Production, Purification And Application Of Ligninolytic Enzymes From Phanerochaete Chrysosporium

Three ligninolytic enzymes, lignin peroxidase(LiP), manganese peroxidase(MnP) and glyoxal oxidase(GLOX) are produced simultaneously by Phanerochaete chrysosporium. Their productivity varied under different culture conditions such as compositions of medium and O2 concentration in overhead air atmosphere.In this thesis, we first studied on GLOX production by cultivating Phanerochaete chrysosporium in C-limited medium in shaking flasks, and optimized the fermentation condition. Then, we studied on producing Lip and MnP and GLOX in bubble column bioreactior. After purification, the ligninolytic enzymes were covalently immobilized on polyurethane foams. Last, the immobilized enzymes was applied to catalyze asymmetric synthesis of sulfoxides from thioanisole.In our study, GLOX could be synthesized by C- and N-limited medium, but it was synthesized more effectively in C-limited medium by the immobilized mycelium on the polyurethane foams. The GLOX's substrates such as methylglyoxal and glyoxal could not induce the synthesis of GLOX, but veratryl alcohol and benzyl alcohol could do well.We found that Fe²⁺ and Cu²⁺ could not but Mn²⁺ could enhance GLOX production. The optimal Mn²⁺ concentration was 2.96×10^-5mol/L. GLOX could be synthesized in air atmosphere, but its productivity was lower than by flushing O₂. The activity reached 29.1U/L in shaking-flask culture after optimization. We found it was more suitable for GLOX production in C-limited medium in bubble column than in shaking flasks, where the same enzyme-producing curve was observed. In the optimal air flux of 1.0 vvm, the maximum GLOX activity appeared on the 5th day of culture. Comparing the effect of O₂ concentration in overhead air atmosphere, we found that 50%⁸0% O₂ could enhance GLOX production by about 20%. GLOX could be produced repeatedly in repeated batch cultures in bubble column. Immobilized P. chrysosporium mycelium could produce LiP and MnP in N-limilted medium in bubble column. With a flux of 0.75vvm of 80% O₂-rich air, the maximum activity of LiP and MnP was 311.8U/L and 68.2U/L, respectively.After ultrafiltrating and salting-out the C-limited broth(GLOX solution), GLOX activity and specific activity reached 233.6 U/L and 283.7 U/g, increasing by 9.6 folds and 1.8 folds respectively. N-limited broth(LiP and MnP solution) was treated by ammonium sulphate salting-out followed by DEAE-Sepharose ion exchange chromatography. After that purification, LiP and MnP activity reached 1137.5U/L and 241.0U/L, being purified by 3.8 folds and 3.0 folds respectively. SDS-PAGE showed the molecular weight of LiP and Mnp was about 42 kDa and 47 kDa respectively, which were the same as reported.As glycosylated enzymes, GLOX, Lip and Mnp could be covalently immobilized on polyurethane foams, their activity recovery yield was 25.6%, 39.1% and 21.4% respectively. Usig enzyme mixture solution and taking advantage of distinctive difference between activity of Lip and Mnp at different pH, we found that LiP catalyzed thioanisole to (R)-sulfoxide with enantiomeric excess of 30.7% and yield of 28.4%; whileas MnP catalyzed thioanisole to (S)-sulfoxide with a yield of 20.8% and e.e. of 36.2% at optimal condition.