Researches On High-efficient Preparation By Microwave Method And Characterization Of Immobilized Trypsin

Immobilized enzyme technology is a technology which developed in 1960 s and could overcome the weaknesses of free enzyme such as poor stability,high cost and low purity products.Immobilized enzymes have showed important application value in many fields.However,the preparation process of immobilized enzyme usually takes long time from hours to days.Nowadays,as the requirement of people's life level is getting higher and higher with the development of science and technology,time-consuming and complicated ways and methods to solve problems are becoming obsolete.People's life style is more and more convenient because of the rapid development of quick determination technologies.Therefore,to explore an efficient preparation method of immobilized enzymes is necessary to satisfy the needs of production and application.In recent years,microwave as an efficient method has been widely applied to scientific researches.But so far few researches have focused on preparing immobilized enzymes under microwave irradiation at home and abroad and to our best knowledge no studies have been done on trypsin.Therefore,immobilized trypsin was prepared by microwave-assisted immobilization method in the present study.The present study includes four parts:(1)The conditions of preparing immobilized trypsin under microwave irradiation with ES-105 resins as the carrier were optimized by Box-Behnken response surface method(RSM).The optimum immobilization conditions were obtained as follows: addition amount of trypsin,pH value,microwave temperature and microwave time were 41 mg per gram resin,6.2,30 ? and 10 min,respectively.The activity of the preparative immobilized trypsin was 322.6 U per gram resin.The immobilized trypsin was stable under the condition of 25 ?~40 ? as well as pH range was 5~7 or 9~10.The immobilized trypsin still could retain 63.2% of its initial activity after reused for 9 cycles.The immobilized trypsin could keep 64.6% of its initial activity with storage at 4 ? after 5 weeks.(2)The conditions of preparing immobilized trypsin under microwave irradiation with ESR-5 resins as the carrier were also optimized by Box-Behnken response surface method(RSM).The optimum immobilization conditions were obtained as follows: addition amount of trypsin,glutaraldehyde concentration,pH value,microwave temperature and microwave time were 26 mg per gram resin,0.53%,9.1,35 ? and 5 min,respectively.The activity of the preparative immobilized trypsin was 390.8 U per gram resin.The immobilized trypsin was stable under the conditions of 25 ?~45 ? and pH range was 8.5~10.0.The immobilized trypsin still could retain 86.7% of its initial activity after reused for 11 cycles,and could keep 63.7% of its initial activity with storage at 4 ? after 5 weeks.(3)The conditions of preparing immobilized trypsin under microwave irradiation with D151 resins as the carrier were optimized by central composite design-response surface method(CCD-RSM).The optimum immobilization conditions were obtained as follows: adsorption time,addition amount of trypsin,glutaraldehyde concentration,pH value,microwave temperature and microwave time were 1.5 h,81 mg per gram resin,1.0%,5.0,29.0 ? and 3.5 min,respectively.The activity of the preparative immobilized trypsin achieved 221.5 U per gram resin.The immobilized trypsin was stable under the conditions of 25 ?~45 ? as well as pH range was 4.0~5.0 or 9.0~10.0.The immobilized trypsin still could retain 73.1% of its initial activity after reused for 8 cycles and could keep 71.1% of its initial activity with storage at 4 ? after 5 weeks.(4)Furthermore,three immobilized trypsin were applied to hydrolyze casein and perilla protein to investigate their enzymatic hydrolysis properties.Results showed that the hydrolysis products of these three immobilized trypsin have a few differences,but no significant differences.In conclusion,the immobilized trypsin with ESR-5 resins as the carrier had the highest enzyme activity and the least amount of enzyme loss.In addition,its operational stability was better than the other two immobilized trypsin,indicating the immobilization way by Schiff chemical reaction with cross linking resins with trypsin was more stable.Its temperature stability was better than immobilized trypsin with ES-105 resins as the carrier,and its pH and storage stabilities showed no significant differences with the others.The hydrolyzed results of casein and perilla protein were similar.Above all,the immobilized trypsin with ESR-5 resins as the carrier was the best among the three immobilized trypsin.