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Partial Purification and Characterization of Peroxidase from Pearl Millet ( P ennisetum G laucum [ L .] R . Br .) Grains
Author(s) -
Goyal Preeti,
Chugh Lakshman K.
Publication year - 2014
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/jfbc.12033
Subject(s) - peroxidase , chemistry , sephadex , size exclusion chromatography , chromatography , enzyme assay , dtnb , enzyme , sodium azide , biochemistry , glutathione
Peroxidase from pearl millet hybrid HHB 94 was purified stepwise by ( NH 4 ) 2 SO 4 fractionation, gel filtration and ion exchange chromatography using Sephadex G ‐100 and DEAE ‐cellulose to near homogeneity. The protocol yielded 41% of peroxidase with 33.2‐fold purification. The purified enzyme preparation has molecular masses of 31 kDa, as determined by gel filtration through Sephadex G ‐100, and 30.4 kDa, as determined by SDS‐PAGE , suggesting that the enzyme is a monomer. The enzyme exhibited maximum activity at pH 5.6 and 30C. It was significantly more stable in the pH range of 6.0–6.8 and at temperature below 35C. Peroxidase showed K m values of 0.10 and 11 mM for o ‐dianisidine and H 2 O 2 , respectively. The activity of peroxidase was highly inhibited by DTT , β‐mercaptoethanol and hydrazine, and moderately by sodium azide, sodium borohydride, oxalic acid, EDTA , DTNB , Mn 2+ , Na + and K + . The enzyme activity was enhanced by Ca 2+ and Fe 3+ . Practical Applications Peroxidase is considered to have an empirical relationship to off‐flavors and odors in foods. Earlier we have shown that peroxidase activity varied in different genotypes, i.e., HHB 94 (high) and 94222A × 78/711 (low), which appeared to be responsible for odor generation in stored pearl millet meal. In 94222A × 78/711, less off‐odor was observed. The enzyme purified from mature pearl millet grains is strongly inhibited by DTT . This new information has been used to assay the enzyme in situ . We have found that in comparison to extractable activity, in situ activity of the enzyme is more strongly correlated with the development of off‐odor in stored flour. Thus, in situ assay is being used to screen germplasm for selecting contrasting genotypes (with respect to off‐odor) that will be used to develop pearl millet hybrids with improved shelf life of flour.