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

Process Biochemistry. 2019; 
JingXiaQiWangQuanLuoYuChenXiang-RuLiaoZheng-BingGuan
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Gene Synthesis Given the inherent codon bias of P. pastoris strains, the GWLF gene was modified according to its preferred codon usage in P. pastoris GS115 (Fig. S1). The codon-optimized gene, containing the restriction sites EcoR I and Avr II, was designed and synthesized by Genscript (Nanjing, China). Then, it was subcloned into pPIC9K for secretory expression in P. pastoris. In order to select efficient transformants, the codon-optimized gene and non-optimized gene were transformed into P. pastorissynchronously to enhance extracellular expression of GWLF. Get A Quote

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... More

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