Prodction, Purification And Immobilization Of A Thermoalkaliphilic Catalase From Thermoascus Aurantiacus

Catalase (EC 1.11.1.6) which presents in all aerobiccellsdecomposes hydrogen peroxide to molecular oxygen and water. It hasbeen applied in a wide field such as bleaching streams in textile and paperindustry, milk disinfection and clinical analysis. In this study, the mediumcomponents of Thermoascus aurantiacus were optimized and theelectrophoresis-grade enzyme was obtained after three prurification steps.The catalase was immobilized onto chitosan beads by the covalentbinding. The immobilized catalase showed higher pH stability andthermostability than the free catalase. And the properties of immobilizedcatalase and its application in removing H2O2 from bleaching streamswere studied. The results showed that this catalase has a potentialapplication in removing residual hydrogen peroxide in industrialbleaching processes.After three step treatments with (NH4)2SO4 fractionnal precipitation,salting-out and DEAE-Sepharose anion-exchange column, athermoalkaliphilic catalase from Thermoascus aurantiacus was purified.We gained 41.3% of the total activity and the specific activity of thecatalase was 4789 U/mg with 4.31 purification fold. The catalasedetermined by SDS-PAGE was composed of two identical subunits andhad a molecular mass of 190 kDa. The Km value for the purified catalase was 40.10 mmol·L-1 and the k2 value was 1.57×104μmol·(min mgprotein)-1. The catalase was highly active over a temperature range from25 to 75℃and a pH range from 7 to 13, having the optimum activity at65℃and pH 12. These results demonstrated that the catalase isthermoalkaliphilic.The immobilized catalase was immobilized onto chitosan supportusing glutaraldehyde as cross-linking agent. The Optimumimmobilization conditions for the immobilization were as follows: 250mg chitosan, cross-lingking temperature 40℃, reaction time 1h, 40ml0.75% glutaraldehyde, catalase 2000 U. The immobilized catalase activitywas 1700 U/g and the acitivity retaining yield of immobilized enzymewas 21.25%. The optimum temperature and pH of the immobilizedcatalase was 75℃and 12, respectively. It retained about 55% of its initialactivity after 13 repeated cycles during the total 130 min reaction time.