Optimization of Growth Condition and Laccase Production by White Rot Fungi

• Main Authors: I-hsi Sai, 賽逸昕
• Other Authors: W.L.Chao
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/c7djn7

碩士 === 東吳大學 === 微生物學系 === 93 === White rot fungi produce three main extracellular enzymes involved in ligninolysis, including laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP). These enzymes are capable of initiating the oxidation of lignin by a free-radical mechanism which also led to the degradation of a wide variety of normally very recalcitrant environmental pollutants. Laccase is a glycosylated polyphenol oxidase which contains four copper ions per molecule and has shown good potential to be used in bioremediation, biopulping, biobleaching, medicine, and textile industry. Forty-five isolates of white rot fungi were screened for their ability to degrade congo red. Based on the decoloration ability, twelve isolates were selected for further study. They were grown in PDB and phosphate buffer culture medium (PBCM) for laccase activity assay. Five isolates were chosen for their high level of laccase production (＞10000 U/L). In order to find out the best nitrogen source for laccase production, we have tested 7 different nitrogen sources in PBCM. With yeast extract and NH4Cl as nitrogen source, isolate TFRI 707 and A 8 have shown the highest laccase production, respectively. When study the effect of temperature on their growth, the data indicated that TFRI 707 and A 8 had higher growth rats on 40 ℃ and 30 ℃respectively. Various factors, including pH (3.5-5.5), metal ions (Mg, Mn, Ca, Cu, Fe, Zn) and inducer (ethanol, veratryl alcohol, ferulic acid, 2,5-xylidine) on laccase production were also studied. The optimal pH for laccase production was 5.5 for both isolates. When Cu supplement was deleted from the medium, decreases in laccase production were observed for both of them. As for the inducers, only veratryl alcohol had a substantial effect on laccase production. With ABTS as the substrate, laccase has an optimum activity at pH 3, and an optimum temperature at 30 ℃. In liquid and semi-solid medium, isolate A8 produce 3 and 4 isoenzymes of laccase, respectively, while 4 and 3 isoenzymes were detected for isolate TFRI 707. Molecular weight was determined using Native PAGE, and laccases secreted by isolate A 8 were between 50-90 kDa, and those excreted by TFRI 707 were between 40-50 kDa. When assaying with SDS-PAGD, the molecular weight of laccases secreted by A 8 were between 50-60 kDa and by TFRI 707 were between 30-40 kDa.