scholarly journals Recombinant cephalosporin-acid synthesase: optimisation of expression in E.coli cells, immobilisation and application for biocatalytic cefazolin synthesis

2015 ◽  
Vol 61 (5) ◽  
pp. 646-651
Author(s):  
M.A. Eldarov ◽  
A.V. Sklyarenko ◽  
M.V. Dumina ◽  
N.V. Medvedeva ◽  
A.A. Jgoun ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Erwinia Carotovora ◽  
Growth Media ◽  
Media Composition ◽  
Specific Enzyme ◽  
Secretion Signal ◽  
Mild Conditions ◽  
Escherichia Coli Cells ◽  
Effective Synthesis ◽  
Efficient Expression

Cephalosporin acid synthetase (CASA) is responsible for specific to synthesis of cephalosporin-acids, its expression in Escherichia coli cells is accompanied by accumulation of unprocessed insoluble precursor. In order to optimize conditions of recombinant CASA production we have studied the effects of several parameters of strain cultivation, including growth media composition, temperature, and inoculation dose. Also plasmids for production of CASA variants with the signal sequence of Erwinia carotovora L-asparaginase (ansCASA) and “leaderless” CASA were created in search of more efficient expression constructs. Removal of the N-terminal secretion signal sequence reduced the production of functionally active CASA more than 10-fold and inhibited strain growth. Insertion of the L-asparaginase signal sequence increased the specific enzyme activity in the resultant recombinant strain. The ansCASA producing strain was used to develop the method of immobilization of the recombinant enzyme on an epoxy-activated macroporous acrylic support. The resultant biocatalyst performed effective synthesis of cefazolin from 3-[(5-methyl-1,3,4-thiadiazol-2-il)-thiomethyl]-7- aminocephalosporanic acid (MMTD-7-ACA) and methyl ester of 1(H)-tetrazolilacetic acid (МETzAA), under mild conditions a transformation level of MMTD-7-ACA to cefazolin of 95% is reached.

Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides

1986 ◽  
Vol 6 (5) ◽  
pp. 1812-1819
Author(s):  
C N Chang ◽  
M Matteucci ◽  
L J Perry ◽  
J J Wulf ◽  
C Y Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Phosphoglycerate Kinase ◽  
Yeast Cells ◽  
Signal Peptidase ◽  
Human Interferon ◽  
Expression Plasmid ◽  
Kinase Gene ◽  
Secretion Signal ◽  
Yeast Invertase

Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.

scholarly journals Cloning of fibA, Encoding an Immunogenic Subunit of the Fibril-Like Surface Structure ofPeptostreptococcus micros

1999 ◽  
Vol 181 (8) ◽  
pp. 2485-2491 ◽  
Author(s):  
B. H. A. Kremer ◽  
J. J. E. Bijlsma ◽  
J. G. Kusters ◽  
J. de Graaff ◽  
T. J. M. van Steenbergen
Keyword(s):  
Amino Acid ◽  
Surface Structure ◽  
Signal Sequence ◽  
Open Reading Frames ◽  
Periodontal Pathogen ◽  
Immunogold Electron Microscopy ◽  
Expression Library ◽  
Gram Positive ◽  
Secretion Signal ◽  
Specific Serum

ABSTRACT Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogenPeptostreptococcus micros. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (RgSm) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the RgSm type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a λTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designatedfibA, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of fibA exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.

The highly efficient expression of the aspartase gene (L-aspartate ammonia-lyase) in Escherichia coli cells

2015 ◽  
Vol 51 (7) ◽  
pp. 751-756 ◽  
Author(s):  
A. D. Novikov ◽  
D. D. Derbikov ◽  
O. V. Shaposhnikova ◽  
T. A. Gubanova ◽  
S. V. Kameneva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Highly Efficient ◽  
Escherichia Coli Cells ◽  
Efficient Expression

Mutational analysis of the flavinylation and binding motifs in two protein targets of the flavin transferase ApbE

