Improvement of enzyme stability via non-covalent complex formation with dextran against temperature and storage lifetime

Abstract

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 (n D /n E 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 o C) applying 60 minutes incubation time for pH 7. The complexes with the molar ratio n D /n HRP : 10 and n D /n GOD : 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 o C; which is considered as a good feature for the usage in practice.

Non-covalent conjugation of cutinase from Fusarium sp. ICT SAC1 with pectin for enhanced stability: Process minutiae, kinetics, thermodynamics and structural study

“…The changes in K m and V max values are due to alterations in the 3-D structure of enzymes leading to modification of the active site of the enzyme or loss of enzyme flexibility. The reduced affinity of cutinase complex with all the tested polysaccharides might be due to the formation of compact and supramolecular rigid structure generated by the interference with polysaccharide [13,22]. Similar findings were reported by Jadhav et al [14] during stabilization of alcohol dehydrogenase with gum Arabic by noncovalent interactions, and Singh et al [18] during interaction of β-cyclodextrin and polyphenol oxidase interactions.…”

“…Cutinase obtained from Fusarium solani was thermally stabilized with trehalose which was attributed to delayed reversible thermal denaturation brought about by shifting the equilibrium between native and denatured towards native state [6,12]. Further, conjugation of glucose oxidase and horseradish peroxidase with dextran [13], β-glucuronidase to polysaccharides [15], alcohol dehydrogenase with dextran and gum Arabic [14] enhanced thermal and pH stability. Pectin has been previously reported not only Page 5 of 52 A c c e p t e d M a n u s c r i p t 5 to have a prominent role in aggregation of glucoamylase and folding but also increase its thermal stability and reusability [19].…”

“…The cutinase solution (1 mg/ml) was mixed with equal volume of polysaccharide solution, mixed gently by inverting the tubes and incubated at 15±2°C for 16 h to interact with each other [13]. The cutinase-polysaccharide solution mixture so formed was used for thermal stability.…”

“…Enzyme stabilization by covalent or non-covalent interaction with polysaccharides is a simple, efficient and widely used strategy that has shown to produce significant improvements in the kinetic stability of enzymes [12][13][14][15]. Such approach augments enzymes stability (pH, thermal, kinetic) at both storage and reaction conditions by providing protective shell in the surrounding vicinity.…”

“…However, covalent conjugation needs laborious procedures to chemically modify the polymers, for instance, oxidation to get aldehyde groups, as per specific requirements [13,16,17].…”