scholarly journals Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Dominic W. S. Wong ◽  
Victor J. Chan ◽  
Amanda A. McCormack ◽  
Ján Hirsch ◽  
Peter Biely
Keyword(s):  
Glucuronic Acid ◽  
Active Form ◽  
Schizophyllum Commune ◽  
Constitutive Expression ◽  
Recombinant Enzyme ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Glucuronoyl Esterase ◽  
Esterase Gene

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were Km 0.25 mM, Vmax 16.3 μM⋅min−1, and kcat 9.27 s−1 with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.

scholarly journals Identification and characterization of YLR328W, theSaccharomyces cerevisiaestructural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme

FEBS Letters ◽  
1999 ◽  
Vol 455 (1-2) ◽  
pp. 13-17 ◽  
Author(s):  
Monica Emanuelli ◽  
Francesco Carnevali ◽  
Maria Lorenzi ◽  
Nadia Raffaelli ◽  
Adolfo Amici ◽  
...  
Keyword(s):  
Recombinant Enzyme ◽  
Gene Encoding ◽  
Identification And Characterization

scholarly journals The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of the nirAGene and Characterization of the Recombinant Enzyme

1999 ◽  
Vol 181 (8) ◽  
pp. 2323-2329 ◽  
Author(s):  
Miguel Prudêncio ◽  
Robert R. Eady ◽  
Gary Sawers
Keyword(s):  
Amino Acid ◽  
Nitrite Reductase ◽  
Recombinant Enzyme ◽  
Leader Peptide ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Blue Copper

ABSTRACT The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed inEscherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

scholarly journals Characterization of the tyramine-producing pathway in Sporolactobacillus sp. P3J

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1841-1849 ◽  
Author(s):  
Monika Coton ◽  
María Fernández ◽  
Hein Trip ◽  
Victor Ladero ◽  
Niels L. Mulder ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Cloning And Expression ◽  
Clear Correlation ◽  
Tyrosine Decarboxylase ◽  
Gene Encoding ◽  
Polycistronic Mrna ◽  
Tyrosine Concentration ◽  
Putative Phage

A sporulated lactic acid bacterium (LAB) isolated from cider must was shown to harbour the tdc gene encoding tyrosine decarboxylase. The isolate belonged to the Sporolactobacillus genus and may correspond to a novel species. The ability of the tdc-positive strain, Sporolactobacillus sp. strain P3J, to produce tyramine in vitro was demonstrated by using HPLC. A 7535 bp nucleotide sequence harbouring the putative tdc gene was determined. Analysis of the obtained sequence showed that four tyramine production-associated genes [tyrosyl-tRNA synthetase (tyrS), tyrosine decarboxylase (tdc), tyrosine permease (tyrP) and Na+/H+ antiporter (nhaC)] were present and were organized as already described in other tyramine-producing LAB. This operon was surrounded by genes showing the highest identities with mobile elements: a putative phage terminase and a putative transposase (downstream and upstream, respectively), suggesting that the tyramine-forming trait was acquired through horizontal gene transfer. Transcription analyses of the tdc gene cluster suggested that tyrS and nhaC are expressed as monocistronic genes while tdc would be part of a polycistronic mRNA together with tyrP. The presence of tyrosine in the culture medium induced the expression of all genes except for tyrS. A clear correlation was observed between initial tyrosine concentration and tyramine production combined with an increase in the final pH reached by the culture. Finally, cloning and expression of the tyrP gene in Lactococcus lactis demonstrated that its product catalyses the exchange of tyrosine and tyramine.

scholarly journals Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme

1996 ◽  
Vol 316 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Douglas S. BURDETTE ◽  
Claire VIEILLE ◽  
J. Gregory ZEIKUS
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Primary Alcohol ◽  
Recombinant Enzyme ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Arrhenius Plots ◽  
Thermoanaerobacter Ethanolicus ◽  
Secondary Alcohol Dehydrogenase

The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (S-ADH) was cloned, sequenced and expressed in Escherichia coli. The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits. The purified recombinant enzyme is optimally active above 90 °C with a half-life of approx. 1.7 h at 90 °C. An NADP(H)-dependent enzyme, the recombinant S-ADH has 1400-fold greater catalytic efficiency in propan-2-ol oxidation than in ethanol oxidation. The enzyme was inactivated by chemical modification with dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis. Zinc was the only metal enhancing S-ADH reactivation after DTNB modification, implicating the involvement of bound zinc in catalysis. Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant S-ADHs were linear from 25 to 90 °C when the enzymes were incubated at 55 °C before assay. Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0 or 25 °C. The observed Arrhenius discontinuity therefore resulted from a temperature-dependent, catalytically significant S-ADH structural change. Hydrophobic cluster analysis comparisons of both mesophilic and thermophilic S-ADH and primary- versus S-ADH amino acid sequences were performed. These comparisons predicted that specific proline residues might contribute to S-ADH thermostability and thermophilicity, and that the catalytic Zn ligands are different in primary-alcohol dehydrogenases (two Cys and a His) and S-ADHs (Cys, His, and Asp).

scholarly journals Cloning and Expression of the algL Gene, Encoding the Azotobacter chroococcum Alginate Lyase: Purification and Characterization of the Enzyme

1999 ◽  
Vol 181 (5) ◽  
pp. 1409-1414 ◽  
Author(s):  
Ana Peciña ◽  
Alberto Pascual ◽  
Antonio Paneque
Keyword(s):  
Gene Sequence ◽  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Azotobacter Chroococcum ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Reading Frame ◽  
Encoding Gene

ABSTRACT The alginate lyase-encoding gene (algL) ofAzotobacter chroococcum was localized to a 3.1-kbEcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

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