Efficient Hydrolysis of Fish Parvalbumin by Marine Bacterial Protease VSP2V-280: Allergen Removal

Abstract

Parvalbumin is a major allergen in fish. However, there is currently no effective and safe way to remove this allergen from fish. In this study, protease gene VSP2V-280 of marine bacteria Virgibacillus sp. SP2 was cloned and expressed. The protease enzyme showed maximum activity at 50°C and pH 10.0. Ca²⁺ and Cu²⁺ promoted the enzyme. The enzyme showed good parvalbumin degradation efficiency in fish. Based on the gel analysis, when 0.3 mg/mL of parvalbumin was incubated with protease VSP2V-280 (30 U/mL) containing 1 mM Ca²⁺ for 3 h, the parvalbumin removal rate reached 97%. The enzyme was further used for parvalbumin removal from Ctenopharyngodon idella, Pelteobagrus fulvidraco, Parabramis pekinensis, and Carassius auratus. The parvalbumin removal rate reached 93% in 4 h at an enzyme dosage of 72 U/mL. The study showed the potential of VSP2V-280 to remove parvalbumin from aquatic products.

Keywords: allergen removal; cloning and expression; enzymatic properties; parvalbumin; protease.

FIGURE 1

Cloning of marine protease VSP2V‐280: (a) amplified products of VSP2V‐280 target gene, (b) pCold I‐VSP2V‐280 clone plasmid, and (c) double digestion of pCold I‐VSP2V‐280 clone plasmid.

FIGURE 2

Purification of marine protease VSP2V‐280 and parvalbumin: (a) band diagram of marine protease VSP2V‐280 eluted using imidazole solutions of different concentrations; (b) band diagram of parvalbumin eluted using imidazole solutions of different concentrations. Here, L1: crude enzyme solution; L2: effluent; L3: 20 mM imidazole elution; L4: 40 mM imidazole elution; L5: 60 mM imidazole elution; and L6: 80 mM imidazole elution.

FIGURE 3

Effect of temperature (20°C–60°C) and pH (4–12) on marine protease VSP2V‐280: (a) effect of temperature on activity of VSP2V‐280, (b) temperature stability of VSP2V‐280, (c) effect of pH on activity of VSP2V‐280, and (d) pH stability of VSP2V‐280.

FIGURE 4

Effect of metal ions (1–5 mM), inhibitors (5 mM), organic solvents (10%), and surfactants (10%) on marine protease VSP2V‐280: (a) effect of metal ions on activity of VSP2V‐280 and (b) effects of inhibitors, organic solvents, and surfactants on protease activity.

FIGURE 5

Parvalbumin degradation analysis by SDS‐PAGE gel assay under different concentrations of enzyme (18–30 U/mL) and presence/absence of Ca²⁺ (final concentration 1 mM): (a, b) 0.3 mg/mL of parvalbumin + enzyme activity of 18 U/mL without Ca²⁺; (c, d) 0.3 mg/mL of parvalbumin + enzyme activity of 18 U/mL with Ca²⁺; (e, f) 0.3 mg/mL of parvalbumin + enzyme activity of 24 U/mL enzyme without Ca²⁺; (g, h) 0.3 mg/mL of parvalbumin + enzyme activity of 24 U/mL with Ca²⁺; (i, j) 0.3 mg/mL of parvalbumin + enzyme activity of 30 U/mL without Ca²⁺; and (k, l) 0.3 mg/mL of parvalbumin + enzyme activity of 30 U/mL with Ca²⁺.

FIGURE 6

Standard ELISA curve for detection of parvalbumin and the residual amount of parvalbumin after digestion of 0.3 mg/mL of parvalbumin by protease (24 U/mL) under different conditions.

FIGURE 7

ELISA test of parvalbumin in fish: (a) parvalbumin content in different fish residues under 54 U/mL enzyme at 0, 1, and 4 h. (b) Residual parvalbumin in C. idella with different enzyme doses at 0, 1, and 4 h.

FIGURE 8

Molecular docking between Ca²⁺ and protease VSP2V‐280: (a) binding position of Ca²⁺ to VSP2V‐280 and (b) result of Ca²⁺ docking with VSP2V‐280.