Strategic optimization of β-galactosidase binding performance in packed bed ion exchange chromatography

Abstract

This study engineered Pichia pastoris to express β-galactosidase (β-gal), and its downstream purification was systematically optimized. Growth conditions were optimized in shake flask and fermenter cultures, with the fermenter achieving an OD₆₀₀ of approximately 45 after 24 h. The biomass was disrupted by high-pressure homogenization (30 kpsi, six cycles, 4 °C) to produce a clarified suspension (25 %, w/v) containing 2.02 × 10³ U/mL β-gal activity and 17.2 mg/mL total protein. STREAMLINE DEAE, a high-density ion-exchange adsorbent, was used to assess β-gal adsorption across pH 4–12, with optimal binding at pH 6 and a maximum static capacity of 5.6 × 10⁵ U/mL. One-factor-at-a-time (OFAT) experiments in packed bed mode evaluated the flow rate, bed height, feedstock concentration, and residence time effects on the 5 % dynamic binding capacity (DBC). Further optimization using fractional factorial design (FFD) and response surface methodology (RSM) produced a second-order predictive model relating DBC to flow rate (F), clarified feed concentration (C₀), and bed height (H). At optimal conditions (F: 2.96 mL/min; C₀: 45 % w/v; H: 16.6 cm), the predicted DBC (1.15 × 10⁵ U/mL) closely matched the experimental value (1.13 × 10⁵ U/mL). This work demonstrates a practical, model-driven approach to optimize high-density enzyme production and purification using packed bed ion-exchange chromatography.