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漆酶是一种分布广泛的多酚氧化酶,在食品、能源和环保等领域具有重要的应用价值.灵芝作为重要的白腐真菌,可以合成分泌包括漆酶在内的多种木质纤维素降解酶,这些酶的活性会随着菌株、发酵条件等不同而不同.以灵芝荣保1号为材料,利用平板实验分析4种重金属(Pb~(2+)、Cd~(2+)、Cu~(2+)和Fe~(2+))在不同浓度下对灵芝菌丝体生长及氧化带形成的影响,用分光光度法测定重金属发酵液中漆酶的活性,并通过实时定量PCR检测重金属离子胁迫下灵芝漆酶同工酶表达的差异性和特异性.结果表明:低浓度Pb~(2+)和Cu~(2+)促进菌丝体生长和氧化带形成,而Cd~(2+)和Fe~(2+)抑制菌丝生长及氧化带形成;Pb~(2+)、Cd~(2+)、Cu~(2+)和Fe~(2+)分别以终浓度为100、20、100、100 mg/kg时对菌丝生长及氧化带的增强作用较大,培养至第11天、第9天、第7天、第7天时,漆酶活性达到最大值,分别为20.83±0.32、15.89±0.21、30.56±0.42、9.44±0.25 U/L,Cu~(2+)促进了发酵液中漆酶酶活,而Pb~(2+)、Cd~(2+)和Fe~(2+)则抑制漆酶酶活.荧光定量PCR结果显示,4种重金属离子在液体培养条件下对15个灵芝漆酶同工酶的转录表达影响存在差异性,其中5个漆酶基因(Glac4、Glac6、Glac10、Glac11和Glac12)转录表达发生上调.综上,灵芝漆酶对重金属离子作用的不同响应表明其具有较复杂的生理功能及调控机制,结果可为进一步阐明漆酶作用机制提供基础.
Laccase is a widely distributed polyphenol oxidase with important applications in food, energy and environmental protection, etc. As an important white rot fungus, Ganoderma lucidum can synthesize and secrete many kinds of lignocellulose, including laccase, Enzymes activity of these enzymes will vary with strains, fermentation conditions, etc. With Ganoderma lucidum No. 1 as the material, four kinds of heavy metals (Pb 2+, Cd 2+, Cu ~ (2+) and Fe ~ (2+)) on the growth of mycelia and the formation of oxidative bands in different concentrations of mycelium, the activity of laccase in heavy metal fermentation broth was determined by spectrophotometry and detected by real-time quantitative PCR The results showed that low concentrations of Pb 2+ and Cu 2+ promoted mycelial growth and oxidative band formation, whereas Cd ~ (-) 2 +, Cd 2+, Cu 2+ and Fe 2+ in the mycelial growth and oxidative band respectively. The final concentration of Pb 2+, At 100, 20, 100 and 100 mg / kg, the mycelial growth and the oxidation zone were greatly enhanced. When cultured to the eleventh day, the ninth day, the seventh day and the seventh day, the laccase activity reached the maximum, Respectively 20.83 ± 0.32,15. 89 ± 0.21, 30.56 ± 0.42 and 9.44 ± 0.25 U / L, respectively. Cu 2+ promoted laccase activity in the fermentation broth, but Pb 2+, Cd 2+ and Fe 2+ +) Inhibited the activity of laccase.Fluorescence quantitative PCR results showed that the four heavy metal ions had different effects on the transcriptional expression of 15 Ganoderma lucidum laccase under liquid culture conditions, of which 5 laccase genes (Glac4, Glac6, Glac10, Glac11 and Glac12) .In summary, different responses of Ganoderma lucidum laccase to heavy metal ions show that they have more complex physiological functions and regulatory mechanisms, and the results may provide a basis for further elucidation of laccase mechanism.