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Epimerase Activity (epimerase + activity)
Selected AbstractsHeterologous gene expression in Lactococcus lactis; expression of the Azotobacter vinelandii algE6 gene product displaying mannuronan C-5 epimerase activityFEMS MICROBIOLOGY LETTERS, Issue 2 2003Janet M. Blatny Abstract The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1,7 can be used to improve the properties of the commercially important polysaccharide alginate that is widely used in a variety of products, such as food and pharmaceuticals. Since lactic acid bacteria are generally regarded as safe, they are attractive candidates for production of the epimerases. A. vinelandii genes are GC-rich, in contrast to those of lactic acid bacteria, but we show here that significant expression levels of the epimerase AlgE6 can be obtained in Lactococcus lactis using the nisin-controlled expression system. A 1200-fold induction ratio was obtained resulting in an epimerase activity of 23,900 dpm mg,1 h,1, using a tritiated alginate substrate. The epimerase was detected by Western blotting and nuclear magnetic resonance spectroscopy analysis of its reaction product showed that the enzyme displayed catalytic properties similar to those produced in Escherichia coli. [source] Characterization of the Saccharomyces cerevisiae galactose mutarotase/UDP-galactose 4-epimerase protein, Gal10pFEMS YEAST RESEARCH, Issue 3 2007Aaron Scott Abstract Saccharomyces cerevisiae and some related yeasts are unusual in that two of the enzyme activities (galactose mutarotase and UDP-galactose 4-epimerase) required for the Leloir pathway of d -galactose catabolism are contained within a single protein,Gal10p. The recently solved structure of the protein shows that the two domains are separate and have similar folds to the separate enzymes from other species. The biochemical properties of Gal10p have been investigated using recombinant protein expressed in, and purified from, Escherichia coli. Protein,protein crosslinking confirmed that Gal10p is a dimer in solution and this state is unaffected by the presence of substrates. The steady-state kinetic parameters of the epimerase reaction are similar to those of the human enzyme, and are not affected by simultaneous activity at the mutarotase active site. The mutarotase active site has a strong preference for galactose over glucose, and is not affected by simultaneous epimerase activity. This absence of reciprocal kinetic effects between the active sites suggests that they act independently and do not influence or regulate each other. [source] The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosaMOLECULAR MICROBIOLOGY, Issue 4 2003Sumita Jain Summary Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide that confers a mucoid phenotype. Alginate is a linear polymer of d -mannuronate (M) and variable amounts of its C-5-epimer, l -guluronate (G). AlgG is a periplasmic C-5-epimerase that converts poly d -mannuronate to the mixed M+G sequence of alginate. To understand better the role and mechanism of AlgG activity, a mutant was constructed in the mucoid strain FRD1 with a defined non-polar deletion of algG . Instead of producing poly mannuronate, the algG deletion mutant secreted dialysable uronic acids, as does a mutant lacking the periplasmic protein AlgK. High levels of unsaturated ends and the nuclear magnetic resonance spectroscopy pattern revealed that the small, secreted uronic acids were the products of extensive polymer digestion by AlgL, a periplasmic alginate lyase co-expressed with AlgG and AlgK. Thus, AlgG is bifunctional with (i) epimerase activity and (ii) a role in protecting alginate from degradation by AlgL during transport through the periplasm. AlgK appears to share the second role. AlgG and AlgK may be part of a periplasmic protein complex, or scaffold, that guides alginate polymers to the outer membrane secretin (AlgE). To characterize the epimerase activity of AlgG further, the algG4 allele of poly mannuronate-producing FRD462 was shown to encode a protein lacking only the epimerase function. The sequence of algG4 has a Ser-272 to Asn substitution in a serine,threonine-rich and conserved region of AlgG, which revealed a critical residue for C-5-epimerase activity. [source] Structure and epimerase activity of anthocyanidin reductase from Vitis viniferaACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2009Mahmoud Gargouri Together with leucoanthocyanidin reductase, anthocyanidin reductase (ANR) is one of the two enzymes of the flavonoid-biosynthesis pathway that produces the flavan-3-ol monomers required for the formation of proanthocyanidins or condensed tannins. It has been shown to catalyse the double reduction of anthocyanidins to form 2R,3R -flavan-3-ols, which can be further transformed to the 2S,3R isomers by non-enzymatic epimerization. ANR from grape (Vitis vinifera) was expressed in Escherichia coli and purified. Unexpectedly, RP-HPLC, LC-MS and NMR experiments clearly established that the enzyme produces a 50:50 mixture of 2,3- cis and 2,3- trans flavan-3-ols which have been identified by chiral chromatography to be 2S,3S - and 2S,3R -flavan-3-ols, i.e. the naturally rare (+)-epicatechin and (,)-catechin, when cyanidin is used as the substrate of the reaction. The first three-dimensional structure of ANR is described at a resolution of 2.2,Å and explains the inactivity of the enzyme in the presence of high salt concentrations. [source] | ||||||||||