Secretory expression and optimization of Bacillus pumilus CotA-laccase mutant GWLF in Pichia pastoris and its mechanism on Evans blue degradation

摘要

In the work Pichia pastoris system was selected to achieve secretory expression of a D501 G/L386 W/G417 L/G57 F mutant of CotA-laccase from Bacillus pumilus (GWLF) with highly improved catalytic efficiency and soluble expression level. The molecular mass of recombinant GWLF was estimated to be 75 kDa, and the active GWLF is a dimeric glycoprotein. A maximal activity of 17,080 +/- 515.30 U/L in the culture supernatant was obtained by high cell density fermentation in a 5 L bioreactor. Biological toxicity test showed that GWLF can efficiently detoxify Evans blue. The mechanism for Evans blue decolorization and detoxification by GWLF was analyzed through liquid chromatography mass spectrometry; the azo bond (-N=N-) was transformed into N-2 instead of toxic aniline compounds, in which water was the only by-product in the degradation process. This study is the first to clarify the mechanism of laccase-catalyzed degradation of Evans blue.