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Purification and partial characterization of acetic acid esterase from malted finger millet (Eleusine coracana, Indaf-15).

Latha, G. Madhavi and Muralikrishna, G. (2007) Purification and partial characterization of acetic acid esterase from malted finger millet (Eleusine coracana, Indaf-15). Journal of agricultural and food chemistry, 55 (3). pp. 895-902. ISSN 0021-8561

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Abstract

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.

Item Type: Article
Uncontrolled Keywords: Acetic acid esterase; ragi; finger millet; purification; homogeneity
Subjects: 600 Technology > 08 Food technology > 21 Cereals > 05 Ragi (Finger Millet)
500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
Divisions: Dept. of Biochemistry
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 07 Aug 2008 11:01
Last Modified: 21 Dec 2016 12:23
URI: http://ir.cftri.com/id/eprint/1892

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