Chimeric allosteric citrate synthases: Construction and properties of citrate synthases containing domains from two different enzymes

Molgat, Gilles Francois; Donald, Lynda J.; Duckworth, Harry W.

doi:10.1016/0003-9861(92)90118-g

Cited by 19 publications

(15 citation statements)

References 31 publications

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“…This is in the range of reported in vitro kinetic data for citrate synthase of E. coli from Pereira et al (1994), who reports a K m of 0.12 mmol l À1 and from Molgat et al (1992), who reports a K m of 0.7 mmol l À1 .…”

Section: Extractionsupporting

confidence: 66%

Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures

2007

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The quantitative estimation of intracellular metabolite concentrations (metabolic profiling) is a prerequisite for a better understanding of biological processes and thus inevitable for the rational improvement of microbial production strains and process design. Since pool sizes of substrates regulate flux through different enzymes, the accurate determination of intracellular metabolite concentrations is necessary to understand in vivo reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates in Escherichia coli K12 was achieved by using a novel in situ rapid sampling and quenching procedure. A new extraction procedure using buffered hot water was established. By use of simultaneous multi-substrate feeding with various ratios of glucose, fructose and acetate during continuous cultivations several metabolic states were induced. Metabolic flux analysis and the newly developed metabolic profiling procedure were used to determine in vivo enzyme kinetics as exemplified for fructose 1,6-bisphosphate aldolase and citrate synthase.

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Section: Extractionsupporting

confidence: 66%

Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures

2007

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“…While the K m value of the chimeric citrate synthases similarly have been found to be lower than those of the parental enzymes (30), substrate affinity decreased by about 2-fold in active human-yeast chimeric phosphoglycerate kinase engineered by domain interchanges (31). However, no significant differences were found between the K m values of parental and chimeric isopropylmalate dehydrogenases (23).…”

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confidence: 95%

Construction of Chimeric β-Glucosidases with Improved Enzymatic Properties

Singh

Hayashi

1995

Journal of Biological Chemistry

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The amino acid sequences of ␤-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show about 40% similarity. The pH/temperature optima and stabilities and substrate specificities of the two enzymes are quite different. C. gilvus ␤-glucosidase exhibits an optimum pH of 6.2-6.4 and temperature of 35°C, whereas the corresponding values for A. tumefaciens are 7.2-7.4 and 60°C, respectively. The substrate specificity of A. tumefaciens enzyme toward different aryl glycosides is broader than C. gilvus enzyme. To analyze these properties further, three chimeric ␤-glucosidases were constructed by substituting segments from the Cterminal homologous region of C. gilvus ␤-glucosidase gene with that of A. tumefaciens. The chimeric enzymes were characterized with respect to pH/temperature activity and stability and substrate specificity. Chimeric enzymes exhibited chromatographic behavior similar to that of C. gilvus enzyme. However, enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45-50°C and pH 6.6 -7.0. K m values of chimeric enzymes for the various saccharides were admixtures of both parental enzymes. These results suggest that the two domains of C. gilvus and A. tumefaciens enzymes probably can fold independently. The homologous C-terminal region in ␤-glucosidase appears to play an important role in determining enzyme characteristics. Changes in the properties on substitution of segments in this region might be related to the enzyme specificity, and ␤-glucosidases with improved properties can be prepared by manipulating this region.The enzyme ␤-glucosidase (EC 3.2.1.21) catalyzes the hydrolysis of alkyl-and aryl-␤-D-glucosides (methyl-␤-D-glucoside and p-nitrophenyl-␤-D-glucoside) as well as glycosides containing only carbohydrate residues (Cellobiose). On the basis of substrate specificity, ␤-glucosidases can be classified as aryl-␤-glucosidases, cellobiases, and those hydrolyzing both aryl-␤-glucosides and oligosaccharides. The last group is often found in cellulolytic microorganisms (1, 2). On the basis of sequence homology, ␤-glucosidases have been divided into two subfamilies (2): BGA (␤-glucosidases and phospho-␤-glucosidases from bacteria to mammals) and BGB (␤-glucosidases from yeasts, molds, and rumen bacteria). It is one of the components of the cellulase enzyme complex required for the hydrolysis of cellulose to glucose by catalyzing the final step which converts cellobiose to glucose (3, 4).The study of these enzymes has been facilitated by the use of recombinant DNA technology (1,5,6). Although a number of cellulase genes including several ␤-glucosidases have been cloned and expressed in both Escherichia coli and Saccharomyces cerevisiae (7-10), their enzymological properties, especially structure-function relationships, have not been well understood, partially because most of the cellulases show little sequence homology. Analysis of structure-function relationships may be facilitated by the formation of chimeric genes/e...

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“…But the metabolic flux of acetate was increased by the decreasing metabolic flux of TCA cycle that resulted from increasing concentration of pyruvate and the ratio of NADH and NAD [18]. The activity of citrate synthase was inhibited by addition of sodium citrate, which led to reducing the metabolic flux of acetyl-CoA to TCA cycle [19]. In L-tryptophan fermentation, the accumulations of acetate, pyruvate, and lactate were decreased.…”

Section: Discussionmentioning

confidence: 98%

Effect of Sodium Citrate on L-tryptophan Fermentation by Escherichia coli

Cheng

Xie

et al. 2015

Lecture Notes in Electrical Engineering

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L-tryptophan is an essential amino acid, and the work for L-tryptophan production by microbial fermentation has important practical significance with the development of L-tryptophan market. In this study, according to the principle of metabolic engineering, the medium of L-tryptophan fermentation by the strain Escherichia coli TRTH was optimized. The accumulation of by-products was decreased and production of desired product was increased by adding sodium citrate to medium. The effect of sodium citrate on L-tryptophan fermentation was investigated in a 30 L fermentor. The results indicated that when L-tryptophan fermentation with the medium containing 2 g/L sodium citrate, as compared with the medium no containing sodium citrate, the biomass, L-tryptophan production and glucose conversion rate were increased by 2.57, 5.32, 4.21 %, and the accumulation of acetate, pyruvate and lactate were decreased by 5.15, 4.89, 5.23 %. With the medium containing 2 g/L sodium citrate for L-tryptophan fermentation, the biomass, L-tryptophan production and glucose conversion rate were 42.7, 35.7 and 18.2 % respectively.

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Chimeric allosteric citrate synthases: Construction and properties of citrate synthases containing domains from two different enzymes

Cited by 19 publications

References 31 publications

Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures

Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures

Construction of Chimeric β-Glucosidases with Improved Enzymatic Properties

Effect of Sodium Citrate on L-tryptophan Fermentation by Escherichia coli

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