Purification and characterization of α-amylase from Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae)

Acanthoscelides obtectus is one of the most notorious pests of stored kidney beans (Phaseolus vulgaris) worldwide. Kidney beans are an important source of food for these insects. α-Amylase is the main carbohydrate-digesting enzyme in animals including insects. In the current study, the biochemical analysis revealed higher α-amylase activity (U/ml) in 3rd and 4th larval instars but decreased gradually in subsequent developmental stages. However, the specific activity (U/mg) interestingly was highest in 1st instar and decreased in further developmental stages. During qualitative analysis of α-amylase using starch-agar and native PAGE, the maximum zone of starch lysis and a prominent band on the gel was observed in 3rd and 4th larval stages. The molecular mass of the native enzyme was also estimated and found to be 30.34 kDa. The crude α-amylase was further purified by ammonium sulfate precipitation, gel filtration on a Sephadex G-75, and ion exchange chromatography on the DEAE cellulose column. The purified amylase was found to be a monomer with a molecular mass of 15 kDa. The specific activity of the purified enzyme increased from 1.74 U/mg in the crude sample to 166.35 U/mg in the final purification step resulting in 95-fold purification with a yield of 11.14%. Further characterization of purified α-amylase revealed a pH optimum of 7.0 and a temperature optimum of 35 °C. Lineweaver-Burk plot analysis revealed K_m and V_max to be 0.09% and 3.3 U/mL, respectively. Oxalic acid, tannic acid, and HgCl₂ significantly inhibited the enzyme, while the Na⁺, Ca^++, and Mg⁺⁺ ions acted as activators. In conclusion, the study revealed, the highest α-amylase activity in 3rd and 4th larval stages of Acanthoscelides obtectus followed by native and SDS PAGE resulting in molecular mass of 30.34 and 15 kDa respectively.