A Study on the Stability and Enzymatic Activity of Yeast Alcohol Dehydrogenase in Presence of the Self-Assembling Block Copolymer Poloxamer 407 (original) (raw)
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Enhanced stability of alcohol dehydrogenase by non-covalent interaction with polysaccharides
Applied Microbiology and Biotechnology, 2014
Non-covalent interaction of alcohol dehydrogenase with polysaccharides was studied using three neutral and three anionic polysaccharides. The process of interaction of alcohol dehydrogenase with gum Arabic was optimized with respect to the ratio of enzyme to gum Arabic, pH, and molarity of buffer. Alcohol dehydrogenase-gum Arabic complex formed under optimized conditions showed 93 % retention of original activity with enhanced thermal and pH stability. Lower inactivation rate constant of alcohol dehydrogenase-gum Arabic complex within the temperature range of 45 to 60°C implied its better stability. Half-life of alcohol dehydrogenase-gum Arabic complex was higher than that of free alcohol dehydrogenase. A slight increment was observed in kinetic constants (K m and V max ) of gum Arabic-complexed alcohol dehydrogenase which may be due to interference by gum Arabic for the binding of substrate to the enzyme. Helix to turn conversion was observed in complexed alcohol dehydrogenase as compared to free alcohol dehydrogenase which may be responsible for observed stability enhancement.
Enzyme immobilization by poly(vinyl alcohol) gel entrapment
Journal of Molecular Catalysis, 1982
A method is presented the formation of poly(vinyI for gel entrapment of enzymes [II, consisting of alcohol) fdrn from enzyme-containing poiy(vinyl alcohol) solution by drying or salt treatment_ The yield of immobilization can be enhanced by adding materials protecting the enzymes (inert proteins, -SH-compounds, etc_) to the gel-forming solution to decrease enzyme leakage. The details of immobilization procedure and some properties of aldolase arrd neutral lactase immobilized in this manner are described.
Enzyme and Microbial Technology, 2007
The present communication reports on changes in the secondary and tertiary structures of native and apo-yeast alcohol dehydrogenase upon heating at 50 • C, as evident from circular dichroism (CD) studies. The presence of sugars provided significant protection with trehalose being the most effective. Exposure of hydrophobic clusters in the protein molecule upon heat denaturation was confirmed by fluorescence studies using 1-anilinonaphthalene-8-sulfonate (ANS) as a hydrophobic reporter probe. All sugars, and especially trehalose, reduced the affinity of both forms of the enzyme for this probe. The effectiveness of sugars in diminishing ANS fluorescence enhancement is in accordance with their ability to protect aggregation of the proteins, reported earlier [Miroliaei M, Nemat-Gorgani M. Sugars protect native and apo yeast alcohol dehydrogenase against irreversible thermoinactivation. Enzyme Microb Technol 2001;29:554-9]. It is concluded that prevention of the mechanisms of irreversible thermoinactivation may occur with retention of the secondary and tertiary structural properties of the proteins.
Process Biochemistry, 2012
The alcohol dehydrogenase (ADH) from Baker's yeast is very active but extremely unstable under several different conditions. Mild immobilization methods such as one-point attachment to agarose activated with cyanogen bromide groups or ionic adsorption to agarose activated with charged groups allow high activity recoveries (80-100%) but do not promote protein stabilization. In contrast, immobilization methods that force the enzyme to be covalently attached at multiple points on the support fully inactivate the enzyme. Herein, we propose an interesting solution to address the dichotomy between activity and stability. We have developed a protocol in which the enzyme is immobilized on agarose activated with glyoxyl groups in the presence of acetyl cysteine, which results in the recovery of 25% of the enzyme activity but increases the thermal stability of the soluble enzyme 50-fold. However, this immobilization technique does not stabilize the enzyme quaternary structure. Hence, a post-immobilization technique using functionalized polymers has been used to cross-link all enzyme subunits. In this method, polycationic polymers (polyethylenimine) cross-link the quaternary structure with a negligible effect on catalytic activity, which results in a derivative that is 5-fold more stable than non-cross-linked derivatives under very dilute and acidic conditions that highly favor subunit dissociation. Therefore, the stability was increased 500-fold for this optimal derivative compared to diluted soluble enzyme, although the relative expressed activity was low (25%). However, the low expressed activity may be overcome by designing immobilized biocatalysts with high volumetric activities.
