Study On Isolation,identification And Preliminary Application Of Laccase-producing Endophytic Fungus From Taxus Wallichiana Var.mairei

Sustainable development and utilization of forest resources is one of the important tasks in the development of forestry industry.The host plant is not isolated individual,but is surrounded and colonized by many microorganisms,including epiphytes and endophytes.Metabolites of endophytic fungi which are related to the growth and improvement resistance of plant,the screening and application of specific endophytic fungi have important research value.This study focused on the screening of laccase endophytic fungi from Taxus wallichiana var.mairei,and 58 endophytic fungi were obtained from the roots,stems and leaves.A strain of endophytic fungus N-31 with laccase activity was screened out and determined by morphology and molecular biology.In order to obtain the maximum laccase activity,further optimized a series of factors including carbon source,nitrogen source,culture temperature,time and initial p H.Finally,the biochemical characteristics and preliminary application of N-31 were investigated.The results were shown below:1.58 endophytic fungi were isolated from Taxus wallichiana var.mairei,of which 37,9 and 12endophytic fungi were isolated from the roots,stems and leaves,respectively.These endophytic fungi belong to Phoma,Pestalotiopsis,Fusarium,Aureobasidium,Paraboeremia and Alternaria.A strain of endophytic fungus with laccase activity was screened by plate chromogenic of guaiacol method,and identified by morphology and molecular biology as Paraboeremia selaginellae.2.The single factors and response surface method were used to optimize the fermentation conditions of laccase producing fungus N-31:2%glucose as carbon source and 1%beef extract as nitrogen source,fermentation temperature 27~oC,fermentation time 4 d and the initial p H 4.6.Under the optimum conditions that laccase activity could reached 105.58 U/L.3.The enzymatic properties and preliminary application of laccase was investigated.The optimal p H of laccase activity at p H 3 and the optimal stability at p H 4.The optimal temperature of laccase activity was 40~oC,and the optimal stability was when the temperature less than 30~oC.After pretreatment with laccase,the yields of six active ingredients including schisandrol A,schisandrol B,schisantherin B,schisandrin A,schisandrin B and schisandrin C of schisandra fruit was increased by 13.63%,18.73%,14.48%,16.18%,24.62%and 22.55%,respectively.Laccase has a certain degradation effect on the sulfamethoxazole.In 7 hours,the degradation rates of sulfamethoxazole with the initial concentrations of10 g/L,20 g/L and 40 g/L were 69.04%,58.27%and 37.80%,respectively.The laccase selected in this paper has good biochemical characteristics,and has the ability of bioconversion assisted extraction and biodegradation of antibiotics,which provides a certain scientific basis for expanding the source and effective use of laccase.