Purification and partial characterization of acetic acid esterase from malted finger millet (Eleusine coracana, Indaf-15).
Acetic acid esterase (EC 3.1.1.6) cleaves the acetyl groups substituted at O-2/O-3 of the xylan backbone of arabinoxylans and is known to modulate their functional properties. To date, this enzyme from cereals has not received much attention. In the present study, acetic acid esterase from 72 h ragi malt was isolated and purified to apparent homogeneity by a four-step purification, i.e., ammonium sulfate precipitation, DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose column chromatography, with a recovery of 0.36% and a fold purification of 34. The products liberated from alpha-NA and PNPA by the action of purified ragi acetic acid esterase were authenticated by ESI-MS and 1H NMR. The pH and temperature optima of the enzyme were found to be 7.5 and 45 degrees C, respectively. The enzyme is stable in the pH range of 6.0-9.0 and temperature range of 30-40 degrees C. The activation energy of the enzymatic reaction was found to be 7.29 kJ mol-1. The apparent Km and Vmax of the purified acetic acid esterase for alpha-NA were 0.04 microM and 0.175 microM min-1 mL-1, respectively. The molecular weight of the native enzyme was found to be 79.4 kDa by GPC whereas the denatured enzyme was found to be 19.7 kDa on SDS, indicating it to be a tetramer. EDTA, citric acid, and metal ions such as Fe+3 and Cu+2 increased the activity while Ni+2, Ca+2, Co+2, Ba+2, Mg+2, Mn+2, Zn+2, and Al+3 reduced the activity. Group-specific reagents such as eserine and PCMB at 25 mM concentration completely inhibited the enzyme while iodoacetamide did not have any effect. Eserine was found to be a competitive inhibitor.
Latha
G Madhavi
GM
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570020, Karnataka, India.
Muralikrishna
G
G
D000115:Acetylesterase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002412:Cations / Q000494:pharmacology; D031748:Eleusine / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D011489:Protein Denaturation; D012639:Seeds / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization
D002412:Cations; D000115:Acetylesterase