Cloning, Site-directed Mutagenesis Of The Laccase Gene From Neurospora Crassa And Its Expression In Pichia Pastoris

Laccases are multicopper phenoloxidases that catalyze the oxidation of a variety of phenolic compounds, with concomitant reduction of O2 to H2O. Interest in laccases has been fueled by their potential uses in detoxification of environmental pollutants, prevention of wine decoloration, paper processing, enzymatic conversion of chemical intermediates, and production of useful chemicals from lignin. Laccase from Neurospora crassa(NCL)is a representative ascomycete laccase. This paper is mainly focused on construction, site-directed mutagenesis, enzymic properties and culture conditions of a laccase-secreting engineered strain from Neurospora crassa.The structure gene of NCL without signal sequence was amplified from genomic DNA of N.crassa by using Successive PCR method. The gene was inserted into the multiple cloning site (MCS) of pPIC9K, a secreting expression vector of Pichia pastoris, so the recombinant plasmid pPIC9K/lacc could be used in extracellular expression of the NCL. The plasmid pPIC9K/lacc was linearized with SacⅠand used to transform P. pastoris KM71. After induction with methanol, the recombinant P. pastoris expressed active NCL enzyme. The maximum activity of laccase reached 2.94U/mL. The molecular weight of the recombinant laccase was about 64.8kD by SDS-PAGE.The mutant gene of NCL was amplified from recombinant plasmid pPIC9K/lacc by using Successive PCR method. The gene was inserted into the multiple cloning site (MCS) of pPIC9K, so the recombinant plasmid pPIC9K/ lacc′could be used in extracellular expression of the NCL. The plasmid pPIC9K/ lacc′was linearized with SacⅠand used to transform P. pastoris KM71. After induction with methanol, the recombinant P. pastoris expressed active NCL enzyme. The maximum activity of laccase reached the same level as its wild-type strain.Both the mutant laccase and the wild-type laccase were purified to apparent electrophoretic homogeneity using Sephadex G-15 and DEAE Sepharose. The molecular weights of the two purified laccases were about 64.8kD by SDS-PAGE. With guaiacol the pH optima of the mutant enzyme and the wild-type laccase were 8.0 and 6.0, respectively. The mutant laccase was stable at pH 3.5 to 9.0, whereas the wild-type laccase was stable at pH 3.5 to 7.5. Compared with the wild-type laccase, the purified enzyme had no changes in other enzymic properties.Fermentation conditions of the mutant strain in shake-flask cultivation were investigated. The maximum biomass(OD600) reached 5.362 when the strain was cultured at 30℃and pH5.5 with 20g/L glucose as carbon source and 20g/L corn steep liquor as nitrogen source. The maximum activity of laccase reached 3.88U/mL when the strain was cultured at 30℃and pH5.5 with 0.5% methanol as carbon source and 20g/L corn steep liquor as nitrogen source.The maximum activity of laccase reached 3.96U/mL when the strain was cultured in a 5 L fermenter.