Efficient Expression of Candida rugosa Lipase CRL1 in Pichia pastoris and its Application for Synthesis of Vitamin E Acetate
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Abstract:
In this study, the optimized gene encoding Candida rugosa lipase CRL1 was synthesized in vitro, cloned into the vector pHKA and expressed in Pichia pastoris, producing a lipase activity of 39.73 U/mL towards p-nitrophenyl butyrate (pNPB) after induction for 120 h in flask fermentation. A new gene encoding GS115/pHKA-AOX1m/αm-CRL1 was obtained by modifying the AOX1 promoter and α-factor signal peptide, which lipase activity achieved 63.63 U/mL. Then the purified CRL1 was isolated from the supernatant of the zymotic fluid, through sequential treatments of ultrafiltration, ammonium sulphate precipitation and anion exchange chromatography. The specific enzyme activity of this purified CRL1 was 984.5 U/mg. The recombined CRL1 was characterized by the optimal temperature and pH at 40 ℃ and 7.5, respectively. Besides, it retained 52.99% of its original activity after incubation at 40 ℃ for 6 h and it was relatively stable in a slightly acidic environment. Furthermore, the crude CRL1 was lyophilized and harvested as an effective biocatalyst to synthesize vitamin E acetate in the solvent-free system. The optimal synthesis was performed with 200 mg D-α-tocopherol, 1 mL acetic anhydride, and 100 mg crude CRL1, at 60 ℃ and a shearing rate of 200 r/min for 6 h, of which more than 97% of D-α-tocopherol was transformed.
