产漆酶半知真菌Myrothecium verrucaria NF-08菌株的分离及产酶研究

Isolation of a Deuteromycete Fungus Myrothecium verrucaria NF-08and Its Laccase Production

【目的】采集凉水国家级自然保护区原始森林土壤样品,分离产漆酶真菌并优化产酶条件,旨在开发产漆酶半知真菌种质资源,提高漆酶产量,为微生物漆酶扩大生产提供菌种资源和条件参数。【方法】以木质素磺酸钠为唯一碳源的无机盐培养基为富集培养基,在愈创木酚-PDA选择性平板上分离产漆酶真菌。利用ABTS[2,2'-连氮-双(3-乙基苯并噻唑-6-磺酸)]和SGZ[4-羟基-3,5-二甲氧基-苯甲醛连氮,即丁香醛联氮]显色反应初筛和摇瓶发酵复筛。确定供试菌株后,利用形态观察结合r DNA-ITS序列分析技术,将菌株鉴定至种。以ABTS法测定供试菌发酵液漆酶活性,以Lowry法测定发酵液总蛋白含量。在考察供试菌株生长、产酶及胞外蛋白总量动态变化基础上,通过单因素试验,研究碳源种类及浓度、氮源种类及浓度、培养基起始p H、装液量、接种量及底物诱导对菌株产漆酶的影响,获得菌株产酶最适培养基组分和培养条件参数。【结果】获得1株发酵周期短、初始酶活高的产酶菌株NF-08。结合形态学特征与r DNA-ITS序列分析结果,鉴定菌株NF-08属于为半知菌亚门疣孢漆斑菌。该菌株在液体发酵培养基中产漆酶活性与菌丝生长和胞外蛋白总量基本同步,发酵第6天达到酶活峰值9.28 U·m L-1。疣孢漆斑菌NF-08产酶最佳碳、氮源分别为4.0%葡萄糖和3.5%蛋白胨,培养基初始p H7.0。最适装液量60 m L·(250 m L)-1,最适接种量4%,最适培养温度和摇床转速分别为30℃和140 r·min-1。没食子酸、阿魏酸和单宁酸显著诱导疣孢漆斑菌NF-08漆酶活性的产生,以没食子酸效果最好。经过培养基组分和培养条件优化以及底物诱导,疣孢漆斑菌NF-08产酶水平达到16.82 U·m L-1,比优化前提高了81.25%。【结论】建立环境样品中产漆酶真菌的分离、纯化及筛选方法,获得了一株发酵周期短、产漆酶活力高的半知真菌疣孢漆斑菌NF-08。该菌株经单因素试验产酶条件优化效果明显,酶活水平提高显著,可为微生物发酵产漆酶提供新的菌株资源,在微生物发酵漆酶工业生产中具有应用潜力。后续对疣孢漆斑菌NF-08漆酶产生、性质及基因表达调控的深入研究,将有助于了解漆酶在半知真菌生活史中的作用及生态学功能,同时拓展半知真菌漆酶的应用领域。

【Objective]】Laccase has been widely used in many industrial fields,such as textile,printing,environment bioremediation and bioelectronics. Microbial laccases,especially from deuteromycetes have become the main laccase resource for theoretical and practical researches due to the short fermentation cycle,mild reaction condition and genetica reconstruction advantage. In this research,soil samples were collected in the primary forest in Liangshui Nature reserve,China in order to isolate laccase-producing fungi and improve the culture conditions. This research focused on the resource exploration of laccase-producing fungi and enhancement of laccase production so as to supply fungal strain and fermentation parameters for enlarging industrial practice of laccase production. 【Method】Lignin-sodium sulfoacid was used as the solecarbon source for the enrichment culture,and guaiacol-PDA selective plates were used to isolate laccase-producing fungi.Two distinctive agents,ABTS [2,2-azino-bis（ 3-ethylbenz-thiazoline-6-sulfonic acid） ] and SGZ（ 4-hy-droxy-3,5-dimethoxybenzaldehyde azine） were both used for primary screening and then shaking fermentation was adopted for secondary screening of laccase-producing fungi. A fungus strain which had a short period of fermentation cycle and high laccase production was obtained and identified as a deuteromycete fungus,Myrothecium verrucaria NF-08 according to morphological characteristics and sequence analysis of r DNA-ITS. The laccase activity was determined by ABTS and the extra-cellular protein was assayed according to Lowry method. Based on the dynamic investigation of laccase activity,fungus growth and protein content,single factor experiments were conducted to investigate effects of the following factors on laccase production,including the categories and concentrations of carbon and nitrogen sources,initial p H value of the medium,inoculum and liquid volume in the flask as well as the induction of laccase substrates. 【Result】The strain NF-08 was isolated,purified and identified as a deuteromycete,Myrothecium verrucaria The laccase production of M.verrucaria NF-08 showed synchronism in concordance with both hypha growth and extracellular protein amount. The laccase activity reached the peak value of 9. 28 U·m L- 1on the 6thday during the fermentation. The optimum carbon and nitrogen source were 4. 0% glucose and 3. 5% peptone,respectively. The best initial p H value of the fermentation medium was 7. 0. The optimum culture conditions were liquid volume 60 m L in a 250 m L flask,inoculum 4%,temperature 30 ℃ and rotation 140 r·min- 1. Gallic acid,ferulic acid and tannin significantly increased the laccase activity produced by M. verrucaria NF-08, especially for gallic acid. As a result of the optimization of medium components,culture conditions as well as substrate induction,the laccase activity reached 16. 82 U·m L- 1which was81. 25% higher than that of the original strain. 【Conclusion】A method for isolating,purifying and screening laccaseproducing fungi from environmental samples was established. A laccase-producing deuteromycete fungus with short fermentation cycle and relative high initial laccase activity was obtained. The culture conditions were remarkably improved and laccase activity produced by M. verrucaria NF-08 was significantly increased. M. verrucaria NF-08 was a new microbial resource for laccase production and had great potential in enlargement fermentation in industrial practice. Furthermore,several meaningful works involved with M. verrucaria NF-08 would be carried on such as enzyme production and characterization,expression and regulation of the laccase gene. This would be beneficial to gain a better understanding of the deuteromycete laccase about the physiological role and ecological function. At the same time,the application fields of the deuteromycete laccase would be widen in the near future.