SCREENING OF PICHIA PASTORIS TRANSFORMANTS PRODUCERS OF THE RECOMBINANT LIPASE PF2001Δ60 USING A LARGE SCALE METHOD
G.C. BREDA1*, M.V.H. MOURA1 AND R.V. ALMEIDA1
1Universidade Federal do Rio de Janeiro, Instituto de Química
Among the biocatalysts, lipases are enzymes of wide industrial application. Our research group seeks to study new enzymes with potential biotechnological applications, such as lipase Pf2001 from the thermophilic archaeal Pyrococcus furiosus that has been cloned and expressed in Pichia pastoris with promising results of production and activity. In this work we performed the insertion of the gene pf2001 into the genome of P. pastoris X-33 based on the construction pPICZαPf with a selection in different concentrations of antibiotic Zeocin (200, 500 and 1000 mg/mL), in order to obtain recombinants with a higher number of copies of the gene. Starting at 151 recombinant strains, it was established a method of analysis in large scale in order to obtain clones with a higher production of the lipase Pf2001 compared to that obtained in previous work of the group. Four new clones were selected with higher activity in extracts containing lipase, 2 of them with a production of at least 37% (27.3 U/L) higher than previously observed.
Financial Support: FAPERJ, CAPES E CNPq ; Area: Biochemistry.
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