scholarly journals New α-l-arabinofuranosidase produced by Streptomyces lividans: cloning and DNA sequence of the abfB gene and characterization of the enzyme

1997 ◽  
Vol 322 (3) ◽  
pp. 845-852 ◽  
Author(s):  
Patrick VINCENT ◽  
François SHARECK ◽  
Claude DUPONT ◽  
Rolf MOROSOLI ◽  
Dieter KLUEPFEL
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequence ◽  
Catalytic Domain ◽  
Expression System ◽  
Prolonged Incubation ◽  
Linker Region ◽  
Xylanolytic Activity ◽  
Homologous Expression

A fully secreted α-l-arabinofuranosidase was cloned from the homologous expression system of Streptomyces lividans.The gene, located upstream adjacent to the previously described xylanase A gene, was sequenced. It is divergently transcribed from the xlnAgene and the two genes are separated by an intercistronic region of 391 nt which contains a palindromic AT-rich sequence. The deduced amino acid sequence of the protein shows that the enzyme contains a distinct catalytic domain which is linked to a specific xylan-binding domain by a linker region. The purified enzyme has a specific arabinofuranose-debranching activity on xylan from Gramineae,acts synergistically with the S. lividansxylanases and binds specifically to xylan. From small arabinoxylo-oligosides, it liberates arabinose and, after prolonged incubation, the purified enzyme exhibits some xylanolytic activity as well.

scholarly journals Cloning and Characterization of vuuA, a Gene Encoding the Vibrio vulnificus Ferric Vulnibactin Receptor

2000 ◽  
Vol 68 (2) ◽  
pp. 526-534 ◽  
Author(s):  
Athena C. D. Webster ◽  
Christine M. Litwin
Keyword(s):  
Transcriptional Regulation ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Outer Membrane ◽  
Dna Sequence ◽  
Vibrio Vulnificus ◽  
Membrane Receptors ◽  
Gene Encoding ◽  
Infant Mouse

ABSTRACT The ability of Vibrio vulnificus to acquire iron from the host has been shown to correlate with virulence. Many iron transport genes are regulated by iron, and in V. vulnificus, transcriptional regulation by iron depends on thefur gene. The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from a V. vulnificus fur mutant had 53% homology with the first 15 amino acids of the mature protein of the Vibrio choleraevibriobactin receptor, ViuA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor of V. vulnificus. Analysis of the DNA sequence of the vuuApromoter region demonstrated a sequence identical to the upstream Fur box of V. cholerae viuA. Northern blot analysis showed that the transcript was strongly regulated by iron. The amino acid sequence of VuuA was 74% identical to the sequence of V. choleraeViuA and was homologous to those of several TonB-dependent outer membrane receptors. An internal deletion of the V. vulnificus vuuA gene resulted in the loss of expression of the 72-kDa protein and the loss of the ability to use transferrin or vulnibactin as a source of iron. This mutant showed reduced virulence in an infant mouse model. Introduction of a plasmid containing the completeviuA coding sequence and 342 bp of upstream DNA into the mutant restored ferric vulnibactin and ferric transferrin utilization to the mutant.

scholarly journals Cloning, sequence, expression and characterization of human beta-mannosidase.

2008 ◽  
Vol 55 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Zahoor Qadir Samra ◽  
Muhammad Amin Athar
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Blot Analysis ◽  
Size Exclusion ◽  
Expression Vectors

Beta-mannosidase (EC 3.2.1.25, MANB) dissects the non-reducing end of N-linked mannose moieties of glycoproteins in eukaryotic cells. The human beta-mannosidase gene was amplified by RT-PCR, cloned and sequenced. The DNA sequence was compared with reported human beta-mannosidase DNA sequence and sixteen nucleotide differences were found. The deduced amino-acid sequence showed that seven codons coded the same amino acids and nine codons coded different amino acids with reference to nucleotide substitution positions but did not affect recombinant MANB enzyme activity. No splice mutation was observed after comparison with reported MANB DNA sequences. A 75% homology of deduced amino-acid sequence was observed with mouse, goat and bovine beta-mannosidase amino-acid sequences. The cloned beta-mannosidase gene was subcloned into pET22b+ and pET28a+ expression vectors to transform the BL21-codon plus cells for expression of recombinant MAN22 and MAN28 enzymes, respectively. The optimized conditions for overexpression of recombinant beta-mannosidase enzyme were induction with 1 mM IPTG for 12 h at 37 degrees C. The expressed beta-mannosidase enzyme was purified to homogeneity by a combination of DEAE-ion exchange and size exclusion chromatography. The molecular mass of MAN22 and MAN28 enzymes is 97 kDa by SDS/PAGE and is confirmed by western blot analysis. The recombinant enzymes are active at 37 degrees C and at pH 5.0 and showed activity with p-nitrophenyl-beta-d-mannopyranoside and not with p-nitrophenyl-alpha-d-mannopyranoside. The K(m) value of enzymes was 2.53 mM. The enzyme activity was inhibited by Zn(2+), Co(2+), Cu(2+), Pb(2+), Ag(1+), iodoacetate, SDS, DMF, DMSO and ethanol. Fe(3+), Ca(2+) Mg(2+), Mn(2+), Triton X-100 and PMSF did not inhibit the enzyme activity. Northern blot analysis showed a transcript of about 3.7 kb in all cells and tissues studied. This is the first report on the expression and characterization of recombinant human MANB enzyme.

