Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).

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The thermal stability of copper/quinone containing amine oxidases from Euphorbia characias latex (ELAO) and lentil seedlings (LSAO) was measured in 100 mM potassium phosphate buffer (pH 7.0) following changes in absorbance at 292 nm. ELAO was shown to be about 10 degrees C more stable than LSAO. The dissociative thermal inactivation of ELAO was studied using putrescine as substrate at different temperatures in the range 47-70 degrees C, and a "conformational lock" was developed using the theory pertaining to oligomeric enzyme. Moreover ELAO was shown to be more stable towards denaturants than LSAO, as confirmed by dodecyl trimethylammonium bromide denaturation curves. A comparison of the numbers of contact sites in inter-subunits of ELAO relative to LSAO led us to conclude that the higher stability of ELAO to temperature and towards denaturants was due to the presence of larger number of contact sites in the conformational lock of the enzyme. This study also gives a putative common mechanism for thermal inactivation of amine oxidases and explains the importance of C-terminal conserved amino acids residues in this class of enzymes.

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Nonenzymatic glycation of biomacromolecules occurs due to the diabetes mellitus and ageing. A number of small molecules, known as chemical chaperones, stabilize protein conformation against thermal and chemically induced denaturation. These compounds are including: polyamines (e.g. spermine and spermidine), amino acids (e.g. lysine) and polyols (e.g. glycerol). In this study the effect of spermidine (Spd), spermine (Spm), and glycerol on glycation, structure and function of lysozyme (LZ), as an extra-cellular protein, by different techniques is investigated. LZ is incubated with or without glucose (50 or 100 mM) in the absence or presence of Spd/Spm/glycerol at 37 °C up to 16 weeks. All the observed changes of glycated-LZ in comparison with the native protein, including: increased fluorescence emission, alteration in the secondary and tertiary structure, and reduced electrophoretic mobility- indicate its structural changes that are accompanied with its reduced activity. Glucose in the presence or absence of Spd induces the protein dimerization, but glucose plus Spm induces its trimmerization. In contrast, glycerol inhibits the LZ glycation and prevents the large changes on its structure and function. Glucose binds lysine residues, decreases the protein positive charges and induces some alterations in its structure and activity. Polyamines also directly bind to LZ, increase its positive charges and hence induce more glycation; more conformational changes, oligomerization and its inactivation in the presence of glucose, but glycerol affect the protein environment and preserve protein from these harmful effects.

Background: L-tryptophan (L-Trp) is a nutritionally essential amino acid that the body uses to synthesize proteins, niacin (vitamin B3) and serotonin (neurotransmitters). Human and animals depend on plants and microorganisms for its supply. Remarkable increasing demand has caused the development of a wide variety of biotechnological methods for its production which work as well for other amino acids.Objectives: The present work reports on the use of Iranian sugar beet molasses as an inexpensive source of L-serine (precursor) and PLP (cofactor) for the production of L-Trp by Escherichia coli (ATCC 11303). Carbon source and buffered condition are also optimized for L-Trp production by E. coli.Materials and Methods: E. coli (ATCC 11303) was used as the microbial source of tryptophan. Batch fermentation was performed with 8L sugar beet molasses medium. The fermentation was carried out at 37 °C with an agitation speed at 250 rpm. The pH was controlled at 7 with 10 N NaOH. The cells were recovered by centrifugation. A 1g mass of E. coli cells (wet mass) was used as the biocatalyst in the reaction medium (100 cc) containing (in grams/100cc): Molasses/glucose=3.5; Indole=0.2; L-Serine=0.35; PLP=0.005; (NH4)2SO4 =0.5 The reaction medium was incubated on rotatory shaker (180 rpm) for 8 h at 37 °C. The reaction was carried out in KH2PO4-K2HPO4 buffering system (pH = 8, 0.1 M). The detection of produced L-Trp was carried out by the use of chromatography methods (HPLC and TLC), and fluorescence analysis.Results: Consequently, the potassium phosphate buffer system was considered as the suitably buffered reaction medium. The concentration of L-Trp produced in reaction medium was 0.3 mM and 0.12 mM for glucose and molasses (a combination of sugar beet and cane molasses), respectively.Conclusions: In conclusion, our present study shows that sugar beet molasses is a source of PLP and L-Ser; and hence, suggests the general application of the approach to produce L-Trp more economically

The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 o C. The thermal inactivation curves were not linear at 52 and 57 o C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 o C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.

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