Study On Thermostable Laccase Production By Recombinant Trichoderma Reesei

Laccase is a type of multi copper-containing polyphenol oxidases capable of oxidizing a wide range of aromatic compounds. It is a kind of environmental friendly green catalyst which has received numerous interests in industrial applications. In this work, a transformant T. reesei ZU-L10with the highest production of laccase was screened from10recombinant transformants by fermentation experiment, the culture conditions were preliminary optimized. Furthermore, the properties of recombinant T. reesei ZU-L10laccase and its application in gold orangeⅡ and Reactive Brilliant Blue KN-R decolorization was investigated. The major results were as follows:The laccase produced by recombinant T. reesei ZU-L10was investigated in shaking flask cultivation with different culture medium and fermentation conditions. The optimized culture medium contained:lactose30g/L, yeast extract5g/L, ammonium tartrate2.8g/L, MgSO4-7H2O0.6g/L, CaCl2·2H2O0.8g/L, KH2PO44g/L, CuSO4·5H2O0.25g/L. The optimized fermentation conditions was:5%inoculum volume, fermentation temperature28℃, initial pH(5.5). The maximum laccase activity was achieved at96h (8.85IU/mL) in shaking flask cultivation, which was6.7times of basal medium (1.32IU/mL).The properties of recombinant T. reesei ZU-L10laccase was studied. SDS-PAGE analysis showed that the recombinant laccase had a molecular mass of around68kDa. The optimum temperature and pH value for the recombinant laccase reaction were65℃and2.5, respectively. The laccase was stablest at pH3.5, approximately70%of the initial enzyme activity was retained after incubation of the laccase at70℃for60min, indicating that it was intrinsically highly thermostable. The recombinant T. reesei ZU-L10laccase could activated by Li+, Zn2+, Mg2+, K+, Co2+, Ba2+and Cu2+while be inhibited by Fe2+, Mn2+, Ca2+and Na+. The apparent Km of ABTS was14.7μM, the Vmax was4.593mM/min. Furthermore, The effects of primary factors on the decolorization of azo dye gold orange Ⅱ and anthraquinone dye Reactive Brilliant Blue KN-R were preliminarily analyzed. Collaborated with redox mediators HOBT, the decolorization rate of100mg/L gold orange Ⅱ can reached90.6%in5h. While without any add redox mediators,87%of100mg/L RBBR was decolorized after5h. The result of decolorization conditions of two dyes were as follows:(1) the appropriate condition of laccase decoloring gold orange Ⅱ were50℃, pH3.5,2mM HOBT and laccase dosage0.2IU/mL;(2) the appropriate condition of laccase decoloring Reactive Brilliant Blue KN-R were50℃, pH3.5, and laccase dosage1.5IU/mL. Excellent thermostability and efficient decolorization suggest that the recombinant T. reesei ZU-L10laccase could be effectively applied to decolorize the recalcitrant dyes from effluents.