Department of Biosciences, Himachal Pradesh University, Shimla 171005, India.
Department of Biosciences, Himachal Pradesh University, Shimla 171005, India.
Int J Biol Macromol. 2024 Oct;278(Pt 4):135009. doi: 10.1016/j.ijbiomac.2024.135009. Epub 2024 Aug 22.
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 and V to be 0.09% and 3.3 U/mL, respectively. Oxalic acid, tannic acid, and HgCl 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.
南美咖啡豆象是世界范围内最臭名昭著的储藏型菜豆害虫之一。菜豆是这些昆虫的重要食物来源。α-淀粉酶是包括昆虫在内的动物中主要的碳水化合物消化酶。在本研究中，生化分析显示，在 3 龄和 4 龄幼虫期，α-淀粉酶活性（U/ml）较高，但在随后的发育阶段逐渐降低。然而，有趣的是，比活力（U/mg）在 1 龄幼虫期最高，并在进一步的发育阶段降低。在使用淀粉琼脂和 native PAGE 进行α-淀粉酶的定性分析时，在 3 龄和 4 龄幼虫期观察到淀粉裂解的最大区域和凝胶上的明显条带。天然酶的分子量也被估算出来，发现为 30.34 kDa。粗α-淀粉酶通过硫酸铵沉淀、Sephadex G-75 凝胶过滤和 DEAE 纤维素柱离子交换层析进一步纯化。纯化的淀粉酶被发现是一种单体，分子量为 15 kDa。纯化酶的比活力从粗样品中的 1.74 U/mg 增加到最后纯化步骤中的 166.35 U/mg，纯化倍数为 95 倍，产率为 11.14%。进一步对纯化的α-淀粉酶进行表征，发现其 pH 最适值为 7.0，温度最适值为 35°C。Lineweaver-Burk 作图分析显示 K 和 V 分别为 0.09%和 3.3 U/mL。草酸、单宁酸和 HgCl 显著抑制该酶，而 Na、Ca 和 Mg 离子则作为激活剂。总之，该研究表明，南美咖啡豆象的 3 龄和 4 龄幼虫期α-淀粉酶活性最高，随后是 native 和 SDS PAGE，分子量分别为 30.34 和 15 kDa。
