Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica.
A cDNA encoding for laccase (Lcc1) was isolated from the ligninolytic fungus Trametes versicolor by reverse transcriptase polymerase chain reaction. The Lcc1 gene was subcloned into the Pichia methanolica expression vector pMETalphaA and transformed into the P. methanolica strains PMAD11 and PMAD16. The extracellular laccase activity of the PMAD11 recombinants was found to be 1.3-fold higher than that of the PMAD16 recombinants. The identity of the recombinant protein was further confirmed by immunodetection using the Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form. The effects of copper concentration, cultivation temperature, pH and methanol concentration in the BMMY on laccase expression were investigated. The laccase activity in the PMAD11 recombinant was up to 12.6 U ml(-1) by optimization.
Guo
Mei
M
Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300222, PR China.
Lu
Fuping
F
Du
Lianxiang
L
Pu
Jun
J
Bai
Dongqing
D
D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D003300:Copper / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D003300:Copper; D042845:Laccase; D000432:Methanol