, kinetic parameters, such as K _m, V _max, and catalytic efficiency, were also determined to understand the kinetic features. The polymer immobilized enzyme was reused to investigate the economic viability of the developed biocatalyst.

     

Diffusion influences in denaturable insolubilized enzyme catalysts

The influence of diffusion on the apparent thermal stability of a reversibly or irreversibly denaturable enzyme is examined theoretically when he enzyme is uniformly distributed in a porous solid. If the overall reaction rate is influenced by diffusion through the catalyst, the insolubilized catalyst is shown to yield an apparently more thermally stable enzyme even though the maximal velocity V_m, and the Michaelis constant, K_m, are the same for the free and insolubilized forms of enzyme. Brief consideration is given to the experimental conditions needed to clearly demonstrate whether insolubilization does or does not effect the thermal stability of the enzyme.     

Accurate kinetic parameter estimation during progress curve analysis of systems with endogenous substrate production

We have identified an error in the published integral form of the modified Michaelis–Menten equation that accounts for endogenous substrate production. The correct solution is presented and the error in both the substrate concentration, S, and the kinetic parameters V_m, K_m, and R resulting from the incorrect solution was characterized. The incorrect integral form resulted in substrate concentration errors as high as 50% resulting in 7–50% error in kinetic parameter estimates. To better reflect experimental scenarios, noise containing substrate depletion data were analyzed by both the incorrect and correct integral equations. While both equations resulted in identical fits to substrate depletion data, the final estimates of V_m, K_m, and R were different and K_m and R estimates from the incorrect integral equation deviated substantially from the actual values. Another observation was that at R = 0, the incorrect integral equation reduced to the correct form of the Michaelis–Menten equation. We believe this combination of excellent fits to experimental data, albeit with incorrect kinetic parameter estimates, and the reduction to the Michaelis–Menten equation at R = 0 is primarily responsible for the incorrectness to go unnoticed. However, the resulting error in kinetic parameter estimates will lead to incorrect biological interpretation and we urge the use of the correct integral form presented in this study. Biotechnol. Bioeng. 2011;108: 2499–2503. © 2011 Wiley Periodicals, Inc.     

The effects of temperature and chorionic gonadotropin on the functional properties of lactate dehydrogenase from oocytes of loach Misgurnus fossilis

Acclimation of loach to relatively low and high temperatures gives rise to changes in the properties of LDH from oocytes. The minimum of apparent K_m values for pyruvate of LDH from immature oocytes of fish acclimated to low temperature was registered at low assay temperatures, whereas adaptation to high temperatures leads to the enzyme showing a minimum at high temperatures. In thermal acclimation of fish the K_m minimum value drifts during 15 days. During oocyte maturation, the K_m minimum drifts during 40 hr. The differences in the kinetic features of LDH from oocytes both at thermal acclimation and during oocyte maturation are not associated with the appearance of new isozymes.     

α-d-Galactosidase immobilized on a soluble polymer

α-d-Galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) from green coffee beans has been immobilized by attachment to cyanogen bromide-activated Dextran T-70. Since this represents the first reported example of the preparation of a water-soluble derivative of an enzyme showing substrate inhibition, the kinetic properties, thermal stability and pH optima were investigated and compared with those of the free enzyme. The K_m, K_s, K_i, V_max, optimum substrate concentration and optimum pH were all lower than those of free enzyme. The enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation. These data, although obtained with the synthetic substrate 4-nitrophenyl-α-d-galactoside, suggest some advantages in using the enzyme conjugate for the removal of terminal α-d-galactopyranosyl groups from the erythrocyte cell surface.     

Directed evolution of <Emphasis Type="Italic">Streptomyces lividans</Emphasis> xylanase B toward enhanced thermal and alkaline pH stability

The thermal and alkaline pH stability of Streptomyces lividans xylanase B was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant thermostability at 70°C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme 3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The K _m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k _cat was slightly decreased, leading to a slightly reduced value of k _cat/K _m, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Our results suggest that DNA shuffling is an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information.