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Studies on starch structure and the differential properties of starch branching enzymes /Andersson, Lena. January 2001 (has links)
Thesis (Ph. D.)--Swedish University of Agricultural Sciences, 2001. / Includes bibliographical references.
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Phosphoenolpyruvate carboxylase and cold acclimation of alfalfaFrank, Scott, 1971- January 1996 (has links)
Phosphoenolpyruvate carboxylase (PEPC) was examined during cold acclimation of seedlings of the freezing-tolerant cultivar (Medicago sativa ssp falcata cv Anik) and the relatively freezing-sensitive cultivar (Medicago sativa cv Trek) of alfalfa. With four days of cold acclimation, PEPC activity increased to 3.5-fold and 2-fold the control levels in Anik and Trek, respectively. This was associated with an increase in the level of a 110 kD PEPC protein and a decrease in the amount of a 120 kD PEPC polypeptide in both cultivars. The role of reversible phosphorylation in regulating PEPC activity was demonstrated by in vitro phosphorylation and dephosphorylation, which caused partial activation and deactivation of PEPC, respectively. In vivo phosphorylation experiments revealed that the 110 kD PEPC subunit is phosphorylated on serine residue(s) during cold acclimation in Anik but not in Trek. Increased PEPC activity could account for the 70% increase in the non-autotrophic or dark fixation of carbon observed in cold acclimated Anik seedlings. A possible role for dark carbon fixation in the cold-induced development of freezing tolerance is through the production of NADPH. Such a source of reducing power may be required for the repair of cold-induced damage and restoration of normal cellular functions.
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Biocatalysis of tyrosinase in chloroform medium using selected phenolic substratesTse, Mara. January 1996 (has links)
The biocatalytic activity of mushroom tyrosinase was optimized in chloroform medium, using five selected phenolic substrates, including catechin (CT), vanillin (VA), chlorogenic acid (CA), p-aminophenol (pAP) and hydroquinone (HQ). The specific activity (SA) of tyrosinase determined as the change in absorbance at the selected wavelength per $ mu$g protein per sec ($ delta$A/$ mu$g protein/sec) in chloroform was much higher than that obtained in aqueous media. The optimal amount of enzymatic protein for tyrosinase biocatalysis in chloroform was found to be 44.0 mg protein/L for CT and VA, 31.6, 180.5 and 90.3 mg protein/L, respectively, for CA, pAP and HQ. The optimal pH for the oxidative activity of tyrosinase in chloroform was 6.0 for all the substrates; however, the optimal temperature for enzymatic activity was 30$ sp circ$C for CT and 25$ sp circ$C for the other four substrates. The use of 1.25 and 6.65 mM catechol in chloroform medium activated the tyrosinase activity maximally by 56.2% and 267.2%, respectively for CT and CA as substrates; however, no effect from catechol (0 to 7 mM) was found with VA, pAP or HQ. In addition, the use of 4.25, 2.25 and 5.39 mM ethylenediamine tetraacetic acid (EDTA) in chloroform, with CT, VA and pAP as substrates, inhibited the tyrosinase activity maximally by 44.3, 84.7 and 67.0%, respectively; however, the use of 4.75 and 1.60 mM EDTA activated the enzyme by 101.9% and 115.9%, respectively, for CA and HQ. The use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) demonstrated the phenolic substrate bioconversion, whereas the spectrophotometric scanning showed the product formation during the enzymatic reaction. (Abstract shortened by UMI.)
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AQX : a novel gene in plant ubiquinone biosynthesisStorey, Benjamin, 1973- January 2002 (has links)
C. elegans worms with mutations in the gene CLK-1 develop slowly and have an extended lifespan. CLK-1 encodes a mitochondrial protein that is responsible for the hydroxylation of 5-demethoxyubiquinone (DMQ), the penultimate step of ubiquinone (Coenzyme-Q or UQ) biosynthesis. Structural homologues of CLK-1 are found in mammals, fruit flies, yeast and some types of bacteria. Interestingly, however, there is no structural homologue of CLK-1 in the Arabidopsis genome and no plant homologue can be found in other sequence databases. Yeast with the CLK-1 homologue COQ7 deleted fail to grow on non-fermentable carbon sources. To identify a plant functional homologue of COQ7/CLK-1, an Arabidopsis cDNA expression library was screened for complementation of a yeast coq7 deletion mutant. A clone was identified that rescued the coq7 respiratory deficiency. Although the sequence of the encoded protein has no structural similarity to proteins in the COQ7/CLK-1 family, it contains a monooxygenase/hydroxylase domain that has sequence similarity with the E. coli DMQ hydroxylase encoded by the UBIF gene. Like the structural homologues of COQ7/CLK-1 found in other eukaryotes, the gene (AQX for 'Alternate Quinone monooXygenase') contains a likely mitochondrial targeting presequence at its N-terminus. HPLC analysis of quinone extracts from rescued cog7 strains does not detect ubiquinone, but instead shows another peak that may be DMQ. It is likely that AQX does not hydroxylate yeast DMQ effectively enough to generate detectable levels of UQ. A unique pathway for UQ biosynthesis in plants is proposed that is defined by AQX and Arabidopsis genes identified on the basis of homology to known E. coli and yeast UQ biosynthesis genes.
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AQX : a novel gene in plant ubiquinone biosynthesisStorey, Benjamin, 1973- January 2002 (has links)
No description available.
