Molecular gene cloning and overexpression of the phytase from Debaryomyces castellii CBS 2923.
The ORF encoding the Debaryomyces castellii CBS 2923 phytase was isolated. The deduced 461-amino-acid sequence corresponded to a 51.2 kDa protein and contained the consensus motif (RHGXRXP) which is conserved among phytases. No signal sequence cleavage site was detected. Nine potential N-glycosylation sites have been predicted. The protein shared 21-69% sequence identities with various phytases of yeast or fungal origin. Heterologous expression of the D. castellii CBS 2923 phytase in the methylotrophic yeast Pichia pastoris was tested under both the P. pastoris inducible alcohol oxidase (AOX1) promoter and the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Maximum production levels obtained were 476 U ml(-1), with the AOX1 expression system and 16.5 U ml(-1) with the GAP one. These productions corresponded to a 320-fold and a 10-fold overexpression of the protein, respectively as compared to the homologous production. The biochemical characteristics of the recombinant phytase were identical to those of the native enzyme.
Ragon
Mélanie
M
Equipe Génie Microbiologique et Enzymatique, SUPAGRO-INRA, 2 place Viala, 34060 Montpellier Cedex 01, France.
Neugnot-Roux
Virginie
V
Chemardin
Patrick
P
Moulin
Guy
G
Boze
Hélène
H
D010832:6-Phytase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D004718:Saccharomycetales / Q000201:enzymology*
D011994:Recombinant Proteins; D010832:6-Phytase