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Recombinant Escherichia Coli Strain (recombinant + escherichia_coli_strain)
Selected AbstractsImproved ,-Glucanase Production by a Recombinant Escherichia coli Strain using Zinc-Ion Supplemented MediumENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2007U. Beshay Abstract In order to investigate the suitability of different metal chelates for affinity chromatography, an expression vector was constructed. It contained a hybrid ,-glucanase as a model protein fused with a His6 -tag and a secretion cassette providing the ability to secrete ,-glucanase into the culture medium. Supplementation of zinc to the medium led to a rapidly increased expression and release of the target protein into the cultivation medium. Results in respect to the supplementation of the commonly used Terrific Broth "TB-medium" with different metal ions are reported with special emphasis on the influence of zinc ions. A concentration of zinc ions in the order of about 0.175 mM led to optimal results. Batch cultivation under well-controlled conditions showed that the growth behavior did not change significantly by adding zinc ions. Growth in a stirred tank bioreactor was much faster in unsupplemented TB-medium compared to shake flask experiments leading to a much higher biomass concentration (15,g/L instead of 3,g/L). The secretion of ,-glucanase under theses conditions started at the transition into the stationary phase and increased to yield an extracellular activity of 1350,U/mL at the end of the fermentation process. An even higher yield of extracellular ,-glucanase (2800,U/mL) was reached when the fermentation was carried out with TB-medium supplemented with 0.175,mM ZnSO4. [source] ChemInform Abstract: Bioconversion of Substituted Styrenes to the Corresponding Enantiomerically Pure Epoxides by a Recombinant Escherichia coli Strain.CHEMINFORM, Issue 7 2001Silvana Bernasconi Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Construction of Recombinant Escherichia coli Catalysts which Simultaneously Express an (S)-Oxynitrilase and Different Nitrilase Variants for the Synthesis of (S)-Mandelic Acid and (S)-Mandelic Amide from Benzaldehyde and CyanideADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 10 2009Olga Sosedov Abstract Recombinant Escherichia coli strains were constructed which simultaneously expressed the genes encoding the (S)-oxynitrilase from cassava (Manihot esculenta) together with the wild-type or a mutant variant of the arylacetonitrilase from Pseudomonas fluorescens EBC191 in a single organism under the control of a rhamnose-inducible promoter. The whole cell catalysts obtained converted benzaldehyde and potassium cyanide in aqueous media at pH,5.2 mainly to (S)-mandelic acid and/or (S)-mandelic amide and synthesized only low amounts of the corresponding (R)-enantiomers. The conversion of benzaldehyde and potassium cyanide (KCN) by a whole-cell catalyst simultaneously expressing the (S)-oxynitrilase and the wild-type nitrilase resulted in a ratio of (S)-mandelic acid to (S)-mandelic amide of about 4:3. This could be explained by the strong nitrile hydratase activity of the wild-type nitrilase with (S)-mandelonitrile as substrate. The relative proportion of (S)-mandelic amide formed in this system was significantly increased by coexpressing the (S)-oxynitrilase with a carboxy-terminally truncated variant of the nitrilase. This whole-cell catalyst converted benzaldehyde and KCN to mandelic amide and mandelic acid in a ratio of about 9:1. The ee of the (S)-mandelic amide formed was calculated to be >95%. [source] Isoprenoid biosynthesis in plants , 2C -methyl- d -erythritol-4-phosphate synthase (IspC protein) of Arabidopsis thalianaFEBS JOURNAL, Issue 19 2006Felix Rohdich The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C -methyl- d -erythritol 4-phosphate from 1-deoxy- d -xylulose 5-phosphate at a rate of 5.6 µmol·min,1·mg,1 (kcat 4.4 s,1). The Michaelis constants for 1-deoxy- d -xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 µm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 µmol·min -1·mg -1. [source] Non-invasive detection of the metabolic burden on recombinant microorganisms during fermentation processes,JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2001Th Bachinger Abstract Heterologous protein production is an important source of therapeutic products. Optimisation of such bioprocesses by adjustment of the expression rate of the heterologous protein to the biosynthetic capacity of the cell metabolism would benefit from an online method for monitoring the metabolic burden. In this study we evaluated the use of a chemical multi-sensor array for this purpose. Fermentations with a recombinant Escherichia coli strain expressing human superoxide dismutase (rhSOD) were monitored by the sensor array. The results of isopropyl-thiogalactopyranoside (IPTG)-induced expression were compared with fermentations with a plasmid-free strain. The overproduction of rhSOD, imposing a high metabolic burden on the plasmid-carrying cells, was distinctly and reproducibly observed by the multi-sensor array. The potential of this