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61.
62.
Tuo Li Annika J. E. Borg Leo Krammer Rolf Breinbauer Bernd Nidetzky 《Biotechnology and bioengineering》2023,120(6):1506-1520
Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4–0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates. 相似文献
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D. K. Vyas C. Moritz D. Peccinini-Seale J. W. Wright W. M. Brown 《Evolution; international journal of organic evolution》1990,44(4):922-932
Restriction endonuclease analyses were performed on mitochondrial DNAs (mtDNAs) representing unisexual parthenogenetic (cytotypes A, B, and C) and bisexual (cytotypes D and E) populations of Amazonian lizards presently regarded as Cnemidophorus lemniscatus. The results of mtDNA cleavage map comparisons among these C. lemniscatus indicated that (1) there was no cleavage site variation among the unisexuals, (2) mtDNAs from the bisexual cytotypes D and E differed in sequence from one another by about 13%, and (3) mtDNAs from cytotypes A–C differed from those of cytotype D by about 5% and from those of cytotype E by about 13%. Higher resolution restriction fragment size comparisons confirmed the high degree of similarity among the unisexual mtDNAs, but identified 12 cleavage site variants among the 13 cytotype D mtDNAs examined. Both cladistic and phenetic (UPGMA) analyses of the data indicate that the unisexual and cytotype D mtDNAs form a single clade, suggesting that a female of cytotype D was the maternal progenitor of the unisexuals. The similarity among the unisexual mtDNAs and the variability among those of cytotype D suggest that the three unisexual cytotypes arose recently from a common maternal lineage. The mtDNA variability observed among cytotype D individuals has a strong geographic component, suggesting that the unisexuals arose from one or a few geographically proximal populations. The mtDNA comparisons also support the conclusion, based on allozyme comparisons (Sites et al., 1990, this issue), that cytotypes D and E, although presently allocated to C. lemniscatus, are separate species. 相似文献
66.
Craig C. Moritz John W. Wright Wesley M. Brown 《Evolution; international journal of organic evolution》1989,43(5):958-968
Mitochondrial DNAs (mtDNAs) of two unisexual, parthenogenetically reproducing species of whiptail lizards (Cnemidophorus velox and C. exsanguis) and their bisexual relatives were compared by restriction-enzyme analysis to assess levels of mtDNA variation and to establish the maternal ancestry of the unisexuals. No cleavage-site differences were found to be diagnostic between C. velox and C. exsanguis mtDNAs, suggesting an ancestry rooted in the same maternal lineage. The mtDNA of the unisexuals is relatively homogeneous, indicating that these lineages are of recent origin. Phylogenetic analysis revealed that the maternal ancestor of both C. velox and C. exsanguis was most probably C. burti stictogrammus, C. costatus barrancorum, or an unidentified taxon closely related to them. In addition, the mtDNA analyses demonstrate conclusively that the triploid species C. velox could not have been formed by the fertilization of an unreduced (diploid) C. inornatus egg, further strengthening the hypothesis that parthenogenesis in Cnemidophorus results from hybridization. 相似文献
67.
C. Moritz S. Donnellan M. Adams P. R. Baverstock 《Evolution; international journal of organic evolution》1989,43(5):994-1003
Variation at 18 allozyme loci was assayed among representatives of the geographically widespread, triploid parthenogenetic form of Heteronotia binoei. A minimum of 52 different genotypes were observed among 143 individuals. Virtually all localities sampled had multiple genotypes among the unisexuals. This represents unusually high genotypic diversity for a unisexual vertebrate. Heterozygosity in the triploids was higher than in diploid bisexual populations of H. binoei. Comparison with the alleles present in the diploid bisexuals confirms that the parthenogens are hybrids and indicates that most of the genotypic diversity stems from repetitive hybrid origins. However, the presence of some alleles unique to the parthenogens suggests that mutation adds to their genetic diversity. The genetic structure of this geographically widespread parthenogen suggests the hypothesis that the persistence and spread of the unisexual lineages is facilitated by genotypic diversity. 相似文献
68.
Mechanisms of the reactions of representative dipeptides (Gly2, Gly-Ala), oligopeptides (Gly3, Gly4) and the polypeptide (poly-Gly)n) in solution and clay suspensions at 85 degrees C were investigated. The reaction products and their yields were analysed and determined by means of HPLC. Interestingly, hydrolysis, where water molecules act as the reactant, was not the main reaction, even for oligopeptides. Formation of cyclic dipeptides prevailed in the reactions of dimers as well as oligopeptides. The breakdown of oligopeptide molecules proceeded via an intramolecular cyclization reaction. For example, the reaction of Gly3 led to the formation of equal amounts of cyclic dipeptide, c(Gly)2 and Gly. The presence of clay (montmorillonite) significantly increased yields in the reactions of dipeptides but it did not have much effect on the reactions of oligopeptides. However, an opposite effect of clay, protection of poly(Gly)n against decomposition, was proven. 相似文献
69.
Richard Griessler Alexandra Schwarz Jan Mucha Bernd Nidetzky 《European journal of biochemistry》2003,270(10):2126-2136
Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate. 相似文献
70.