Stability Of Yeast Alcohol Dehydrogenase Research Articles

Enhancing Catalytic Activity and Stability of Yeast Alcohol Dehydrogenase by Encapsulation in Chitosan-Calcium Phosphate Hybrid Beads

A kind of calcium phosphate-mineralized chitosan beads (chitosan–CaP) was prepared via a one-pot method by adding droplets of Ca2+-containing chitosan aqueous solution into phosphate-containing sodium tripolyphosphate aqueous solution. The chitosan beads formed immediately coupled with in situ precipitation of calcium phosphate on the surface. The antiswelling properties of hybrid beads were greatly improved with the swelling degree as low as 5%. The morphology of the resultant chitosan–CaP hybrid beads was observed by scanning electron microscopy (SEM). Yeast alcohol dehydrogenase (YADH) was encapsulated in the hybrid beads with an about 40% lower enzyme leakage compared with that in the pure chitosan beads. The optimal temperature and pH value for enzymatic conversion catalyzed by YADH immobilized in the chitosan–CaP beads were 30 °C and 7.0, respectively, which were identical to those for free YADH. The immobilized YADH displayed obviously higher thermal stability, pH stability, recycling stability, an...

Biosilica-coated κ-carrageenan microspheres for yeast alcohol dehydrogenase encapsulation

In this study, a novel polysaccharide/inorganic hybrid biocomposite was prepared through biomineralization-inspired process for efficient immobilization of yeast alcohol dehydrogenase (YADH). YADH-encapuslated κ-carrageenan microspheres (KCM) were first coated with chitosan, which was used to guide and catalyze the biomimetic formation of silica, and then coated with silica derived from tetraethoxysiliane (TEOS). Scanning electron microscopy (SEM) equipped with a energy dispersive X-ray spectrometer (EDX) was employed to investigate the composition and thickness of silica film. Compared with KCM, the silica-coated κ-carrageenan microspheres (SKCM) containing YADH exhibited improved anti-swelling and catalytic properties. Enzyme leakage of YADH in KCM was detected to be 63.9% after 1 h, while the enzyme leakage in SKCM was as low as 18.2%. The KCM adsorbed water promptly and after 1 h maximum water uptake can be as high as 1576 wt%, while the water uptake of SKCM was only 143 wt% even after 48 h. The optimum pH and temperature for encapsulated YADH were pH 6.5 and 25°C, which were just the same as those for free YADH, but the encapsulated YADH exhibited broader pH and temperature ranges for high activity. Furthermore, the relative activity of YADH in KCM declined almost to zero after 8 recycles, while the relative activity of YADH in SKCM still maintained more than 50%. The significantly increased recycling stability of YADH in SKCM may be attributed to the effective inhibition of enzyme leakage by the compact biosilica layer.

Kinetic Analysis about the Effects of Neutral Salts on the Thermal Stability of Yeast Alcohol Dehydrogenase

The effects of salts on the rate constants of inactivation by heat of yeast alcohol dehydrogenase (YADH) at 60.0 degrees C were measured. Different effects were observed at low and high salt concentrations. At high concentrations, some salts had stabilizing effects, while others were destabilizing. The effects of salts in the high concentration range examined can be described as follows: (decreased thermal stability) NaClO(4) < NaI = (C(2)H(5))(4)NBr < NH(4)Br < NaBr = KBr = CsBr = (no addition) < (CH(3))(4)NBr < KCl < KF < Na(2)SO(4) (increased thermal stability). The decreasing effect of NaClO(4) on YADH controlled the thermal stability of the enzyme absolutely and was not compensated by the addition of Na(2)SO(4), a salt which stabilized the enzyme. However, Na(2)SO(4) compensation did occur in response to the decrease in thermal stability caused by (C(2)H(5))(4)NBr. The rate constants of inactivation by heat (k (in)) of the enzyme were measured at various temperatures. Effective values of the thermodynamic activation parameters of thermal inactivation, activation of free energy (DeltaG (double dagger)), activation enthalpy (DeltaH (double dagger)), and activation entropy (DeltaS (double dagger)), were determined. The thermal stability of YADH in 0.8 M Na(2)SO(4) increased more than that of pyruvate kinase from Bacillus stearothermophilus, a moderate thermophile. The changes in the values of DeltaH (double dagger) and DeltaS (double dagger) were great and showed a general compensatory tendency, with the exception of in the case of NaClO(4). The temperature for the general compensation effect (T (c)) was approximately 123 degrees C. With Na(2)SO(4), the thermal stability of YADH at a temperature below T (c) was greater than that in the absence of salt due to the higher values of DeltaH (double dagger) and DeltaS (double dagger), respectively, and thus was an example of low-temperature enzymatic stabilization. With (C(2)H(5))(4)NBr, the thermal stability of YADH at a temperature below T (c) was lower than that in the absence of salt due to the lower values of DeltaH (double dagger) and DeltaS (double dagger), respectively, and thus was an example of low-temperature enzymatic destabilization. But with NaClO(4), the changes in the values of DeltaH (double dagger) and DeltaS (double dagger) were small and the thermal stability of YADH was thus an example of high-temperature enzymatic destabilization.