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Phosphoenolpyruvate carboxylase and cold acclimation of alfalfaFrank, Scott, 1971- January 1996 (has links)
No description available.
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Biocatalysis of tyrosinase in chloroform medium using selected phenolic substratesTse, Mara. January 1996 (has links)
No description available.
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Protoplast isolation and plant regeneration in Bambara groundnut : a platform for transient gene expressionAyeleso, Taiwo Betty January 2016 (has links)
Thesis (MTech (Agriculture))--Cape Peninsula University Of Technology, 2016. / Bambara groundnut (Vigna subterranea), a dicotyledonous plant is a legume which has a potential to contribute to food security and nutrition. Protoplasts are naked plant cells lacking cell walls. Viable protoplasts are potentially totipotent. Therefore, when given the correct stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated through the tissue culture process. Protoplast systems are valuable and versatile cell based systems that are useful in observing cellular processes and activities.
In this study, the isolation of protoplast from the leaves of Bambara groundnut plant was extensively optimised. The factors affecting protoplast isolation considered in this study were ages of plant material, mannitol concentration, combinations and concentrations of enzymes and duration of incubation. Effects of ages of Bambara groundnut plant (4, 6, 8, 10 weeks), molarities of mannitol (0.4 M, 0.5 M. 0.6 M and 0.7 M), concentration and combination of enzymes (1%, 2% and 4% cellulase, 0.5% and 1% macerozyme and, 0.5% and 1% pectinase) at different incubation duration (4, 18, 24, 42 hours) were investigated. Overall, it can be deduced from this study that the optimal protoplast yield (4.6 ± 0.14×105ml-1/gFW) and viability (86.5 ± 2.12%) were achieved by digesting the leaves of four week old Bambara groundnut plant with 2% cellulase and 0.5 % macerozyme with 0.5M mannitol for 18 hours. Freshly isolated protoplasts were then cultured at different densities of 1 × 104 - 2 ×106 protoplasts/ml using MS in three different culture (Liquid, agar and agarose bead) methods. First cell division was observed only in liquid medium. With several attempts, no division was achieved in the agar and agarose bead methods, division also did not progress in the liquid medium and hence, plant regeneration from Bambara groundnut protoplasts could not be achieved in this study. Consequently, a further study is underway to compare the proteomic profiles of freshly isolated protoplasts and cultured protoplasts in order to gain insights into the expression of proteins that could perhaps be contributing to the difficulty in regenerating Bambara groundnut plant through protoplast technology. The present study is novel because it is the first study to optimise the various factors that could affect protoplast isolation from the leaves of Bambara groundnut and thus developed an efficient protocol for protoplasts isolation from leaves of Bambara groundnut for cell manipulation studies.
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Screening, purification and characterisation of an active Hydroxynitrile Lyase (Nitrilase) from indigenous South African PlantsMopai, Kgaugelo Lydia January 2013 (has links)
Thesis (MSc. (Biochemistry)) -- University of Limpopo, 2013 / Hydroxynitrile lyases (HNLs) are enzymes that catalyse enantioselective cleavage of the substrate in a reaction and are also used as important industrial biocatalysts for the synthesis of chiral cyanohydrins. The aim of the study was to screen indigenous South African plants for potential hydroxynitrile lyase activity, purify and biochemically characterise the active hydroxynitrile lyase(s) from the selected plants. Several indigenous plants were randomly collected, identified and screened for HNL activity. The plant parts (leaves, seeds or fruits) were processed using established experimental protocols in order to obtain the crude enzyme extracts. The enzymatic conversion of benzaldehyde and potassium cyanide to mandelonitrile was optimised and consequently used for the screening of HNL activity. Enzyme activity was detected in the crude enzyme extracts of Kalanchoe spp and Senecio spp and these were then designated as Ks and Sb, respectively. Ammonium sulphate fractionation, DEAE Toyopearl 650M and Concanavalin A chromatography techniques were then used in the purification process of the active crude enzyme extracts. Subsequently, two purified active fractions were isolated from each plant species with molecular masses estimated at 64.64 kDa and 64.06 kDa for the KsHNL enzymes and 70.60 kDa and 74.04 kDa for SbHNL enzymes. The optimum temperature and pH of all the isolated enzymes were determined as 50°C and pH 5, respectively. The experimental Km and Vmax values of the enzymes were respectively determined to be 0.33 and 0.73 mM and 1.238 and 1.948 μM/min for KsHNL; while that for SbHNL enzymes were 5.86 and 0.22 mM and 9.741 and 1.905 μM/min. The effect of additives and metal ions (viz., DTT, DEP, mercury chloride, magnesium chloride and zinc chloride) was determined. The experimental data obtained alluded to the notion that both KsHNL and SbHNL enzymes may contain the cysteine and serine residues next to their active sites and that a histidine residue may be involved in the catalytic activities of both the isolated KsHNL enzymes and one of the SbHNL enzymes. All the isolated enzymes from the two plant species did not seem to contain an FAD group. These findings compared favourably to the theoretical type II HNLs, although with a slight difference in that they displayed high molecular weights. Kalanchoe spp and Senecio spp are the two indigenous South African plants that were found to contain active HNLs. The isolated HNLs from the two plants have a potential to be xv
purified to homogeneity, cloned and overexpressed into robust recombinant enzymes that can be used for large scale industrial applications.
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