Purification And Laccase From Huaier And The Function Of Laccase Mediator System On Reactive Dyes Degradation

Laccase is a polyphenol oxidase and widely distributed in plants,fungi,bacteria,insects,etc.It has a wide range of substrates and can catalyze the oxidative decomposition of aromatic and non-aromatic compounds such as lignin,phenols and amines.Since its oxidation process and products do not produce by-products of environmental pollution,it is called“green catalyst”and is widely used in environmental protection,papermaking,textile,food processing,drug improvement and biomonitoring.It has been reported in the literature that laccases combining with certain small molecule compounds,so called laccase mediator system?LMS?,have more application value for environmental protection and lignin degradation.In this study,wild Huaier fruiting bodies collected from Populus canadensis were used as experimental materials.Further studies included pure culture strain isolation,identification,optimum culture conditions for mycelia,optimal laccase fermentation conditions,laccase purification,enzymatic properties,and cloning,degradation towards reactive dyes by the laccase mediator systems.The specific research results are as follows:1.Combined with morphological analysis and ITS?internal transcribed spacer?identification,the XS-01 strain was identified as Perenniporia robiniophila,and the optimal carbon source for mycelial growth was starch?4.2±0.1 mm/d?and maltose?4.2±0.1 mm/d?,the optimal nitrogen source is:Yeast extract?4.2±0.1 mm/d?,the optimum growth factor is:VB₁?3.5±0.1 mm/d?,the optimum C/N ratio range is 30/1-60/1,the optimum temperature is:32°C?5.8±0.1 mm/d?,and the optimum growth pH is 7?5.9±0.1 mm/d?.The optimal laccase conditions for fermentation were 5%inoculum.XS-01 was inoculated into PD medium supplemented with 1.0 mmol/L Cu²⁺,and cultured at 28°C for96 h to obtain maximum enzyme activity with 8.3×10⁴ IU.2.Crude laccase solution of XS-01 strain was obtained under the above fermentation condition and followed centrifugal filtration.Subsequently,enzyme solution was purified by DEAE-Sepharose,SP-Sepharose,Q-Sepharose and Superdex 75 gel filtration chromatography.The purified laccase was determined as a single-subunit protein of 66 kDa by SDS-PAGE and gel filtration chromatography methods.The final recovery rate and purification factor were 3.19%and 70.49,respectively.The purified laccase demonstrated an optimal temperature range of 50-60°C with considerable high thermostability.It possessed an optimal pH value of pH 2.2,but quite sensitive in the pH range of2.2-2.6 with about 40-53%activity loss after 1 h incubation.The K_m and V_maxax were determined to be54.3?mol/L and 5.8?mol/min respectively at 50°C,pH 2.2,and ABTS as the substrate.The purified laccase was inhibited by the tested metal ions except K⁺and Na⁺in the ions concentration of 1.25-10mmol/L.Fe³⁺was determined to be the highest inhibitor of the laccase with 51.22%activity loss after 1h incubation.A 859 bp of partial core region of the laccase gene was obtained using Cu?and Cu?as primers.It demonstrates the higher similarity of 74.63%towards the laccase gene of Dichomitus squalens LYAD-421 SS1.3.The purified laccase possessed degradation activities towards reactive brilliant blue?RBB?,reactive brilliant orange?RBO?,and reactive black?RB?with degradation rate of 27%,10%and 2%,respectively.During the laccase mediator system?LMS?assay,acetosyringone?AS?and syringaldehyde?SA?were determined to be the best mediators of the laccase towards the three reactive dyes.The optimal reaction condition was determined to be 50°C,pH 5.0,the mediator concentration of 0.1mmol/L.After 0.5 h incubation,the degradation rate of L+AS towards the three dyes was 75%,79%,and 91%,respectively.That of L+SA was 65%,77%,92%,respectively.Toxicity tests were carried out on the degradation products of the three dyes under optimal conditions with ryegrass,Escherichia coli and Staphylococcus aureus.The products of the three reactive dyes had no obvious toxicity towards the two bacteria.