Effect of Glu235Ser on enzyme performance and in vitro characterization of human β-glucocerebrosidase in a Pichia pastoris.

Gaucher disease (GD) is the most common lysosomal storage disorder and is caused by inherited deficiency of β-glucocerebrosidase (GCase), resulting in glucocerebroside accumulation in multiple organs. Enzyme replacement therapy (ERT) is associated with complex and costly manufacturing processes, which has motivated the development of scalable expression systems. This study investigates recombinant production of human GCase using the Pichia pastoris GlycoSwitch® platform. The human β-glucocerebrosidase (gba) gene was cloned and expressed to generate both wild-type (WT) and mutant (Glu235Ser) GCase as functional glycoproteins. Both enzymes were purified and characterized using biochemical, kinetic, and computational analyses. The mutant exhibited higher catalytic efficiency, showing ~20 and ~10-fold increases in kcat/KM toward pNPG and 4-MUG, respectively, compared with WT. Both enzymes retained a similar optimal pH (8.0), while the mutant showed higher residual activity at elevated temperatures, retaining 50-60% activity with an optimal range of 60-70 °C. Structural modeling of the Glu235Ser variant suggested that catalysis might involve a His-Asp dyad mechanism. Modeling also indicated that the boat conformation of pNPG is both energetically favorable and catalytically preferred. In vitro assays indicated approximately 2-fold higher activity retention of the mutant under acidic and short-term plasma incubation conditions. In PMA-differentiated U937 macrophage-like cells, the mutant showed increased cellular uptake and prolonged intracellular activity compared to the wild-type enzyme; however, these effects may not be solely attributed to the mutation and may also be influenced by differences in glycosylation or other factors.