Study On Screening,Identified And Fermented Production Technology Of Novel Laccaseproducing Fungal Endophytes From Pigeon Pea [Cajanus Cajan(L.) Millsp.]

Laccase,a member of enzymes known as the polyphenol oxidases,can catalyze oxidation of an array of substrates.Currently laccases are extensively employed in food industry,paper industry and dye decolorization.In this study,three laccase-producing fungal endophytes were screened from 185 strains isolated from pigeon pea[Cajanus cajan（L.）Millsp.].The morphological and molecular identification were also studied.The fermentation conditions were optimized based on the activity of the laccase,and then the properties of the laccase and the application of the laccase in the dye decolorization were investigated.The effects of dye decolorization including azo dye Congo red and triphenylmethane Crystal violet by the fungal laccase were investigated to broaden the application of laccase in industry.Conclusions were summarized below:1.Three laccase-producing fungal endophytes were isolated from pigeon pea and carried on the classification and identification.Three fungal endophytes could produce laccase in the differential medium,because laccase can catalyze oxidation of guaiacol as the chromogenic reaction of red brown.They were named as R-26,R-37,R-45,respectively.Furthermore,morphology and molecular identification indicated that the three endophytic fungi are most closely related to Myrothecium verrucaria.The results showed that the activity of the laccase from strain R-37 was significantly higher than that from strain R-26 and from strain R-45,respectively.Therefore,the strain R-37 was used as the experimental strain.2.The optimum fermentation conditions of endophytic fungus R-37 were obtained by optimization of the fermented medium,the composition of fermented medium and the fermented conditions;R-37 was selected to optimize the fermentation conditions for laccase production effected by different single factors,such as different fermented medium,carbon sources,nitrogen sources,fermented time,fermented temperature and the initial pH value of the medium.The results showed that the composition of the optimum fermentation medium was as follows:glucose 20 g/L,peptone 10 g/L,CuSO₄·5H₂O 0.10 mmol/L,MgSO₄·7H₂O 1.5 g/L,KH₂PO₄3.0 g/L,potato juice 200 g/L,VB₁ 0.01 g/L.Fermented time,fermented temperature and the initial pH value of the medium were selected to central composite design and response surface optimization.The best fermentation conditions were as follows:fermented time 5 days,temperature 30℃,pH value 6.22.The maximum activity of laccase from R-37 was 16.52±0.18 U/m L under the above conditions.3.The enzymatic properties of laccase produced by R-37 isolated from pigeon pea were studied.The decolorization of different dyes including Congo red and Crystal violet by the laccase was also studied;After studying the properties of the laccase,its optimum temperature is 45℃and thermal stability is best at temperatures of 25⁴5℃;its optimum pH is 4.5 and the stability of the laccase is best at pH values of 4.5.The decolorization of different dyes including Congo red and Crystal violet by this laccase was also studied.The results showed that ABTS can significantly improve the decolorization rate of dyes.In the absence of ABTS,the laccase showed decolorization capability toward Congo red and Crystal violet,with 51.14±0.13%and33.42±0.17%of dyes decolorization respectively,after 24 h of incubation.However,in the presence of ABTS,the laccase showed more effective decolorization capability toward Congo red and Crystal violet,with 89.11±0.14%and 75.34±0.27%of dyes decolorization respectively,after 24 h of incubation.The optimum decolorization conditions of Congo red were as follws:dye concentration 50 mg/L,ABTS 0.01 mmol/L,laccase 1.5 U/mL,temperature 35℃,pH 4.5 and reaction time 4 h.The decolorization ratio reached 96.33±0.13%under above conditions.The optimum decolorization conditions of Crystal violet were as follws:dye concentration 25 mg/L,ABTS 0.01 mmol/L,laccase 1.5 U/m L,temperature35℃,pH 4.5 and reaction time 8 h.The decolorization ratio reached 81.36±0.28%under above conditions.In summary,the fungal endophyte R-37 producing laccase was isolated from pigeon pea;the best fermented technology conditions of endophytic fungus with high laccase production were obtained by optimizing the fermented medium and culture conditions.Additionally,the effects of dye decolorization including azo dye Congo red and triphenylmethane Crystal violet by the fungal laccase were investigated.Therefore,this study has laid a solid foundation for the effective use of pigeon pea endophytic fungi and fungal laccase,and also provided a new path and method for the research and development of laccase in industry.