Cooperation of enzymes involved in carbohydrate digestion of Colorado potato beetle (Leptinotarsa decemlineata, Say)

Colorado potato beetle (Leptinotarsa decemlineata, Say) is the main pest of Solanaceae and its survival is mainly dependent on the carbohydrate digestion. Characterizing the gut enzymes may help us with finding effective inhibitors for plant protection. Activity measurements revealed that gut extracts contain alpha- and beta-glucosidase in addition to alpha-amylase. For larvae, amylase activity was detected only in gut saturated with nutrients. Leptinotarsa decemlineata alpha-amylase (LDAmy) had optimum pH of 6.0 and was active under 30-40 degrees C temperature measured on a selective alpha-amylase substrate, 2-chloro-4-nitrophenyl-4-O-alpha-D-galactopyranosyl-maltoside. HPLC analysis demonstrated dimer, trimer, and tetramer reducing end amylolytic products from 2-chloro-4-nitrophenyl-maltoheptaoside substrate in similar ratio than that of during porcine pancreatic alpha-amylase (PPA) catalyzed hydrolysis. The 4,6-O-benzylidene-modified substrate (BzG7PNP) is very stable toward hydrolysis by exo-glycosidases, therefore is very useful to monitor the digestion catalyzed by alpha-amylases exclusively. Similarly to PPA active site, three glycon and two aglycon binding sites are suggested for LDAmy based on the pattern of early hydrolysis products of BzG7PNP. The observed similarity between LDAmy and PPA raises the possibility of using known inhibitors of mammalian alpha-amylases to protect the potato plant from attack of Colorado potato beetle.