Overexpression and Phylogenetic Analysis of a Thermostable α-Glucosidase from Thermus thermophilus

2014 ◽  
Vol 1004-1005 ◽  
pp. 841-848
Author(s):  
Xun Li ◽  
Hua Xiang Gu ◽  
Hao Shi ◽  
Fei Wang
Keyword(s):  
Phylogenetic Trees ◽  
Expression Vector ◽  
Thermus Thermophilus ◽  
Glycoside Hydrolases ◽  
Neighbor Joining ◽  
E Coli ◽  
Optimal Activity ◽  
Evolution Analysis ◽  
Terminal Amino ◽  
Ph Range

The α-glucosidase geneaglfromThermus thermophilusHB8 was cloned into expression vector pBV220. The phylogenetic trees of α-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. Evolution analysis indicated the α-glucosidase fromT. thermophileHB8 was distant from the other glycoside hydrolases 4 and 31 α-glucosidases. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of theaglwas increased 30-fold. The recombinant AGL was purified by the heat treatment, and had a molecular mass of 61 kDa. The optimal activity was at pH 7.8 and 95°C over a 10 min assay. The purified enzyme was stable over a pH range of 5.4-8.6, and had a 1-h half life at 85°C. Kinetic experiments at 90°C withp-nitrophenyl-α-D-glucoside as substrate gave aKm, andVmaxof 0.072 mM and 400 U/mg. Thus, this report provides an industrial means to produce the recombinant α-glucosidase inE. coli.

Cleavage of immunoglobulin G by excretory–secretory cathepsin S-like protease of Spirometra mansoni plerocercoid

Parasitology ◽  
1994 ◽  
Vol 109 (5) ◽  
pp. 611-621 ◽  
Author(s):  
Y. Kong ◽  
Y.-B. Chung ◽  
S.-Y. Cho ◽  
S.-Y. Kang
Keyword(s):  
Immunoglobulin G ◽  
Cathepsin L ◽  
Immunoblot Analysis ◽  
Cathepsin S ◽  
Terminal Amino Acid ◽  
Optimal Activity ◽  
Spirometra Mansoni ◽  
Terminal Amino ◽  
Secretory Products ◽  
Ph Range

When immunoglobulin G (IgG) was incubated with Spirometra mansoni plerocercoid (sparganum), it was cleaved into Fab and Fc fragments. Fab/c fragments were also hydrolysed. The digestion was accelerated by dithiothreitol (DTT), indicating that cleavage of IgG heavy chain was due to a cysteine protease secreted into the medium. The responsible enzyme, of Mr 27 (± 0·8) kDa, was purified by a series of thiopropyl affinity, Sephacryl S-300 HR and DEAE-anion exchange chromatographies, either from worm extracts or from excretory–secretory products (ESP). The purified, thiol-dependent protease showed an optimal activity at pH 5·7 with 0·1 M sodium acetate but was active over the pH range 4·5–8·0. Its activity was inhibited completely by 10−5 M L-trans-epoxysuccinylleucylamido(4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA), but by only 53% using the specific cathepsin L inhibitor, Z-Phe-Phe-CHN2 (5 × 10−5 M). Partial NH2-terminal amino acid sequence was Leu-Pro-Asp-Ser-Val-Asn-Trp-Arg-Glu-Gly-Ala-Val-Thr-Ala-Val which showed 80% homology to human cathepsin S. Immunoblot analysis showed that sera from infected patients exhibited IgE antibody reaction. It is proposed that cleavage of immunoglobulin by an excreted–secreted, cathepsin S-like, allergenic protease is a mechanism of immune evasion used by the sparganum.

scholarly journals Cloning of aminoglycoside phosphotransferase (APH) gene from antibiotic-producing strain of Bacillus circulans into a high-expression vector, pKK223-3. Purification, properties and location of the enzyme

1990 ◽  
Vol 268 (3) ◽  
pp. 671-677 ◽  
Author(s):  
M Sarwar ◽  
M Akhtar
Keyword(s):  
Expression Vector ◽  
Gene Sequence ◽  
Signal Sequence ◽  
Subcellular Location ◽  
High Expression ◽  
Bacillus Circulans ◽  
E Coli ◽  
Aminoglycoside Phosphotransferase ◽  
Terminal Amino

