Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst |
| |
| Authors: | Marcelo Victor Holanda Moura Giulia Pontes da Silva Ant?nio Carlos de Oliveira Machado Fernando Araripe Gon?alves Torres Denise Maria Guimar?es Freire Rodrigo Volcan Almeida |
| |
| Institution: | 1. Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.; 2. Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brasil.; Weizmann Institute of Science, ISRAEL, |
| |
| Abstract: | Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mgdcw and >80 U/mgdcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase. |
| |
| Keywords: | |
|
|
