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Production of 7-methylxanthine from Theobromine by Metabolically Engineered E. coli

In this work, a novel biocatalytic process for the production of 7-methylxanthines from theobromine, an economic feedstock has been developed. Bench scale production of 7-methlxanthine has been demonstrated. The biocatalytic process used in this work operates at 30 OC and atmospheric pressure, and is environmentally friendly. The biocatalyst was E. coli BL21(DE3) engineered with ndmB/D genes combinations. These modifications enabled specific N7- demethylation of theobromine to 7-methylxanthine. This production process consists of uniform fermentation conditions with a specific metabolically engineered strain, uniform induction of specific enzymes for 7-methylxanthine production, uniform recovery and preparation of biocatalyst for reaction and uniform recovery of pure 7-methylxanthine.

   Many E. coli BL21(DE3) strains metabolically engineered with single and/or multiple ndmB/D genes were tested for catalytic activity, and the best strains which had the higher activity were chosen to carry out the N-demethylation reaction of theobromine. Strain pBD2dDB had the highest activity for the production of 7-methylxanthine from theobromine. That strain was used to find the optimum amount of cells required to achieve complete conversion of theobromine to 7-methylxanthine within two hours. It was found that the optimum concentration of pBD2dDB strain to achieve 100% conversion of 0.5 mM theobromine to 7-methylxanthine was 5 mg/mL. The cell growth of pBD2dDB strain was studied using two different growth media, (Luria-Bertani Broth and Super Broth). Super broth was found to be the best medium to produce the highest amount of cell paste (1.5 g). Subsequently, the process was scaled up in which 2 L reaction volume was used to produce 7-methylxanthine (100% conversion) from 0.5 mM theobromine catalyzed by pBD2dDB strain. The reactions was carried out at 30 oC and 250 rpm shaker speed, and the reaction medium was 50 mM potassium phosphate buffer (pH=7). 7-methylxanthines was separated by preparative chromatography with high recovery, and the product solution was collected, purified by drying at 120-140 oC for 4 hours and, recovered (127 mg). Purity of the isolated 7-methylxanthine was comparable to authentic standards with no contaminant peaks, as observed by HPLC, LC-MS, and NMR. 

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Publication Date
Tue Feb 01 2011
Journal Name
Biochemical Engineering Journal
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Publication Date
Sun Apr 22 2018
Journal Name
Acs Biomaterials Science & Engineering
Engineered coatings for titanium implants to present ultra-low doses of BMP-7

The ongoing research to improve the clinical outcome of titanium implants has resulted in the implementation of multiple approaches to deliver osteogenic growth factors accelerating and sustaining osseointegration. Here we show the presentation of human bone morphogenetic protein 7 (BMP-7) adsorbed to titanium discs coated with poly(ethyl acrylate) (PEA). We have previously shown that PEA promotes fibronectin organization into nanonetworks exposing integrin- and growth-factor-binding domains, allowing a synergistic interaction at the integrin/growth factor receptor level. Here, titanium discs were coated with PEA and fibronectin and then decorated with ng/mL doses of BMP-7. Human mesenchymal stem cells were used to investigate cellular resp

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Publication Date
Wed May 10 2017
Journal Name
Ibn Al-haitham Journal For Pure And Applied Sciences
Genetic Relationship Between Heavy Metals Resistance and β- Lactamase Production In E. Coli and Staphylococcus Aureus

  This study is a trail to know if the genes controlling some of heavy metals resistance ( lead, zinc, cadmium, cromium) in two types of pathogenic bacteria  E. coli  as gram negative bacteria and S. aureus as gram positive bacteria, present on the β-lactamase plasmid.     Ten isolates of each bacterial types which produced β-lactamase enzyme, were cultivated in the presence of acridine orange. The growing in the presence of acridine orange resulted in loss of the β-lactamase genes in S. aureus and E. coli, and loss of the heavy metals resistance in S. aureus, while the resistance of E. coli against heavy metals still without any change.   The results indicate that the genes for heavy

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Publication Date
Wed Dec 01 2021
Journal Name
Iraqi Journal Of Veterinary Sciences
RAPD-PCR and phylogenetic analysis of E. coli isolated from human and cattle urinary tract infections

This study aimed at isolating uropathogenic Escherichia coli from urinary tract infections (UTIs) of human and cattle to examine the molecular diversity and phylogenetic relationship of the isolates. A total of 100 urine samples were collected from UTIs of human and cattle. The isolates identification was done using routine diagnostic methods and confirmed by Vitek2. Antimicrobial susceptibility was tested against 10 antimicrobials. Random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) was applied to identify the genetic diversity among E. coli isolates from human and animal origin by using five different octamer primers. The gelJ software for the phylogenetic analysis created Dendrograms. Out of 50 human urine samples, E.

