Screening Of Edible Fungi With High Laccase Production And Study On Enzymatic Properties Of Laccase From Ganoderma Sinense

Laccase is a kind of copper-containing polyphenol oxidase,and its only by-product is water,which is a natural and green catalyst.Therefore,laccases are widely used in food,medicine,cosmetics,bioremediation,and biosensors.Laccase is widely distributed in fungi,bacteria,plants,and insects.White-rot fungi are the main source of laccase.The edible fungi in white-rot fungi have good food safety,which can meet the safety requirements of laccase-producing microorganisms in the food industry.Laccase from edible fungi also has the advantages of easy access（extracellular enzyme）and high redox potential,and has great application potential in food,feed,medicine,and other fields.Therefore,in this study,we screened strains with high laccase production from 55 edible fungi,then optimized the optimal media and culture conditions for laccase production,isolated and purified the laccase from fermentation broth,and studied the enzymatic properties of laccase.The main results are as follows:First of all,based on NCBI（National Center of Biotechnology Information）database,Uniprot database（Unified Protein Database）,and literature databases were searched for edible fungi containing laccase gene sequences（or amino acid sequences）or producing laccase.Based on the search results,edible fungi were collected for subsequent experiments.The actual collected edible fungi were screened for preliminary screening（plate assay）and secondary screening（submerged fermentation）,and the results were as follows.A total of 97 edible fungi with laccase gene sequences were screened from the NCBI database,and 93 edible fungi with laccase amino acid sequences were screened from Uni Pprot database.A total of 74 edible fungi producing laccase have been reported.55 edible fungi were actually collected.The plate assay was used for the preliminary screening of 55 edible fungi,51 of which produced laccase.Then,51 strains of edible fungi were screened by submerged fermentation.The Ganoderma sinense strain Gs-1 with high laccase activity was used in subsequent experiments.Secondly,the optimal media and culture conditions for laccase production of G.sinense Gs-1 were optimized by single factor experiments and response surface experiment design,and the results were as follows.The optimal liquid media for strain Gs-1 was glucose 19.21g/L,tryptone 2.0 g/L,Cu²⁺1.08 mmol/L,and soybean meal 32.59 g/L.Under the optimized conditions,the laccase activity of G.sinense reached 6787.58 U/L.The optimal culture conditions of strain Gs-1 were temperature 25.5℃,p H 4.9,loading volume 100 m L,and fermentation time 10.1 days.Under the optimized conditions,the laccase activity of G.sinense reached 9278.52 U/L.Finally,the crude laccase was purified by ammonium sulfate salting out,dialysis bag dialysis,and anion exchange chromatography to obtain the laccase isoenzyme.The specific activity of the isoenzyme was 44.57 U/mg.The purification multiple was 8.21 times,and the recovery rate was 15.44%.A single laccase,Lac3,with a molecular weight of about 61.22k Da,was recovered from Native-PAGE electrophoresis.The enzymatic properties of crude laccases,their isoenzymes,and Lac3 were studied.Using guaiacol as the substrate,the optimal temperature of the three laccase samples was 50℃,and the optimal p H was 4.5.They had good stability at 30-40℃and p H 5.0-9.0.When the concentration of metal ions was 0.5 and1.0 mmol/L,Cu²⁺promoted the activities of the three laccases,while only Ni²⁺promoted the activities of the isoenzymes and Lac3 enzymes.The activities of the three laccases were completely inhibited by the presence of Fe²⁺.The relative activity of the three laccases was more than 60%after incubation with methanol,ethanol,glycerin,isopropanol,DMSO,acetone,and other organic solvents（30%）at room temperature for 24 h,indicating that they had good tolerance to organic solvents.With guaiacol as the substrate,the Mi constant K_mvalues of crude laccase,isoenzyme,and Lac3 were 0.17,0.15,and 0.13 mmol/L,respectively,and the maximum reaction rates V_max were 7.82,5.95,and 9.73μmol/min,respectively.Ultraviolet spectrum analysis showed that the isoenzyme and Lac3 had no absorption peak at610 nm,but had a weak shoulder absorption peak at 280 nm,indicating that the isoenzyme and Lac3 might be yellow laccases.Infrared spectrum analysis showed that the contents of secondary structure in the isoenzyme wereβ-fold 17.60%,random coil 33.75%,α-helix31.58%,andβ-angle 17.06%,respectively.The content of secondary structure in Lac3 wasβ-fold 20.39%,random coil 36.81%,α-helix 27.11%,andβ-angle 15.68%,respectively.