title: Molecular cloning, characterization and expression of lipoxygenase 2 (lox-2) isozyme from Indian soybean [Glycine max (L.) Merrill] cv. Pusa 16.
abstract: The consumption of soybean is limited worldwide, despite being highly nutritious and having versatile uses due to the presence of grassy, beany and rancid off-flavour. The lipoxygenase-2 (LOX-2) is the key enzyme responsible for the production of volatiles released from the beans, which cause off-flavour in soy products. In this study, a 2.6-kb full-length lox2 gene (NCBI accession No. JQ929619.1) was isolated and cloned from soybean (Glycine max L. Merril) cv. Pusa 16. The cloned cDNA sequence of lox2 gene showed the complete open reading frame (ORF) of a putative protein, having 866 amino acids with start codon present at the foremost position and stop codon at the end. The theoretical pI of predicted protein was 6.22. A hydropathy profile calculated from the amino acid sequence resembled those of dicot LOXs, suggesting conservation of the secondary structure of these enzymes. The LOX-2 showed conserved six Histidine residues within a span of 520 to 590 amino acid position, a signature element for the enzyme activity. The lox2 gene was expressed using pET vector in prokaryotic expression system. The recombinant LOX-2 protein was purified after induction with IPTG (isopentyl thiogalactoside). A prominent band of 97 kDa was observed, when affinity purified fractions were analyzed by SDS-PAGE. The purified protein was characterized for the enzyme activity, substrate preference and K(m). Inhibitor studies with natural antioxidant molecules present in soybean revealed alpha-tocopherol to be the most effective inhibitor of LOX-2.
authors.affiliation: Division of Biochemistry, Indian Agricultural Research Institute, New Delhi 110012.
authors.forename: Somnath
authors.initials: S
authors.lastname: Mandal
authors.forename: Nandita
authors.initials: N
authors.lastname: Sahana
authors.forename: A P
authors.initials: AP
authors.lastname: Rajarani
authors.forename: I M
authors.initials: IM
authors.lastname: Santha
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007194:India; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D008084:Lipoxygenase