Characterization Of Laccase From Leptographium Qinlingensis Associated With Dendroctonus Armandi

The effect of different culture conditions on laccase activity of Leptographium qinlingensis was studied. The laccase derived from L. qinlingensis was purified and characterized in enzymology. Meanwhile, DNA of L. qinlingensis was extracted, and laccase gene fragment was obtained by PCR. The laccase gene fragment was sequenced and analysised.1. The effect of different culture conditions on the laccase activity of L. qinlingensis was investigated by single-factor experiment. The results shown that laccase activity of L. qinlingensis were significantly increased by maltose (as carbon source) and yeast (as nitrogen source). The laccase activity was induced by copper ions that reached 292.36 IU/L under the medium containing 0.6 mM Cu²⁺, however, the laccase activity was inhibited by copper ions exceeding 0.9 mM. Furthermore, the laccase activity was induced by phloem powder of host trees (Pinus armandi Fr.). The peak of laccase activity occurred at the 11th day. The results showed that different culture conditions have remarkable effect on laccase activity of L. qinlingensis. The maximum laccase activity was 681.82 IU/L in the medium containing maltose as carbon source, yeast as nitrogen source, 0.6 mM Cu²⁺ and 6 g/L phloem powder of host trees at the 11th day.2. The crude enzyme with laccase of L. qinlingensis was concentrated by dialyzing and was purified by DEAE-cellulose (DE-52) chromatographic column (1.6 cm×26 cm). The high-activity laccase was obtained from the elution liquor. The results showed that the laccase activity was 5209.38 IU/L, the specific activity of laccase was 193.84 IU/mg, and the enzymic purity increases 25.63 fold.3. It was studied that the laccase optimal reaction temperature, the laccase thermal stability, the laccase optimal reaction pH, and the laccase pH stability. The variance analysis of the effect of different metal ions on laccase activity was carried on with the software SAS (V8.1). The results revealed that the laccase residual activity reached 90% at 35℃to 45℃, and the laccase reaction did not fit in the condition of high-temperature. The laccase is stable at 25℃to 45℃, and the higher the temperature and the longer the stay warm, the less stable the laccase. The laccase of L. qinlingensis optimal reaction pH is 4.4, the further pH get to 4.4, the lower the laccase activity. The laccase is stable in a pH range from 3.6 to 4.4. 1 mM Cu²⁺ or 1 mM Mg²⁺ was no significantly influence for the laccase activity, however, 10 mM Cu²⁺ or 10 mM Mg²⁺ was extremely significant influence on the laccase activity. Besides, both 1 mM and 10 mM Co²⁺,Mn²⁺,Ca²⁺,Na+,Fe²⁺ were extremely significant influence on the laccase activity.4. DNA of L. qinlingensis was extracted by improved CTAB method, and laccase gene fragment was obtained by PCR. The laccase gene fragment was sequenced and analysised. A comparison of the nucleotide sequences of the aim fragment and the reported laccase gene in GenBank was carried out by Blastn. The nucleotide sequences of the aim fragment and laccase gene nucleotide sequences of others reported 6 fungi strains were analyzed by ClustalX 2.0.10. The evolutionary tree of 7 fungi strains was drawn. Indicated that the aim fragment was closely related to Calocera viscose (EU882517.1) and Trichoderma hamatum (FJ040333.1), and was far related to Inocybe (EU88251.1), Penicillium chrysogenum (XM 002567796.1), Aspergillus niger (XM₀013892), Nectria (XM 003046649.1). Most of the fungi of high similarity with the aim fragment were found in rotten wood and plant residues, so it is relative to lignin degradation.