scholarly journals Nghiên cứu biểu hiện và tinh chế -glucuronidase có nguồn gốc vi khuẩn dạ cỏ dê trong tế bào Esccherichia coli

2018 ◽  
Vol 34 (2) ◽  
Author(s):  
Huyen Do
Keyword(s):  
Glucuronic Acid ◽  
Bromothymol Blue ◽  
Birchwood Xylan ◽  
E Coli

α-1,2-Glucuronidase GH67 là enzyme có khả năng phân cắt chuỗi bên 4-O-methylglucuronic acid ((Me)GlcA) trong glucuronoarabinoxylan để làm tăng chuyển hóa lignocellulose thành đường đơn cho lên men sản xuất cồn và các chất có giá trị từ sinh khối thực vật. Trong nghiên cứu này, gen Gglc1 dài 1908 nucleotide mã hóa cho enzyme α-1,2-glucuronidase GH67 trưởng thành có nguồn gốc từ vi khuẩn trong dạ cỏ dê đã được biểu hiện thành công trong chủng E. coli Roseta. Hầu hết enzyme được biểu hiện dưới dạng tan khi chủng tái tổ hợp được nuôi cấy trong môi trường TB, PE có 0,05 mM IPTG ở 25oC, 30oC. Enzym được tinh chế thành công bằng sắc ký ái lực với độ tinh sạch 90%. Hoạt tính của enzyme đã được đánh giá sơ bộ dựa trên sự chuyển hóa (Me)GlcA trong birchwood xylan thành 4-O-methyl-D-glucuronic acid và D-glucuronic acid làm thay đổi pH dung dịch và được phát hiện bằng bromothymol blue. Enzyme sẽ được tiếp tục đánh giá tính chất để xem xét khả năng bổ sung vào hỗn hợp enzyme cho chuyển hóa hiệu quả sinh khối thực vật thành đường.

scholarly journals Entropy-driven binding of gut bacterial β-glucuronidase inhibitors ameliorates irinotecan-induced toxicity

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Hsien-Ya Lin ◽  
Chia-Yu Chen ◽  
Ting-Chien Lin ◽  
Lun-Fu Yeh ◽  
Wei-Che Hsieh ◽  
...  
Keyword(s):  
Cancer Metabolism ◽  
Glucuronic Acid ◽  
Intestinal Epithelial Cells ◽  
Primary Treatment ◽  
Water Molecules ◽  
Intestinal Epithelial ◽  
E Coli ◽  
Severe Diarrhea ◽  
Coordinated Water ◽  
Consequent Increase

AbstractIrinotecan inhibits cell proliferation and thus is used for the primary treatment of colorectal cancer. Metabolism of irinotecan involves incorporation of β-glucuronic acid to facilitate excretion. During transit of the glucuronidated product through the gastrointestinal tract, an induced upregulation of gut microbial β-glucuronidase (GUS) activity may cause severe diarrhea and thus force many patients to stop treatment. We herein report the development of uronic isofagomine (UIFG) derivatives that act as general, potent inhibitors of bacterial GUSs, especially those of Escherichia coli and Clostridium perfringens. The best inhibitor, C6-nonyl UIFG, is 23,300-fold more selective for E. coli GUS than for human GUS (Ki = 0.0045 and 105 μM, respectively). Structural evidence indicated that the loss of coordinated water molecules, with the consequent increase in entropy, contributes to the high affinity and selectivity for bacterial GUSs. The inhibitors also effectively reduced irinotecan-induced diarrhea in mice without damaging intestinal epithelial cells.

Stability and Hydrolysis of Desomorphine-Glucuronide

2019 ◽  
Vol 43 (7) ◽  
pp. 536-542 ◽  
Author(s):  
Jessica Winborn ◽  
Sarah Kerrigan
Keyword(s):  
Glucuronic Acid ◽  
Helix Pomatia ◽  
Sample Treatment ◽  
E Coli ◽  
Hydrolysis Method ◽  
Flight Mass Spectrometry ◽  
The Stability ◽  
Hydrolysis Of ◽  
Additional Sample ◽  
Analgesic Potency

AbstractDesomorphine, the principal opioid in Krokodil, has an analgesic potency approximately ten-times that of morphine. Similar to other opioids, during phase II metabolism it undergoes conjugation with glucuronic acid to form desomorphine-glucuronide. Although hydrolysis of conjugated species is sometimes required prior to analysis, desomorphine-glucuronide has not been fully investigated. In this study, six hydrolysis procedures were optimized and evaluated. Deconjugation efficiencies using chemical and enzymatic hydrolysis were evaluated and stability in aqueous solution was assessed. Acid hydrolysis was compared with five β-glucuronidase sources (BGTurbo™, IMCSzyme™, Escherichia coli, Helix pomatia and Patella vulgata). At optimal conditions, each hydrolysis method produced complete hydrolysis (≥96%). However, under simulated challenging conditions, P. vulgata was the most efficient β-glucuronidase for the hydrolysis of desomorphine-glucuronide. Both BGTurbo™ and IMCSzyme™ offered fast hydrolysis with no need for sample cleanup prior to liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS) analysis. Hydrolysates using E. coli, H. pomatia and P. vulgata underwent additional sample treatment using β-Gone™ cartridges. Additionally, the stability of free and conjugated drug was evaluated at elevated temperature (60°C) in aqueous solutions between pH 4 and 10. No degradation was observed for either desomorphine or desomorphine-glucuronide under any of the conditions tested.

