Study On The Ligninolytic Enzymes From Pleurotus Eryngii

Pleurotus eryngii (P. eryngii) is a fungus belonging to Eumycophyta, Eubasidiomycetes, Agaricales, Pleurotaceae, pleurotus. As a kind of white rot fungi, it has been reported to have the capability to degrade lignin, and is therefore considered a model organism having extensive biotechnological applications. With remarkably oxidizing ability, degradation capacity and substrate non-specificity; ligninolytic enzymes involved in lignin biodegradation can oxidize various substrates including aromatic pollutants, heterocycle compounds, synthetic dyes and so on. In this work, using P. eryngii GIM 5.280 as starting strain, the mutagenesis of protoplast were performed for the screening of high-yield strains of ligninolytic enzymes with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol as substrates. And then the production conditions in liquid submerged fermentation of ligninolytic enzymes of P. eryngii were optimized. Furthermore, the purification procedure of ligninolytic enzymes of P. eryngii was established, and purified enzymes were used to investigate their enzymatic properties, catalysis behaviors, dye decoloring ability, and lignin degradation ability. These studies will enhance our knowledge of the ligninolytic system of this edible mushroom. Furthermore, it will also clarify the potential applications of ligninolytic enzymes of P. eryngii in the environmental protection, paper pulp bleaching, processing of food and other industrial applications.In order to screen P. eryngii strains of high-yield ligninolytic enzymes, the preparation conditions of P. eryngii protoplasts were investigated in details. Results showed that the highest production rate of protoplast could be obtained by using 0.6 mol/L(M) mannitol,4% helicase,2% lywallzyme, and digesting for 3 hours at 30℃, pH 5.5. The protoplast numbers of P. eryngii reached to 1.82×108/mL. The optimal medium for protoplast regeneration of P. eryngii was SYM medium, using 0.6 mol/L sucrose as osmotic stabilizers, and 0.7% agar as upper-layer plate. The highest regeneration rate was 0.35%.The UV and 60Coγirradiation mutagenesis of P. eryngii protoplasts, and serial subcultivations were performed for screening high-yield strains of ligninolytic enzymes. The results indicated that the strain 007 (We named this strain as P. eryngiico007.) of P. eryngii exhibited outstanding genetic stability after four successive generations. Furthermore, the ligninolytic enzyme activity of this strain was up to 110U/mL, a 54.3% enhancement over that of the original strain. The optimum production conditions of ligninlytic enzymes of P. eryngiiCo007 in liquid culture was investigated through single factor experiment and response surface method, and the result were listed as follows: the compositions of optimal fermentation medium were glucose 30g/L, MgSO4·7H2O 0.5g/L, KH2PO4 4g/L, yeast extract 2g/L, tryptone 2g/L, straw 11g/L, CuSO4 4.01mmol/L, Tween 800.84g/L; The optimal cultivating conditions were 15%(v/v) inoculation volume,26℃and pH 5.63. The ligninolytic enzymes were prepared by cultivating P. eryngiiCo007 in a 3-liter fermentor under the conditions of 26℃,200 rpm, and aeration with 0.5 liter·min-1. Proteins with ligninolytic enzyme activity were purified from 10-day-old liquid cultures of P. eryngii, which exhibited maximal enzymatic activity (324.72 U/mL).The ligninolytic enzymes were separated and purified from the culture broth of Pleurotus eryngiiCo60-7 strain. An efficient purification procedure was establised, which was mainly composed of the precipitation of ammonium sulfate and ion exchange chromatography. In the precipitation of ammonium sulphate, two-stage precipitations were applied. In the first-stage precipitation,35% saturation of ammonium sulfate was used to remove the hybridproteins and in second-stage of precipitoation,75% saturation of ammonium sulfate was used to capture the ligninolytic enzymes. Next, the crude ligninllytic enzymes were purified by DEAE-SepharoseTM Fast Flow (DEAE FF) ion-exchange chromatography under three-step elution. Finally, a 76.2% activity yield was obtained with a purification factor of 10.3. The results of SDS PAGE and Native PAGE showed that three ligninolytic enzymes were present in the elution after DEAE FF ion-exchange chromatography, and these enzymes were named