Production And Immobilization And Application Of Catalase From Thermoascus Aurantiacus

Extracellular catalase secreted by Thermoascus aurantiacus was studied in this thesis.Catalase can decompose H₂O₂ to H₂O and O₂ efficiently.It is widely used in food disinfection,clinical analysis, medical diagnosis and industry of textile,paper and pulping.In this thesis, production conditions of catalase by Thermoascus aurantiacus were optimized,Catalase in the broth was primary purified for enzymatic properties study and immobilization.Macroporous adsorption resins were chosen as the immobilized carrier,and the immobilization conditions,the properties of immobilized catalase and its application in removing H₂O₂ were studied.The shake flask fermentation experiments showed that,in seed incubation period,corn starch and peptone were the best carbon and nitrogen source respectively.Optimized conditions were:200rpm rotation rate,liquid load was 200mL per 500mL flask.In basic fermentation period,10m/L ethanol and 20g/L dextrin and 1Og/L peptone were used as carbon source and nitrogen source.Optimized conditions were:initial pH of medium was 7.0,liquid load was 90mL/500mL,and rotation rate was controlled by stages,the maximum catalase activity was up to 2267U/mL.In 3L fermentor,the optimum condition was 6%(v/v) inoculum size,200rpm agitation rate.After 24h incubation,2mL ethanol (16mL total volume) were added once 12h,and the fermentation time was extended to 116h,maximum enzyme activity was up to 2532U/mL.A filtration-ultrafiltration-ammonium sulfate salting-out -dialysis process were used for the primary purification of catalase in the broth,the enzyme recovery and protein recovery were 59.3%and 30.2% respectively,the final enzyme specific activity was 4608U/mg,the purification fold was 1.96.The enzyme's optimum catalysis reaction conditions were 30℃,pH7.0,and its thermal and alkali resistance were good.The storage stability of purified enzyme was enhanced,when proper glycerol or gelatin was added into the ultrafiltration enzyme liquid.AB-8 macroporous adsorption resin was chosen to be the catalase immobilization carrier.Glutaraldehyde was the cross-linking agent. Optimum immobilization conditions:35℃absorption temperature,6h absorption time,970U/g(carrier) enzyme dosage at pH7.0,and 0.2% glutaraldehyde,2h cross-linking time,at 4℃.45.2%enzyme recovery was obtained.The optimum reaction condition for immobilized catalase was 35℃at pH7.0.Both the thermal and alkali resistance of immobilized catalase were enhanced,operation and storage stabilities was promoted since remaining enzyme activity was 60%after 10 times uses.Both free catalase and immobilized catalase have high efficiency in removing H₂O₂, while they were able to remove H₂O₂ completely in 20min.