Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties

Enzyme and Microbial Technology
Ara Itzel Pérez de los SantosMaría Eugenia Hidalgo-Lara

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 h...Continue Reading

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Citations

Feb 26, 2019·Frontiers in Bioengineering and Biotechnology·David RodriguesAdalberto Jr Pessoa
Feb 27, 2017·Brazilian Journal of Microbiology : [publication of the Brazilian Society for Microbiology]·Cesar Andres Diaz AriasAdalberto Pessoa Junior
Feb 18, 2021·Journal of Food Biochemistry·Letícia Mara RasboldAlexandre Maller

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