Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Physico-chemical characterization of amylases of L. decemlineata and their inhibitors from plants
Structural and functional properties of enzymes and inhibitors involved in pathological processes are intensively investigated in recent years (Pradhan et al, 2014; Selvaraj et al, 2014). A major role in the interaction between insects and plants play hydrolytic enzymes of phytophagon and their inhibitors from plants (Louis et al, 2013). Amylases are considered as one of the most active carbohydrases of Colorado potato beetle (Leptinotarsa decemlineata Say). Properties of some amylases of Colorado potato beetle are known, but the study of the structure of enzymes and their specific plants inhibitors remains an urgent task. In this study we investigated the physico-chemical properties and molecular composition of amylases of Colorado potato beetle and their inhibitors from the leaves of the potato.
For chromatographic purification of insects amylases and their protein inhibitors from plants the affinity sorbent based on polyacrylamide gel was used. As a ligand was used starch.
According to 2D-electrophoresis results, amylases of Colorado potato beetle are presented by four proteins (or groups of proteins with similar isoelectric point) with a molecular weight in the range from 30 to 50 kDa. Apparently, these proteins are alpha-amylases, including well-known representatives of the molecular weight of 40-60 kDa. Perhaps among the studied proteins are also presented low-molecular forms of beta-amylases or fragments of these molecules, containing the substrate binding site.
Amylases of Colorado potato beetle are active in a relatively wide range of pH. Enzymes carry out the hydrolysis of starch in the pH interval from 6 to 8, maximal activity is observed at neutral pH. By this property, amylases of the beetles do not differ essentially from described in the literature amylolytic enzymes of microorganisms and various animals, including insects and mammals (Horvathova, Janecek, Sturdik, 2000). Amylolytic enzymes of Colorado potato beetle are most active in the range from 30 to 40 °C. When exposed to temperatures above 60 °C the enzyme activity is almost completely lost.
For the selection of inhibitors of amylases from plant tissue as affine ligands were used early purified enzymes. Inhibitors of amylases have relatively large diversity of molecular content, which presents by five proteins with isoelectric point in the pH range from 5 to 6. Molecular weight of amylases inhibitors is 15-25 kDa, which corresponds to the characteristic of many known amylases of plant and microbial origin (Feng et al, 1996).
Selvaraj C, Singh SK (2014) Validation of potential inhibitors for SrtA against Bacillus anthracis by combined approach of ligand-based and molecular dynamics simulation J Biomol Struct Dyn. 32(8), 1333-1349.
Louis J, Peiffer M, Ray S, Luthe D and Felton G (2013) Host-specific salivary elicitor(s) of European corn borer induce defenses in tomato and maize New Phytologist 199(1), 66 - 73. Horvathova V, Janecek S, Sturdik E. (2000) Amylolytic enzymes: their specificities, origins and properties Biologia, Bratislava 55(6), 605 - 615.
Feng G, Richardson M, Shun Chen M, Kramer K, Morgan T, Reeck G. (1996) Alpha-amylase inhibitors from wheat: Amino acid sequences and patterns of inhibition of insect and human alpha-amylases Insect Biochemistry and Molecular Biology 26(5), 419 - 426.
Vyacheslav O. Tsvetkov
Department of Biology