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.
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.
Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
Marcelo Victor Holanda
MV
Moura
Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
Giulia Pontes
GP
da Silva
Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
Antônio Carlos de Oliveira
AC
Machado
Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brasil.
Fernando Araripe Gonçalves
FA
Torres
Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
Denise Maria Guimarães
DM
Freire
Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
Rodrigo Volcan
RV
Almeida
D002175:Candida / Q000201:enzymology / Q000235:genetics*; D002384:Catalysis; D061505:Cell Surface Display Techniques; D019295:Computational Biology / Q000379:methods; D004795:Enzyme Stability; D015870:Gene Expression; D017930:Genes, Reporter; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D020798:Two-Hybrid System Techniques
D008049:Lipase