Study of digestive enzyme activities and partial characterization of digestive proteases in a freshwater teleost, Labeo rohita, during early ontogeny

Abstract

Digestive enzymes of freshwater fish Labeo rohita (family Cyprinidae; class Actinopterygii; infraclass Teleostei; order Cypriniformes) were studied during ontogenic development. Amylase, protease and lipase activities showed a polynomial relationship with age of rohu, whereas trypsin, chymotrypsin and lipase activities exhibited exponential trends with the age of rohu larvae. SDS-PAGE of crude enzyme extract revealed that the proteases of higher molecular weight (MW) appeared during early ontogeny, whereas low MW proteases were observed at a later stage. Substrate SDS-PAGE supported the quantitative study of protease activities as evidenced with the increasing number and intensity of activity bands with the age of fish. The number of protease activity bands observed in 4, 10, 12 and 24 DAH (days after hatching) larvae were 5, 7, 8 and 9, respectively. Inhibition of protease activities with soybean trypsin inhibitor (SBTI) (58.6–81.2%), phenyl methyl sulphonyl fluoride (PMSF) (55.6–70%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (41.1–52.3%) and N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (27.9–44.5%) indicated the presence of serine proteases, trypsin- and chymotrypsin-like enzymes in rohu larvae. Inhibition with SBTI and TPCK showed power, TLCK and ethylenediaminetetraacetic acid showed exponential, whereas PMSF showed polynomial relationships during the study period.