Studies On Activity, Purificaton And Property Of Laccase From Pleurotus Eryngii Var. Tuoliensis

Laccases (p-diphenol: oxygen oxidoreductases, EC1.10.3.2) are blue multi copper-containingphenol, play an important role of lignin degradation and are closely related to the growth of P. eryngiivar. tuoliensis. In order to clarify the effects of environmental conditions on laccase activity, growth anddevelopment of P. eryngii var. tuoliensis in solid culture or liquid culture, the mycelia growth rate,laccase activity, biomass and biological efficiency were measured under different temperatures andinitial pH treatment conditions. Moreover, to clear the property of laccase of P. eryngii var. tuoliensis,isolation and purification of laccase from the fermentation broth were carried. Finally, the results are asfollows.⑴By single factor experiment, the laccase activity, mycelia growth rate and biomass of P. eryngiivar. tuoliensis were measured under four different temperatures treatments, and the results showed thatthe biomass increased gradually with the temperature increasing in liquid culture from18℃to27℃,but the order of laccase activity after13d is18℃>21℃>24℃>27℃. In solid state media, the order ofmycelia growth rate was24℃>21℃>27℃>18℃; when the substrate in test tubes were covered withmycelium, the order of laccase activity is18℃>24℃>21℃>27℃.(2) Cultivation experiments were finished under four different temperature treatment conditions,the results showed that the order of the average yield of single packet is21℃>24℃>18℃>27℃.There is an average yield difference of26.8g between the highest and the lowest, and the biologicalefficiency of the two treatments is63.04%and55.37%, respectively. Difference of its average yield issignificant. Cultivation period of18℃treatment is longer than that of the other three treatments about20d.The optimum culture temperature during the growth of P. eryngii var. tuoliensis mycelia is between21℃~24℃.(3) By single factor experiment, the laccase activity, mycelia growth rate and biomass of P. eryngiivar. tuoliensis were examined under six initial pH treatment conditions. The results were the optimuminitial pH was5.91for laccase activity and6.16for the growth of mycelia under solid culture conditions.Under liquid culture conditions, laccase activity increased gradually with the initial pH increasing from5.48to6.71. Change of biomass first increased, and then tended to be stable, and finally decreased.(4) Cultivation experiments were finished under six different initial pH treatment conditions in thispaper. The results showed that the average yield of per experimential bag and biological efficiency ofpH5.91treatment was263.03g and75.15%, respectively; and which of the initial pH6.36treatmentwas215.11g and62.73%, respectively. There is no significance among the six treatments. Takeproduction and cultivation factor into consideration, the initial pH value of5.91is the best treatment.(5) By adding different kinds of inducer (including CuSO4, p-coumaric acid, vanillin) to GYPmedium, the effect of inducer on P. eryngii var. tuoliensis laccase activity were tested. The results wereas follows: both the type and concentration level of the inducers have effect on the P. eryngii var.tuoliensis laccase activity. The induction of copper was the most significant and laccase activity reached maximum when its concentration was100μM. The effect of p-coumaric acid was the second, and whenthe best induction concentration was200μM. Vanillin produced the minimal effect on laccase, andlaccase activity reached the highest value at100μM.(6) An extracellular laccase (designed as PnLac) from the culture broth of P. eryngii var. tuoliensis00485myceliua was isolated through ion exchange chromatography on DEAE-cellulose, CM-cellulose,and SP-Sepharose, and gel filtration on Superdex75. The enzyme was purified99.3-fold from crudeextract with a16.6%yield. SDS-PAGE showed PnLac was a monmeric protein of65kDa. Three aminoacid sequences were obtained after tryptic digestion of PnLac, and they exhibited some similarity tothose of laccases from the mushrooms Pleurotus ostreatus, Lentinus sajur-caju and Pleurotus eryngii byNBCI-BLAST. Its optimum pH and temperature were3.0and50℃for ABTS [2,2′-azinzo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt], respectively. The laccase activity was inhibitedby Ca2+and Hg2+while increased by Cu2+and Mn2+. The Km and Vmax values of PnLac was0.17mmol/L and1.76OD/min/U, respectively.