The aminoglycoside phosphotransferase gene from a butirosin-producing strain of Bacillus circulans was cloned in a high-expression vector (pKK223-3) to give the recombinant plasmid pMS5. Escherichia coli harbouring the plasmid, E. coli JM103[pMS5], was characterized, and several features of the expression of the phosphotransferase were studied. The phosphotransferase activity was best expressed in a medium lacking glucose, and the highest levels of the enzyme were found between 12 and 24 h of growth. The induction of the phosphotransferase expression with isopropyl beta-D-thiogalactopyranoside (inducer) was found to be undesirable as the overproduction of the enzyme led to the killing of the bacteria. The subcellular location of the phosphotransferase, and also the site in vivo of the phosphorylation of neomycin, was found to be in the cytoplasm. The phosphotransferase was purified to homogeneity in good yield (17 mg of purified protein/3 litres of culture) and was shown to be a monomer of Mr 30,000-32,000. The N-terminal amino acid sequence was in agreement with that predicted from the gene sequence and confirmed the absence of any signal sequence. The regiospecificity of the phosphotransferase reaction was studied by m.s. and by 1H-, 13C- and 31P-n.m.r. using ribostamycin as the substrate, and it was found that the antibiotic was phosphorylated at the 3′-hydroxy group.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Cloning of Salmonella enterica Serovar Enteritidis Fimbrial Protein SefA as a Surface Protein in Escherichia coli Confers the Ability To Attach to Eukaryotic Cell Lines

2009 ◽  
Vol 75 (20) ◽  
pp. 6622-6625 ◽  
Author(s):  
Douglas L. Rank ◽  
Mahdi A. Saeed ◽  
Peter M. Muriana
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Expression Vector ◽  
Surface Protein ◽  
Surface Expression ◽  
Eukaryotic Cell ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Western Blot Assay ◽  
E Coli ◽  
Fimbrial Protein

ABSTRACT The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

Studies on diphosphopyridine nucleotidase and platelet damaging factor in an extracellular product of Listeria monocytogenes

1970 ◽  
Vol 16 (10) ◽  
pp. 909-916 ◽  
Author(s):  
I. H. Siddique ◽  
L. C. Ying ◽  
R. A. Chung
Keyword(s):  
Listeria Monocytogenes ◽  
Hemolytic Activity ◽  
Virulent Strain ◽  
Optimal Activity ◽  
Extracellular Product ◽  
Heat Labile ◽  
Ph Range

Hemolysin preparations from a virulent strain of Listeria monocytogenes were chromatographed on Sephadex G-100 and Sephadex DEAE A-50 columns. Three types of activities were identified: DPNase activity, hemolytic activity, and platelet-damaging activity. The separation of the peak with DPN-destroying activity from the peaks with hemolytic and platelet-damaging activities provided evidence that the factor in the solutions responsible for the destruction of DPN was distinct from that causing hemolysis and platelet-damage. The DPNase factor was found to be non-dialyzable, to be heat labile, and to have optimal activity in the pH range of 6.8–7.4.

Probing structure–activity relationships in bactericidal peptide βpep-25

2008 ◽  
Vol 414 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Ruud P. M. Dings ◽  
Judith R. Haseman ◽  
Kevin H. Mayo
Keyword(s):  
Amino Acid Residues ◽  
Neutralization Activity ◽  
Bacterial Membranes ◽  
E Coli ◽  
Structure Activity Relationships ◽  
Optimal Activity ◽  
Structure Activity ◽  
Cationic Residues ◽  
Β Sheet ◽  
And Staphylococcus Aureus

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure–activity relationships in the bacterial membrane disruptor βpep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of βpep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel β-sheet structure is the bioactive conformation of βpep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, βpep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.

scholarly journals Potential Probiotic Enterococcus faecium OV3-6 and Its Bioactive Peptide as Alternative Bio-Preservation

Foods ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2264
Author(s):  
Thiwanya Choeisoongnern ◽  
Sasithorn Sirilun ◽  
Rungaroon Waditee-Sirisattha ◽  
Komsak Pintha ◽  
Sartjin Peerajan ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Ribosomal Proteins ◽  
Starter Culture ◽  
Heat Stability ◽  
Fermented Food ◽  
E Coli ◽  
Active Peptide ◽  
Wide Ph Range ◽  
Small Intestinal ◽  
Ph Range

Probiotic Enterococcus faecium OV3-6 and its secreted active peptide were characterized and investigated. The strain survived in simulated gastric and small intestinal conditions at 88.16% and 94.33%, respectively. The safety assessment revealed that the strain was shown α-hemolysis and susceptible to most clinically relevant antibiotics, but intermediate sensitivity to erythromycin and kanamycin was found. It does not harbor any virulence genes except for the efaAfm gene. Both of its living cells and the cell-free supernatants (CFS) of the strain significantly reduced the adhesion of E. coli and S. Typhi on Caco-2 cells. The strain can regulate the secretion of pro and inflammatory cytokines, IL-6 and IL-12 and induce the secretion of anti-inflammatory IL-10 of the Caco-2 cell. The strain can prevent the growth of Gram-positive strains belonging to the genera Bacillus, Carnobacterium, Listeria, and Staphylococcus. It also presented the entP gene that involves the production of bacteriocin named enterocin P. The antimicrobial peptide was matched 40% with 50S ribosomal proteins L29 (7.325 kDa), as revealed by LC-MS/MS. This active peptide exhibits heat stability, is stable over a wide pH range of 2−10, and maintains its activity at −20 and 4 °C for 12 weeks of storage. Altogether, E. faecium OV3-6 thus has potential for consideration as a probiotic and bio-preservative for applied use as a fermented food starter culture and in functional food or feed industries.

