scholarly journals Expression of a modified xylanase in yeast

2009 ◽  
Author(s):  
◽  
Nokuthula Peace Mchunu
Keyword(s):  
Culture Media ◽  
Paper Industry ◽  
Methylotrophic Yeast ◽  
Thermomyces Lanuginosus ◽  
Host Proteins ◽  
E Coli ◽  
Gap Promoter ◽  
Naturally Occurring ◽  
Adh2 Promoter ◽  
Suitable Expression

Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

1992 ◽  
Vol 68 (05) ◽  
pp. 539-544 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Jack Henkin ◽  
Victor Gurewich
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cell ◽  
Human Gene ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Glycosylation Site ◽  
Clot Lysis ◽  
Cell Culture Media ◽  
E Coli ◽  
The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

scholarly journals Antimicrobial efficacy of a new tri-antibiotic combination against resistant endodontic pathogens: An in-vitro study

2020 ◽  
Vol 23 (4) ◽  
pp. 8p ◽  
Author(s):  
Prasanna T. Dahake ◽  
Sudhindra M Baliga
Keyword(s):  
Root Canal ◽  
Pathogenic Bacteria ◽  
Culture Media ◽  
In Vitro Study ◽  
Vitro Study ◽  
New Combination ◽  
Antimicrobial Efficacy ◽  
E Coli ◽  
Background Removal

Background: Removal of all the pathogenic bacteria from the root canal system is of prime importance for the success of endodontic therapy. Objective: The study aimed to determine the antimicrobial efficacy of three antibiotics and their new combination against selected endodontic pathogens. Methods: In this in-vitro study, we used bacterial strains associated with the refractory endodontic condition and determined MIC and MBC of Clindamycin (C), Metronidazole (M), Doxycycline (D) as well as their combination CMD. We cultured Candida Albicans, Pseudomonas Aeruginosa, Escherichia Coli, Enterococcus Faecalis, Streptococcus Mutans, Bacillus Subtilis subsp. spizizenii, Actinomyces Actinomycetemcomitans on selective culture media. We analyzed the data using paired 't' test, one-way ANOVA, and Tuckey's HSD post hoc test. Results: Clindamycin inhibited the growth of C. Albicans (90%) and S. Mutans (90%) significantly and P. Aeruginosa, E. Coli, E. Faecalis, B. Subtilis, and A. Actinomycetemcomitans were resistant to it. Metronidazole did not inhibit any of the bacteria. Doxycycline inhibited C. Albicans (90%), P. Aeruginosa (90%), and S. Mutans (90%) significantly while E. Coli, E. Faecalis, B. Subtilis, and A. Actinomycetemcomitans were resistant to it. The combination of CMD inhibited all the microbes significantly. However, at bactericidal concentrations of CMD, E. Faecalis (p = 0.024), B. Subtilis (p = 0.021) and A. Actinomycetemcomitans (p = 0.041) were eliminated significantly, while C. Albicans (p = 0.164), P. Aeruginosa (p = 0.489), E. Coli (p = 0.106) and S. Mutans (p = 0.121) showed resistance. Conclusion: Combination CMD can be used against resistant endodontic pathogens to achieve predictable endodontic results.KEYWORDSAntimicrobial agents; Clindamycin; Doxycycline; Metronidazole; Root canal therapy.    

scholarly journals Microbial Synthesis of Non-Natural Anthraquinone Glucosides Displaying Superior Antiproliferative Properties

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2171 ◽  
Author(s):  
Trang Nguyen ◽  
Ramesh Pandey ◽  
Prakash Parajuli ◽  
Jang Han ◽  
Hye Jung ◽  
...  
Keyword(s):  
Cell Growth ◽  
Biological Activities ◽  
Uterine Cervical Cancer ◽  
Cell Growth Inhibition ◽  
Microbial Synthesis ◽  
E Coli ◽  
Anticancer Effects ◽  
Naturally Occurring ◽  
Spectroscopic Analyses ◽  
Proliferative Assay

Anthraquinones, naturally occurring bioactive compounds, have been reported to exhibit various biological activities, including anti-inflammatory, antiviral, antimicrobial, and anticancer effects. In this study, we biotransformed three selected anthraquinones into their novel O-glucoside derivatives, expressing a versatile glycosyltransferase (YjiC) from Bacillus licheniformis DSM 13 in Escherichia coli. Anthraflavic acid, alizarin, and 2-amino-3-hydroxyanthraquinone were exogenously fed to recombinant E. coli as substrate for biotransformation. The products anthraflavic acid-O-glucoside, alizarin 2-O-β-d-glucoside, and 2-amino-3-O-glucosyl anthraquinone produced in the culture broths were characterized by various chromatographic and spectroscopic analyses. The comparative anti-proliferative assay against various cancer cells (gastric cancer-AGS, uterine cervical cancer-HeLa, and liver cancer-HepG2) were remarkable, since the synthesized glucoside compounds showed more than 60% of cell growth inhibition at concentrations ranging from ~50 μM to 100 μM. Importantly, one of the synthesized glucoside derivatives, alizarin 2-O-glucoside inhibited more than 90% of cell growth in all the cancer cell lines tested.

