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

• Published in: Biocatalysis and biotransformation 2015-11, Vol.33 (5-6), p.260-269
• Main Authors: Xiao, Xiao, Liu, Zhenyu, Chen, Yuan, Wang, Guilong, Li, Xiang, Fang, Zhonggang, Huang, Shuangshuang, Liu, Zimin, Yan, Yunjun, Xu, Li
• Format: Article
• Language: English
• Subjects: Candida rugosa; Candida rugosa lipase LIP1; co-expression; Olea; Pichia pastoris; resolution; Vitreoscilla; Vitreoscilla hemoglobin
• ISSN: 1024-2422; 1029-2446
• DOI: 10.3109/10242422.2016.1168815

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 620 U/mL, which was 1.69-fold greater than that of the recombinant strain GS115/9Klip1 # 139 (365 U/mL) which only expressed LIP1 from a single promoter, and 1.37-fold greater than that of the recombinant strain GS115/9Klip1FZlip1 # 39 (450 U/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 ± 379.5 U/mL, which was 2.65-fold greater than that of GS115/9Klip1 # 139 (2820 ± 112 U/mL) and 1.82-fold greater than that of GS115/9Klip1FZlip1 # 39 (4100 ± 205 U/mL) in 10 L fermenters. The conversion ratio (C) and enantiomeric excess (ee s ) of racemic ibuprofen by immobilized CRL LIP1 reached 35.10 and 31.63%, respectively.