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Publication Date
Fri Jan 26 2024
Journal Name
Iraqi Journal Of Science
Effect of seed oil Ricinus communis on E. coli isolated from Recurrent Urinary Tract Infections

This study was conducted in Wasit governorate for the period between February 2012 to February 2013 to determine the impact of Castor seed oil at different concentrations 100, 75, 50 and 25% in inhibition of the growth of E. coli isolated from 52 male and female patients (2-70 yrs) attending Al Zahra Hospital and Al Karama Hospital in Kut city. Oil was extracted from seeds of Castor had the ability to inhibit E. coli isolated from patients presented with recurrent urinary tract infections . Zone of inhibition accomplishing was 9.06 mm in diameter.HPLC analysis revealed that the content of α-linolenic in Castor seed oil (18.90 μg\ml) was higher than other fatty acids followed by oleic. Perhaps this is why it able to inhibit E. coli; whi

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Publication Date
Thu Jul 20 2023
Journal Name
Ibn Al-haitham Journal For Pure And Applied Sciences
Detection of antibiotic resistance of the phylogenetic group E among E. coli bacteria isolated from diarrheal cases in children under five years

 

From 50 stool samples collected from children with diarrhea of both sexes who visited various hospitals in Baghdad, 26 isolates of E.coli were found to belong to the phylogenetic group E. The findings revealed that the percentage of E.coli for thephylogenetic group E is (52%) , making it the dominant group among the other phylogenetic groups. The findings demonstrated that 100% of the E.coli isolates from phylogenetic group E are resistant to penicillin, and only 15% are resistant to imipenem. Multi-drug resistance (MDR) was found to be 15%, while XDR reached 85%. The results of thephylogenetic group for the remaining species of isolates in this study were group A (2/50 and by 4%), gr

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Publication Date
Mon Aug 01 2016
Journal Name
Enzyme And Microbial Technology
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Publication Date
Tue Feb 01 2022
Journal Name
Baghdad Science Journal
An Epidemiological and Multidrug Resistance Study for E. coli Isolated from Urinary Tract Infection (Three Years of Study)

Uropathogenic E. coli (UPEC) is problematic and still the leading cause of urinary tract infections worldwide. It is developed resistance against most antibiotics. The investigation, surveillance system, and efficient strategy will facilitate selecting an appropriate treatment that could control the bacterial distribution. The present study aims to investigate the epidemiology and associated risk factors of uropathogenic E. coli and to study their antibiotic resistance patterns. 1585 midstream urine specimens were collected from symptomatic urinary tract infections (UTI) patients (225 males and 1360 females) admitted to Zakho emergency hospital, Zakho, Kurdistan Region, Iraq from January 2016 until the end of December 2

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Publication Date
Wed Aug 09 2017
Journal Name
Ibn Al-haitham Journal For Pure And Applied Sciences
Determination of Optimum Cultural Conditions for the Production of Cytosine Deaminase From Escherichia coli

    The study involved isolation and characterization of E.coli from patient’s infected with diarrhea , in order to study the ability of the bacteria to produce cytosine deaminase (CD). Result showed eight isolates of E.coli which showed adifference in the production of (CD) and the isolate of E. coli E33 was the beast of its production of CD than the other’s and the value of the specific activity was 4.920  u/mg protein , when grown in the medium which contains 1% glycerol ,3% peptone as a source of Carbon and Nitrogen respectively with pH 8.   The optimum cultural condition‘s for the production of CD from E. coli E33 was studied the result‘s  showed that the isolate gave the

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Publication Date
Thu Jan 21 2010
Journal Name
Iraqi Journal Of Veterinary Medicine
Production and Partial Purification of Heat-Stable Enterotoxin (A) Produced by Enterotoxigenic Escherichia coli

A total of (25) stool samples were collected from children and adults (2- 4) years old suffering from diarrhea to isolate E. coli strains that produce heat-stable enterotoxin a (STa), and after performing microscopic examination, cultural characterization and biochemical identification only (11) isolates showed positive E. coli. STa activity was estimated by using suckling mouse assay (SMA) and from these (11) isolates only (5) showed STa activity and the one with the highest STa activity was selected for large scale production of STa, which was followed by partial purification using ion-exchange chromatography (normal phase) using DEAE sephadex A-50 column. After purification and determination of protein concentration by using the standard

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