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Title:	Purification and characterization of NAD+-citral dehydrogenase from Polygonum minus Huds. leaves
Authors:	Nik Rashida Nik Abdul Ghani (P66149)
Supervisor:	Maizom Hassan, Dr.
Keywords:	Insect hormones Polygonum Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia
Issue Date:	17-May-2016
Description:	Plant-derived alarm pheromones such as geraniol, citral and geranic acid have been considered as potential non-pesticides for sustainable crop protection and established as attractive targets for the development of integrated pest management. In plants, conversion of geraniol to geranial is catalyzed by geraniol dehydrogenase and subsequently, geranial is oxidized to geranic acid probably by citral dehydrogenase (citral-DH). Isolation and characterization of citral-DH from Polygonum minus are important for understanding this pathway, their organization and regulation. This will provide the necessary first step in genetic engineering of plants to improve purity of pheromone compound produced. The objectives of this study are to purify and characterize citral-DH, and to develop a kinetic model for the integrated network in geraniol metabolic pathway. Stable, highly active cell-free extract was obtained using extraction buffer containing 0.1 M tricine-NaOH (pH 7.5), 10% (w/v) polyvinylpolypyrrolidone, 50% (w/w) amberlite XAD-4 with 2.5 mM - mercaptoethanol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 25% (v/v) sucrose and 5 mM thiourea. The enzyme preparation was separated into two activity peaks using GigaCap Q Toyopearl 650M column chromatography at pH 7.5, suggesting the existence of two citral-DH isoenzymes. Both isoenzymes were purified to homogeneity through a combination of ion exchange, affinity and gel filtration chromatographies. The optimal conditions for activity of both isoenzymes were 35 °C and pH 9.5. The isoelectric points for citral-DH I and II were 4.2 and 7.1, respectively. Sulfhydryl agents, chelating agents, and metal ions inhibited the enzyme activity. Both isoenzymes appeared to exist as heterodimers. Citral-DH isoenzymes showed specificity towards citral, α-methyl cinnamaldehyde, trans-cinnamaldehyde and Sperillyl aldehyde. Other allylic, non-allylic, aromatic and aliphatic aldehydes were not oxidized. The Km values for citral of citral-DH I and citral-DH II were 0.45 mM and 0.56 mM, respectively. The amino acid sequences of tryptic peptides of both isoenzymes analyzed by MALDI-TOF/TOF MS/MS spectrometry showed no significant similarity with those reported geranial dehydrogenases and other aldehyde dehydrogenases. A kinetic model for the integrated network in geraniol metabolic pathway was constructed on the basis of the bibliomic data from BRENDA database for geraniol synthase and geraniol-DHs; and in vitro kinetic parameter values for both citral-DH isoenzymes. Variant of the methods for oxidation mathematical models were carried out using COPASI 4.11 software. Sensitivity analysis from output graphs showed that the behavior of the systems indicated some robustness. This systems biology approach of enzyme kinetics suggested that each citral-DH I and citral-DH II isoenzymes, followed a Michaelis-Menten mechanism. The purification and characterization in this present study will serve as a basis to provide new information on recombinant technology of the enzymes and useful molecular tool in manipulating plant semiochemicals emission. The deployment of transgenic plant in integrated pest management by overproducing alarm pheromone of geranic acid opens a novel strategy to control a variety of insect pests.,'Certification of Master's/Doctoral Thesis' is not available,Master of Science
Pages:	94
Call Number:	QL494.5.N535 2016 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Institute of Systems Biology / Institut Biologi Sistem (INBIOSIS)

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