title: Biochemical properties of glycosylation and characterization of a histidine acid phosphatase (phytase) expressed in Pichia pastoris.
abstract: Phytases catalyze the cleavage of phosphate groups from phytic acid. Here, we have studied the effects of glycosylation on the properties of Aspergillus japonicus C03 phytase expressed in Pichia pastoris. The enzyme ORF of 1338 nucleotides was cloned from genomic DNA, and encoded a secreted mature protein of 446 amino acids, which included the sequence motif RHGXRX and dipeptide HD, classifying the phytase as a histidine acid phosphate. After transformation and 72h of induction, P.pastoris GS115 expressed a 75kDa protein showing 526U/mg phytase activity and 143mg/L of protein. The amino acid sequence showed 8 and 3 potential N- and O-glycosylation sites, respectively. Analysis by ESMS showed two glycoform masses of 75,467 and 72,793, which after deglycosylation decreased to 54,327 and 54,128, respectively, indicating a carbohydrate content of 27-30%. A single GlcNAc was assigned at Asn6, Asn38, Asn84, Asn99, Asn209, Asn218, Asn355 and Asn367. The recombinant phytase showed maximum activity at 50°C, a half-life of 40min, and farUVCD spectroscopy indicated a secondary structure rich in α-helix. Thermal denaturation analyses reveal the melting temperature varied from 50°C at pH 6 to a maximum of 66°C at pH 3 and pH 4.
authors.affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brazil; Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14040-901 Ribeirão Preto, SP, Brazil.
authors.forename: Raquel
authors.initials: R
authors.lastname: Fonseca-Maldonado
authors.affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brazil.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Maller
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Marcelle-Coutu Pavilion 2950, Montréal, Canada.
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneil
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Marcelle-Coutu Pavilion 2950, Montréal, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14049-901 Ribeirão Preto, SP, Brazil.
authors.forename: Carla
authors.initials: C
authors.lastname: Botelho-Machado
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14049-901 Ribeirão Preto, SP, Brazil.
authors.forename: Richard John
authors.initials: RJ
authors.lastname: Ward
authors.affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14040-901 Ribeirão Preto, SP, Brazil. Electronic address: polizeli@ffclrp.usp.br.
authors.forename: Maria de Lourdes Teixeira de Moraes
authors.initials: Mde L
authors.lastname: Polizeli
mesh_terms: D010832:6-Phytase / Q000378:metabolism*; D000135:Acid Phosphatase; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D000135:Acid Phosphatase; D010832:6-Phytase
keywords: Aspergillus japonicus C03; Glycosylation; Molecular structure; Phytase; Pichia pastoris