| In order to obtain the species with high extracellular laccase-productivity, colorful reactions of 13 species of ectomycorrhizal fungi were studied by using the selective method of guaiacol. At the same time, conditions for laccase-production and characteristic of laccase from the fungi which was the best fungi for laccase production——Amanita vaginata were studied too. And the oxidation of phenol by A. vaginata and its laccase was also tested. Effect of phenol on the biochemical change of pines inoculated with or without Amanita vaginata was evaluated. The main points of this dissertation are as follows:1. The selection of laccase production strainThe study of selective method of guaiacol found that 8 species of ECMF (A. vaginata, Suillus lactifluus, Spathularia flavida, Boletus luridus, Suillus laricinus, Phylloporus bellus, Suillus bovinus and Tian Taishan 5# (unidentified strain)) had the ability to produce extracellular laccase. The study of spot tests for laccase enzyme and determination of laccase-activity showed that A. vaginata was the best one in 13 species of ectomycorrhizal fungi to produce laccase, with activity of 13.25 U/L on the eleventh day.2. Effects of culture conditions on laccase production by A. vaginataThe activity of laccase from A. vaginata reached highest at 25℃, with 17.94 U/L on the thirteenth day. And it reached 12.90 U/L (highest) on the eleventh day, when PDA liquid medium was used as culture medium. The maximum activities of laccase and mycelial dry weight were obtained, when glucose and peptone were used as carbon and nitrogen sources respectively, C/N ratio was 4:1, and the initial pH value of medium was 6.0. The activity of laccase increased when Cu2+ (0.5 mM) and Mg2+ (5 mM) existed.3. The enzymatic characteristic of temperature and pH for laccase from A. vaginataEnzymatic characteristic of laccase from A. vaginata indicated that the optimal reaction temperature was 25℃and the optimal reaction pH value was 3.0. The enzyme had a high thermal stability: residual activity was 58% of the origin after 60 min at 65℃; and after 20 min at 75℃, the activity was only left 4% of the origin. And the laccase showed a high stability in acidic conditions: residual activity was above 75% of the origin after 120 min at pH 2.0, 3.0, 4.0; and it was reduced as time prolonged at pH 5.0, 6.0; after 15min at pH 7.0, the activity was only left 8.5% of the origin. 4. The oxidation of phenol by A. vaginata and its laccaseThe growth effect, tolerance and laccase activitiy of A. vaginata under different concentrations of phenol were studied at the condition of pure culture. The oxidation ratios of phenol by A. vaginata and its laccase were also examined. The results showed that various phenol concentrations had inhibition on the growth of studied ectomycorrhizal fungi and the secretion of laccase. The inhibition increased as the concentration of phenol increased. The oxidation ratios of phenol by A. vaginata and its laccase increased as time prolonged. And the oxidation ratios decreased with the increasing of phenol concentration.5. Effect of phenol on the biochemical change of pines inoculated with or without A. vaginataPine (Pinus tabulaeformis) were grown in pots containing sterilized soil with six phenol levels (0, 100, 200, 300, 400 and 500 mg/kg) and inoculated with or without ectomycorrhizal fungi——A. vaginata. The results showed that mycorrhizae inoculation promoted plants growth including height, diameter at ground level, dry weight and root activity of the seedling under the existence of phenol. The height of mycorrhizal seedlings was promoted 8.5% compared to non-mycorrhizal pines at 0 mg/kg of phenol, the diameter at ground level was promoted 7.78% at 200 mg/kg of phenol, the dry weight of mycorrhizal seedlings was promoted 20.29% at 200 mg/kg of phenol, and the root activity of mycorrhizal seedlings was higher than non-mycorrhizal ones, which was promoted 95.02% at 400 mg/kg of phenol. Chlorophyll content, proline content, SOD, POD and PPO activity of mycorrhizal seedlings were higher than that of non-mycorrhizal seedlings. The Chlorophyll content and POD activity of mycorrhizal seedlings were higher than that of non-mycorrhizal ones, which were promoted 21.67% and 114.37% respectively, at 400 mg/kg phenol. The proline content and SOD activity of mycorrhizal seedlings were promoted 191.22% and 90.4% respectively, which was compared to non-mycorrhizal pines, at 0 mg/kg of phenol. PPO activity of was promoted 45.42% at 100 mg/kg of phenol for mycorrhizal seedlings, which was compared to non-mycorrhizal ones.
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