Streamlined production of immobilized D-psicose 3-epimerase via secretion in Pichia pastoris: a new paradigm for industrial D-psicose production

BACKGROUND: D-psicose, a rare sugar with significant health benefits, holds great promise as a low-calorie sweetener. Its synthesis requires the enzyme called D-psicose 3-epimerase (DPEase), which converts D-fructose into D-psicose. This study focuses on an alternative protein expression system for secretion DPEase production, using Pichia pastoris KM71. The gene encoding DPEase from Bacillus sp. KCTC 13219 was codon-optimized and fused downstream of the α-factor signal peptide. A one-step purification and immobilization method was developed by directly binding crude DPEase to a His-tag affinity column, enhancing both enzyme stability and reusability. RESULTS: The recombinant DPEase was successfully expressed in P. pastoris and efficiently secreted into the culture medium, simplifying downstream processing. The purified DPEase exhibited optimal activity at pH 6.0 and 60 °C, demonstrating remarkable thermostability and maintaining over 80% relative activity across a broad pH range (pH 5.0-11.0) and temperature range (35-70 °C). Purification with 200 mM imidazole elution resulted in a 12.54-fold increase in the purification factor, achieving a specific activity of 3.65 Units/mg. The maximum D-psicose conversion rate of purified DPEase was 17.03% at 120 min reaction with 10% (w/v) D-fructose. The developed DPEase immobilization system showed high binding efficiency, facilitating one-step purification and immobilization for ready-to-use DPEase column. The immobilized enzyme could be reused up to five cycles, maintaining 83.38% relative activity, highlighting the potential of this system for efficient D-psicose production. CONCLUSIONS: This study successfully developed a prototype system for extracellular DPEase production in a recombinant microorganism. This streamlined enzyme purification and immobilization, significantly reducing the DPEase production costs. The recombinant DPEase exhibited remarkable stability across a wide range of pH and temperature. This broad stability makes the enzyme highly promising for industrial-scale D-psicose production, resulting in reduced energy costs and simplified synthesis process. The DPEase demonstrated desirable properties for various D-psicose conversion conditions, and the immobilized enzyme exhibited efficient reusability. These findings support the potential application of this system for large-scale production of D-psicose, a rare sugar with promising uses in the food and pharmaceutical industries.