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Scientia Silvae Sinicae ›› 2015, Vol. 51 ›› Issue (1): 80-87.doi: 10.11707/j.1001-7488.20150109

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

Gao Dongni1,2, Fan Xiaoxu1,2, Zhao Dan1,2   

  1. 1. Key Laboratory of Microbiology Life Science Department, Heilongjiang University Harbin 150080;
    2. Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education Harbin 150500
  • Received:2013-11-17 Revised:2014-06-04 Online:2015-01-25 Published:2015-01-23

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Abstract:

[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 sole carbon source for the enrichment culture,and guaiacol-PDA selective plates were used to isolate laccase-producing fungi. Two distinctive agents, ABTS 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 rDNA-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 pH 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 ·mL-1 on the 6th day during the fermentation. The optimum carbon and nitrogen source were 4.0% glucose and 3.5% peptone, respectively. The best initial pH value of the fermentation medium was 7.0. The optimum culture conditions were liquid volume 60 mL in a 250 mL 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 ·mL-1 which was 81.25% higher than that of the original strain. [Conclusion]A method for isolating, purifying and screening laccase-producing 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.

Key words: deuteromycete, Myrothecium verrucaria, laccase, laccase-producing condition

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