Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.
Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.
Department of Applied Microbiology, Centre for Chemistry and Chemical Engineering, Lund Institute of Technology/Lund University, P.O. Box 124, Sweden.
Feng
F
Hong
Nina Q
NQ
Meinander
Leif J
LJ
Jönsson
D004795:Enzyme Stability; D005285:Fermentation / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D042845:Laccase; D000432:Methanol / Q000494:pharmacology*; D010088:Oxidoreductases / Q000096:biosynthesis*; D010843:Pichia / Q000187:drug effects / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D013696:Temperature; D014183:Transplantation, Heterologous
D011994:Recombinant Proteins; D010088:Oxidoreductases; D042845:Laccase; D000432:Methanol