as Peco60-7-1dⅠ, Peco60-7-1dⅡ, and Peco60-7-1dⅢ, respectively. Using N-terminal sequence determination and peptide mapping analysis, Peco60-7-1dⅠ, Peco60-7-ldⅡ, and Peco60-7-1dⅢwere found to be similar to the aryl-alcohol oxidase, laccase and versatile peroxidase isoenzyme, respectively. Therefore, they were named as AAO-Peco60-7,Lac-Peco60-7 and VP-Peco60-7.AAO-Peco60-7 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, DEAE FF chromatography, SephacrylTM S-200 High Resolution chromatography, and SourceTM 15Q chromatography with a activity yield of 25.5% and a purification factor of 38.2. The molecular mass of AAO-Peco60-7 was estimated to be 70kDa by SDS-PAGE and its isoelectric point was 4.2. The optimal pH and temperature for oxidizing ABTS was 3.0 and 70℃respectively. The Vmax and Km values of the enzyme for ABTS were 102.04 U/mg and 214.96μmol. The enzyme was relatively stable at pH3.6, and 88% enzymatic activity was remained after incubating for 24 h. And the enzyme was also stable from 4℃to 60℃, and the survival enzymatic activity was over 80% after incubating for 50 min.0.1 mM various metal ions had no pronounced effects on enzyme activity of AAO-Peco60-7, However, the activity of the enzyme was strongly inhibited by Fe3+, Fe2+, Ag+ and Ca2+.Lac-Peco60-7 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, DEAE FF chromatography and the gel slice of Native-PAGE. The molecular mass of Lac-Peco60-7 was estimated to be 62kDa by SDS-PAGE and its isoelectric point was 4.5. The optimal pH and temperature for ABTS were 4.0 and 60℃, respectively. The Vmax and Km values of the enzyme for ABTS were 227.27U/mg and 252.33μmol. The enzyme was relatively stable at pH 4.6, and 85% enzymatic activity was remained after incubating for 24 h. And the enzyme was also stable at 50℃, and the survival enzymatic activity was over 40% after incubating for 40 min.0.1mM various metal ions had different effects on enzyme activity, the enzymatic activity of Lac-Peco60-7 was strongly promoted by Zn2+ and Cu2+, however, its activity was inhibited by Ag+ and Fe2+VP-Peco60-7 was purified by ammonium sulfate precipitation, DEAE FF chromatography and SephacrylTM S-200 High Resolution chromatography with a activity yield of 19.1% and a purification factor of 27.0. The molecular mass of VP-Peco60-7 was estimated to be 40 kDa by SDS-PAGE and its isoelectric point was 4.1. The optimal pH and temperature for ABTS were 3.0 and 50℃, respectively. The Vmax and Km values of the enzyme toward substrate ABTS were 188.68U/mg and 203.09μmol. The enzyme was relatively stable following with 50% survival activity after incubating for 24 h at pH 4.0 and over 52% survival activity after incubating for 60 min at 30℃.0.1 mM various metal ions had different effects on enzyme activity, the enzymatic activity of Lac-Peco60-7 was promoted by Zn2+ and Cu2+, however, its activity was strongly inhibited by Fe3+, Fe2+ and Mn2+ Ligninolytic enzymes separated from DEAE FF were used to decolorize three dyes, including bromchlorphenol blue, methylene blue and Congo red, which could be classified as triphenylmethane, heterocyclic and disazo dyes. The results showed that the purified ligninolytic enzymes had the best decoloring effect for bromphenol blue, with a 99.3% decolorization rate after treating for 24 h. And the decolorization rate of Congo red and methylene blue were 53.7% and 11.5% after treated for 24 hours, respectively. The optimal decolorization conditions were as follows:decolorizing temperature was 30℃, decolorizing pH was 4.0, H2O2 concentration was 5μmol/L, concentration of ligninolytic enzyme was 20 U/mL, concentration of bromophenol blue is 25μmol/L. The decolorization rate of bromphenol blue reached up to 85.61% after treated for 2 hours. The adding of ABTS had no pronounced effect on decolorization rate of bromphenol blue.The study on sodium lignosulfonate biodegradation by ligninolytic enzymes purified from DEAE FF showed that about 82.13% of sodium lignosulfonate could be degraded in the system containing 100 mg/L sodium lignosulfonate,1 mmol/L ABTS,20 U/mL ligninolytic enzyme, and 50 mmol/L sodium acetate buffer (pH4.6) for 8 hours at 40℃. The absorption peak at the 273nm was obviously decreased by UV-VIS spectrogram (200-800nm), which was also demonstrated that the ABTS could clearly improve the biodegradation of sodium lignosulfonate.