To investigate the effects of several carbohydrates on the biodegradation of lignin by Pleurotus ostreatus, the strain was cultured on the media formulated with individually added carbohydrates such as cellulose, cellobiose, glucose, xylan, and xylose. Fractionation of the lignin biodegradation products by Sephadex G-75 column chromatography revealed that polymerization was more apparent rather than depolymerization in the absence of carbohydrates. However, depolymerization and lignin biodegradation were promoted by the addition of carbohydrates.
Several inducers such as gallic acid, ferulic acid, 2,5-xylidine, cycloheximide and puromycine were added respectively for the higher production of polyphenol oxidase and it was found that ferulic acid was the most effective inducer.
Extracellular polyphenol oxidase was purified by ammonium sulfate fractionation, protamine sulfate precipitation, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 gel permeation chromatography. The molecular weight of the enzyme was estimated to be 58,000 by SDS-polyacrylamide gel electrophoresis, and the isoelectric point was 3.75. The optimum temperature for the enzyme activity was about 45℃, and the pH optimum was 6.5. The enzyme was found to be stable at temperature below 35℃ and able to oxidize various phenolic compounds containing at least one hydroxyl group regardless of its position.
After the reaction of ferulic acid with the purified polyphenol oxidase, the reaction products were separated by TLC and HPLC and analyzed by H-NMR, Mass and IR spectroscopy. The products were found to be styrenoid dimer with two benzene ring called 2-(3´-hydroxy-4´-methoxyphenyl)-4-hydroxy-5-methoxystyrene and styrenoid trimer.
Glucose oxidizing enzymes were purified using Sephadex G-200 chromatography, DEAF-Sephadex A-50 ion exchange chromatography and CM-Sephadex chromatography. Glucose dehydrogenase and glucose oxidase activity were detected in the same position during the column chromatography, and polyacryamide gel electrophoretogram indicated that one enzyme showed bifuctional activities. The optimum temperature for their activities was about 60℃ and the pH optima for glucose oxidase and glucose dehydrogenase activity were 4.5 and 6.0 respectively. The activities of these two enzymes showed different heat stability: the half life of oxidase activity was 2 hours at 40℃, while dehydrogenase was 1 hour at 80℃.
In order to investigate the function of glucose oxidase and glucose dehydrogenase, the reaction mixture of polyphenol oxidase, glucose oxidizing enzymes and ferulic acid was incubated in the presence and absence of O₂ and the reaction products were also analyzed by TLC. In the presence of O₂, the substrate was converted synergetically to the products when glucose oxidizing enzymes were added with polyphenol oxidase and ferulic acid. In the view point of electron tranfer system involved in lignin metabolism, the results suggest that the polymerization of lignin was promoted by supplying H₂O₂ from glucose oxidase to polyphenol oxidase-glucose oxidizing enzyme system in the presence of O₂. In the absence of O₂, however, no reaction can proceeded due to the inhibition of polyphenol oxidase.
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