Optimization Of Fermentation Conditions For Laccase Production By Trametes Sp. LS-10C

Laccase is a type of polyphenol oxidases wih high redox potential,capable of catalyzing of the phenol compounds.In the presence of certain mediators that is a kind of small molecular compounds,laccase is also capable of oxidizing non-phenolic substances.Thus,laccase has good application prospects in many fields such as environment,food improvement,pulping and pulp bleaching,dyeing and printing,wastewater treatment,bio-energy and chemical medicine.In this study,a strain LS-10C of high laccase-producing screened by the guaiacol colorimetric method,the fermentation medium was optimized,the fed-batch fermentation process of Trametes sp.LS-10C was studied preliminarily for improving laccase production,the basic enzymatic properties obtained by the isolated and purified laccase basic properties of the use of enzymatic separation and purification of laccase,and its application in the field of azo dyes decolorization,which would,as wished,provide theoretical foundation for industrial production and application of the laccase.This paper firstly used guaiacol colorimetric method for screening a strain LS-10C wih a high yield of laccase from the nature rapidly,and the strain was identified as Trametes sp.by morphological observation,5.8S rDNA-ITS homology and phylogenetic tree constructing.Then,laccase activity as screening index,the fermentation medium of Trametes sp.LS-10C was optimized through the single factor experiment and uniform test design,and the optimal fermentation medium listed was as follows:glucose 14.4 g/L,bean dregs 10.1 g/L,bran 100 g/L,NH4Cl 3 g/L,KH2PO41.4 g/L,CuSO4-5H2O 1 g/L,NaCl 1 g/L,MgSO4 0.8 g/L,CaCl2 0.5 g/L,natural pH value.With the optimized medium,under the conditions of 30?,200 r/min,and 50 mL/250 mL volume,laccase activity reached 595.15 U/mL,which was about 35.66-fold higher than that obtained in the initial fermentation medium,was reached at the fermentation time of 8 day.With compared to the related published works,Trametes sp.LS-10C has the characteristics of fast enzyme production rate,short fermentation time,and high enzyme activity,etc.Based on the shaking flask fermentation,effects of flowing materials such as copper sulfate and glucose into the 10 L bioreactor on the laccase production were studied preliminarily.And the optimized parameters of laccase production from the strain were obtained by the submerged fermentation,which listed as follows:adding copper sulfate and glucose simultaneously,controlling the amount of reducing sugar to 0.05%,the ending concentration of Cu2+ controlled from 3.5 mmol/L to 4.0 mmol/L,stirring speed 300 r/min,medium volume 70%,ventilation volume 5 L/min,fermentation temperature 30 ?,natural pH value.Under the conditions mentioned above,laccase activity of LS-10C reaches the maximum value 868.27 U/mL on the 8th day that is about 152.11%as much as the unoptimized level.The lacccse produced by Trametes sp.LS-10C isolated and purified by ammonium sulfate precipitation and anion-exchange chromatography to obtaine the electrophoresis pure laccase with specific activity of 450.55 U/mg,the purification fold of 2.36 and 58.94%of the crude extract.The results of Native-PAGE and SDS-PAGE showed that the molecular weight of the laccase is about 65 kDa,and there is one kind of laccase isozyme.The optimum reaction temperature and pH of the purified laccase is 40 ?and 4.0,respectively.The laccase has good stability when the of temperature below 50? and pH range from 5.0 to 7.0.The laccase activity could be promoted by Cu2+ and Mg2+,and strong inhibited by Fe3+,Fe2+and SDS,inhibition rates were 81.18%,95.70%and 93.65%,respectively.The Km and Vm of ABTS catalyzed by the laccase from Trametes sp.LS-10C was 66.97 ?mol/L and 11.35 mmol/(L-min)respectively,which showed the enzyme has a strong affinity to ABTS.The purified laccase from Trametes sp.LS-10C was applied to the azo dyes decolorization to investigate the effects of the mediator,laccase dosage,dye concentration,temperature and pH on 3 kinds of direct dyes(Direct Blue 86,Direct Orange 26 and Direct Red 31,etc.)by single factor experiment.The results showed that the azo dyes decolorization efficiency was different between laccase and laccase-mediator system.Direct Blue 86 decolorization efficiency is higher when used LS-10C laccase alone,while the laccase-ABTS system plays a significant role in promoting the decolorization efficiency of Direct Orange 26 and Direct Red 31.Three kinds of direct dyes have higher decoloration rate when laccase worked under the conditions of enzyme dosage 10 U/mL,and dyes concentration from 75 mg/L to 150 mg/L.Different dyes have different optimum decoloring temperature and pH.For example,the optimum decoloring temperature of Direct Orange 26 and Direct Red 31 range from 35? to 45 ?,while Direct Blue 86 range from 45 ? to 60 ?.The optimum decoloring pH value of Direct Red 31,Direct Blue 86 and Direct Orange 26 are 4.0,4.5 and 5.0,respectively.The laccase catalyzed Direct Blue 86,Direct Orange 26 and Direct Red 31 decoloring 12 hours under the optimum conditions could obtain the average decolorization rates 70.14%and 77.22%and 88.36%,respectively.