Isolation And Identification Of Pycnoporus Sanguineus SYBC-L1 And Its Laccase Production By Fermentation

Laccase (EC1.10.3.2, benzenediol: oxygen oxidoreductase) is a oxidoreductase belonging to the multinuclear copper-containing oxidase and can be investigated for a variety of applications including detoxification of harmful substances, bleaching of paper pulp, decolorization of textile dyes, transformation of lignins or their derivatives and improvement of food properties. In this study, a new laccase-producing strain was isolated and identified. The fermentation medium and condition for laccase production were optimized. The physiological characteristics of the strain for laccase production were also investigated. Laccases produced by Pycnoporus sanguineus SYBC-L1 were purified and characterized. The applications in decolotization and degradation of textile dyes were studied. The results were as follows:A white-rot fungus SYBC-L1, which produced laccase, was isolated using PDA screening medium. According to morphological and molecular biological identification, the strain was belonged to Pycnoporus sanguineus, located in phylum Basidiomycota, order Polyporales, family Polyporaceae, genus Pycnoporus and designated as Pycnoporus sanguineus SYBC-L1. The GenBank accession numbers of the 18S and 5.8S rDNA gene sequence were EU888830 and EU888831, respectively.The fermentation process for laccase production by P. sanguineus SYBC-L1 in submerged fermentation was studied by using some industrial and agricultural by-products. The fermentation medium and conditions were preliminarily optimized by one-factor-at-a-time method, followed by further optimization via response surface methodology. The obtained optimal parameters were glucose 50 g/L, wheat bran 52.5 g/L, soybean meal powder 15 g/L, NH4H2PO4 1.0 g/L, CuSO4·5H2O 2.0 mmol/L, KH2PO4 0.5 mmol/L, NaCl 17 mmol/L, ferulaic acid 26.1μmol/L, initial medium pH 4.0. Under the optimal fermentation condition, the maximum laccase activity reached 61.25 U/ml, which was approximately 87 folds than that in the basal medium. The kinetic models of submerged fermentation for mycelial growth, laccase production and substrate consumption were developed based on the experimental results. Based on previous results, the fermentation process was enlarged in 7 L bioreactor and the laccase yield was 55 U/ml after cultivation for 10 d.The utilization of an environmental waster of water hyacinth by P. sanguineus SYBC-L1 was investigated. The fermentation medium were optimized by response surface methodology using water hyacinth as the substrate. The optimal medium were water hyacinth 25.1%, sawdust 13.9%, CuSO4·5H2O 1.5 mmol/L, gallic acid 40μmol/L, initial moisture 65%, initial medium pH 6.0. Under the optimal fermentation condition, the maximum laccase activity reached 32.02 U/g dry substrate, which was approximately 4.5 folds than that in the basal medium. This strategy not only increased the value of water hyacinth's utilization, but also contributed to the further use of water hyacinth.The physiological characteristics of P. sanguineus SYBC-L1 during the laccase producing process were investigated. It was found that when the yield of laccase was high, the oxidation level was relatively high, the contents of malondialdehyde, H2O2 and ascorbic acid were increased and the activities of SOD and CAT were enhanced, indicating that the strain was under oxidative stress during the laccase production period. The oxidation stress study on P. sanguineus SYBC-L1 was performed by using Fenton reagent, the result revealed that H2O2 and Fe2+ with the ratio of 10:1 could significantly promote the laccase production. It was supposed that laccase secretion might be regulated by reactive oxygen, which could induce the gene expression for laccase production.Two laccase isozymes (LacI and LacII) were purified by using (NH4)2SO4 fractionation, DEAE-cellulose anion-exchange chromatography and Sephadex G-100 filtration. SDS-PAGE showed that both LacI and LacII were monomeric proteins with the molecular mass of 55.89 and 63.07 kDa, respectively. LacI and LacII respectively contained 36.92% and 13.58% carbohydrates. The UV–vis spectrum of the purified laccases both showed a broad shoulder near 330 nm. The optimal pH values were observed in the acidic region and the catalytic temperatures were broad. LacI and LacII both showed not only high catalytic activity at high or low temperature, but also good stabilities toward pH and temperature. LacI and LacII were strongly inhibited by SDS, NaN3,β-mercaptoethanol and KI, but slightly enhanced by EDTA and metal ions of Na+, Cu2+, Mg2+ and Zn2+ at low concentration. LacI and LacII showed a wide range of substrates and the best substrates were both found to be ABTS. The Km values of LacI and LacII was 0.0166 and 0.0435 mmol/L and the catalytic efficiencies was 19640.36 and 31172.64 S-1·mM-1, respectively, for ABTS as substrate. Through UPLC/MS/MS and protein sequences analysis, LacI and LacII were showed to be identical with laccases from genus Pycnoporus.The decolorization and degradation of dyes by P. sanguineus SYBC-L1 laccase were invistegated. Anthraquinone dyes were the laccase substrates and could be directly degraded by P. sanguineus SYBC-L1 laccases, however, azo dyes were not the laccase substrates and could be degraded in the presence of some redox mediators. The decolorization rates could be enhanced by adding some small molecular weight redox mediators. The decolorization rates of RBBR, acid red 1 and reactive black 5 were 94.91%, 90.87% and 72.15%, respectively, after 20 min incubation by adding syringaldehyde, while in the presence of HBT, the decolorization rates of acid blue 129 and reactive blue 4 were 81.73% and 92.18%, respectively, after 60 min incubation. The biodegradations were monitored by HPLC and the results showed some degradation products were generated in the degradation process. Phytotoxicity study revealed that the biodegraded products had no toxicity on wheat germination, but only slightly inhibited on the length of the plumule and radical.