Thermal stabilization of amylolytic enzymes by covalent coupling to soluble polysaccharides

Abstract

The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application.

Unusual properties of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin

“…Improved thermal stability of enzymes immobilized on solid carriers has been observed by many researchers. Such improvement has been explained as the reduced mobility of the enzyme after immobilization, 7,8,40 in support of our conclusion.…”

Engineering of protein thermodynamic, kinetic, and colloidal stability: Chemical Glycosylation with monofunctionally activated glycans

“…More specifically, chemical glycosylation represents an often explored methodology to modulate protein stability (Broersen et al, 2004;Dellacherie et al, 1983;Lenders and Crichton, 1984;Levitsky et al, 1999;Srivastava, 1991;Vegarud and Christensen, 1975;Wartchow et al, 1995). However, mechanistic details of protein stabilization by glycans within these applications remain uncertain due to a lack of systematic and statistically meaningful exploration between the nature and amount of glycan bound to proteins, and their resulting physicochemical properties upon glycosylation.…”

A novel chemoenzymatic glycosylation strategy: application to lysozyme modification

“…The loss of enzymic activity after chemical modification is a phenomenon frequently encountered in the literature dealing with this topic [1,2,4,7,8,10,12,18,19]. It can be justified by enzyme conformational changes or steric hindrances to the enzyme-substrate contact provoked by modifiers, especially when large molecules are involved.…”

“…Among the many compounds which can be used as modifiers, glycosides are outstanding as regards both the number of reports and the improvements in enzyme functionality (i.e. thermostability) achieved [1][2][3].…”