scholarly journals EXPLORATION OF BROTH CHICKEN GUT AS GROWTH MEDIUM OF Escherichia coli BL21 pET-Endo FOR Endo-β-1,4-D-Xylanase PRODUCTION

2017 ◽  
Vol 13 (2) ◽  
Author(s):  
Anak agung Istri ratnadewi Dewi ◽  
Moch Yoris Alidiona ◽  
Agung Budi Santoso ◽  
Ika Oktavianawatia
Keyword(s):  
Enzyme Purification ◽  
Large Scale ◽  
Incubation Temperature ◽  
Specific Activity ◽  
Hydrolytic Enzyme ◽  
Optimal Growth ◽  
Good Alternative ◽  
Gene Encoding ◽  
Xylanase Production ◽  
E Coli

Endo-β-1,4-D-xylanase is a hydrolytic enzyme that breakdown the 1.4 chain of xylan polysaccharide. We have succes to transform the plasmid pET-Endo gene encoding endo-1,4-β-D-xylanase from Bacillus sp. Originaly from termites abdominal to E. coli BL21. The clone was ready for large scale of enzyme production. To reduce porduction cost we look for subtitute medium for the expensive Luria Berthani broth. Chicken guts broth is good alternative while rich of protein and very cheap.  Growth of E. Coli BL21 in Chicken guts broth and LB broth (as control) was observed by Optical Density (OD) with spectrofotometer. Concentration of glucose added in broth and incubation temperature was varied. The result showed that optimal growth was in addition of 1,5% glucose and incubated at  37 <sup>o</sup>C for 16 hours. This optimal condition was used  for E. coli BL21 pET-Endo for xylanase production. Enzyme purification have done by Ni-NTA affinity chromatography. Highest protein yield was 0,076 mg/ml obtained in 100 mM imidazole elucidation. Xylanase characteization were : activity 0,042 U/ml, specific activity 0,556 U/ μg, purification factor 3,16 times and molecular weight ± 30.000 Dalton

scholarly journals EXPLORATION OF BROTH CHICKEN GUT AS GROWTH MEDIA OF Escherichia coli BL21 pET-Endo FOR ENDO-Β-1,4-D-XYLANASE PRODUCTION

2017 ◽  
Vol 13 (2) ◽  
pp. 191
Author(s):  
Anak Agung Istri Ratnadewi ◽  
Moch. Yoris Alidion ◽  
Agung Budi Santoso ◽  
Ika Oktavianawatia
Keyword(s):  
Large Scale ◽  
Incubation Temperature ◽  
Specific Activity ◽  
Hydrolytic Enzyme ◽  
Optimal Growth ◽  
Good Alternative ◽  
Growth Media ◽  
Gene Encoding ◽  
Xylanase Production ◽  
E Coli

<p>Endo-β-1,4-D-xylanase is a hydrolytic enzyme that breakdown the 1.4 chain of xylan polysaccharide. We have succes to transform the plasmid pET-Endo gene encoding endo-1,4-β-D-xylanase from Bacillus sp. originally from termites abdominal to E. coli BL21. The clone was ready for large scale of enzyme production. To reduce production cost, we look for subtitute media for the expensive Luria Berthani broth. Chicken guts broth is good alternative while rich of protein and very cheap. The content of N dissolved chicken guts broth reaches 87 % of LB broth. Growth of E. Coli BL21 in Chicken guts broth and LB broth (as control) was observed by Optical Density (OD) using spectrofotometer. Concentration of glucose added in broth and incubation temperature was varied. The result showed that optimal growth was in addition of 1.5 % glucose and incubated at  37 <sup>o</sup>C for 16 h. This optimal condition was used to grow E. coli BL21 pET-Endo for xylanase production. Enzyme purification was done by Ni-NTA affinity chromatography. Highest protein yield was 0.076 mg/mL obtained in 100 mM imidazole elucidation. The activity and specific activity of xylanase were estimated as 0.042 U/mL and 0.556 U/µg, respectively. The purification factor was 3.16 time and the molecular weight of enzyme was ± 30, 000 Dalton</p>

