Li_2020_Protein.Expr.Purif__105804

Lipase from Rhizomucor miehei (RML) is a versatile biocatalyst used in food industry, fine chemicals, and biodiesel production. Yeast surface display allows direct application of lipase in form of whole-cell biocatalyst, avoiding purification and immobilization process, but the protease of the host cell may affect the activity of displayed lipase. Herein, we used the protease-deficient Pichia pastoris, PichiaPink(TM) to efficiently display RML. RML gene, GCW21 gene and alpha-factor gene were co-cloned into plasmid pPink LC/HC and transformed into protease-deficient P. pastoris. After inducible expression for 96 h, the lipase activity of displayed RML reached 121.72 U/g in proteinase-A-deficient P. pastoris harboring high-copy plasmid, which exhibited 46.7% higher than recombinant P. pastoris without protease defect. Displayed RML occurred the maximum activity at pH 8.0 and 45 degC and the optimal substrate was p-nitrophenyl octanoate. Metal ions Li(+), Na(+), K(+), and Mg(2+) of 1-10 mM had activation towards displayed RML. Displayed RML was effectively improved in PichiaPink(TM) protease-deficient system, which may promote the further research and development of the industrial application of RML.