Abstract
Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (kcat/Km) and the Michaelis–Menten constant (Km) of immobilized lipoxygenase were 21% and 49% of kcat/Km and Km of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest.
Highlights
Soybean lipoxygenase (LOX, EC.1.13.11.12) catalyzes the first step in the octadecanoid pathway, which produces diverse oxylipin derivatives including jasmonic acid (JA)
The results revealed wide variation in immobilization efficiency, from 49.6% to 92.4% and 24.6% to 51.32% for GDA-linked rice husk silica (RHS) and epichlorohydrin/polyethylene glycol 8000 (ECH-PEG)-linked RHS, respectively, and that immobilization efficiency was influenced by the molecular size of the immobilized proteins and the hydrophobicity of the solvent in which the protein was dissolved [33]
The results suggest that low catalytic efficiency following immobilization or adsorption of soybean LOX on RHS may be unavoidable at a low concentration of substrate
Summary
Soybean lipoxygenase (LOX, EC.1.13.11.12) catalyzes the first step in the octadecanoid pathway, which produces diverse oxylipin derivatives including jasmonic acid (JA). The primary substrates of LOX are α-linolenic acid (LnA) and linoleic acid (LA), which are released from the chloroplast membrane by phospholipase C and converted by LOX into their corresponding hydroperoxyl derivatives [1,2,3,4]. JA is a ubiquitous plant stress hormone whose synthesis is triggered by various biotic and abiotic environmental stimuli. It has been suggested that JA and its analogues protect plants from damaging insects [5] and that they have potential as noncytotoxic chemotherapeutic agents [6,7,8]. Biosynthesis of JA is initiated by 13-LOX, which oxygenates LnA into 13-hydroperoxy octadecatrienoic acid (13-HPOT). Allene oxide synthase (AOS) converts 13-HPOT to an unstable 12,13-epoxy octadecatrienoic acid (12,13-EOT), which is metabolized to 12-oxo-phytodienoic acid (12-OPDA) by allene oxide cyclase (AOC). 12-OPDA is reduced to 3-oxo-2-(2 pentenyl)cyclopentane-1-octanoic acid (OPC 8:0) by 12-oxo-phytodienoate reductase (OPR), which is converted to JA by three cycles of β-oxidation (Scheme 1)
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