Purification and characterization ofThermobifida fuscaxylanase 10B

2004 ◽  
Vol 50 (10) ◽  
pp. 835-843 ◽  
Author(s):  
Jeong H Kim ◽  
Diana Irwin ◽  
David B Wilson
Keyword(s):  
Enzyme Purification ◽  
Major Product ◽  
Corn Fiber ◽  
Thermobifida Fusca ◽  
Purification And Characterization ◽  
Gene Encoding ◽  
E Coli ◽  
Pure Cellulose ◽  
C Terminus ◽  
Xylanase Enzyme

Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 °C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl α-D-arabinofuranoside, p-nitrophenyl-β-D-xylobioside, p-nitrophenyl-β-D-xyloside, and p-nitrophenyl-β-D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.Key words: xylanase, enzyme purification, Thermobifida fusca, family 10 hydrolase.

scholarly journals Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

2004 ◽  
Vol 70 (6) ◽  
pp. 3292-3297 ◽  
Author(s):  
Gerard M. Gibbs ◽  
Barrie E. Davidson ◽  
Alan J. Hillier
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
Scale Production ◽  
Gene Encoding ◽  
Strong Activity ◽  
E Coli ◽  
Large Scale Production ◽  
Bacterial Cell Membrane

ABSTRACT Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.

Construction of a Novel Recombinant Escherichia coli Strain Capable of Producing 1,3–propanediol

2011 ◽  
Vol 396-398 ◽  
pp. 2499-2502 ◽  
Author(s):  
Xiang Hui Qi ◽  
Qi Guo ◽  
Yu Tuo Wei ◽  
Hong Xu ◽  
Ri Bo Huang
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Gel Filtration ◽  
Coli Strain ◽  
Optimal Temperature ◽  
Recombinant Enzymes ◽  
Microbial Conversion ◽  
Gene Encoding ◽  
E Coli ◽  
Nickel Chelate

1, 3-propanediol (1, 3-PD) is biologically synthesized by glycerol dehydratase (GDHt) and 1, 3-propanediol dehydrogenase (PDOR). In present study, the gldABC gene, encoding GDHt from Klebsiella pneumoniae and the yqhD gene, encoding PDOR isoenzyme from E.coli BL21 were cloned and co-expressed in E.coli JM109 using plasmid pSE380. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration to study the properties. Optimal temperature and pH of recombinant GDHt with specific activity of 85.8 U/mg were 45 °C and 9.0; and optimal temperature and pH of recombinant YqhD with specific activity of 80.0 U/mg were 37 °C, 7.0. The microbial conversion of 1,3-PD from glycerol by this recombinant E. coli strain was studied and the production of 1,3-PD was about 28.0 g/l.

Characterization and Functional Expression of Xylose Isomerase from Thermus thermophilus

2013 ◽  
Vol 641-642 ◽  
pp. 919-922
Author(s):  
An Gen Lu ◽  
Ze Xi Yang ◽  
Fei Wang ◽  
Lang Xu ◽  
Wen Ying Deng ◽  
...  
Keyword(s):  
Fossil Fuels ◽  
Specific Activity ◽  
Thermus Thermophilus ◽  
Xylose Isomerase ◽  
Functional Expression ◽  
Future Application ◽  
Gene Encoding ◽  
E Coli ◽  
Rate Limiting ◽  
Pentose Sugars

Ethanol produced from hexose and pentose sugars hydrolysated by lignocellulose is an environment-friendly alternative to fossil fuels. Xylose isomerase is the major rate-limiting enzyme in the ethanol synthesis biologically pathway of xylose fermentation. In present study, xylA gene encoding xylose isomerase was cloned from Thermus thermophilus and overexpressed in E. coli BL21. Purified recombinant enzyme was used to study the enzymatic characterization. Specific activity of recombinant PDOR was 19.6 U/mg. Optimal temperature and pH were 80 °C, 8.0, respectively. Km and Vmax values were 15.9 mM, 22.8 U/mg. This research may form a basis for the future application of xylose isomerase.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

Overexpression and Characterization of the Gene Encoding 1,3-Propanediol Oxidoreductase of Citrobacter freundii

