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 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

Gene cloning, protein purification, and enzymatic properties of multicopper oxidase, fromKlebsiellasp. 601

2008 ◽  
Vol 54 (9) ◽  
pp. 725-733 ◽  
Author(s):  
Yang Li ◽  
Jiao Yin ◽  
Guosheng Qu ◽  
Luchao Lv ◽  
Yadong Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Kinetic Studies ◽  
Multicopper Oxidase ◽  
Coli Strain ◽  
Gene Encoding ◽  
E Coli ◽  
C Terminus ◽  
Optimal Ph

A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2′-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat/Kmvalues of 0.49 mmol·L–1, 1.08 × 103s–1, and 2.23 × 103s–1·mmol–1·L, respectively, for DMP, 5.63 mmol·L–1, 6.64 × 103s–1, and 1.18 × 103s–1·mmol–1·L for ABTS, and 0.023 mmol·L–1, 11 s–1, and 4.68 × 102s–1·mmol–1·L for SGZ.

scholarly journals Identification of a New Class of Cytochrome P450 from a Rhodococcus sp

2002 ◽  
Vol 184 (14) ◽  
pp. 3898-3908 ◽  
Author(s):  
Gareth A. Roberts ◽  
Gideon Grogan ◽  
Andy Greter ◽  
Sabine L. Flitsch ◽  
Nicholas J. Turner
Keyword(s):  
Cytochrome P450 ◽  
Cytochromes P450 ◽  
Pcr Primers ◽  
Soluble Form ◽  
Flavin Mononucleotide ◽  
Gene Encoding ◽  
Binding Motifs ◽  
E Coli ◽  
New Class ◽  
C Terminus

ABSTRACT A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of E. coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.

Purification and characterization of two xylanases from Chaetomium thermophile var. coprophile

1989 ◽  
Vol 35 (9) ◽  
pp. 836-842 ◽  
Author(s):  
Ramesh K. Ganju ◽  
Paul J. Vithayathil ◽  
S. K. Murthy
Keyword(s):  
Amino Acids ◽  
Enzyme Purification ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Larch Wood ◽  
Hydrolysis Products ◽  
Xylanase Enzyme ◽  
Temperature Optima

Two xylanases (I and II) out of several extracellular xylanases produced by the thermophilic fungus Chaetomium thermophile var. coprophile were purified to homogeneity by a combination of ion exchange and gel filtration chromatographic procedures. They had molecular weights of 26 000 (xylanase I) and 7000 (xylanase II). The temperature optima for xylanase I and II were 70 and 60 °C, and they were optimally active at pH 4.8–6.4 and 5.4–6.0, respectively. Xylanase I was found to be comparatively more stable than xylanase II at higher temperatures. Amino acid composition indicated that xylanase I contained high amounts of glycine, threonine, and low amounts of histidine and sulphur-containing amino acids. Each enzyme released different hydrolysis products from larch wood xylan. Xylanase I produced mainly xylobiose and xylotriose whereas xylanase II produced mainly xylobiose.Key words: Xylanase, enzyme purification, characterization, Chaetomium thermophile.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 926
Author(s):  
Maria C. Martins ◽  
Susana F. Fernandes ◽  
Bruno A. Salgueiro ◽  
Jéssica C. Soares ◽  
Célia V. Romão ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Reductase Activity ◽  
Conserved Residues ◽  
Mutant Proteins ◽  
E Coli ◽  
C Terminus ◽  
Bona Fide ◽  
Oxygen Reductase ◽  
Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

scholarly journals Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

2012 ◽  
Vol 11 (8) ◽  
pp. 1055-1066 ◽  
Author(s):  
Matthias Kretschmer ◽  
Jana Klose ◽  
James W. Kronstad
Keyword(s):  
Ustilago Maydis ◽  
Short Chain Fatty Acids ◽  
Phytopathogenic Fungi ◽  
Metabolic Adaptation ◽  
In Planta ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Content Type ◽  
C Terminus ◽  
Fungal Phytopathogens

ABSTRACTAn understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogenUstilago maydis. However,mfe2mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in thehad1gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog,had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of anmfe2Δ mutant. We also show that short-chain fatty acids induce cell death inU. maydisand that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms byU. maydisthat includes potential metabolic contributions to proliferationin plantaand an effect on virulence-related morphogenesis.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

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