scholarly journals Microbiological quality of commercially available poultry feeds sold in Bangladesh

2017 ◽  
Vol 3 (1) ◽  
pp. 52-60
Author(s):  
Nigar Sultana ◽  
Md Atiqul Haque ◽  
Md Mostafizer Rahman ◽  
Mir Rowshan Akter ◽  
Mst Deloara Begum ◽  
...  
Keyword(s):  
Public Health ◽  
Methylene Blue ◽  
Culture Media ◽  
Microbiological Quality ◽  
Control Measures ◽  
Nutrient Agar ◽  
Poultry Feed ◽  
Salmonella Spp ◽  
Isolation And Identification ◽  
E Coli

The study was conducted aiming at the isolation and identification of pathogens from poultry feed manufactured by four different brands namely A (broiler starter), B (broiler finisher), C (layer starter) and D (layer finisher) sold in retail stores of Rangpur city of Bangladesh. All these samples were collected from four randomly chosen outlets and analyzed by culturing in different culture media such as Nutrient broth (NB), Nutrient agar (NA), Salmonella-Shigella (SS) agar, Eosin methylene blue (EMB) agar, MacConkey agar, Triple sugar iron (TSI) agar slant, Motility, Indole, Urease (MIU) and Saboraud Dextrose agar (SDA) media. The bacterial agents were isolated and examined under light microscope for their gross morphological and conventional biochemical characteristics. The bacteriological analyses were done at the Microbiology Laboratory of Hajee Mohammad Danesh Science and Technology University, Dinajpur during the period of January to June, 2014. Total bacterial colonies of all the samples were counted separately according to the American Public Health Association, using nutrient agar medium for total viable count (TVC), Eosine methylene blue (EMB) agar media for total E. coli count (TEC) and Salmonella-Shigella agar for TSC (total salmonella count). Saboraud Dextrose agar (SDA) media was used for detection of fungus. The virulence effect of the organism present in feed were observed by inoculating the organism in poultry. Recorded result showed that average TVC of feed sample A, B, C and D were 5.45x106, 3.28x105, 5.14x106 and 4.53x105 CFU/gm (colony forming unit per gram) respectively. TEC of feed sample A, B, C and D were recorded 6.25x105, 8.26x103, 5.52x105 and 5.65x104 CFU/gm respectively. TSC of feed sample A, B, C and D were recorded 3.15x104, 2.68x103, 4.46x103 and 1.19x104 CFU/gm respectively. The highest TVC, TEC and TSC were found in broiler starter (feed sample A) and lowest TVC, TEC and TSC were found in broiler finisher (feed sample B). Fungal count was 1.85x105 CFU/ gm in layer finisher (feed sample D) could be as a result of their high pathogenecity as reported by researchers elsewhere. These organisms can cause several poultry and farm animal infections specially mycotoxicosis having public health significance to both human and poultry. The presence of high numbers of E. coli and Salmonella spp. in poultry feed were indicative of poor hygienic practices during manufacture, post process contamination and unsatisfactory transportation and reservation. Therefore reinforce the need for preventive control measures, hygienic handling and processing of feeds to reduce the risk of potential human health hazards.Asian J. Med. Biol. Res. March 2017, 3(1): 52-60

scholarly journals Evaluation of the Antibacterial Activity of a Geopolymer Mortar Based on Metakaolin Supplemented with TiO2 and CuO Particles Using Glass Waste as Fine Aggregate

Coatings ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 157 ◽  
Author(s):  
Ruby Gutiérrez ◽  
Mónica Villaquirán-Caicedo ◽  
Sandra Ramírez-Benavides ◽  
Myriam Astudillo ◽  
Daniel Mejía
Keyword(s):  
Culture Media ◽  
Raw Materials ◽  
Silicate Solution ◽  
Fine Aggregate ◽  
X Ray Diffraction ◽  
Glass Waste ◽  
Homogeneous Surface ◽  
E Coli ◽  
Study Results ◽  
Antibacterial Surfaces

Metakaolin-based geopolymer cements were produced by alkaline activation with a potassium hydroxide and potassium silicate solution. To produce the geopolymer composites, 10 wt.% titanium oxide (TiO2) and 5 wt.% copper oxide (CuO) nanoparticles were used. The geopolymer mortar was prepared using glass waste as fine aggregate. The raw materials and materials produced were characterized by X-ray diffraction, electron microscopy, and Fourier-transform infrared spectroscopy techniques. Likewise, the geopolymer samples were characterized to determine their physical properties, including their density, porosity, and absorption. The photocatalytic activity of the materials was evaluated by activating the nanoparticles in a chamber with UV–Vis light during 24 h; then, different tests were performed to determine the growth inhibition of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa bacteria in nutrient agar for times of up to 24 h. The study results showed that a geopolymer mortar containing glass waste as fine aggregate (GP-G) exhibited a water absorption 56.73% lower than that of the reference geopolymer paste without glass (GP). Likewise, glass particles allowed the material to have a smoother and more homogeneous surface. The pore volume and density of the GP-G were 37.97% lower and 40.36% higher, respectively, than those of the GP. The study with bacteria showed that, after 24 h in the culture media, the GP-G mortars exhibited a high inhibition capacity for the growth of P. aeruginosa from solutions of 10−4 mL and in solutions of 10−6 mL for E. coli and S. aureus. These results indicate the possibility of generating antibacterial surfaces by applying geopolymer composite.

