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Title:	Purification and characterisation of a novel NAD+-farnesal dehydrogenase from Polygonum minus leaves
Authors:	Ahmad Faris Seman @ Kamarulzaman (P63581)
Supervisor:	Maizom Hassan, Dr.
Keywords:	Juvenile hormones Insect hormones Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia
Issue Date:	17-Feb-2016
Description:	Juvenile hormones (JH) are important for regulating both development and reproduction in an insect's life. Since JH analogs may keep the insects in an immature and potentially injurious stage longer than normal, a chemical, which would shut off JH production, could be more useful. Furthermore, the sesquiterpenoid JH III and its biosynthetic precursors were identified in sedges Cyperus iria. It was suggested that the JH III pathway found in the sedges is similar to the insect pathway. Therefore, studies on the enzymes in JH III biosynthetic and metabolic pathways should be helpful in the discovery of more effective analogs and for the establishment of new means of pest management. In order to elucidate JH III biosynthetic pathway in the plant, we investigate the enzymes participating in this sesquiterpene metabolism pathway in Polygonum minus. Farnesal dehydrogenase (FalDH) is responsible to catalyse the oxidation of farnesal into farnesoic acid in the biosynthetic pathway of JH III and has yet to be isolated and characterised from any organism. The objectives of the study were to purify and determine the physicochemical and biochemical properties of FalDH and to model the kinetic simulation of the enzyme. FalDH was extracted from P. minus leaves by using polyvinylpolypyrrolidone, Amberlite XAD-4, sucrose, 2-mercaptoethanol, thiourea, EDTA and phenylmethylsulfonylflouride in tricine-NaOH buffer (pH 7.5, 0.1 M) at 4 °C. The enzyme was successfully purified to homogeneity by ion-exchange chromatographies on Toyopearl Gigacap Q and Toyopearl Gigacap S, and affinity chromatography on Toyopearl AF-Blue HC, followed by size exclusion chromatography (SEC) on Toyopearl TSK-Gel G3000SW. FalDH exist as monomer with molecular weight of 70 kDa. The enzyme was relatively stable up to 40°C, but was rapidly inactivated at temperatures above 45°C. The optimum temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The isoelectric point was 6.6. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ions. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methylcinnamaldehyde was oxidised at lower activity. Aromatic and cyclic aldehydes, such as p-cumic aldehyde, carvone, and perillyl aldehyde, and aliphatic aldehydes were not dehydrogenated. The Km values for farnesal, trans- cinnamaldehyde, citral, α- methylcinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. Farnesal analogues with a hydroxyl or a methoxy group at the para position of the benzene ring strongly inhibit FalDH activity. The inhibition study suggests that it may be possible to design more potent analogue for FalDH inhibition in the biosynthetic pathway of JH. The kinetic simulation using integrated data of catalysis and inhibition kinetic parameter of FalDH enzyme was successfully modelled with COPASI software. It simulates the correlation of concentration of inhibitors and metabolite involved when a single reaction event happens.,'Certification of Master's/Doctoral Thesis' is not available,Master of Science
Pages:	93
Call Number:	QL494.5A376 2017 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Institute of Systems Biology / Institut Biologi Sistem (INBIOSIS)

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