title: Tannase sequence from a xerophilic Aspergillus niger Strain and production of the enzyme in Pichia pastoris.
abstract: Tannin acyl hydrolases, or tannases (EC 3.1.1.20), are enzymes with potential biotechnological applications. In this work, we describe the gene and amino acid sequences of the tannase from Aspergillus niger GH1. In addition, we engineered Pichia pastoris strains to produce and secrete the enzyme, and the produced tannase was characterized biochemically. The nucleotide sequence of mature tannase had a length of 1,686 bp, and encodes a protein of 562 amino acids. A molecular model of mature A. niger GH1 tannase showed the presence of two structural domains, one with an α/β-hydrolase fold and one lid domain that covers the catalytic site, likely being residues Ser-196, Asp-448, and His-494 the putative catalytic triad, which are connected by a disulfide bond between the neighboring cysteines, Cys-195 and Cys-495. A 120-ml shake flask culture with a constructed recombinant P. pastoris strain showed extracellular tannase activity at 48 h induction of 0.57 U/ml. The produced tannase was N-glycosylated, consisted of two subunits, likely linked by a disulfide bond, and had an optimum pH of 5.0 and optimum temperature of 20 °C. These biochemical properties differed from those of native A. niger GH1 tannase. The recombinant tannase could be suitable for food and beverage applications.
authors.affiliation: Facultad de Ciencias Biológicas, Instituto de Biotecnología, Universidad Autónoma de Nuevo León (UANL), Av. Universidad S/N, Col. Ciudad Universitaria, 66455, San Nicolás De Los Garza, NL, Mexico.
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Fuentes-Garibay
authors.forename: Cristóbal Noé
authors.initials: CN
authors.lastname: Aguilar
authors.forename: Raúl
authors.initials: R
authors.lastname: Rodríguez-Herrera
authors.forename: Martha
authors.initials: M
authors.lastname: Guerrero-Olazarán
authors.forename: José María
authors.initials: JM
authors.lastname: Viader-Salvadó
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C022711:tannase