Effects of mixed reverse micellar structure on stability and activity of yeast alcohol dehydrogenase

The effects of Brij30 concentration on the stabilities and activities of yeast alcohol dehydrogenase (YADH) at various ω 0 values have been studied. Generally, the residual activity of YADH decreased rapidly first and then approached to a steady value. By the addition of Brij30, it was found that the stability of YADH could be enhanced significantly at ω 0=10. However, at ω 0=20, the stability of YADH was not remarkably improved and under some conditions was even lower than that in the absence of Brij30. By the investigation on the hydrodynamic diameter of mixed reverse micelles and its distribution via dynamic light scattering, it was suggested that the structure of mixed reverse micelles and the stability of YADH were determined by four important factors, including the surface charge density, bound water, reverse micellar size, and the entrapment of water by hydrophilic–hydrophilic interaction of Aerosol OT (AOT) and Brij30. The effects of these four factors on the stability of YADH at various ω 0 values and Brij30 concentration have been discussed in detail. When they were reduced, the stability of YADH might be improved. In addition, it was found that the activity of YADH in AOT/Brij30 mixed reverse micelles might be enhanced at appropriate Brij30 concentrations and ω 0 values. According to the hydrodynamic diameter of mixed reverse micelles and its distribution, three main factors were suggested. They were the hydrophobic and electrostatic interactions between enzyme and surfactants, the reverse micellar size, and the bound degree of water molecules. An optimal reverse micellar size and the reductions of the other two factors would lead to the enhancement of enzyme activity.

Effect of zinc ions on conformational stability of yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase preparations were prepared with the conformational zinc ion removed (Apo-I YADH) and with both the conformational and catalytic zinc ions removed (Apo-II YADH). The unfolding of Apo-I YADH and Apo-II YADH during denaturation in urea solutions was then followed by fluorescence emission, circular dichroism, and second-derivative optical spectroscopies. Compared with the native enzyme, Apo-I YADH incurred some slight unfolding, and its stability against urea was markedly decreased, while Apo-II YADH incurred marked unfolding but contained residual ordered structure even at high urea concentrations. The results show that native YADH is more conformationally stable against urea denaturation than Apo-I YADH, indicating that the conformational Zn(2+) plays an important role in stabilizing the conformation of the YADH molecule. However, unfolding of the region around the conformational zinc ion is shown not to be the rate limited step in the unfolding of the molecule by the fact that the unfolding and inactivation rate constants of native and Apo-I YADH are the same. It is suggested that the catalytic zinc ion is more important in maintaining the structure of YADH. YADH lost its cooperative unfolding ability after the zinc ions were removed. The shape of the transition curves of Apo-I YADH suggests the existence of an unfolding intermediate. For both native and Apo-I YADH, inactivation occurs at much lower urea concentrations than that needed to produce significant conformational changes of the enzyme molecule. At urea concentration above 4 M, the inactivation rate constants are much higher than those of the fast phase of the reaction of unfolding. These results support the suggestion of flexibility at the active site of the enzyme (C. L. Tsou (1986) Trends Biochem. Sci., 11, 427-429; (1993) Science, 262, 308-381).

The effect of heparin and pentosan polysulfate on the thermal stability of yeast alcohol dehydrogenase.

Heparin and pentosan polysulfate as organic polyanions inhibit yeast alcohol dehydrogenase (YADH). The aim of this study was to determine the effect of heparin and pentosan polysulfate on the thermostability of alcohol dehydrogenase. Spectral and kinetic analyses showed that these compounds increase the thermal stability of the enzyme and eliminate entirely thermal aggregation. The thermostabilizing effect of unfractionated heparin and pentosan polysulfate was accelerated in the presence of NAD+. The addition of NAD+ (11 microM) to the incubation medium decreased the inhibition of the YADH activity in the presence of pentosan polysulfate (1.32 microM). Moreover, 38% of the residual activity of YADH was found after a 5-min incubation at 70 degrees C. These findings indicate that heparinoids not only modulate the enzyme activity but also can prevent the protein's thermal denaturation.

Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase is a tetrameric enzyme containing zinc. Initially we confirmed the presence of two zinc atoms per subunit. Incubation of the enzyme with increasing concentrations of dithiothreitol, a method for partial chelation, allowed first the reduction of four disulphide bridges per enzyme, but eventually was sufficient to chelate the structural zinc atom without having any effect on the zinc located in the active site. The enzyme activity was not affected but the enzyme became very sensitive to heat denaturation. Chelation by EDTA was also performed. Given its location at an external position in the globular protein, protected in each subunit by one disulphide bridge, the results establish that the second zinc atom present on each enzymic subunit plays a prominent conformational role, probably by stabilizing the tertiary structure of yeast alcohol dehydrogenase. Recovery experiments were performed by incubation of the native enzyme, or the dithiothreitol-treated enzyme, with a small amount of Zn2+. A stabilization effect was found when the structural zinc was re-incorporated after its removal by dithiothreitol. In all cases a large increase in activity was also observed, which was much greater than that expected based on the amount of re-incorporated zinc atom, suggesting the re-activation of some inactive commercial enzyme which had lost some of its original catalytic zinc atoms.

Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307 degrees C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.

Behavior of Enzymes in the Presence of Additives Influence of Alcohóls, Polyols, and Sugars on Activity and Stability of Yeast Alcohol Dehydrogenase

L'effet stabilisant de differentes molecules est etudie sur l'alcool deshydrogenase de levure. Ces molecules sont des alcools avec des longueurs de chaines carbonees et/ou des nombres de groupements hydroxyles differents (monoalcool, dialcool, polyalcools avec un rapport OH/C egal a 1, c'est-a-dire des sucres). Ces experiences permettent d'etablir des relations entre stabilisation de l'enzyme et microenvironnement modifie par ces additifs

Rapid purification of yeast alcohol dehydrogenase

Making use of the unusual stability of yeast alcohol dehydrogenase in the presence of ethanol, a simple, rapid procedure for isolating this enzyme in high yield is presented. Once-crystallized enzyme is obtained within 5 h of commencing the procedure; this is undegraded and substantially free of proteolytic activity.