Preparation Of MMS Carriers And Their Application In The Immobilization Of Enzymes

The technology of immobilization enzyme have been developing fast since 1960s. In the recent decades, research on usable immobilization carriers, for example, the magnetic microspheres (MMS) carrier has been focused much attention in this field. MMS consists of two parts, the magnetic nucleus and macromolecule shell. In this paper several novel MMS carriers are prepared by introducing some organic functional groups on the magnetic nucleus via simple chemical modification and entrapment, and their application in immobilizing laccase and lipase has been evaluated. Firstly, some new MMS composite carriers (M1-M5) were prepared. M1 was prepared by co-precipitation of ferrous and ferric hydroxides, M2 by co-precipitation of 3-mercaptopropioic acid, ferrous and ferric hydroxides, and M3 by co-precipitation of N-(aminoethyl)-3-mercaptopropoinamide, ferrous and ferric hydroxides. M4 was prepared by the entrapment with 3-aminopropyltriethoxysilane (APTES) and tetraethoxysilane, and M5 by the entrapment with APTES-MA and tetraethoxysilane. Secondly, M1, M2 and M3 were applied to immobilize laccase. The experimental results clearly show that the immobilization efficiency are 9.62%,62.1% and 64.1%, and the loading amount are 0.0415mg/g,0.457mg/g and 0.480mg/g, respectively for these three carriers. Among the three carriers, M3 composite carrier shows the best immobilization efficiency. Enzymatic research indicates that the laccase immobilized on M3 composite carrier shows the best activity in the buffer of pH 6.8 (7.4 for free enzyme), ion concentration of 25 mmol/L. However, the limit for M3 is that the activity of the laccase immobilized on M3 is low. In the experimental, laccase is obtained from one fungi strain that can produce laccase efficiently. The strain is screened from soil samples and named as L1. The effect of the culture conditions on activity of L1 producing laccase is studied, and the optimal culture conditions are: PYGM culture medium, phenol as a revulsant, the culture time 7 days and the temperature 30℃. The characterization of laccase produced from this fungus was also investigated, and the optimum pH was 8.0 and the optimum temperature 33℃. The chloride ion is observed to be an efficient inhibitor for this laccase. The laccase shows an appreciable activity for degrading phenol between the concentrations of 50-250 mg/L. Thirdly, M4 and M5 were applied for immobilizing PPL. The experimental results clearly show that the immobilization efficiency of PPL on M4 is above 86%, but the activity reclaim only 35%-50%. The immobilization efficiency of PPL on M5 is 88.1% and the activity reclaim 59.4%. The ideal immobilation conditions are as follows: the buffer: pH 7.0, the temperature: 20℃, the best stirring time: 90 min, and the optimal ratio of carrier to PPL about 100mg/g. Meanwhile the PPL immobilized on M5 shows high reaction activity in the buffer of pH 7.0 and the temperature of 37℃. Finally, the catalytic hydrolysis of 2-phenylpropyl acetate by PPL and PPL immobilized on M5 was studied. As a result, the reaction conversions are 53% and 40%, respectively for PPL and PPL immobilized on M5. An interesting result is that the e.e. value (76%) of the alcohol by PPL immobilized on M5 is higher than that by PPL (52%). Moreover, the catalytic activity of the PPL immobilized on M5 almost doesn't decline after cycled 4 times successively. All the results demonstrate that M1, M2 and M3 magnetic carriers are suboptimal to immobilize laccase. However, M4 and M5 carriers are efficient carriers for immobilizing PPL. Moreover, the performance of M5 is better than that of M4.