Behavior of Hemorrhagic Escherichia coli O157:H7 During the Manufacture of Cottage Cheese

1992 ◽  
Vol 55 (5) ◽  
pp. 379-381 ◽  
Author(s):  
MARLENE M. AROCHA ◽  
MELINDA MCVEY ◽  
SUSAN D. LODER ◽  
JOHN H. RUPNOW ◽  
LLOYD BULLERMAN
Keyword(s):  
Escherichia Coli ◽  
Glucuronic Acid ◽  
Skim Milk ◽  
Enterohemorrhagic Escherichia Coli ◽  
Cottage Cheese ◽  
Escherichia Coli O157 ◽  
Phenol Red ◽  
E Coli ◽  
Heat Treated ◽  
And Control

The ability of enterohemorrhagic Escherichia coli O157:H7 to grow and survive during the manufacture of Cottage cheese was determined. Pasteurized skim milk artificially contaminated with E. coli O157:H7 was used to make Cottage cheese by the washed curd method. E. coli O157:H7 was enumerated by surface plating samples on MacConkey sorbitol agar with 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid cyclohexylammonium salt (MSA-BCIG) and incubating at 42°C for 24 h. The heat treated samples were previously inoculated into a modified EC broth with novobiocin and incubated static at 35°C for 24 h. Sorbitol and β-glucuronidase negative colonies were picked from MSA-BCIG, spread on Levine eosin methylene blue agar plates and phenol red sorbitol agar plates with 4-methylumbelliferyl-β-D-glucuronide (PRS-MUG) added for confirmation. E. coli O157:H7 increased 100-fold in numbers during the manufacturing process, but death occurred during cooking of the curd and whey. The pH and acidity did not halt the growth of this pathogen during the manufacture of the cheese; furthermore, the values of these parameters were the same between the contaminated and control samples.

THE EFFECT OF DIHYDROSTREPTOMYCIN ON THE FORMATION OF ADAPTIVE ENZYMES BY A STRAIN OF ESCHERICHIA COLI

1955 ◽  
Vol 33 (1) ◽  
pp. 792-796
Author(s):  
Susan M. Roote ◽  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Glucuronic Acid ◽  
E Coli ◽  
Enzyme Formation

The adaptive formation of enzymes for the oxidation of L-arabinose, lactose, and D-glucuronic acid was inhibited by dihydrostreptomycin in a strain of Escherichia coli which would not grow in the presence of this antibiotic. Adaptive enzyme formation was not inhibited in E. coli variants which would grow in the presence of dihydrostreptomycin or which required the antibiotic for growth.

scholarly journals Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

2008 ◽  
Vol 191 (5) ◽  
pp. 1565-1573 ◽  
Author(s):  
Sang-Hwal Yoon ◽  
Tae Seok Moon ◽  
Pooya Iranpour ◽  
Amanda M. Lanza ◽  
Kristala Jones Prather
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Pseudomonas Syringae ◽  
Glucuronic Acid ◽  
Glucaric Acid ◽  
Michaelis Constant ◽  
Turnover Number ◽  
Reading Frame ◽  
E Coli ◽  
Uronate Dehydrogenase

ABSTRACT Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K m ) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k cat = 1.9 × 102 s−1 on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.

THE EFFECT OF DIHYDROSTREPTOMYCIN ON THE FORMATION OF ADAPTIVE ENZYMES BY A STRAIN OF ESCHERICHIA COLI

1955 ◽  
Vol 33 (5) ◽  
pp. 792-796 ◽  
Author(s):  
Susan M. Roote ◽  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Glucuronic Acid ◽  
E Coli ◽  
Enzyme Formation

The adaptive formation of enzymes for the oxidation of L-arabinose, lactose, and D-glucuronic acid was inhibited by dihydrostreptomycin in a strain of Escherichia coli which would not grow in the presence of this antibiotic. Adaptive enzyme formation was not inhibited in E. coli variants which would grow in the presence of dihydrostreptomycin or which required the antibiotic for growth.

Biochemical characterization of the β-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461

2001 ◽  
Vol 358 (2) ◽  
pp. 457-464 ◽  
Author(s):  
Paula VIDEIRA ◽  
Arsénio FIALHO ◽  
Roberto A. GEREMIA ◽  
Christelle BRETON ◽  
Isabel SÁ-CORREIA
Keyword(s):  
Glucuronic Acid ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Enzymic Activity ◽  
Gellan Gum ◽  
Open Reading Frames ◽  
Sphingomonas Paucimobilis ◽  
E Coli ◽  
Repeat Units

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929–939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH2 and -CO2H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH2 (GelKNH2) and -CO2H (GelKCOOH) halves were overexpressed in Escherichia coli. GelK and GelKNH2 were present in both the soluble and membrane fractions of E. coli, whereas GelKCOOH was only present in the soluble fraction. GelK catalysed the transfer of [14C]glucuronic acid from UDP-[14C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by β-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PPi-lipid moiety is needed for enzymic activity. Recombinant GelKNH2 and GelKCOOH did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.

Endotoxin Alteration of the Mucopolysaccharide Blood Vessel Coating in Rats

1974 ◽  
Vol 32 ◽  
pp. 148-149
Author(s):  
D. E. Philpott ◽  
A. Takahashi
Keyword(s):  
Skeletal Muscle ◽  
Endothelial Cells ◽  
Salivary Gland ◽  
Carotid Artery ◽  
Basement Membrane ◽  
Coronary Vessels ◽  
Ruthenium Red ◽  
E Coli ◽  
Old Rats ◽  
The Brain

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.

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