Study On Characteristics And Application On Bagasse Ethanol Production Of Laccase From Pleurotus Eryngii

Laccase(EC 1.10.3.2,β-diphenoloxidase)is a kind of copper polyphenol oxidase.It has the ability to degrade lignin and phenolic compounds.Due to its wide range of catalytic substrates,laccases have widespread applications ranging from bioenergy,food,papermaking,textile and so on.Laccases are mainly derived from White-rot fungus in the nature.Compared with most of White-rot fungus,Pleurotus Eryngii has bigger ability to produce laccase.At present,Pleurotus Eryngii has the second highest yield of the edible fungi factory production in China.With the yield increasing daily,factories produced more and more fungus chaff which need effective way to deal with.In this dissertation,the ability of Pleurotus Eryngii to product laccase,enzymology characters of Pleurotus Eryngii laccase and its application on bagasse ethanol production were studied.The purpose of the research is to try to solve the production problems of laccase,to find a new cheap source of laccase production,to solve the li-miting factor of low efficiency of ethanol production from bagasse,and to create a new way for Pleurotus Eryngii chaff’s recycling,which can not only reduce the po-llution of the environment,but also effectively improve the utilization of resources with good ecological effect.The research contents were as below:1 Effects of different nutriational conditions on the growth rate of hyphae and laccase production of Pleurotus EryngiiThe growth rate of hyphae,laccase activity and the expression of laccase gene of Pleurotus Eryngii under different nutritional conditions were determined and analyzed.Under the conditions of low nitrogen medium,mycelium grew well but had low laccase activity.Under the conditions of high nitrogen and low carbon medium,mycelium grew bad but had the highest laccase activity.It was suggested that nitrogen source can promote fungal laccase production and carbon source can promote the growth of hypha.Under the conditions of high inorganic salts medium,mycelium also grew bad,but with high laccase activity.It was suggested that inorganic salt can also promote the laccase production.Under the conditions of low carbon,nitrogen and inorganic salts medium,mycelium grew quickly and sparsely,with low laccase acti-vity.Under the conditions of tween 20 medium,the growth rate of hyphae was the worst,with no laccase activity.Laccase showed an transcription pattern by real-time fluorescent quantitative PCR:Low nitrogen medium>Complete nutrition medium>High nitrogen and low carbon medium>High inorganic salt medium>Tween 20 me-dium>Low carbon,nitrogen and inorganic salt medium.The result showed that the exocellular laccase activity and laccase genes expression of Pleurotus Eryngii have no direct relevance at the transcriptional level.2 Purification and characterization of laccaseLaccase derived from Pleurotus Eryngii chaff was purified and characterized:Using RO water as leaching solution,the ratio of solid to liquid of 1:7,shaker tempe-rature 25℃,shaker time 1 h and shaker rate 130 r/min to extract crude enzyme fluid,and then,laccase was purified by ammonium sulfate fractional precipitation(30%to 80%),dialysis,vacuum freeze drying and DEAE-sepharose anion-exchange chroma-tography.Ultimately,laccase was purified up to 10.5 times and showed a relative mo-lecular mass of 44.3 kDa by SDS-PAGE.The optimum temperature was 40 ℃ and the optimum pH was 4.0.The enzymatic activity was stable in pH 4.6 and 10-20℃.With the final concentration of 0.05 mM,Mg2+,Fe3+,Ag+,Na+,Ca2+,Ba2+,Zn2+,K+,NH4+and Mn2+all showed the promoting effect of laccase.Magnesium ions effect was most obvious,and the enzyme activity could be enhanced by 21.31%.However,Fe2+st-rongly inhibited laccase activity up to 98.36%.The values of kinetic parameters Vmax and Km for purified laccase were 0.991(mM-min)and 0.087(mM).3 The application of laccase in ethanol production from bagasseUsing Pleurotus Eryngii chaff to extract crude enzyme liquid to make crude lac-case powder.Bagasse,degraded by laccase powder and fermented to produce ethanol was studied.With laccase treatment,total phenol degradation rate was 33.51%in the hydrolysate,and glucose and xylose concentration were respectively 5.41 g/L and 2.44 g/L in the sugarcane residue.Ethanol yield in laccase-treated fermentation were all higher than that of controls(P<0.05),with at least 10%of the improvement.The experiment group of bagasse had the largest increase by 1.57 times.It proves that lac-case extracted from Pleurotus Eryngii can not only degrade phenolic compounds to reduce its toxic effect of fermentation,but also effectively degrade lignin of bagasse to produce reducing sugar,which could promote the yeast growth and ethanol production.