Quantitative fluorometric analysis of the protective effect of chitosan on thermal unfolding of catalytically active native and genetically-engineered chitosanases

We have taken advantage of the intrinsic fluorescence properties of chitosanases to rapidly and quantitatively evaluate the protective effect of chitosan against thermal denaturation of chitosanases. The studies were done using wild type chitosanases N 174 produced by Streptomyces sp. N 174 and SCO produced by Streptomyces coelicolor A3(2). In addition, two mutants of N 174 genetically engineered by single amino acid substitutions (A 104L and K 164R) and one consensus (N 174-CONS) chitosanase designed by multiple amino acid substitutions of N 174 were analyzed. Chitosan used had a weight average molecular weight (Mw) of 220 kDa and was 85% deacetylated. Results showed a pH and concentration-dependent protective effect of chitosan in all the cases. However, the extent of thermal protection varied depending on chitosanases, suggesting that key amino acid residues contributed to resistance to heat denaturation. The transition temperatures (T,,,) of N174 were 54 degrees C and 69.5 degrees C in the absence and presence (6 g/I) of chitosan, respectively. T,, were increased by 11. 6 degrees C (N 174-CONS), 13.8 degrees C (CSN-A 104L), 15.6 degrees C (N 174-K 164R) and 25.2 degrees C (SCO) in the presence of chitosan (6 g/l). The thermal protective effect was attributed to an enzyme-ligand thermostabilization mechanism since it was not mimicked by the presence of anionic (carboxymethyl cellulose, heparin) or cationic (polyethylene imine) polymers, polyhydroxylated (glycerol, sorbitol) compounds or inorganic salts. Furthermore, the data from fluorometry experiments were in agreement with those obtained by analysis of reaction time-courses performed at 61 degrees C in which case CSN-A 104L was rapidly inactivated whereas N 174, N 174CONS and N 174-K 164R remained active over a reaction time of 90 min. This study presents evidence that (1) the fluorometric determination of T,, in the presence of chitosan is a reliable technique for a rapid assessment of the thermal behavior of chitosanases, (2) it is applicable to structure-function studies of mutant chitosanases and, (3) it can be useful to provide an insight into the mechanism by which mutations can influence chitosanase stability. (c) 2007 Elsevier B.V All rights reserved.