The Separation And Application Of Laccase From Agaricus Bisporus

Laccase?EC 1.10.3.2?is a copper-containing polyphenol oxidase,which has been widely found in plants,bacteria,higher and fungi,insects.It has widely applied in food industry,paper industry,organic synthesis,industrial dyes and medical fields.In addition,it has also attracted people attention in environmental protection.The present work showed that the laccase were extracted from the fruiting of Agaricus bisporus and the enzymatic properties of laccase were analyzed using 2,2'-Azinobis-3-ethylbenzthiazoline-6-sulphonate?ABTS?as a substrate.The specific activity of the laccase was 56.58 U/mg protein,the recovery yield was 3.9%and the purification fold was 2.8 after homogenization,filtration,ammonium sulfate precipitation and anion exchange chromatography.When the property of laccase was determined by ABTS,the optimal conditions were pH 2.4 and 65?.And the stability of the laccase was stable under the conditions of pH 2.4 at 30?.The enzyme activity could be increased by Cu2+,but inhibited by Fe2+.Under the optimum condition,the value of Km and Vmax was 2.94 mmol/L and 0.20 mmol/?L·min?for ABTS,respectively.The effects of pH on the oxidation of catechins and acetaminophen were studied by using laccase from Agaricus bisporus.At 30 ?,the optimum pH for the laccase to catechins and acetaminophen was 3.0 and 2.2,respectively.Under reaction conditions of 30 ?,pH 3.0,1 U/mL laccase for 96h,catechins or the same concentration of acetaminophen was added alone,and the residual of catechins or acetaminophen percentage was 3%and 21%,respectively.When the reaction system contained catechins and acetaminophen,the result showed that the laccase prefer to catalyze the oxidation of catechins rather than acetaminophen.The immobilization conditions of laccase and the optimum of loading amounts of laccase were determined and the enzymatic properties of immobilized laccase were studied.The amino-functionalized silica nanoparticles were prepared by adding 0.04 g SiO₂ nanoparticles,4 mL anhydrous ethanol and 80 ?L 3-aminopropyltriethoxysilane?APTS?at 30 ? for 2 h.Using glutaraldehyde as a crosslinker,laccase was added to the suspension of the amino-functionalized nano-SiO₂ particles at 4 ? overnight for making immobilized laccase.The immobilization yield experiment indicated that the optimum value was 6 U laccase protein/g of SiO₂ nanoparticles.The optimum pH and temperature for the immobilized laccase were 2.2 and 65 ?,respectively,which is similar to the free laccase.The kinetic parameter of the immobilized laccase for ABTS showed that Km was 0.88 mmol/Land Vmax was 8.32 × 10-3 mmol/?L·min?.Compared with the free enzyme,it was more easily bound to ABTS.At 30 ? and pH 2.2,the immobilized laccase was more stable.The recycle times of the immobilized laccase was 5-10 times.