Over-expression of active Candida rugosa lip1 in Pichia pastoris via high cell-density fermentation and its application to resolve racemic ibuprofen

To improve the productivity of Candida rugosa lipase (CRL) and alleviate respiration limitations during high cell-density fermentation, codon-optimized CRL LIP1, and Vitreoscilla hemoglobin (VHb) were co-expressed in Pichia pastoris. The activity of the recombinant strain that expressed LIP1 and VHb from dual promoters, named GS115/9Klip1FZvgb-lip1(#)1, toward olive oil reached 620U/mL, which was 1.69-fold greater than that of the recombinant strain GS115/9Klip1(#)139 (365U/mL) which only expressed LIP1 from a single promoter, and 1.37-fold greater than that of the recombinant strain GS115/9Klip1FZlip1(#)39 (450U/mL), which only expressed LIP1 from two promoters, in shaking flasks. With FM22 as the basic medium and methanol/D-sorbitol (1:1, v/v) as an inducer, the maximum activity of GS115/9Klip1FZvgb-lip1(#)1 reached 7490 +/- 379.5U/mL, which was 2.65-fold greater than that of GS115/9Klip1(#)139 (2820 +/- 112U/mL) and 1.82-fold greater than that of GS115/9Klip1FZlip1(#)39 (4100 +/- 205U/mL) in 10L fermenters. The conversion ratio (C) and enantiomeric excess (ee(s)) of racemic ibuprofen by immobilized CRL LIP1 reached 35.10 and 31.63%, respectively.