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Synthetic Gene (synthetic + gene)
Selected AbstractsCassette mutagenesis of lysine 130 of human glutamate dehydrogenaseFEBS JOURNAL, Issue 11 2001An essential residue in catalysis It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5,-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH. [source] Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applicationsLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2009L. Poidevin Abstract Aims:, The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications. Methods and Results:, The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6·5 and at 50,60°C. Furthermore, EstA remained stable at pH 6,8 and below 50°C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP). Conclusion:, The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates. Significance and Impact of the Study:, Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated. [source] Nutritional Value of Cassava for Use as a Staple Food and Recent Advances for ImprovementCOMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY, Issue 3 2009Julie A. Montagnac ABSTRACT:, Cassava is a drought-tolerant, staple food crop grown in tropical and subtropical areas where many people are afflicted with undernutrition, making it a potentially valuable food source for developing countries. Cassava roots are a good source of energy while the leaves provide protein, vitamins, and minerals. However, cassava roots and leaves are deficient in sulfur-containing amino acids (methionine and cysteine) and some nutrients are not optimally distributed within the plant. Cassava also contains antinutrients that can have either positive or adverse effects on health depending upon the amount ingested. Although some of these compounds act as antioxidants and anticarcinogens, they can interfere with nutrient absorption and utilization and may have toxic side effects. Efforts to add nutritional value to cassava (biofortification) by increasing the contents of protein, minerals, starch, and ,-carotene are underway. The transfer of a 284 bp synthetic gene coding for a storage protein rich in essential amino acids and the crossbreeding of wild-type cassava varieties with Manihot dichotoma or Manihot oligantha have shown promising results regarding cassava protein content. Enhancing ADP glucose pyrophosphorylase activity in cassava roots or adding amylase to cassava gruels increases cassava energy density. Moreover, carotenoid-rich yellow and orange cassava may be a foodstuff for delivering provitamin A to vitamin A,depleted populations. Researchers are currently investigating the effects of cassava processing techniques on carotenoid stability and isomerization, as well as the vitamin A value of different varieties of cassava. Biofortified cassava could alleviate some aspects of food insecurity in developing countries if widely adopted. [source] Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transferFEMS MICROBIOLOGY LETTERS, Issue 2 2007Petra Louis Abstract Butyrate producers constitute an important bacterial group in the human large intestine. Butyryl-CoA is formed from two molecules of acetyl-CoA in a process resembling ,-oxidation in reverse. Three different arrangements of the six genes coding for this pathway have been found in low mol% G+C-content Gram-positive human colonic bacteria using DNA sequencing and degenerate PCR. Gene arrangements were strongly conserved within phylogenetic groups defined by 16S rRNA gene sequence relationships. In the case of one of the genes, encoding ,-hydroxybutyryl-CoA dehydrogenase, however, sequence relationships were strongly suggestive of horizontal gene transfer between lineages. The newly identified gene for butyryl-CoA CoA-transferase, which performs the final step in butyrate formation in most known human colonic bacteria, was not closely linked to these central pathway genes. [source] Prostacyclin inhibits endothelial cell XIAP ubiquitination and degradationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Jun-Yang Liou To understand the role of prostacyclin (PGI2) in protecting endothelial cells from apoptosis, we evaluated the effects of carbaprostacyclin (cPGI2) on H2O2 -induced human umbilical vein endothelial cell (HUVEC) apoptosis. cPGI2 suppressed H2O2 -induced annexin V-positive cells in a concentration- and time-dependent manner. Pre-treatment of HUVEC with 50 µM cPGI2 for 4 h produced the maximal anti-apoptotic effect. Authentic PGI2 generated by adenoviral transfer of PGI2 synthetic genes exerted a similar protective effect. cPGI2 inhibited Smac/DIABLO release from mitochondria, caspase 3 activation, focal adhesion protein degradation, and cell detachment. cPGI2 selectively protected X-linked inhibitor of apoptosis protein (X-linked IAP, XIAP) from H2O2 -induced ubiquitination, and preserved XIAP protein levels. PD-98059 but not H-89 abrogated the protective action of cPGI2. cPGI2 increased ERK phosphorylation which was blocked by PD-98059. HUVEC stably transfected with dominant negative Ras abrogated XIAP preservation by cPGI2 while constitutive active Ras increased ERK phosphorylation and protected XIAP from degradation. Our results demonstrate for the first time that PGI2 inhibits XIAP ubiquitination and degradation via the Ras/MEK-1/ERK signaling pathway. Preservation of XIAP proteins represents a key mechanism by which PGI2 protects endothelial cells from oxidant-induced apoptosis. J. Cell. Physiol. 212:840,848, 2007. © 2007 Wiley-Liss, Inc. [source] | ||||||||||