title: Gene encoding a novel invertase from a xerophilic Aspergillus niger strain and production of the enzyme in Pichia pastoris.
abstract: β-Fructofuranosidases or invertases (EC 3.2.1.26) are enzymes that are widely used in the food industry, where fructose is preferred over sucrose, because it is sweeter and does not crystallize easily. Since Aspergillus niger GH1, an xerophilic fungus from the Mexican semi-desert, has been reported to be an invertase producer, and because of the need for new enzymes with biotechnological applications, in this work, we describe the gene and amino acid sequence of the invertase from A. niger GH1, and the use of a synthetic gene to produce the enzyme in the methylotrophic yeast Pichia pastoris. In addition, the produced invertase was characterized biochemically. The sequence of the invertase gene had a length of 1770 bp without introns, encodes a protein of 589 amino acids, and presented an identity of 93% and 97% with invertases from Aspergillus kawachi IFO 4308 and A. niger B60, respectively. A 4.2 L culture with the constructed recombinant P. pastoris strain showed an extracellular and periplasmic invertase production at 72 h induction of 498 and 3776 invertase units (U), respectively, which corresponds to 1018 U/L of culture medium. The invertase produced had an optimum pH of 5.0, optimum temperature of 60 °C, and specific activity of 3389 U/mg protein, and after storage for 96 h at 4 °C showed 93.7% of its activity. This invertase could be suitable for producing inverted sugar used in the food industry.
authors.affiliation: Universidad Autónoma de Nuevo León, UANL, Facultad de Ciencias Biológicas, Instituto de Biotecnología, 66450 San Nicolás de los Garza, Nuevo León, Mexico; DIA-UAdeC/School of Chemistry, Universidad Autónoma de Coahuila, 25280 Saltillo, Coahuila, Mexico.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Veana
authors.affiliation: Universidad Autónoma de Nuevo León, UANL, Facultad de Ciencias Biológicas, Instituto de Biotecnología, 66450 San Nicolás de los Garza, Nuevo León, Mexico.
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Fuentes-Garibay
authors.affiliation: DIA-UAdeC/School of Chemistry, Universidad Autónoma de Coahuila, 25280 Saltillo, Coahuila, Mexico.
authors.forename: Cristóbal Noé
authors.initials: CN
authors.lastname: Aguilar
authors.affiliation: DIA-UAdeC/School of Chemistry, Universidad Autónoma de Coahuila, 25280 Saltillo, Coahuila, Mexico.
authors.forename: Raúl
authors.initials: R
authors.lastname: Rodríguez-Herrera
authors.affiliation: Universidad Autónoma de Nuevo León, UANL, Facultad de Ciencias Biológicas, Instituto de Biotecnología, 66450 San Nicolás de los Garza, Nuevo León, Mexico.
authors.forename: Martha
authors.initials: M
authors.lastname: Guerrero-Olazarán
authors.affiliation: Universidad Autónoma de Nuevo León, UANL, Facultad de Ciencias Biológicas, Instituto de Biotecnología, 66450 San Nicolás de los Garza, Nuevo León, Mexico. Electronic address: jose.viadersl@uanl.edu.mx.
authors.forename: José María
authors.initials: JM
authors.lastname: Viader-Salvadó
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D045604:Extracellular Fluid / Q000201:enzymology; D005632:Fructose / Q000096:biosynthesis; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005813:Genes, Synthetic; D005947:Glucose / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods; D019897:Periplasm / Q000201:enzymology; D010843:Pichia; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017385:Sequence Homology; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D043324:beta-Fructofuranosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D005632:Fructose; D013395:Sucrose; D043324:beta-Fructofuranosidase; D005947:Glucose
keywords: Aspergillus niger GH1 strain; Pichia pastoris; Recombinant invertase; Synthetic gene; β-Fructofuranosidases