A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour

Abstract

This study reports on the purification and characterization of a digestive α-amylase from blue crab (Portunussegnis) viscera designated Blue Crab Amylase (BCA). The enzyme was purified to homogeneity by ultrafiltration, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with the final purification fold of 424.02, specific activity of 1390.8 U mg−1 and 27.8% recovery. BCA, showing a molecular weight of approximately 45 kDa, possesses desirable biotechnological features, such as optimal temperature of 50 °C, interesting thermal stability which is enhanced in the presence of starch, high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), high specific activity, quite high storage and broad pH range stability. The enzyme displayed Km and Vmax values, of 7.5 ± 0.25 mg mL−1 and 2000 ± 23 μmol min−1 mg−1 for potato starch, respectively. It hydrolyzed various carbohydrates and produced maltose, maltotriose and maltotetraose as the major end products of starch hydrolysis. In addition, the purified enzyme was successfully utilized for the improvement of the antioxidant potential of oat flour, which could be extended to other cereals. Interestingly, besides its suitability for application in different industrial sectors, especially food industries, the biochemical properties of BCA from the blue crab viscera provide novel features with other marine-derived enzymes and better understanding of the biodegradability of carbohydrates in marine environments, particularly in invasive alien crustaceans.

Toward a More Comprehensive View of α-Amylase across Decapods Crustaceans: New Insights from Carnivorous

“…Studies in the crabs Carcinus maenas [60], N. granulata [10], and M. spinosissimus [53], in the spiny lobster P. argus [24], in the crayfish Cherax quadricarinatus [61], in three species of penaeid shrimps [13] and in other invertebrates [62][63][64] have showed enhancements in α-amylase activity when CaCl2 concentration increases up to a maximum and then decrease. However, exceptions occurred, as in the lobster Homarus americanus, where no effect of calcium was reported [55], while in the crab Portunus segnis, only minor effect of calcium was reported [65]. Calcium binding sites are important structural features of amylase enzymes, and it is well known that this ion is important for the activity and stability of α-amylases.…”

“…In the spiny lobster, this enzymatic activity is strongly affected at alkaline pH values [18]. Crustacean α-amylases are known to be divided into two groups, one with optimal pH below 6.3 including isopods, amphipods and Astacura, and other groups with higher pH optimum comprising shrimps and brachyurans [54,65,67,68]. A variety of optimal pH values for α-amylase also occur in insects, other very diverse group of invertebrates.…”

“…Although the α-amylase of the tropical king crab M. spinosissimus was stable at a high temperature (> 50 o C) [53], αamylase activity from the tropical lobster P. argus is compromised above 30 °C [24] as in other crustaceans. Yet, in the crab P. segnis, which is tolerant of a wide range of temperatures from 13 °C to 30 °C, the enzyme is also highly stable at 50 o C [65]. Less variation has been observed in optimal temperature [i.e.…”

“…lobster P. argus, 50 °C [24]; shrimp F. californiensis, 30-40 °C [54]; and different species of crab, e.g. Scylla serrata, 50 o C [68], N. granulate, 30-40 °C [10]; and P. segnis 50 o C [65]. In summary, the relationship between stability and habitat temperature is not clear, probably because this feature mostly depends on the conserved architecture of the enzyme among crustaceans.…”

“…potato, wheat, maize) is also a source of variation. So, in the crab P. segnis, Km for α-amylase was reported to be 7.5 mg mL -1 for potato starch [65] but the catalytic activity was lower toward other starches.…”