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 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>

scholarly journals Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

2006 ◽  
Vol 72 (5) ◽  
pp. 3418-3428 ◽  
Author(s):  
Hideo Kawaguchi ◽  
Alain A. Vert�s ◽  
Shohei Okino ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Xylose Isomerase ◽  
Mineral Medium ◽  
Gene Encoding ◽  
Xylose Consumption ◽  
E Coli ◽  
High Copy Number Plasmid ◽  
Sugar Mixture ◽  
Pentose Sugar

ABSTRACT The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vert�s, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

scholarly journals Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae

2009 ◽  
Vol 75 (8) ◽  
pp. 2304-2311 ◽  
Author(s):  
Dawid Brat ◽  
Eckhard Boles ◽  
Beate Wiedemann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Xylose Fermentation ◽  
Thermus Thermophilus ◽  
Xylose Isomerase ◽  
Functional Expression ◽  
Yeast Cells ◽  
Highly Active ◽  
Excellent Starting Point ◽  
Starting Point

ABSTRACT In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

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.

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 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.

scholarly journals Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hbγ-ECS1) in Latex Production inHevea brasiliensis

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Wei Fang ◽  
Luo Shi Qiao ◽  
Wu Ming ◽  
Qiu Jian ◽  
Yang Wen Feng ◽  
...  
Keyword(s):  
Specific Activity ◽  
Rubber Tree ◽  
Natural Rubber Latex ◽  
Cloning And Expression ◽  
E Coli ◽  
Glutamylcysteine Synthetase ◽  
Commercial Source ◽  
Rate Limiting

Rubber tree is a major commercial source of natural rubber. Latex coagulation is delayed by thiols, which belong to the important type of antioxidants in laticifer submembrane, and is composed of glutathione (GSH), cysteine, and methionine. The rate-limiting enzyme,γ-ECS, plays an important role in regulating the biosynthesis of glutathione under any environment conditions. To understand the relation betweenγ-ECS and thiols and to correlate latex flow with one-time tapping and continuous tapping, we cloned and derived the full length of oneγ-ECS from rubber tree latex (Hbγ-ECS1). According to qPCR analysis, the expression levels ofHbγ-ECS1were induced by tapping and Ethrel stimulation, and the expression was related to thiols content in the latex. Continuous tapping induced injury, and the expression ofHbγECS1increased with routine tapping and Ethrel-stimulation tapping (more intensive tapping). According to expression in long-term flowing latex, the gene was related to the duration of latex flow.HbγECS1was expressed inE. coliRosetta using pET-sumo as an expression vector and the recombinant enzyme was purified; then we achieved 0.827 U/mg specific activity and about 66 kDa molecular weight. The present study can help us understand the complex role ofHbγ-ECSin thiols biosynthesis, which is influenced by tapping.

scholarly journals Molecular Cloning and Functional Expression inLactobacillus plantarum 80 of xylT, Encoding thed-Xylose–H+ Symporter ofLactobacillus brevis

1998 ◽  
Vol 64 (12) ◽  
pp. 4720-4728 ◽  
Author(s):  
Stéphane Chaillou ◽  
Yeou-Cherng Bor ◽  
Carl A. Batt ◽  
Pieter W. Postma ◽  
Peter H. Pouwels
Keyword(s):  
Xylose Isomerase ◽  
Proton Motive Force ◽  
Motive Force ◽  
Affinity Constant ◽  
Transport Activity ◽  
Gene Encoding ◽  
Kinase Gene ◽  
E Coli ◽  
Xylose Transport ◽  
Xylulose Kinase

ABSTRACT A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding d-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the d-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to thed-xylose–H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the d-xylose–Na+ symporter ofTetragenococcus halophila, XylE (57%), and to thel-arabinose–H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the d-xylose repressor protein) as a probe revealed the existence of a xylR homologue inL. brevis which is not located with thexyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream ofxylA and xylT and by the requirement ofd-xylose for the induction of d-xylose isomerase, d-xylulose kinase, and d-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of d-glucose andd-xylose, the d-xylose isomerase andd-xylulose kinase activities were reduced fourfold and thed-xylose transport activity was reduced by sixfold, suggesting catabolite repression by d-glucose ofd-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked d-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of d-xylose in L. plantarum80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of d-glucose. The apparent affinity constant of XylT ford-xylose was approximately 215 μM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-d-glucose inhibited the transport ofd-xylose by XylT competitively, with aKi of 220 μM.

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.

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