煤附生真菌产漆酶菌株的分离鉴定及产酶特性研究

Identification and production of laccase-producing fungal strainisolated from coal

从煤炭样品中筛选到一株产漆酶活性菌株,经菌体形态观察和ITS序列分析,鉴定为Trichoderma asperellum W03。菌株所产漆酶的最适反应pH为3.5-4.5,最适反应温度45℃,类似于白腐真菌漆酶。液态发酵条件的均匀设计实验表明,适宜的发酵培养基组成为:土豆200.00g/L、葡萄糖9.36g/L、米糠粉37.44g/L、硝酸钾4.00g/L、KH2PO43.20g/L、MgSO4·7H2O2.00g/L、CuSO4·5H2O0.005g/L、初始pH8.0;在33℃、180r/min、50mL/250mL的摇瓶培养条件下,棘孢木霉W03在孢子接种培养后48h、84h产酶量较高,分别处在菌体的快速生长期和衰亡期;菌体产酶受Cu2+、联苯胺诱导,而受1-萘酚、愈创木酚和2,4-D抑制。

Laccase-producing strain screened from low-rank coal was identified as Trichoderma asperellum W03 according to its mycelia morphology and ITS sequence analysis. The laccase has an optimum pH of 3.5-4.5 and an optimum temperature of 45℃, which is similar to that from white rot fungi. Components of laccase-producing medium （g/L） optimized by uniform design were 200.00g/L potato, 9.36g/L glucose, 37.44g/L rice bran powder, 4.00g/L KNO3, 3.20g/L KH2PO4, 2.00g/L MgSO4·7H2O and 0.005g/L CuSO4·5H2O. The peak of laccase production appeared in the 48h and 84h corresponding to the logarithmic phase and decline phase, respectively, when Trichoderma asperellum W03 was submerged in shaking-flask with liquid volume 50mL/250mL under constant temperature of 33℃ and vibration of 180r/min. The laccase production in Trichoderma asperellum W03 was significantly improved by addition of some substance such as potato extract, rice bran, Cu2＋ or benzidine in the medium, butobviously inhibited by 1-naphthol, guaiacol and 2,4-D. In addition, the suitable initial pH for laccase production was 8.0.