Laccase Production By Pycnoporus Sp. SYBC-L3and Its Applications

Laccase （EC1.10.3.2） is a blue copper-containing oxidoreductase, capable ofpolymerizing, degrading and transforming a large variety of phenolic and non-phenoliccompounds. During one-electron transfer among copper atoms within active center, molecularoxygen is concomitantly reduced to water. In view of its wide spectrum of catalytic substrates,laccase is being increasingly found its applications in numerous fields such as food industry,textile dying and decolorization, biosynthesis and bioremediation as well as biosensor.Laccase is mostly produced by fungi in nature and fungal laccases represent the relativelyhigh catalytic capability among various lassase sources. Overview of published literaturereveals that in-depth applications of laccase can be restricted by low laccase yields with highproduction input. Screening potential wild-type fungus from nature with hyper-ability oflaccase excretion can hereof significantly contribute to full applications of this enzyme inindustrial and environmental aspects. The following research conducted a series of studiesbeginning with isolation of a white-rot fungus and ending with application of laccase by thisfungus, within which optimizing laccase production in flask level, conducting fermentationprocesses in various bioreactors, characterizing laccase properties and involving the fungusinto lignocellulosic biomass pretreatment and textile discharge treatment were also included.Below are the main findings from our research.1) A laccase-screting wild-type fungus isolated from a rotten wood was characterizedbased on its moyphological characteristics and molecular identification techniques anddesignated as Pycnoporus sp. SYBC-L3. Laccase production by this fungus was thenenhanced by optimizing some major medium components coupled with fermentationparameters and the favorable culture conditions are: initial pH value of5.0, seed age of3d,inoculum of10%and rotation rate of200rpm; the optimized medium consists of: maltose14g/L, soybean meal6g/L, KH₂HPO₄1g/L, MgSO₄·7H₂O0.5g/L, Na₂HPO₄·7H₂O0.2g/L, Cu²⁺1.5mmol/L, vanilla1mmol/L, Mn²⁺0.2mmol/L which finally yielded the highest laccaseactivity up to45U/mL,57-fold higher than the un-optimized one.2) Laccase excretion abilities of Pycnoporus sp. SYBC-L3were further tested invarious bioreactors in light of the flask-obtained investigation. The highest laccase activity,110U/mL, was obtianed in a50L stirred bioreactor. The crude laccase exhibited promisingproperties in terms of high catalytic activity under broad temperatures ranging from0-100oCwith optimum T of55oC within acidic pH spectra. It displayed inconceivable relative activityof as high as40%and80%at0oC and100oC, respectively. Long half-lives were obtained forlaccase under all varying temperatures assayed, especially at room temperature. Enzymecocktail of the culture broth was then subjected to nano-LC-MS/MS for predominantextracellular protein identification, which suggested that at least13differetn proteins existedin the fungal culture broth with three iso-laccases as the major ones. Crude laccase powderwas successfully prepared using spray-dryer with activity recovery of approximatedly80%.3) Pure laccase （designated as lac-L） was harvested by a three-step purification process,resulting in a purification fold of4.67and activity recovery of28%. Lac-L has an estimated molecular weight of58kDa and pI of4.7. N-terminal amino sequence of lac-L wasdetermined as AIGPVADLTLTNAAV. Optimal T and pH of lac-L were substrate-dependentbut generally in the range of T50-70oC and pH3-5. ABTS was proved to be the best substrate,followed by DMP, guaiacol and SGZ. Lac-L was tolerant to most metal ions but dramticallydepressed by Ag⁺, Fe²⁺and Fe³⁺at even low concentrations. Sr²⁺imparted weak stimuluseffect on lac-L activity. Stamina profiles of lac-L on elevated temperatures suggested thatlac-L was as excellent as or higher than other thermostable fungal laccases. The stabilities oflac-L towards high temperature and pH as well as deactivation by some metal ions wereelucidated using CD specturm technique.4) Energy crop switchgrass is one of the most promising lignocellulosic feedstocks forbioethanol production, in which cellulose conversion is a rate-limiting step. Fungalpretreatment by Pycnoporus sp. SYBC-L3was performed herein to enhance subsequentsaccharification and simultaneously produce useful enzymes as a co-product. The funguscaused dramatic changes in chemical composition in pretreated switchgrass over time. Thehighest lignin removal and saccharification efficiency was attained for cultivation time36dwhile the peak enzyme activity occurred at54d. SEM associated with FTIR techniquesdemonstrated that fungal pretreatmetn formed numorous pores or pits on the biomass surfaceand altered the chemical compositions of switchgrass, thereafter the recalcitrance coupledwith intensity of this biomass significantly reduced.5) Treatment and re-utilization of wastes have been foci worldwide in recent years.Fungal cultivation of Pycnoporus sp. SYBC-L3in textile effluent was for the first timeattempted to achieve both color removal and laccase production. The favorable concentrationof textile for maximum fungal growth and laccase activity was shown to be80%. Amendmentof80%textile effluent with12g/L lignocellulosic biomass Phragmites australis enhancedlaccase production, under which the peak activity reached5.6U/mL and70%color removaloccurred in this culture sytem. This novel scheme provides an alternative of management oftexteile effluent as well as other environmental pollutants control.6) Soil water repellency （SWR） is a re-occurring phenomenon on sandy soil on golfcourt and thus induce serious turfgrass death. The crude enzyme extract （CEE）, derived fromthe fungal pretreatment of switchgrass as above mentioned, was firstly employed to mitigateSWR seveity of sampled soils from variously located golf courts. All collected soils presentedwater repellency to varying extents and returned to hydrophilic status following with the CEEtreatment. Effective alleviation of hydrophobicity were achieved in1-d treatment with nofurther significant changes observed in the following4-d treatment. High residual enzymeactivities, around50%or higher, remained in the filtrate of enzymatic soil treatment for5days. The possible mechanisms for alleviation of SWR with enzymatic treatment were thusdiscussed. Potentially, laccase enzyme liquid might serve as a biological agent for preventionand amelioration of turfgrass soils in golf court management.