2014 ◽  
Vol 636 ◽  
pp. 121-124
Author(s):  
Xiang Hui Qi ◽  
Jing Fei Zhu ◽  
Yan Luo ◽  
Jing Lin ◽  
Xu Wang ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Raw Material ◽  
Biological Synthesis ◽  
Citrobacter Freundii ◽  
Gene Encoding ◽  
E Coli ◽  
Nickel Chelate ◽  
Future Work

Glycerol can be biologically converted to 1,3-propanediol (1,3-PD), a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. 1,3-propanediol oxidoreductase (PDOR) is the rate-limiting enzyme in 1,3-PD synthesis biologically pathway. In present study, the dhaT gene encoding PDOR was cloned from Citrobacter freundii and overexpressed in E. coli BL21. The recombinant enzyme was purified by nickel-chelate chromatography combined with gel filtration to study the enzymatic characterization. Specific activity of recombinant PDOR was 55.2 U/mg. Km and Vmax values were 8.9 mM, 40.2 U/mg respectively. Holoenzyme of PDOR maybe a decamer of which a monomer has a molecular mass of 43 kDa. This research may form a basis for the future work on biological synthesis of 1, 3-PD.

Cloning and expression of DL-2-haloacid dehalogenase gene from Burkholderia cepacia

2000 ◽  
Vol 42 (7-8) ◽  
pp. 261-268 ◽  
Author(s):  
Y. Ohkouchi ◽  
H. Koshikawa ◽  
Y. Terashima
Keyword(s):  
Burkholderia Cepacia ◽  
Specific Activity ◽  
Wild Strain ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Haloacid Dehalogenase ◽  
E Coli ◽  
Vector Systems ◽  
Lac Promoter ◽  
Acetic Acids

Burkholderia cepacia strain KY, which can utilize a herbicide 2,4-D as a sole carbon and energy source, catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids. We have cloned the gene encoding DL-2-haloacid dehalogenase, and obtained a recombinant plasmid (pUCDEXL) containing approximately 4.5 kbp insert. In both of B. cepacia strain KY and this clone E. coli JM109/pUCDEXL, DL-2-haloacid dehalogenase was induced significantly with monohalogenated acetic acids, such as chloroacetate, bromoacetate and iodoacetate. This dehalogenase was also overexpressed in E. coli using three different promoters. In pET vector systems with T7 lac promoter, a large amount of dehalogenase was selectively expressed, but some parts of the protein were accumulated in the form of inclusion bodies. This problem was overcome to carry on growth and induction at 22°C, and at the same time, the maximum specific activity of dehalogenase was reached at 12.6 U/mg, 500-fold higher activity than in wild strain, B. cepacia strain KY grown with 2,4-D.

scholarly journals Production and Partial Purification of Heat-Stable Enterotoxin (A) Produced by Enterotoxigenic Escherichia coli

2010 ◽  
Vol 34 (1) ◽  
pp. 122-129
Author(s):  
Rajiha I. Al-Nuaimy
Keyword(s):  
Large Scale ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Enterotoxigenic Escherichia Coli ◽  
Partial Purification ◽  
Scale Production ◽  
E Coli ◽  
Standard Curve ◽  
Heat Stable ◽  
Large Scale Production

A total of (25) stool samples were collected from children and adults (2- 4) years oldsuffering from diarrhea to isolate E. coli strains that produce heat-stable enterotoxin a (STa),and after performing microscopic examination, cultural characterization and biochemicalidentification only (11) isolates showed positive E. coli. STa activity was estimated by usingsuckling mouse assay (SMA) and from these (11) isolates only (5) showed STa activity andthe one with the highest STa activity was selected for large scale production of STa, whichwas followed by partial purification using ion-exchange chromatography (normal phase)using DEAE sephadex A-50 column. After purification and determination of proteinconcentration by using the standard curve of bovine serum albumin, the concentration oftoxin-protein was estimated as (1.08) mg/ml. The specific activity varied from (350) U/mgprotein at the first step of purification to (2366.6) U/mg protein at the final step, while thefinal purification of the toxin was about (6.76) fold and with a yield of (18.25) %.

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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