Large-scale Enzymatic Synthesis of 12-Ketoursodeoxycholic Acid from Dehydrocholic Acid by Simultaneous Combination of 3α-Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni and 7β-Hydroxysteroid Dehydrogenase from Collinsella aerofaciens

2012 ◽  
Vol 67 (10) ◽  
pp. 1037-1044 ◽  
Author(s):  
Daniel Bakonyi ◽  
Astrid Wirtz ◽  
Werner Hummel
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Hydroxysteroid Dehydrogenase ◽  
Coli Strain ◽  
Optically Active ◽  
Carbonyl Groups ◽  
Dehydrocholic Acid ◽  
Crude Product ◽  
High Yields ◽  
Pseudomonas Testosteroni

12-Keto-UDCA is an important optically active component for the drug ursodeoxycholic acid (UDCA). Starting from the three-keto compound dehydrocholic acid, the carbonyl groups at position 3 and 7 have to be reduced stereo- and regioselectively. In this case we applied two hydroxysteroid dehydrogenases for this purpose, the NAD-dependent 3α-HSDH from Pseudomonas testosteroni and the NADP-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Both enzymes can be produced in high yields by an Escherichia coli strain as recombinant proteins. In order to avoid impurities by the 7a-hydroxysteroid dehydrogenase of Escherichia coli, a mutant strain with an inactivated 7a-enzyme was applied for producing the three enzymes. For bioconversion, the dehydrogenases can be used as crude enzyme samples and are applied simultaneously. A 1.8 L batch of 100mM DHCA incubated at pH = 8.0 and 25°C resulted in 80 g crude product with a quite high purity of ≥ 99:5% as judged by HPLC analysis.

Optimization of 7α-hydroxysteroid dehydrogenase production by Escherichia coli 080

1990 ◽  
Vol 36 (10) ◽  
pp. 725-727 ◽  
Author(s):  
V. Prabha ◽  
Meenakshi Gupta ◽  
K. G. Gupta
Keyword(s):  
Escherichia Coli ◽  
Oxygen Consumption ◽  
Oxygen Consumption Rate ◽  
Enzyme Production ◽  
Consumption Rate ◽  
Ethylenediaminetetraacetic Acid ◽  
Brain Heart Infusion ◽  
Hydroxysteroid Dehydrogenase ◽  
Coli Strain ◽  
Optimal Conditions

7α-Hydroxysteroid dehydrogenase (EC 1.1.1.159) production by Escherichia coli strain 080 was highest when the organism was grown in brain heart infusion broth at pH 6.5 for 72–96 h with shaking at 37 °C. The oxygen consumption rate had a strong effect on the production of this constitutive enzyme. Glucose and lactose at 0.2–0.4%, detergents, and ethylenediaminetetraacetic acid were found to increase the enzyme production. Key words: 7α-hydroxysteroid dehydrogenase, Escherichia coli 080, optimal conditions for 7α-hydroxysteroid dehydrogenase production.

scholarly journals Molecular evolution of the Escherichia coli chromosome. I. Analysis of structure and natural variation in a previously uncharacterized region between trp and tonB.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 345-358
Author(s):  
A Stoltzfus ◽  
J F Leslie ◽  
R Milkman
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Natural Variation ◽  
Genetic Recombination ◽  
Coli Strain ◽  
Open Reading Frames ◽  
Dna Rearrangements ◽  
E Coli ◽  
Wild Strains ◽  
Reading Frames

Abstract We present the sequence of a 3500-bp region of the Escherichia coli strain K12 chromosome lying between the tryptophan operon and the tonB gene. Analysis of the sequence yields six open reading frames that have properties characteristic of genes for proteins. The reading frames are closely spaced, and putative transcription units and control sites compose over 95% of the DNA. The sequences of several wild strains of E. coli have been determined for a large segment of the region described. Comparison of these sequences reveals the effects of base substitutions, DNA rearrangements, and recombination. In the regions presumably expressed as polypeptides, most of the natural variation results from synonymous substitutions. However, the DNA rearrangements identified have end points within the open reading frames and disrupt them in a variety of ways. The effects of genetic recombination between strains, recently found to be significant on a large scale in E. coli, are also apparent in the region between trp and tonB.

