产木质纤维素降解酶真菌的筛选及产酶特性

Screening and enzymatic characterization of the fungal strains producing lignocellulose-degrading enzymes

摘要:

【背景】利用微生物处理秸秆引起研究者的广泛关注。【目的】筛选生长速度快、木质纤维素降解酶活性强的真菌菌株，用于植物秸秆降解和高效利用。【方法】从自然界采集的样品中分离纯化真菌菌株，利用PDA-愈创木酚和PDA-羧甲基纤维素钠平板初筛，再经过液体发酵检测漆酶酶活、羧甲基纤维素酶酶活及菌丝生长速率复筛目的菌株，通过内转录间隔区(internal transcribed spacer, ITS)测序法对目的菌株进行鉴定，对目的菌株产漆酶和羧甲基纤维素酶活力进行测定及酶学性质研究。【结果】从样品中分离纯化到18株真菌，通过初筛筛选出9株产木质纤维素降解酶真菌菌株，再经过复筛，筛选出一株产漆酶、羧甲基纤维素酶活力高、菌丝生长快的菌株M1，经过分子生物学鉴定M1为糙皮侧耳(Pleurotus ostreatus)，其漆酶酶活为(243.59±1.11) U/mL，羧甲基纤维素酶酶活为(36.03±0.63) U/mL。在5 d的培养期内，菌丝生长速率为(9.43±0.32) mm/d。对菌株M1的发酵粗酶液的酶学性质进行了检测分析，结果表明，所产的漆酶在pH 5.0-6.5相对酶活为90%以上，在pH 5.5的介质中最为稳定，在低于55 ℃环境稳定性较高，相对酶活90%以上；该菌所产羧甲基纤维素酶在pH 5.5-6.5相对酶活为90%以上，在pH 6.0的介质中最为稳定，在低于60 ℃环境相对稳定性较高，相对酶活90%以上。【结论】本研究筛选到的菌株M1具有较高的漆酶和羧甲基纤维素酶活性，具有降解木质素和纤维素的潜力，为植物秸秆的生物降解提供了有效的菌种资源。

Abstract:

[Background] Microbial degradation of straw has attracted increasing attention of researchers. [Objective] To screen out the fungal strains with fast growth and strong activities of degrading lignin and cellulose for the efficient utilization of straw. [Methods] The fungal strains were isolated from the samples collected in the natural environments. PDA-guaiacol and PDA-carboxymethylcellulose were used for preliminary screening and then the activities of laccase and carboxymethyl cellulose (CMCase) of the selected strains were determined by liquid fermentation. The target strain was obtained by re-screening based on mycelial growth rate and identified by sequencing of internal transcribed spacer (ITS). Furthermore, the laccase and CMCase activites of the target strain was determined and the enzymatic properties were studied. [Results] A total of 18 strains of fungi were isolated from the samples, among which 9 strains producing lignocellulose-degrading enzymes were selected through preliminary screening. After re-screening, strain M1 with high activities of laccase and CMCase and fast mycelial growth was screened out as the target strain and identified as Pleurotus ostreatus. The strain had the laccase and CMCase activities of (243.59±1.11) U/mL and (36.03±0.63) U/mL, respectively, as well as the mycelial growth rate of (9.43±0.32) mm/d during the 5 days of culture. The relative activity of laccase produced by strain M1 was the most stable at pH 5.5 and over 90% in a pH range of 5.0 to 6.5 and below 55 ℃. The relative activity of CMCase produced by strain M1 was the most stable at pH 6.0 and over 90% in a pH range of 5.5 to 6.5 and below 60 ℃. [Conclusion] P. ostreatus M1 had high laccase and CMCase activities and the potential of degrading lignin and cellulose. This study provides a promising fungal strain for the biodegradation of straw.