Purification and characterization of ferulic acid esterase from malted finger millet (Eleusine coracana, Indaf-15).
Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5'-OH group of arabinosyl residues of arabinoxylans and is known to modulate their functional properties. In this study, ferulic acid esterase from 96 h finger millet malt was purified to apparent homogeneity by three-step purification with a recovery of 3% and a fold purification of 22. The substrate p-nitrophenylferulate (PNPF) was synthesized and used to assay this enzyme spectrophotometrically. The products liberated from ragi and wheat water-soluble polysaccharides by the action of purified ragi ferulic acid esterase were identified by ESI-MS. The pH and temperature optima of the enzyme were found to be 6.0 and 45 degrees C, respectively. The pH and temperature stabilities of the enzyme were found to be in the range of 5.5-9.0 and 30 degrees C, respectively. The activation energy of the enzymatic reaction was found to be 4.08 kJ mol(-1). The apparent K m and V max of the purified ferulic acid esterase for PNPF were 0.053 microM and 0.085 unit mL(-1), respectively. The enzyme is a monomer with a molecular mass of 16.5 kDa. Metal ions such as Ni(2+), Zn(2+), Co(2+), and Cu(2+) and oxalic and citric acids enhanced the enzyme activity. The enzyme was completely inhibited by Fe(3+). Group specific reagents such as p-chloromercuric benzoate and iodoacetamide inhibited the enzyme, indicating the possible presence of cysteine residues in the active site pocket.
Latha
G Madhavi
GM
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570 020, Karnataka, India.
Srinivas
P
P
Muralikrishna
G
G
D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D002412:Cations; D003373:Coumaric Acids / Q000378:metabolism; D002523:Edible Grain / Q000201:enzymology*; D031748:Eleusine / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D013379:Substrate Specificity
D002412:Cations; D003373:Coumaric Acids; D004791:Enzyme Inhibitors; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase