Isolation and characterization of endochitinase and exochitinase of Setaria cervi.
Chitin metabolism has been shown to have a role in the development of parasitic nematodes including filarial parasites and the enzymes associated with chitin metabolism have been considered as potential vaccine and drug target. Chitinases are members of the enzyme superfamily of glycoside hydrolases, which are characterized by the ability to hydrolyze glycosidic bonds in chitin chain by either an endolytic or an exolytic mechanism. In the present study, we have demonstrated the chitinase (exochitinase and endochitinase) activity in different stages of Setaria cervi (bovine filarial parasite) and have also purified and characterized the endochitinase from microfilarial stage of the parasite. The chitinase activity has been detected in adult and microfilarial stages of S. cervi using the fluorescent substrates. The S. cervi adult stage was found to have high activity of exochitinase (28.72±0.25 nmol/min/mg) while microfilarial stage showed high activity of endochitinase (24.40±0.25 nmol/min/mg). Native polyacrylamide gel electrophoresis, followed by staining of enzyme activity with fluorescent substrates, revealed single isoenzymic form of exochitinase in adults and endochitinase in microfilariae of S. cervi. The endochitinase from S. cervi microfilariae was purified employing chitin affinity matrix and DEAE-Sephacel ion-exchange chromatography. The enzyme was purified about 55 fold with an enzyme recovery of 22.33%. The purified enzyme exhibited a doublet of protein bands on SDS-PAGE at 65-70 kDa. The closantel (chitinase inhibitor) strongly inhibited the enzyme activity of S. cervi microfilariae endochitinase with a Ki value of 4.3±0.18 μM.
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226031, India.
Piyush
P
Dravid
Amity University Uttar Pradesh, Lucknow Campus, Lucknow 226028, India.
Deep C
DC
Kaushal
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow 226031, India.
Jitendra K
JK
Saxena
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226031, India. Electronic address: nuzhatkaushal@hotmail.com.
Nuzhat A
NA
Kaushal
D000818:Animals; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006596:Hexosaminidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008842:Microfilariae / Q000201:enzymology / Q000378:metabolism; D012458:Salicylanilides / Q000378:metabolism; D017182:Setaria Nematode / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism
D012458:Salicylanilides; D002686:Chitin; C079639:1,4-beta-chitobiosidase; D006596:Hexosaminidases; D002688:Chitinases; C023342:closantel
Bovine filarial parasite; Chitinase; Endochitinase; Exochitinase; Filariasis; Setaria cervi