%0 Journal Article %1 Ertesvag1994 %A Ertesvag, H. %A Doseth, B. %A Larsen, B. %A Skjak-Braek, G. %A Valla, S. %D 1994 %J J. Bacteriol. %K ; Acid Acids Alginates/metabolism Amino Analysis Analysis, Azotobacter Bacterial/*genetics Base Calcium/metabolism Carbohydrate Cloning, DNA Data Epimerases/biosynthesis/*genetics Escherichia Gene Genes, Glucuronic Gov't Hexuronic Homology, Isomerism Library Magnetic Molecular Non-U.S. Nucleic Proteins/biosynthesis Recombinant Repetitive Research Resonance Sequence Sequences, Spectroscopy Support, coli/genetics vinelandii/enzymology/*genetics %N 10 %P 2846-53 %T Cloning and expression of an Azotobacter vinelandii mannuronan C-5-epimerase gene. %U http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?cmd=prlinks&dbfrom=pubmed&retmode=ref&id=8188585 %V 176 %X An Azotobacter vinelandii mannuronan C-5-epimerase gene was cloned in Escherichia coli. This enzyme catalyzes the Ca(2+)-dependent epimerization of D-mannuronic acid residues in alginate to the corresponding epimer L-guluronic acid. The epimerase gene was identified by screening a bacteriophage EMBL3 gene library of A. vinelandii DNA with a synthetic oligonucleotide probe. The sequence of this probe was deduced after determination of the N-terminal amino acid sequence of a previously reported extracellular mannuronan C-5-epimerase from A. vinelandii. A DNA fragment hybridizing against the probe was subcloned in a plasmid vector in E. coli, and the corresponding recombinant plasmid expressed intracellular mannuronan C-5-epimerase in this host. The nucleotide sequence of the gene encoding the epimerase was determined, and the sequence data showed that the molecular mass of the deduced protein is 103 kDa. A module consisting of about 150 amino acids was repeated tandemly four times in the C-terminal part of the deduced protein. Each of the four repeats contained four to six tandemly oriented nonameric repeats. The sequences in these motifs are similar to the Ca(2+)-binding domains of functionally unrelated secreted proteins reported previously in other bacteria. The reaction product of the recombinant epimerase was analyzed by nuclear magnetic resonance spectroscopy, and the results showed that the guluronic acid residues were distributed in blocks along the polysaccharide chain. Such a nonrandom distribution pattern, which is important for the commercial use of alginate, has previously also been identified in the reaction product of the corresponding enzyme isolated from A. vinelandii.

@article{Ertesvag1994, __markedentry = {[phpts:6]}, abstract = {An Azotobacter vinelandii mannuronan C-5-epimerase gene was cloned in Escherichia coli. This enzyme catalyzes the Ca(2+)-dependent epimerization of D-mannuronic acid residues in alginate to the corresponding epimer L-guluronic acid. The epimerase gene was identified by screening a bacteriophage EMBL3 gene library of A. vinelandii DNA with a synthetic oligonucleotide probe. The sequence of this probe was deduced after determination of the N-terminal amino acid sequence of a previously reported extracellular mannuronan C-5-epimerase from A. vinelandii. A DNA fragment hybridizing against the probe was subcloned in a plasmid vector in E. coli, and the corresponding recombinant plasmid expressed intracellular mannuronan C-5-epimerase in this host. The nucleotide sequence of the gene encoding the epimerase was determined, and the sequence data showed that the molecular mass of the deduced protein is 103 kDa. A module consisting of about 150 amino acids was repeated tandemly four times in the C-terminal part of the deduced protein. Each of the four repeats contained four to six tandemly oriented nonameric repeats. The sequences in these motifs are similar to the Ca(2+)-binding domains of functionally unrelated secreted proteins reported previously in other bacteria. The reaction product of the recombinant epimerase was analyzed by nuclear magnetic resonance spectroscopy, and the results showed that the guluronic acid residues were distributed in blocks along the polysaccharide chain. Such a nonrandom distribution pattern, which is important for the commercial use of alginate, has previously also been identified in the reaction product of the corresponding enzyme isolated from A. vinelandii.}, added-at = {2011-11-04T13:47:04.000+0100}, author = {Ertesvag, H. and Doseth, B. and Larsen, B. and Skjak-Braek, G. and Valla, S.}, authoraddress = {UNIGEN Center for Molecular Biology, University of Trondheim, Norway.}, biburl = {https://www.bibsonomy.org/bibtex/2626a666b29179600631f32ce003e802e/pawelsikorski}, interhash = {84080076a81f80c8a1cc1d894144f0d6}, intrahash = {626a666b29179600631f32ce003e802e}, journal = {J. Bacteriol.}, keywords = {; Acid Acids Alginates/metabolism Amino Analysis Analysis, Azotobacter Bacterial/*genetics Base Calcium/metabolism Carbohydrate Cloning, DNA Data Epimerases/biosynthesis/*genetics Escherichia Gene Genes, Glucuronic Gov't Hexuronic Homology, Isomerism Library Magnetic Molecular Non-U.S. Nucleic Proteins/biosynthesis Recombinant Repetitive Research Resonance Sequence Sequences, Spectroscopy Support, coli/genetics vinelandii/enzymology/*genetics}, language = {eng}, medline-da = {19940620}, medline-dcom = {19940620}, medline-edat = {1994/05/01}, medline-fau = {Ertesvag, H ; Doseth, B ; Larsen, B ; Skjak-Braek, G ; Valla, S}, medline-gs = {algE}, medline-is = {0021-9193 (Print)}, medline-jid = {2985120R}, medline-jt = {Journal of bacteriology.}, medline-lr = {20041117}, medline-mhda = {1994/05/01 00:01}, medline-own = {NLM}, medline-pl = {UNITED STATES}, medline-pmid = {8188585}, medline-pst = {ppublish}, medline-pt = {Journal Article}, medline-pubm = {Print}, medline-rn = {0 (Alginates) ; 0 (Hexuronic Acids) ; 0 (Recombinant Proteins) ; 576-37-4 (Glucuronic Acid) ; 7440-70-2 (Calcium) ; 9005-32-7 (alginic acid) ; EC 5.1.3 (Carbohydrate Epimerases) ; EC 5.1.3.- (mannuronan c-5-epimerase)}, medline-sb = {IM}, medline-si = {GENBANK/L39096}, medline-so = {J Bacteriol. 1994 May;176(10):2846-53.}, medline-stat = {MEDLINE}, number = 10, owner = {phpts}, pages = {2846-53}, timestamp = {2011-11-04T13:47:10.000+0100}, title = {Cloning and expression of an Azotobacter vinelandii mannuronan C-5-epimerase gene.}, url = {http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?cmd=prlinks\&dbfrom=pubmed\&retmode=ref\&id=8188585}, volume = 176, year = 1994 }