scholarly journals Characterization of a New and Efficient Polyvalent Phage Infecting E. coli O157:H7, Salmonella spp., and Shigella sonnei

2021 ◽  
Vol 9 (10) ◽  
pp. 2105
Author(s):  
Su-Hyeon Kim ◽  
Damilare Adeyemi ◽  
Mi-Kyung Park
Keyword(s):  
Burst Size ◽  
Genomic Analysis ◽  
Cesium Chloride ◽  
Shigella Sonnei ◽  
Health Concern ◽  
Lytic Phage ◽  
Salmonella Spp ◽  
E Coli ◽  
Significant Public Health ◽  
Ph Range

Ongoing outbreaks of foodborne diseases remain a significant public health concern. Lytic phages provide promising attributes as biocontrol agents. This study characterized KFS-EC3, a polyvalent and lytic phage, which was isolated from slaughterhouse sewage and purified by cesium chloride density centrifugation. Host range and efficiency of plating analyses revealed that KFS-EC3 is polyvalent and can efficiently infect E. coli O157:H7, Salmonella spp., and Shigella sonnei. KFS-EC3 had a latent time of 20 min and burst size of ~71 phages/infected cell. KFS-EC3 was stable and infectious following storage at a pH range of 3 to 11 and a temperature range of −70°C to 60°C. KFS-EC3 could inhibit E. coli O157:H7 growth by 2 logs up to 52 h even at the lowest MOI of 0.001. Genomic analysis of KFS-EC3 revealed that it consisted of 167,440 bp and 273 ORFs identified as functional genes, without any genes associated with antibiotic resistance, virulence, allergenicity, and lysogenicity. This phage was finally classified into the Tequatrovirus genus of the Myoviridae family. In conclusion, KFS-EC3 could simultaneously infect E. coli O157:H7, S. sonnei, and Salmonella spp. with the lowest MOI values over long periods, suggesting its suitability for simultaneous pathogen control in foods.

A Dual-Expression Vector Allowing Expression in E. coli and P. pastoris, Including New Modifications

2003 ◽  
pp. 31-42
Author(s):  
Angelika Lueking ◽  
Sabine Horn ◽  
Hans Lehrach ◽  
Dolores J. Cahill
Keyword(s):  
Expression Vector ◽  
E Coli ◽  
Dual Expression

scholarly journals Analysis of crystalline and solution states of ligand-free spermidine N-acetyltransferase (SpeG) from Escherichia coli

2019 ◽  
Vol 75 (6) ◽  
pp. 545-553 ◽  
Author(s):  
Ekaterina V. Filippova ◽  
Steven Weigand ◽  
Olga Kiryukhina ◽  
Alan J. Wolfe ◽  
Wayne F. Anderson
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Crystal Structures ◽  
Coenzyme A ◽  
Amino Group ◽  
Acetyl Coenzyme A ◽  
E Coli ◽  
Ligand Free ◽  
Acetyl Group ◽  
Terminal Amino

Spermidine N-acetyltransferase (SpeG) transfers an acetyl group from acetyl-coenzyme A to an N-terminal amino group of intracellular spermidine. This acetylation inactivates spermidine, reducing the polyamine toxicity that tends to occur under certain chemical and physical stresses. The structure of the SpeG protein from Vibrio cholerae has been characterized: while the monomer possesses a structural fold similar to those of other Gcn5-related N-acetyltransferase superfamily members, its dodecameric structure remains exceptional. In this paper, structural analyses of SpeG isolated from Escherichia coli are described. Like V. cholerae SpeG, E. coli SpeG forms dodecamers, as revealed by two crystal structures of the ligand-free E. coli SpeG dodecamer determined at 1.75 and 2.9 Å resolution. Although both V. cholerae SpeG and E. coli SpeG can adopt an asymmetric open dodecameric state, solution analysis showed that the oligomeric composition of ligand-free E. coli SpeG differs from that of ligand-free V. cholerae SpeG. Based on these data, it is proposed that the equilibrium balance of SpeG oligomers in the absence of ligands differs from one species to another and thus might be important for SpeG function.

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