Enzymatic preparation of glycerophosphatilcholine catalyzed by combinational phospholipases: a comparative study of concerted versus stepwise catalysis

Abstract

Glycerophosphatilcholine (GPC) is widely applied in medical, pharmaceutical, food and cosmetic industries. Due to the lack of natural resources, enzymatic preparation of GPC has been explored in recent years. This study aimed to investigate and compare the effects of different addition methods of combinational phospholipases (PLA₁ and PLA₂) and various process parameters (time, temperature, pH, substrate concentrate, enzyme load, and stirring rate) on the preparation of GPC. The results showed that compared with concerted catalysis, the catalytic efficiency of adding PLA₂ and then PLA₁ (PLA₂ → A₁) was higher, whereas that of adding PLA₁ and then PLA₂ was lower. The main reason might be that the method of PLA₂ → A₁ could reduce acyl migration and the competition between PLA₁ and PLA₂, which was beneficial to improve the GPC yield and shorten the reaction time. This paper could provide a novel approach for the future preparation of GPC catalyzed by combinational phospholipases.

Fig. 1. The action sites of four phospholipases. PLA₁, phospholipase A₁; PLA₂, phospholipase A₂; PLB phospholipase B; PLC, phospholipase C; PLD, phospholipase D.

Fig. 2. Schemes of GPC enzymatic preparation from PC via PLA₁ + A₂ (A), PLA₁ → A₂ (B) and PLA₂ → A₁ (C).

Fig. 3. Calibration curves for quantification of GPC and PC.

Fig. 4. A general scheme of this study.

Fig. 5. Time-course of PC conversion in enzymatic hydrolysis catalyzed by PLA₁ (■) and PLA₂ (●). Reaction conditions: temperature, 40 °C, pH, 5.5, substrate concentration, 10%, PLA₁ load, 1.50 wt%, PLA₂ load, 1.50 wt%, stirring rate, 500 rpm.

Fig. 6. Effect of reaction time of method I (PLA₁ + A₂) (A), method II (PLA₁ → A₂) (B) and method III (PLA₂ → A₁) (C) on the GPC yield (■) and PC conversion (○) in enzymatic hydrolysis. Reaction conditions: pH, 5.5, substrate concentration, 10%, PLA₁ load, 0.75 wt%, PLA₂ load, 0.75 wt%, temperature, 40 °C, stirring rate, 500 rpm.

Fig. 7. Effect of temperature on the GPC yield (A) and PC conversion (B) in enzymatic hydrolysis. Method I (PLA₁ + A₂) (■), method II (PLA₁ → A₂) (●), method III (PLA₂ → A₁) (▲). Reaction conditions: pH, 5.5, substrate concentration, 10%, PLA₁ load, 0.75 wt%, PLA₂ load, 0.75 wt%, reaction time of method I (PLA₁ + A₂), 60 min, reaction time of PLA₁ and PLA₂ in method II (PLA₁ → A₂), 30 and 150 min, reaction time of PLA₂ and PLA₁ in method III (PLA₂ → A₁), 60 and 60 min, stirring rate, 500 rpm.

Fig. 8. Effect of pH on the GPC yield (A) and PC conversion (B) in enzymatic hydrolysis. Method I (PLA₁ + A₂) (■), method II (PLA₁ → A₂) (●), method III (PLA₂ → A₁) (▲). Reaction conditions: substrate concentration, 10%, PLA₁ load, 0.75 wt%, PLA₂ load, 0.75 wt%, reaction time of method I (PLA₁ + A₂), 60 min, reaction time of PLA₁ and PLA₂ in method II (PLA₁ → A₂), 30 and 150 min, reaction time of PLA₂ and PLA₁ in method III (PLA₂ →A₁), 60 and 60 min, stirring rate, 500 rpm, temperatures of method I (PLA₁ + A₂) and method III (PLA₂ → A₁), 40 °C, temperature of method II (PLA₁ →A₂), 60 °C.

Fig. 9. Effect of substrate concentration on the GPC yield (A) and PC conversion (B) in enzymatic hydrolysis. Method I (PLA₁ + A₂) (■), method II (PLA₁ → A₂) (●), method III (PLA₂ → A₁) (▲). Reaction conditions: PLA₁ load, 0.75 wt%, PLA₂ load, 0.75 wt%, reaction time of method I (PLA₁ + A₂), 60 min, reaction time of PLA₁ and PLA₂ in method II (PLA₁ → A₂), 30 and 150 min, reaction time of PLA₂ and PLA₁ in method III (PLA₂ → A₁), 60 and 60 min, stirring rate, 500 rpm, temperatures of method I (PLA₁ + A₂) and method III (PLA₂ → A₁), 40 °C, temperature of method II (PLA₁ → A₂), 60 °C, pH of method I (PLA₁ + A₂) and method III (PLA₂ → A₁), 5.5, pH of method II (PLA₁ → A₂), 6.5.

Fig. 10. Effect of enzyme load on the GPC yield (A) and PC conversion (B) in enzymatic hydrolysis. Method I (PLA₁ + A₂) (■), method II (PLA₁ → A₂) (●), method III (PLA₂ → A₁) (▲). Reaction conditions: reaction time of method I (PLA₁ + A₂), 60 min, reaction time of PLA₁ and PLA₂ in method II (PLA₁ → A₂), 30 and 150 min, reaction time of PLA₂ and PLA₁ in method III (PLA₂ → A₁), 60 and 60 min, stirring rate, 500 rpm, temperatures of method I (PLA₁ + A₂) and method III (PLA₂ → A₁), 40 °C, temperature of method II (PLA₁ → A₂), 60 °C, pH of method I (PLA₁ + A₂) and method III (PLA₂ → A₁), 5.5, pH of method II (PLA₁ →A₂), 6.5, substrate concentration, 10%.

Fig. 11. Effect of stirring rate on the GPC yield (A) and PC conversion (B) in enzymatic hydrolysis. Method I (PLA₁ + A₂) (■), method II (PLA₁ → A₂) (●), method III (PLA₂ → A₁) (▲). Reaction conditions: reaction time of method I (PLA₁ + A₂), 60 min, reaction time of PLA₁ and PLA₂ in method II (PLA₁ → A₂), 30 and 150 min, reaction time of PLA₂ and PLA₁ in method III (PLA₂ → A₁), 60 and 60 min, temperatures of method I (PLA₁ + A₂) and method III (PLA₂ → A₁), 40 °C, temperature of method II (PLA₁ → A₂), 60 °C, pH of method I (PLA₁ + A₂) and method III (PLA₂ → A₁), 5.5, ph of method II (PLA₁ →A₂), 6.5, substrate concentration, 10%, PLA₁ load, 0.75 wt%, PLA₂ load, 0.75 wt%.