Induction, Dye Decolorization And Molecular Cloning Of Laccase From The Coprinus Comatus

Laccases are blue multi copper-containing phenol oxidases. Its ability to degrade ligin, removal of toxic compounds and decolourization of various industrial dyes brings laccases a wide application prospect in environmental protection, pulp and paper production and textile industry.The static and shaking cultivation methods were used for Coprinus comatus laccase production. The laccase activity of 0.55 IU/ml was obtained on the 21 day but it just took 7 days to reach the same activity at rotary shaker and the activity can reach to 1.61 IU/ml on the 19 day. It was concluded that shaking cultivation method can reduce the enzyme production time and the laccase activity was three times higher than the static cultivation. So shaken culture is more conducive for Coprinus comatus to produce laccase. The proportion of carbon and nitrogen source was another key factor for the Coprinus comatus laccase production. In this experiment four different media LNHC (low nitrogen and high carbon), HNLC (high nitrogen and low carbon), LNLC (low nitrogen and low carbon) and HNLC (high nitrogen and high carbon) were prepared to study its effects on laccase production. The result was that the LNHC medium showed the highest activity and HNLC medium produced more isoenzymes than other media.Base on the results, LNHC and HNLC were selected to produce laccase with different inducer. LNHC culture medium can lead to highest laccase activity by o-Toluidine (3.25IU/ml). HNLC culture medium can lead to laccase isoforms by o-Toluidine (2mM) and Tannic acid (0.8mM).The crude enzymes from Coprinus comatus cultivated with LNHC and HNLC media were used to study the decolourization for dyes. The reaction mixture for decolourization consist of three types of dyes respectively (50mg/ml), crude laccase (0.5IU/mL) by different inducers and 1-hydroxybenzotriazole (HBT) (final concentration,0.1g/L) in Acetate buffer (0.1mol/L, pH4.5) in a final volume of 2 mL. It can be concluded that the crude enzymes cultivated by different media showed higher activity in Anthraquinone dyes and Triphenylmethane dyes than Azo dyes removal. Decolorization percentage of Brilliant Blue R,Bromophenol blue,Reactive Orange1 and Congo red were 71%,78%,14% and 4% without the addition of redox mediators. In addition, it was demonstrated that the crude enzymes containing more laccase isoforms in the HNLC medium induced by o-Toluidine or Hydroxybenzoic acid showed higher decolorization rate to Anthraquinone dyes than the crude enzymes in the LNHC medium.Laccase induced by ferulic acid or copper in the HNLC medium with more isoforms showed lower decolorization ratio than the crude enzymes in the LNHC medium. With the addition of HBT, decolorization rate increased significantly. Gene specific primers are designed based on the sequence of Coprinus comatus which we have cloned in the earlier stage experiment. SMARTTM RACE cDNA Amplification Kit was used to get the full-length cDNA of the laccase named lac3. The full length of the lac3 cDNA was 1968bp, containing an open reading frame of 1554bp encoding for 535 amino acids, among which the former 18 amino acids belong to the signal peptide. The theoretical pI and the molecular mass are 5.29 and 50064.87 Dalton. EasySelectTM Pichia Expression was used for the heterogenous expression of lac3. PPICZaB was used as expression vector and KM71H (Muts, Arg+) as host strain. The laccase Lac3 activities were not detected in both MM plates (with Cu2+ and ABTS) and shaken-flask cultivation (cultured in BMMY).