Purification of 7α-hydroxysteroid dehydrogenase from Escherichia coli strain 080

1990 ◽  
Vol 36 (2) ◽  
pp. 131-135 ◽  
Author(s):  
Vijay Prabha ◽  
Meenakshi Gupta ◽  
D. Seiffge ◽  
K. G. Gupta
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Key Words ◽  
Ammonium Sulfate ◽  
Gel Electrophoresis ◽  
Enzyme Preparation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Coli Strain

Purification studies of 7α-hydroxysteroid dehydrogenase (7α-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 °C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54 000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera. Key words: 7α-hydroxysteroid dehydrogenase, Escherichia coli.

scholarly journals Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

2015 ◽  
Vol 81 (18) ◽  
pp. 6276-6284 ◽  
Author(s):  
Chin Giaw Lim ◽  
Lynn Wong ◽  
Namita Bhan ◽  
Hila Dvora ◽  
Peng Xu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Colorimetric Assay ◽  
Cost Effective ◽  
Coli Strain ◽  
Water Soluble ◽  
Natural Food ◽  
Scale Production ◽  
Terrestrial Plants ◽  
Content Type

ABSTRACTAnthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (+)-catechin inEscherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identifiedE. colitransporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.

scholarly journals Iron Acquisition of Urinary Tract Infection Escherichia coli Involves Pathogenicity in Caenorhabditis elegans

2021 ◽  
Vol 9 (2) ◽  
pp. 310
Author(s):  
Masayuki Hashimoto ◽  
Yi-Fen Ma ◽  
Sin-Tian Wang ◽  
Chang-Shi Chen ◽  
Ching-Hao Teng
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Caenorhabditis Elegans ◽  
Large Scale ◽  
Iron Acquisition ◽  
Infection Model ◽  
High Throughput Analysis ◽  
E Coli ◽  
C Elegans ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

scholarly journals A simple and efficient automated microvolume radiosynthesis of [18F]Florbetaben

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ksenia Lisova ◽  
Jia Wang ◽  
Philip H. Chao ◽  
R. Michael van Dam
Keyword(s):  
Large Scale ◽  
Solid Phase ◽  
Acetonitrile Solution ◽  
Synthesis Methods ◽  
Pet Tracers ◽  
Molar Activity ◽  
Alternative Approaches ◽  
High Yields ◽  
Sufficient Concentration

Abstract Background Current automated radiosynthesizers are generally optimized for producing large batches of PET tracers. Preclinical imaging studies, however, often require only a small portion of a regular batch, which cannot be economically produced on a conventional synthesizer. Alternative approaches are desired to produce small to moderate batches to reduce cost and the amount of reagents and radioisotope needed to produce PET tracers with high molar activity. In this work we describe the first reported microvolume method for production of [18F]Florbetaben for use in imaging of Alzheimer’s disease. Procedures The microscale synthesis of [18F]Florbetaben was adapted from conventional-scale synthesis methods. Aqueous [18F]fluoride was azeotropically dried with K2CO3/K222 (275/383 nmol) complex prior to radiofluorination of the Boc-protected precursor (80 nmol) in 10 μL DMSO at 130 °C for 5 min. The resulting intermediate was deprotected with HCl at 90 °C for 3 min and recovered from the chip in aqueous acetonitrile solution. The crude product was purified via analytical scale HPLC and the collected fraction reformulated via solid-phase extraction using a miniature C18 cartridge. Results Starting with 270 ± 100 MBq (n = 3) of [18F]Fluoride, the method affords formulated product with 49 ± 3% (decay-corrected) yield,> 98% radiochemical purity and a molar activity of 338 ± 55 GBq/μmol. The miniature C18 cartridge enables efficient elution with only 150 μL of ethanol which is diluted to a final volume of 1.0 mL, thus providing a sufficient concentration for in vivo imaging. The whole procedure can be completed in 55 min. Conclusions This work describes an efficient and reliable procedure to produce [18F]Florbetaben in quantities sufficient for large-scale preclinical applications. This method provides very high yields and molar activities compared to reported literature methods. This method can be applied to higher starting activities with special consideration given to automation and radiolysis prevention.

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