High-level expression and characterization of Galactomyces geotrichum (BT107) lipase I in Pichia pastoris.
The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by PCR from genomic DNA, sequenced and cloned into a Pichia pastoris expression vector. Clones containing multiple copies of lipI integrated in their genome were analyzed to achieve high-level expression of the recombinant lipase I. One strain with four or more copies of the expression cassette was able to produce more than 200mg/L of extracellular heterologous protein. The lipase I was partially purified using anion exchange chromatography and its activity on monounsaturated (triolein) and polyunsaturated (triEPA) triglycerides was analyzed by a novel HPLC-MS assay.
Fernández
Layla
L
Department of Biotechnology, Puleva Biotech, Camino de Purchil, 66, 18004-Granada, Spain.
Pérez-Victoria
Ignacio
I
Zafra
Alberto
A
Benítez
Pedro L
PL
Morales
Juan C
JC
Velasco
Javier
J
Adrio
José L
JL
D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal / Q000235:genetics; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012440:Saccharomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase