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In this study, we overcame the limitations of single-enzyme system catalysis by codisplaying Candida rugosa lipase 1 (CRL1) and Rhizopus oryzae lipase (ROL) on the cell surfaces of the whole-cell catalyst Pichia pastoris to produce biodiesel from tallow seed oil. We screened double antibiotic-resistant strains on tributyrin plates, performed second electroporation based on single-displayed ROL on GS115/KpRS recombinants and single-displayed CRL1 on GS115/ZCS recombinants and obtained an ROL/CRL1 codisplay on P. pastoris GS115 surfaces. The maximum activity of the codisplaying GS115/pRCS recombinant was 470.59 U/g dried cells, which was 3.9-fold and 1.3-fold higher than that of single-displayed ROL and CRL1, respectively. When self-immobilized lipases were used as whole-cell catalysts, the rate of methyl ester production from GS115/pRCS harboring ROL and CRL1 was 1.4-fold higher than that obtained with single-displayed ROL. Therefore, biodiesel catalysis by synergetic codisplayed enzymes is an alternative biodiesel production strategy.

Codisplay of Rhizopus oryzae and Candida rugosa Lipases for Biodiesel Production