Purification and Characteristics of Beta-1, 4-Glucanase Occel9 of Oxya chinensis

Aims: Oxya chinensis is a very serious paddy pest and causes major loss in rice crop. The objectives of the study were (1) Studying the molecular basis of plant biomass degradation by Oxya chinensis. (2) The role of Neem (Azadirachta indica) derived compounds, Azadirachtin and Saponins was also studied as enzyme inhibitors with special reference to biopesticides.
Place of Study: Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore.
Methodology: Oxya chinensis insects were collected from local fields and dried. The dried insects were studied for the purification and characterization of cellulose hydrolyzing enzyme activity. Enzyme was purified using ion-exchange chromatography. Spectroscopic study was carried out to identify the signature pattern of the protein. The role of purified neem (Azadirachta indica) derived compounds Saponins and Azadirachtin was also studied as enzyme inhibitors with special reference to Biopesticides.
Results: Total soluble protein was isolated from salivary gland and gut and the presence of cellulase activity endo-1, 4-ß-glucanase (OcCel9) in each tissue was indicated. After purification both enzymes appeared at same position on zymogram, containing 2.0% carboxymethyl cellulose as substrate, after non-denaturing PAGE. The beta-1,4-β-glucanase (OcCel9) activity from the gut and salivary gland had the same Km values. The pH and temperature profiles of Oxya chinensis beta-1,4-glucanase indicated optimum pH at 3.0 and optimum temperature at 50ºC. SDS-PAGE analysis indicated that the enzyme has two subunits/isozymes of molecular mass 60 and 55 kDa respectively. The spectroscopic analysis of the isolated protein showed that it had homology with rice, 1, 4-beta-D-glucanase. An internal amino acid sequence of the rice enzyme (OcCel9) revealed that this enzyme belongs to glycosyl hydrolase family 9 (Glycosylhydrolases family 9 active sites signature 2). Neem, Azadirachtaindica derived saponins were able to inhibit OcCel9 on substrate agar plates.
Conclusion: OcCel9 was successfully purified and is a very active cellulose hydrolyzing enzyme and can be of significant technological importance. Moreover the enzyme can be studied as a potential target for producing novel biopesicides.