One of the primary sources of enzyme instability is protein oxidative modification triggering activity loss or denaturation. We show here that the side chain of Cys108 is the main site undergoing ...stress-induced oxidation in Trigonopsis variabilis D-amino acid oxidase, a flavoenzyme employed industrially for the conversion of cephalosporin C. High-resolution anion-exchange chromatography was used to separate the reduced and oxidized protein forms, which constitute, in a molar ratio of about 3:1, the active biocatalyst isolated from the yeast. Comparative analysis of their tryptic peptides by electrospray tandem mass spectrometry allowed unequivocal assignment of the modification as the oxidation of Cys108 into cysteine sulfinic acid. Cys108 is likely located on a surface-exposed protein region within the flavin adenine dinucleotide (FAD) binding domain, but remote from the active center. Its oxidized side chain was remarkably stable in solution, thus enabling the relative biochemical characterization of native and modified enzyme forms. The oxidation of Cys108 causes a global conformational response that affects the protein environment of the FAD cofactor. In comparison with the native enzyme, it results in a fourfold-decreased specific activity, reflecting a catalytic efficiency for reduction of dioxygen lowered by about the same factor, and a markedly decreased propensity to aggregate under conditions of thermal denaturation. These results open up unprecedented routes for stabilization of the oxidase and underscore the possible significance of protein chemical heterogeneity for biocatalyst function and stability.
Abstract For simulations of flow and microbial conversion reactions, related to modeling of simultaneous extraction and fermentation process in a single sugar beet cossette a software package ...OpenFOAM was used. The mass transfer of the components (sucrose, glucose, fructose and ethanol) in the studied system was controlled by the convection and diffusion processes. Microbial conversion rates and yield coefficients were experimentally determined and/or estimated by mathematical simulation. Dimensions of the model sugar beet cossette (SBC) were: average length of cosettes 40.10 mm, average thickness 3.32 mm and average width 3.5 mm, and represented in the model as a square-shape cross-section mathematical simulation. Dimensions of the model sugar beet cossette (SBC) were: average length of cosettes 40.10 mm, average thickness 3.32 mm and average width 3.5 mm, and represented in the model as a square-shape cross-section used to study the mass transfer and microbial conversion rates on the scale of single sugar beet cossette in the short time scales (up to 25 s). This model can be used for simulation of extractant flow around single sugar beet cossette as well as for description of simultaneous extraction and fermentation process in the studied system.
Abstract The aim of this work was the adaptation of a Gas Chromatographic-Flame Ionization Detector (GC-FID) method for detection and quantification of extracellular free amino acids in demineralized ...water, De Mann Rogosa Sharpe (MRS) medium and corn grits (CG) withdrawn during lactic acid fermentation. In order to analyze free amino acids by the GC-FID method it was necessary to convert free amino acids to volatile compounds. This was accomplished by derivatization of free amino acids with ethylchlor formate in aqueous medium followed by extraction of volatile free amino acid esters with chloroform. It was proven that the combination of derivatization and extraction procedure with developed GC-FID method gave accurate, reproducible and sensitive analytical results. Quantification of 15 (Ala, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Asn, Met, Pro, Lys, His, Asp and Glu) out of 20 ethoxycarbonyl-ethyl esters of free amino acids in demineralized water and MRS medium was achieved by established methods. In corn grits medium all of the above mentioned 15 amino acids, except His, were quantified with this GC-FID method. The established method was efficiently verified in monitoring of extracellular free amino acid concentration during lactic acid production with Lactobacillus rhamnosus DSM 20021 T in MRS medium and Lactobacillus amylovorus DSM 20531 T in corn grits medium.
Batch cultivation of monoculture of Lactobacillus sp. and two-strain mixed culture of Lactobacillus sp. and Lactobacillus amylovorus DSM 20531.sup.T was carried out with the aim of producing L-(+)- ...and D-(-)/L-(+)-lactic acid to be implemented in poly(lactic acid) polymer production. Metabolic capacity of two Lactobacillus strains to ferment different carbon sources (glucose, sucrose or soluble starch) during cultivation in MRS medium at 40°C, in a laboratory-scale stirred tank bioreactor was defined. Lactobacillus sp. showed similar affinity towards mono- and disaccharide substrates, which were homofermentatively converted mostly to L-(+)-lactic acid. L. amylovorus DSM 20531.sup.T has been characterized as a D/L-lactate producer and it is capable of conducting simultaneous saccharification and fermentation. Due to the interaction of Lactobacillus sp. with L. amylovorus DSM 20531.sup.T, starch was hydrolysed and fermented to the mixture of L-(+)- and D-(-)-lactic acid. Modified LuedekingPiret kinetics used for the description of substrate utilization, growth of mono- and mixed cultures and production of lactic acid stereoisomers showed good agreement with experimental data. Key words. monoculture, mixed culture, Lactobacillus sp., batch fermentation, L-(+)- and/or D-(-)-lactic acid, bacteriocin, amensalism, mathematical model
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