【作者】 李翠；

【导师】 唐明；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2010， 硕士

【摘要】 本研究采用愈创木酚筛选法从13种外生菌根真菌中筛选具有产漆酶能力的菌种,研究最佳产漆酶菌种的产酶条件和酶的特性、以及该菌漆酶对苯酚的氧化作用;同时研究苯酚胁迫下,接种最佳产漆酶菌种灰鹅膏菌(Amanita vaginata)对油松(Pinus tabulaeformis)苗木生理生化特性的影响。得出以下结论:1.产漆酶菌株的筛选采用愈创木酚筛选法筛选出灰鹅膏菌(Amanita vaginata)、乳黄粘盖牛肝菌(Suillus lactifluus)、黄地勺菌(Spathularia flavida)、褐黄牛肝菌(Boletus luridus)、灰环粘盖牛肝菌(Suillus laricinus)、美丽褶孔牛肝菌(Phylloporus bellus)、粘盖牛肝菌(Suillus bovinus)和天台山5#(未鉴定菌株)8种外生菌根真菌具有产漆酶能力;漆酶点试试验和漆酶活性大小测定结果表明以灰鹅膏菌产漆酶能力最佳,培养11 d时其酶活性为13.25 U/L。2.培养条件对灰鹅膏菌产漆酶的影响灰鹅膏菌产酶培养条件测定表明,温度为25℃时漆酶活性最高,培养13 d时高达17.94 U/L;在PDA液体培养基中酶活性最高,培养11 d时为12.90 U/L;以葡萄糖为C源、蛋白胨为N源、C/N为4/1、初始pH为6.0时,酶活性和菌丝体干重最高;添加0.5 mmoL/L Cu2+和5 mmoL/L Mg2+时,可增强酶活性。3.灰鹅膏菌漆酶的温度和pH酶学特性灰鹅膏菌漆酶特性研究表明,该酶最适反应温度为25℃、pH为3.0,具有较高的热稳定性,65℃保温60 min剩余酶活为58%;该酶在酸性条件下表现出较强的稳定性,在pH2.0、3.0、4.0保持120 min后,仍保留有75%以上的酶活性;pH5.0和6.0时漆酶活性随着时间的延长下降较快;pH7.0时保持15 min剩余酶活仅为8.5%。4.灰鹅膏菌及其漆酶对苯酚的氧化作用在纯培养条件下,研究了灰鹅膏菌对苯酚的耐受性和苯酚胁迫下漆酶活性,测定了灰鹅膏菌及其漆酶对苯酚的氧化率,结果表明:不同浓度苯酚对灰鹅膏菌生长和漆酶有一定的抑制作用,且随着苯酚浓度的增大抑制作用增强,灰鹅膏菌和其分泌的漆酶对苯酚的氧化率均随着时间的延长而增大,且随着苯酚浓度的升高呈现下降趋势。5.苯酚胁迫下接种灰鹅膏菌对油松生理生化的影响苯酚胁迫下,接种灰鹅膏菌增加了油松苗木的株高、地径、干重和根系活力,苯酚浓度为0 mg/kg时接种幼苗株高比对照时提高8.5%、苯酚浓度为200 mg/kg时地径提高7.78%、苯酚浓度为300 mg/kg时干重增加20.29%,苯酚浓度为400 mg/kg时根系活力增加95.02%;接种外生菌根真菌后,油松叶绿素含量、根部游离脯氨酸含量、SOD、POD和PPO活性均高于非菌根化油松幼苗,在苯酚浓度为400 mg/kg时接种油松叶绿素总含量比对照提高21.67%、POD活性提高114.37%,苯酚浓度为0 mg/kg时游离脯氨酸含量接种比对照提高191.22%、SOD活性提高90.4%,在苯酚浓度为100 mg/kg时接种灰鹅膏菌油松根部PPO活性比对照提高45.42%。更多还原

【Abstract】 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.更多还原