scholarly journals The Compromised Anti-microbial Function of Mesenchymal Stem Cells in Diabetic Patients

2017 ◽  
Vol 2 (3) ◽  
pp. 2473011417S0004
Author(s):  
Zijun Zhang ◽  
Lew Schon ◽  
Young Cho
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bacterial Infection ◽  
Conditioned Medium ◽  
Culture Media ◽  
Cell Metabolism ◽  
Diabetic Group ◽  
Diabetic Patients ◽  
Paracrine Factors ◽  
E Coli

Category: Basic Sciences/Biologics Introduction/Purpose: Diabetic foot infection (DFI), including skin infection and osteomyelitis, is a severe complication of late- stage diabetes. Mesenchymal stem cells (MSCs) facilitate bacterial clearance. In bacterial infection, MSCs, via paracrine mediators, regulate the host cell metabolism and inflammatory response. Particularly, MSCs augment the antibacterial function of neutrophils. It is generally believed that hyperglycemia in diabetes is toxic to MSCs/progenitors and detrimental to their regenerative function. It is unknown, however, whether the antibacterial function of MSCs is compromised in diabetes. Methods: Bone marrow samples from 6 diabetic and 4 non-diabetic patients (approved by IRB) were used for MSC isolation. 1. MSCs from both diabetic and non-diabetic patients were treated with lipopolysaccharides (LPS), a bacterial wall component, for 6 hours. The tissue culture media were collected as conditioned medium. E. coli from a single colony were cultured with addition of the conditioned medium generated by either diabetic or non-diabetic MSCs and inoculated on LB-agar plates overnight. Bacterial colonies were counted. 2. Human macrophages were isolated from umbilical cord blood and co-cultured with either diabetic or non-diabetic MSCs, in a trans-well system, for 24 hours. The macrophages were then cultured with heat-inactivated E. coli for one hour. After extensive washing, macrophage and bacteria were stained with Pappenheim method. Bacterial phagocytosis of macrophages, after co- cultured with diabetic or non-diabetic MSCs, was assessed under a microscope. Results: There was no statistical difference in the number of E. coli colonies when regular medium produced by diabetic and non- diabetic MSCs was added into the bacterial culture. When the diabetic and non-diabetic MSCs were treated with LPS and the conditioned medium was collected and added into bacterial cultures, E. coli colonies increased in the diabetic group, about 3 fold, as compared with the non-diabetic group (p < 0.05). Macrophages were counted in defined areas of the Petri dishes and designated as infected or uninfected, according to the presentation of bacterial bodies or not. While the infection rate of macrophages co-cultured with non-diabetic MSCs was 85% (±5.5%), it was 70% (±6.6%) when macrophages were co-cultured with diabetic MSCs (p = 0.006). Conclusion: MSCs-produced paracrine factors suppressed the growth of E. coli but diabetic and non-diabetic MSCs had no difference in such a function. Activation with LPS did not augment the non-diabetic MSCs but weakened diabetic MSCs in suppression of bacterial growth. MSCs regulate macrophages in bacterial phagocytosis. Diabetic MSCs, however, had a limited role in regulation of macrophages. This study demonstrated that MSCs in diabetic patients are compromised in anti-bacterial infection. The results not only deepen the understanding of bacterial infection in diabetes but also open up new strategy to control bacterial infection in diabetic patients.

scholarly journals Natural Genetic Transformation of Clinical Isolates of Escherichia coli in Urine and Water

2002 ◽  
Vol 68 (1) ◽  
pp. 440-443 ◽  
Author(s):  
Markus Woegerbauer ◽  
Bernard Jenni ◽  
Florian Thalhammer ◽  
Wolfgang Graninger ◽  
Heinz Burgmann
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Current Knowledge ◽  
Antibiotic Resistance Genes ◽  
Aquatic Environments ◽  
Wild Type ◽  
E Coli ◽  
Naturally Occurring ◽  
Natural Genetic Transformation ◽  
Type Strains

ABSTRACT Transfer of plasmid-borne antibiotic resistance genes in Escherichia coli wild-type strains is possible by transformation under naturally occurring conditions in oligotrophic, aquatic environments containing physiologic concentrations of calcium. In contrast, transformation is suppressed in nitrogen-rich body fluids like urine, a common habitat of uropathogenic strains. Current knowledge indicates that transformation of these E. coli wild-type strains is of no relevance for the acquisition of resistance in this clinically important environment.

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