| The concept of a novel immobilized enzyme (called "recorded enzyme") is first described by this dissertation. Compared to the ordinary immobilized enzyme, a ligand is added into preparation process to enduce enzyme conformation and the final enzyme conformation is rigid. Then its preparation process and the catalytic characteristic in water and organic solvent were investigated, SEM was utilized to straight token the state of recorded enzyme in the end.The first part of this dissertation emphasizes on exploring the process of recorded enzyme preparation. Lipase and lauric acid were added into a copolymer system, which contained four different kinds of PEG200(400)-di(meth)acrylate ester and trimethylolpropane trimethacrylate. The polymerization was initiated by ultraviolet irradiation and lauric acid was extracted by neat organic solvent. Then four recorded lipase polymers of locking lipase conformation could be obtained. When actone was selected for washout solvent, the maximal extraction rate of 86.31% can obtained, as well as the leak of lipase was keep in a small range. When function monomer and crosslinked agent were mixed by the scale of 2:1 (mass scale) and irradiation time was 45s, four recoreded lipases shown the excellent resistant to swelling behavior in six common solvents, especially in water and heptane, which is full consistent with the anticipant assumption for recored lipase carrier.The second part presents the catalytic characterization of recorded lipase in water and organic solvent. The activity of four recoreded lipases in water was obtained by olive oil hydrolyzation. Theirs optimization reaction time was 15 min, and the best activity (6.07 U/g) was attained by Da (PEG400-diacrylate as function monomer), Its activity yield was 171%. At the same time, the results shown that the monomer containing methyl could easily form super branch polymer, which will increase the mass-transfer barriers lead to the decrease of activity and yield. In addition, theirsstability were enhanced greatly. Recorded lipases were able to retain more than 75 %> 50 % and 70 % of their initial activities under the circumstance of strong acid(pH=2.40^ strong alkaline(pH= 12.00) and high temperature(80°C), respectively. Moreover, Because of the thermal unstable of carrier from 153°C, Da inactivate around 160°C. After immersing recorded lipases into precipitation denaturants for 30 days, recorded lipases were able to retain at least 50 % of their initial activities. All these illuminate the rigidity conformation is advantageous for the activity and stability when the catalytic reaction occur in water. The activity in organic solvent (heptane) was measured by esterify reaction of lauric acid and n-butanol. Da was selected for model for its high activity. The ativity was 14.46 U/g for 24h at 40 °C and activity yield was only 33.72%. Rising temperature could up activity and yield to 16.42 U/g and 43.52%, respectively. In the further research displayed that added the pore-causing solvent (toluene) to reaction system for decrease mass-transfer barriers was no sense, because toluene would make lipase inactivate. But add water into heptane was able to enhance enzyme activity. When the content of water was l%(v/v), the recorded lipase activity reach the best activity 38.23 U/g after catalyzing 24h, which was 2.64 times compared to anhydrous system. If continued to increase water content, the esterify activity decrease sharply and inactivate at the content of 10%(v/v). All these further shown that the flexibility exploded by water was crucial to the lipase activiy. The best activity and stability can be obtained by keep the lipase active conformation and increase some flexibility at the same time. The reaction mechanism of recorded lipase according to the Ping-Pong theory, the equation of,. . ... , 20.390[A][B]kinetics could be express by v =3It was found that Da also has adsorptive ability to lauric acid. Its equilibrium adsorption amount was 30.22(mg-g"1) at 40°C, which was 1.35 times compared to 30 °C, and its absorption yield was 70.46%. The adsorption behavior accorded with Langmuir law, its kL-0.024(mg>g"1-min'1); The pore-causing solvent (toluene) was advantageous for the adsorption amount to 40.04(mg-g"1) and yield to 93.35%, here kL=0.033(mg-g"1-min'1). But water was unamiable to adsorption process. When thewater content was only 0.5% (v/v) at 40°C, the adsorption amount decrease about 1 times, when up to 5%(v/v), there are no adsorption ability exist. This phenomenon is ascribed to the water alter the rigidity action point lead to weaken the adsorption.The last part refers to the direct token of the surface of Da by SEM. Some exiguous power existed in truncation surface and pore interior. Its size was ten or some tens times of lipase molecular. But these powers can not be found in carrier surface. It was speculated that these powers may be the polycoin of Upases.
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