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

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 INVA_AOX1 and INVB_AOX1. The expression levels of INVA_AOX1 (1660U/mg) and INVB_AOX1 (1993U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVA_AOX1 and INVB_AOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (k_cat/K_M) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVA_AOX1 and INVB_AOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVA_AOX1 and D-INVB_AOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164s^-1mM^-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 INVA_AOX1, INVB_AOX1, D-INVA_AOX1 and D-INVB_AOX1. The invertase activity of both enzymes increased in 80% (INVA_AOX1) and 20% (INVB_AOX1) in the presence of Mn²⁺ at 1mM and 5mM, respectively. INVA_AOX1 and INVB_AOX1 were highly active at sucrose concentrations of up to 400 and 300mM, respectively; however, the tolerance to sucrose decreased to 300mM for D-INVA_AOX1. Our findings suggest that glycosylation of INVA_AOX1 and INVB_AOX1 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.