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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Biochemical properties of glycosylation and characterization of a histidine acid phosphatase (phytase) expressed in Pichia pastoris

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Author(s):
Fonseca-Maldonado, Raquel [1, 2] ; Maller, Alexandre [2] ; Bonneil, Eric [3] ; Thibault, Pierre [3] ; Botelho-Machado, Carla [4] ; Ward, Richard John [4] ; Teixeira de Moraes Polizeli, Maria de Lourdes [1]
Total Authors: 7
Affiliation:
[1] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, BR-14040901 Ribeirao Preto, SP - Brazil
[2] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, BR-14049900 Ribeirao Preto, SP - Brazil
[3] Univ Montreal, Inst Res Immunol & Canc, Montreal, PQ - Canada
[4] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, BR-14040901 Ribeirao Preto, SP - Brazil
Total Affiliations: 4
Document type: Journal article
Source: Protein Expression and Purification; v. 99, p. 43-49, JUL 2014.
Web of Science Citations: 14
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 72 h of induction, P. pastoris GS115 expressed a 75 kDa protein showing 526 U/mg phytase activity and 143 mg/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 degrees C, a half-life of 40 mm, and farUVCD spectroscopy indicated a secondary structure rich in a-helix. Thermal denaturation analyses reveal the melting temperature varied from 50 degrees C at pH 6 to a maximum of 66 degrees C at pH 3 and pH 4. (C) 2014 Elsevier Inc. All rights reserved. (AU)