INVESTIGADORES
CERMINATI Sebastian
artículos
Título:
Pichia pastoris engineering for the production of a modified phospholipase C
Autor/es:
ELENA, CLAUDIA; RAVASI, PABLO; CERMINATI, SEBASTIÁN; PEIRÚ, SALVADOR; CASTELLI, MARÍA EUGENIA; MENZELLA, HUGO
Revista:
PROCESS BIOCHEMISTRY - (Print)
Editorial:
ELSEVIER SCI LTD
Referencias:
Lugar: Amsterdam; Año: 2016 vol. 51 p. 1935 - 1944
ISSN:
1359-5113
Resumen:
Crude vegetable oils are refined to remove impurities that adversely impact in their stability, color and flavor. In recent years, enzymatic degumming methods using phospholipase C (PLC) enzymes provide an environmentally friendly process for phospholipids removal with improved oil recovery yields.In this study, Pichia pastoris was used as the expression system for the production of PLC-Y, a modified PLC enzyme derived from Bacillus cereus. Production of secreted PLC-Y driven by the methanol inducible AOX1 promoter was optimized by genetic strain engineering which included gene codon optimization, generation of multi-copy chromosomal integrations and the co-expression of helper factors supporting protein folding, processing and secretion processes. In addition, tunable promoters directing helper factor expression were tested.In batch cultures, a strain harboring seven integrated copies of the PLC-Y expression cassette and co-expressing the HAC1 transcription factor under an attenuated AOX1 promoter showed a 6.2 fold increase in the production titers compared to the strain harboring a single gene copy. A fed-batch fermentation process developed using this engineered strain produced 4.5 g/l of this enzyme. The results presented in this work show the viability of using PLC-Y for oil degumming and provide a manufacturing process for its cost effective production.