non-invasive method of non-specific metabolic burden monitoring is demonstrated by the results of the study. © 2001 Society of Chemical Industry [source] Simple enzymatic procedure for l -carnosine synthesis: whole-cell biocatalysis and efficient biocatalyst recyclingMICROBIAL BIOTECHNOLOGY, Issue 1 2010Jan Heyland Summary , -Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with , -peptides. Recently, , -aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of , -peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of , -peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant ,,, -dipeptide l -carnosine (, -alanine- l -histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a , -peptidase, could be used directly as whole-cell biocatalysts for the synthesis of l -carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l -carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation of l -carnosine to a concentration of 3.7 g l,1. [source] The effect of heating rate on Escherichia coli metabolism, physiological stress, transcriptional response, and production of temperature-induced recombinant protein: A scale-down studyBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Luis Caspeta Abstract At the laboratory scale, sudden step increases from 30 to 42°C can be readily accomplished when expressing heterologous proteins in heat-inducible systems. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decreases as the scale increases. In this work, the transcriptional and metabolic responses of a recombinant Escherichia coli strain to temperature-induced synthesis of pre-proinsulin in high cell density cultures were examined at different heating rates. Heating rates of 6, 1.7, 0.8, and 0.4°C/min were tested in a scale-down approach to mimic fermentors of 0.1, 5, 20, and 100 m3, respectively. The highest yield and concentration of recombinant protein was obtained for the slowest heating rate. As the heating rate increased, the yield and maximum recombinant protein concentration decreased, whereas a larger fraction of carbon skeletons was lost as acetate, lactate, and formate. Compared to 30°C, the mRNA levels of selected heat-shock genes at 38 and 42°C, as quantified by qRT-PCR, increased between 2- to over 42-fold when cultures were induced at 6, 1.7, and 0.8°C/min, but no increase was observed at 0.4°C/min. Only small increases (between 1.5- and 4-fold) in the expression of the stress genes spoT and relA were observed at 42°C for cultures induced at 1.7 and 6°C/min, suggesting that cells subjected to slow temperature increases can adapt to stress. mRNA levels of genes from the transcription,translation machinery (tufB, rpoA, and tig) decreased between 40% and 80% at 6, 1.7 and 0.8°C/min, whereas a transient increase occurred for 0.4°C/min at 42°C. mRNA levels of the gene coding for pre-proinsulin showed a similar profile to transcripts of heat-shock genes, reflecting a probable analogous induction mechanism. Altogether, the results obtained indicate that slow heating rates, such as those likely to occur in conventional large-scale fermentors, favored heterologous protein synthesis by the thermo-inducible expression system used in this report. Knowledge of the effect of heating rate on bacterial physiology and product formation is useful for the rational design of scale-down and scale-up strategies and optimum recombinant protein induction schemes. Biotechnol. Bioeng. 2009;102: 468,482. © 2008 Wiley Periodicals, Inc. [source] Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3-hydroxypropionic acid from glycerol,BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Chelladurai Rathnasingh Abstract 3-Hydroxypropionic acid (3-HP) is a commercially valuable chemical with the potential to be a key building block for deriving many industrially important chemicals. However, its biological production has not been well documented. Our previous study demonstrated the feasibility of producing 3-HP from glycerol using the recombinant Escherichia coli SH254 expressing glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH), and reported that an "imbalance between the two enzymes" and the "instability of the first enzyme DhaB" were the major factors limiting 3-HP production. In this study, the efficiency of the recombinant strain(s) was improved by expressing DhaB and AldH in two compatible isopropyl-thio-,-galactoside (IPTG) inducible plasmids along with glycerol dehydratase reactivase (GDR). The expression levels of the two proteins were measured. It was found that the changes in protein expression were associated with their enzymatic activity and balance. While cloning an alternate aldehyde dehydrogenase (ALDH), ,-ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH-BGK1 showed the highest level of 3-HP production (2.8,g/L) under shake-flask conditions. When an aerobic fed-batch process was carried out under bioreactor conditions at pH 7.0, the recombinant SH-BGK1 produced 38.7,g 3-HP/L with an average yield of 35%. This article reports the highest level of 3-HP production from glycerol thus far. Biotechnol. Bioeng. 2009; 104: 729,739 © 2009 Wiley Periodicals, Inc. [source] | |||||||||||||