title: Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties.
abstract: Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVAAOX1 and INVBAOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (kcat/KM) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVAAOX1 and INVBAOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVAAOX1 and D-INVBAOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s(-1) mM(-1), respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35-45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVAAOX1, INVBAOX1, D-INVAAOX1 and D-INVBAOX1. The invertase activity of both enzymes increased in 80% (INVAAOX1) and 20% (INVBAOX1) in the presence of Mn(2+) at 1 mM and 5 mM, respectively. INVAAOX1 and INVBAOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVAAOX1. Our findings suggest that glycosylation of INVAAOX1 and INVBAOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses.
authors.affiliation: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Av. Instituto Politécnico Nacional No. 2508, D.F. Mexico CP 07360, Mexico.
authors.forename: Ara Itzel
authors.initials: AI
authors.lastname: Pérez de los Santos
authors.affiliation: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Av. Instituto Politécnico Nacional No. 2508, D.F. Mexico CP 07360, Mexico.
authors.forename: Maribel
authors.initials: M
authors.lastname: Cayetano-Cruz
authors.affiliation: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Av. Instituto Politécnico Nacional No. 2508, D.F. Mexico CP 07360, Mexico.
authors.forename: Marina
authors.initials: M
authors.lastname: Gutiérrez-Antón
authors.affiliation: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Av. Instituto Politécnico Nacional No. 2508, D.F. Mexico CP 07360, Mexico.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Santiago-Hernández
authors.affiliation: Centro de Investigación en Biotecnología Aplicada-IPN, Km 1.5 Carretera Estatal Tecuexcomac-Tepetitla, Tepetitla, Tlaxcala 90700, Mexico.
authors.forename: Miguel
authors.initials: M
authors.lastname: Plascencia-Espinosa
authors.affiliation: Departamento of Alimentos y Biotecnología, Facultad de Química, UNAM, C.P. 04510 D.F. Mexico, Mexico.
authors.forename: Amelia
authors.initials: A
authors.lastname: Farrés
authors.affiliation: Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Av. Instituto Politécnico Nacional No. 2508, D.F. Mexico CP 07360, Mexico. Electronic address: ehidalgo@cinvestav.mx.
authors.forename: María Eugenia
authors.initials: ME
authors.lastname: Hidalgo-Lara
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D017355:Zymomonas / Q000201:enzymology / Q000235:genetics; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; C089498:invB protein, Zymomonas; D013395:Sucrose; D043324:beta-Fructofuranosidase
keywords: AOX1 promoter; Effect of glycosylation; Intracellular/extracellular invertases; Pichia pastoris; Protein expression; Zymomonas mobilis