Cloning and Characterization of the Polyhydroxybutyrate Depolymerase Gene of Pseudomonas stutzeri and Analysis of the Function of Substrate-Binding Domains

1999 ◽  
Vol 65 (1) ◽  
pp. 189-197 ◽  
Author(s):  
Takeshi Ohura ◽  
Ken-Ichi Kasuya ◽  
Yoshiharu Doi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Fusion Proteins ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Pseudomonas Stutzeri ◽  
Linker Region ◽  
Binding Characteristics ◽  
Binding Domains

ABSTRACT The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZPst ) of Pseudomonas stutzeriwas cloned and sequenced. phaZPst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B fromClostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S -transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.

scholarly journals Amino acid sequence and characterization of a protein inhibitor of protein kinase C.

1990 ◽  
Vol 265 (8) ◽  
pp. 4583-4591 ◽  
Author(s):  
J D Pearson ◽  
D B DeWald ◽  
W R Mathews ◽  
N M Mozier ◽  
H A Zürcher-Neely ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Amino Acid ◽  
Protein Kinase ◽  
Amino Acid Sequence ◽  
Protein Inhibitor ◽  
Kinase C

scholarly journals Analysis of Htra Gene from Zebrafish (Danio Rerio)

2015 ◽  
Vol 10 (2) ◽  
Author(s):  
M. Murwantoko ◽  
Chio Oka ◽  
Masashi Kawaichi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Danio Rerio ◽  
Serine Proteases ◽  
Catalytic Domain ◽  
Fruit Fly ◽  
Wide Range ◽  
Igf Binding ◽  
Bacterial Homolog

HtrA which is characterized by the combination of a trypsin-like catalytic domain with at least one C-terminalPDZ domain is a highly conserved family of serine proteases found in a wide range of organisms. However theidentified HtrA family numbers varies among spesies, for example the number of mammalian, Eschericia coli,fruit fly-HtrA family are 4, 3 and 1 gene respectively. One gene is predicted exist in zebrafish. Since no completeinformation available on zebrafish HtrA, in this paper zebrafish HtrA (zHtrA) gene was analyzed. The zHtrA isbelonged to HtrA1 member and predicted encodes 478 amino acids with a signal peptide, a IGF binding domain,a Kazal-type inhibitor domain in the up stream of HtrA-bacterial homolog. At the amino acid sequence the zHtrA1showed the 69%, 69%, 68%, 54% and 54% with the rat HtrA1, mouse HtrA1, human HtrA1, human HtrA3 andmouse HtrA4 respectively. The zHtrA1 is firstly expressed at 60 hpf and mainly in the vertebral rudiments in thetail region.

scholarly journals Purification and partial characterization of a lectin from Caesalpinia tinctoria Domb, ex Dc fruits

2003 ◽  
Vol 15 (2) ◽  
pp. 119-122 ◽  
Author(s):  
Marli Lourdes de Oliveira ◽  
Leila Maria Beltramini ◽  
Salvatore Giovanni de Simone ◽  
Maria Helena Nasser Brumano ◽  
Rosemeire Aparecida Silva-Lucca ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Terminal Amino Acid ◽  
Saline Extract ◽  
Hydrophobic Residues ◽  
Terminal Amino ◽  
Helix Conformation ◽  
Far Ultraviolet ◽  
Terminal Amino Acid Sequence

A lectin was isolated from the pod saline extract of Caesalpinia tinctoria by dialoconcentration on Centripep-10 and affinity chromatography on chitin column. The purified lectin was partially characterized with respect to its biochemical and structural properties. It contains 8.3 % of carbohydrate and exhibited an agglutinating activity against human erythrocytes (ABO groups). Its amino acid composition was characterized by a great number of acidic and hydrophobic residues and the estimated molecular mass was 12.5 kDa. The presence of only one N-terminal amino acid sequence (D¹-V-P-A-Y-V-Y-V-H-F10-G-F-G-E-E-H-R -D-V-F20-D), showed the homogeneity of the purified lectin. The far-ultraviolet circular dichroism (CD) spectrum of lectin indicated that it contains 10 % a-helix, 38 % b-sheet, 28 % unordered form and 6 % of P II (poly-L-proline II helix conformation).

scholarly journals Characterization of two newly isolated Staphylococcus aureus bacteriophages from Japan belonging to the genus Silviavirus

2020 ◽  
Vol 165 (10) ◽  
pp. 2355-2359
Author(s):  
Naoya Kitamura ◽  
Eri Sasabe ◽  
Shigenobu Matsuzaki ◽  
Masanori Daibata ◽  
Tetsuya Yamamoto
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequences ◽  
Related Gene ◽  
Bacteriophage Therapy ◽  
Dna Rearrangements ◽  
Site Specific

Abstract Two Staphylococcus aureus bacteriophages, KSAP7 and KSAP11, were isolated from sewage and characterized. Based on morphology and DNA sequences, they were assigned to the genus Silviavirus, subfamily Twortvirinae, family Herelleviridae, whose members are hypothesized to be suitable for bacteriophage therapy. The KSAP7 and KSAP11 genomes were 137,950 and 138,307 bp in size, respectively. Although their DNA sequences were almost identical, evidence of site-specific DNA rearrangements was found in two regions. Changes in the number of PIEPEK amino acid sequence repeats encoded by orf10 and the insertion/deletion of a 541-bp sequence that includes a possible tail-related gene were identified.

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