Abstract
Improvement of enzyme stability via non-covalent complex formation with dextran against temperature and storage lifetime The optimal methodology to prepare the novel modified enzyme, polymer-enzyme complex, was developed to give a high catalytic activity in aqueous solution. The non-covalent complexes of two different enzymes (horseradish peroxidase and glucose oxidase) were prepared with various molar ratios (nD/nE 0,05; 0,1; 1; 5; 10; 15; 20) by using 75kDa dextran. The thermal stabilities of the obtained complexes were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time for pH 7. The complexes with the molar ratio nD/nHRP: 10 and nD/nGOD: 5 showed the highest thermal stability. Its activity was very high (ca. 1,5-fold higher activity than pure enzyme for HRP-dextran complexes) and almost the same between applying one hour incubation time and without incubation, and could also be measured at high temperatures (70, 80 °C). We finally succeeded in preparing dextran-enzyme complexes which showed higher activity than pure enzyme in aqueos solution at all temperatures for pH 7. In addition, the mentioned complexes at pH 7 had very long storage lifetime compared to purified enzyme at +4 °C; which is considered as a good feature for the usage in practice.
Highlights
Protein-polysaccharide complexes are important in industrial applications such as micro- and nano-encapsulation processes[1,2,3,4], the design of multi-layers structures, the formation and the stabilization of food emulsions[6], the formation of new food gels[12] and the recovery of proteins from industrial by-products[8,9,10]
We found that the molar ratio of the polymer/ enzyme in the preparation stage governed the activity of the polymer-enzyme complex
Ε: Molar absorption coefficient of o-dianisidine (11.300 M-1 cm-1) t: Incubation time (10 minute) CHRP: HRP concentration (0.0025 mg/ml) A460: Absorbance at 460 nm Thermal and storage stabilities of pure enzyme and complexes were evaluated with the activities determined according to the procedure described above
Summary
Protein-polysaccharide complexes are important in industrial applications such as micro- and nano-encapsulation processes[1,2,3,4], the design of multi-layers structures, the formation and the stabilization of food emulsions[6], the formation of new food gels[12] and the recovery of proteins from industrial by-products[8,9,10]. Supra-molecular structures formed by non-covalent interactions between protein and polysaccharides (complexes, aggregated complexes and coacervates) have represented a very active research area in the last 3 – 4 years. The formation of complexes between neutral biopolymers is less studied, but the curiosity of scientists should modify this situation in the few years[11, 12]. Non-covalent enzyme-dextran complexes have been reported by de la Casa et al.[28, 29]. They have been analyzed as the effect of the modification by dextrans of the microenvironment of Candida rugosa lipase. The non-covalently-formed complexes of HRP and GOD with dextran have been proposed as an alternative modified enzyme towards industrial application and storage stability. Storage lifetimes of purified enzymes and the complexes with nD/nHRP:[10]; nD/nGOD:[5], respectively, were studied
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