The Production, Properties And Applications Of Myrothecium Verrucaria NF-05Laccase

Laccase (p-diphenol:dioxygen oxidoreductase, EC1.10.3.2) is a kind of multiphenolic oxidoreductase which belongs to the family of blue multicopper oxidoreductases. In this research, we isolated a laccase-producing fungus strain and reported a comprehensive investigation on the biochemical, spectral, enzymatic and electrochemical properties of the purified laccase protein. Further more, we also made some practical research on optimization and inducement of laccase production, declorisation and degradation of dyes together with transformation of phenols and amines. The major techniques and results of this research are as follows:(1) Isolation and identification of laccase-producing strainA fungus named NF-05with high laccase-producing ability was selected from soil sample. When cultured at30℃on a PDA plate for6days, the colony of NF-05was large, flat, rounded, with short and velvety white hyphae, white color on the surface and brown on the back. On the tenth day, light yellow spores emerged then turned to dark green and agglomerated. Strain NF-05was morphologically identified to deuteromycotina, Myrothecium. Combining with rDNA-ITS sequencing (544bp) and BLAST analysis (NCBI accession number HM347341), NF-05was identified as M.verrucaria.(2) Optimization of laccase productionM.verrucaria NF-05reached the highest enzyme activity at8.38U/ml on the fifth day in basal medium, partially coupling with hyphae growing. Variance and multiple comparison analysis based on single factor experiments illustrated the optimum medium components and cultivation conditions were:4%glucose,3%peptone, initial pH value7.0, liquid volume60ml/250ml, inoculum4%,30℃and140rpm. Gallic acid and Fe3+significantly increased laccase production of NF-05. Laccase activity reached to12.82U/ml owing to single factor experiments. Response surface methodology was performed for further optimization. Plackett Burman design indicated the significant effects of glucose, Cu2+and gallic acid on laccase production process. Central composite design was preceded based on steepest ascent experiment. A fitting equation was achieved and testified with the maximum laccase activity at19.82U/ml, which was2.37fold of the initial value. The optimum concentration of glucose, Cu2+and gallic acid were26.47g/L,236.3μM and138.4μM, respectively.(3) Inducement of extracellular laccaseVariance and multiple comparison analysis illustrated that Cu2+in range of 0.1-4.0mM significantly increased laccase production of NF-05. The laccase activity was43.23U/ml with0.2mM Cu2+as inducers. Sixteen tested phenols remarkably induced laccase-producing process, especially ferulic acid with the enzyme activity of234.15U/ml.3,3'-dimethylbiphenyl-4,4'-diamine was the best inducer with the enzyme activity at258.11U/ml among all the14amines which exerted significant inducement on extracellular laccase of NF-05. Another six substrates also increased laccase production, the best among which was sodium lignosulphonate. The laccase activity was267.92U/ml which was the highest level of microbial laccase so far. Four synthetic dyes showed remarkable positive effects for laccase inducement particularly Ponceau S.(4) Purification and identification of target proteinTarget protein was purified from the fermentation liquid of strain NF-05via salting out of (NH4)2SO4,anion change chromatography of DEAE-sepharose fast flow and filtration chromatography of sephadexG-75gel. SDS-PAGE and native-PAGE revealed that the purified protein was a monomer with a molecular weight of66kDa which oxidized ABTS to green band. The first ten amino acid sequences were APQISPQYPM. Together with the peptide analysis based on matrix-assisted laser desorption/ionization time of fight mass spectrometry, the purified protein was identified as laccase protein.(5) Spectral propertiesThe average numbers of copper and fermium ions were3.08±0.3and0.95±0.2per protein molecule respectively, resulting in the ratio of3:1. The ultraviolet-visible scan figure among300-650nm showed no absorption peak. Further more, there was no obvious signal in electron paramagnanetic resonance detection. In view of the "silence" spectral properties, it could be deduced that the lack of absorption in visible light range and absence of EPR signal probably resulted from the existence of incomplete oxidation state of copper (Cu+), which had a fully occupied electron configuration of d10and no d-d transition could take place. The incomplete oxidation of metal ions might confer the easiness of electron transfer in active center of the laccase and also render the protein extra high activity. Consequently, NF-05laccase was a white laccase with new metal composition and spectral properties.(6) Enzymatic propertiesThe optimum catalyze condition of NF-05laccase was pH4.0,40℃. Na+, Mn2+Cu2+and Zn2+significantly increased enzyme activity at specific concentration. The protein maintained more than50%activity in presence of5%tested organic solvent. Regular protein inhibitors such as L-cysteine, SDS, DTT and Na3N almost totally inhibited enzyme activity at the concentration of1mM. Halogen anions were special inhibitors for NF-05laccase. NF-05laccase showed great intimacy to ABTS with a Km value of85.9μM.(7) Decolorization and degradation of synthetic dyesAmong azo dyes, more than90%of orange I and methyl orange was decolorized by NF-05laccase without mediators. Decolorization rate of orange G6reached to more than90%in TE, VA or HBT mediated system; ACE could well mediate the decolorisation system of amaranth. Mediators could increase the decolorisation rate for anthraquinone dyes to a less extent. The decolorisation rate of basic fuchsin was59.33%, which was the highest among all tested arylmethane dyes without mediators. Malachite green was the second best arylmethane dyes decolorized by NF-05laccase. PZ mediated system could increase the decolorization rate from48.68%to91.42%. NF-05laccase could not efficiently decolorize the tested dyes of other structure types. The decolorization systems under higher enzyme concentration and without mediators involvement were scanned under400-700nm. The specific absorption peaks of orange I, eriochrome black T, fuchsin basic and phenol red were almost totally degraded within10min.(8) Transformation of phenols and aminesComparison studies of catalyzing phenols and amines by NF-05laccase and control commercial oxidoreductases (laccase and bilirubin oxidase) were conducted. The reaction was coupled with4-aminoantipyrine and referenced by phenol. NF-05laccase exerted higher transforming efficiency of14phenols,15amines and6other type substrates than control enzymes.(9) Electrochemical propertiesUnder the nitrogen saturation condition, M.verrucaria NF-05could directly absorb on GP and GC electrodes. Obvious redox peak was observed which indicated the occurrence of direct electron transfer and quasi-reversible electrochemical reaction. On GP electrode, the Epa and Epc were-0.026V and0.028V, respectively.△Ep was54mM and E0' was1mV. On GC electrode, thea and Epc were-0.027V and0.01V, respectively.△Ep was37mM and E0' was-8.5mV. The transmormfation efficiencies of peroxide electrocatalyzed by M.verrucaria NF-05lascase were higher than control commercial enzymes on both GP and GC electrodes.