Annals of the New York Academy of Sciences, 1988
The use of enzymatic reactions in biotechnological processes may be constrained by rapid inactivation of the biocatalysts involved.' The different ways to stabilize enzymes have attracted considerable attention.2 Many enzyme immobilization procedures have been proposed to avoid protein unfolding and aggregati~n.~ On the other hand, immobilization often leads to an increase in.diffusion restraint and, thus, it cannot be applied conveniently in industrial processes involving nowNewtonian reaction media. Recently, newly developed techniques such as protein engineering and directed mutagenesis have been used to limit enzyme inactivation' and to study the thermostability mechanism.' Another possible approach to increase enzyme longevity is to use additives such as sugars and polyols that preserve the biocatalyst activity by modulating its microenvironment.6 The exact mechanism of enzyme protection by additives is not yet perfectly known and, therefore, a better understanding of protein-solvent interactions is needed. Mainly, the influence of carbon chain length has been studied.' Uedaira et a1.* also have correlated the protective effect of a sugar to its hydroxyl radical position. The addition of these solutes has been reported to prevent the enzyme from unfolding by strengthening hydrogen bonds or by strengthening hydrophobic interactions (or both). Solute-solvent interactions may be approached through the thermodynamical water activity concept (a,)? When comparing the previously described it becomes obvious that the efficiency of an additive to preserve enzymatic activity is dependent both on its chemical character and on the enzyme itself. It is then of interest to focus on the understanding of stabilization mechanisms.
Novel method of enzymes stabilization on crosslinked thermosensitive carriers
Enzyme and Microbial Technology, 2006
In this paper an immobilization of invertase on thermosensitive copolymers of N-isopropylacrylamide and 2-hydroxyethyl methacrylate or glycidyl methacrylate modified by aminolysis is evaluated. The method is based on the swelling properties of stimuli-sensitive polymers, which work like a pump that sucks up the enzyme on cooling and then on subsequent crosslinking of the enzyme. The attention was focused on the properties of the carrier-enzyme systems, particularly on the effect of crosslinking on their stability. Activity of TG8-NH 2 carrier was very low and independent on concentration of glutaraldehyde used, but carriers TH8 and TH8-NH 2 were more active, especially when 1.0 and 2.5 vol.% of glutaraldehyde were used. It was also observed, that preparations crosslinked by glutaraldehyde were more stable than preparations without crosslinking agent.
Study on the thermal stability of α-amylase modified by maleic anhydride copolymer
International Journal of Biological Macromolecules, 1994
The thermal inactivation of mesophilic Bacillus subtilis =-amylase modified by maleic anhydride/vinyl acetate copolymer has been studied at different polymer/enzyme ratios in the pH range of relevance to enzymatic catalysis. Enzymatic activity measurements combined with circular dichroism measurements were used to determine the enzyme thermostability. The apparent first-order rate constants and activation energies of thermo-inactivation affected by addition of Ca 2+ ions as well as by modification have been calculated. The modified ~-amylase exhibited sufficiently high catalytic activity with enhanced resistance to the thermal unfolding process.
Lactam-amide graft copolymers as novel support for enzyme immobilization
Journal of Applied Polymer Science, 2002
A graft random copolymer of N-vinyl-2-pyrrolidone and N,NЈ-dimethylacrylamide onto polypropylene was synthesized using a simultaneous gamma radiation technique from a 60 Co source, so that the hydrogel poly(propylene-g-vinylpyrrolidoneco-N,NЈ-dimethylacrylamide) [PP-g-(VP-co-DMAM)], thus produced by grafting, could be used as a support for enzyme immobilization. The grafted spheres showed very good swelling behavior in water due to the incorporation of hydrophilicity in the PP spheres. The influence of pH and temperature on as well as the determination of the kinetic parameters, K M and V max , for both immobilized and soluble invertase were determined. PP-g-(VP-co-DMAM) grafting onto the PP spheres caused a significant change in the water content of the support and was more pronounced for the spheres with a high degree of grafting. A porous structure of the polymeric spheres was observed by scanning electron microscopy (SEM). The porous structure contributed to the reaction rate decrease due to diffusional effects, as shown by the larger K M value observed for immobilized invertase relative to the free enzyme. The enzyme affinity for the substrate (K M /V max) remains quite good after immobilization. The thermal stability of immobilized invertase was significatively higher than that of the free enzyme and a displacement of 20°C was observed for the immobilized enzyme.