Stabilization of a raw starch saccharifying amylase from Aspergillus carbonarius using anionic and cationic polysaccharides

Amylases are applied in numerous industries, including bio-fuel for hydrolysis of starchy substrates to fermentable sugars but their use is limited due to instability under reaction environments. Chemical modification is a simple method of protein stabilization, highly successful when properly designed and optimized. Carboxymethyl cellulose (CMC), dextran, chitosan and β-cyclodextrin were used to modify a raw starch saccharifying amylase produced from Aspergillus carbonarius. Modified lysine residues was determined using trinitrobenzene sulphonic acid method, the phenol sulphuric acid method was used to determine increased carbohydrate content, and fluorescence measurements were carried out using LS 55 spectofluorimeter. Amylase activity assay was by the dinitrosalicylic acid method. Use of β-cyclodextrin resulted to the highest number (10) of modified lysine residues and increased carbohydrate content of 80.8%. While the optimum pH of chitosan and β-cyclodextrin was 5, same as the native enzyme, modification with CMC and dextran altered the pH to 7. pH stability of the modified amylases increased, and most acid stable was the CMC-modified. Using chitosan and dextran, optimum temperature increased from 30^oC to 60 ^oC, and using β-cyclodextrin it was 40^oC.  Most thermostable was the chitosan modified amylase. Modification led to the alteration of the kinetic constants of the amylase. Increase in spectra mass was observed in all cases of polysaccharide modification. This is probably due to conformational changes in enzyme structure which resulted in aromatic residues been situated in non-polar, less exposed aqueous environment.