2019 ◽  
Vol 366 (22) ◽  
Author(s):  
Yulia V Bertsova ◽  
Marina V Serebryakova ◽  
Victor A Anashkin ◽  
Alexander A Baykov ◽  
Alexander V Bogachev
Keyword(s):  
Mutational Analysis ◽  
Vibrio Harveyi ◽  
Binding Pocket ◽  
Serine Residue ◽  
Specific Enzyme ◽  
Protein Targets ◽  
Binding Motifs ◽  
Nadh:Quinone Oxidoreductase ◽  
Escherichia Coli Cells

ABSTRACT Many flavoproteins belonging to three domain types contain an FMN residue linked through a phosphoester bond to a threonine or serine residue found in a conserved seven-residue motif. The flavinylation reaction is catalyzed by a specific enzyme, ApbE, which uses FAD as a substrate. To determine the structural requirements of the flavinylation reaction, we examined the effects of single substitutions in the flavinylation motif of Klebsiella pneumoniae cytoplasmic fumarate reductase on its modification by its own ApbE in recombinant Escherichia coli cells. The replacement of the flavin acceptor threonine with alanine completely abolished the modification reaction, whereas the replacements of conserved aspartate and serine had only minor effects. Effects of other substitutions, including replacing the acceptor threonine with serine, (a 10–55% decrease in the flavinylation degree) pinpointed important glycine and alanine residues and suggested an excessive capacity of the ApbE-based flavinylation system in vivo. Consistent with this deduction, drastic replacements of conserved leucine and threonine residues in the binding pocket that accommodates FMN residue still allowed appreciable flavinylation of the NqrC subunit of Vibrio harveyi Na+-translocating NADH:quinone oxidoreductase, despite a profound weakening of the isoalloxazine ring binding and an increase in its exposure to solvent.

scholarly journals From a Basic Microalga and an Acetic Acid Bacterium Cellulose Producer to a Living Symbiotic Biofilm

Materials ◽  
2019 ◽  
Vol 12 (14) ◽  
pp. 2275
Author(s):  
Vítor Nóbrega ◽  
Marisa Faria ◽  
Antera Quintana ◽  
Manfred Kaufmann ◽  
Artur Ferreira ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Acetic Acid Bacterium ◽  
Growth Media ◽  
Media Composition ◽  
Symbiotic Interaction ◽  
Microalgae Biomass ◽  
The Subject ◽  
Living Material ◽  
First Time

Bacterial cellulose (BC) has recently been the subject of a considerable amount of research, not only for its environmentally friendly biosynthesis, but also for its high potential in areas such as biomedicine or biomaterials. A symbiotic relationship between a photosynthetic microalga, Chlamydomonas debaryana, and a cellulose producer bacterium, Komagataeibacter saccharivorans, was established in order to obtain a viable and active biofilm. The effect of the growth media composition ratio on the produced living material was investigated, as well as the microalgae biomass quantity, temperature, and incubation time. The optimal temperature for higher symbiotic biofilm production was 30 °C with an incubation period of 14 days. The high microalgae presence, 0.75% w/v, and 60:40 HS:BG-11 medium (v/v) induced a biofilm microalgae incorporation rate of 85%. The obtained results report, for the first time, a successful symbiotic interaction developed in situ between an alkaline photosynthetic microalga and an acetic acid bacterium. These results are promising and open a new window to BC living biofilm applications in medical fields that have not yet been explored.

scholarly journals Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria

1980 ◽  
Vol 186 (1) ◽  
pp. 13-19 ◽  
Author(s):  
S D Shukla ◽  
J M Turner
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Mycobacterium Smegmatis ◽  
Lipid Synthesis ◽  
Erwinia Carotovora ◽  
Radioactive Isotopes ◽  
Growth Media ◽  
Lipid Fractions ◽  
Base Moiety ◽  
Aldehyde Dehydrogenase Activity

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.

scholarly journals Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

2012 ◽  
Vol 78 (9) ◽  
pp. 3469-3472 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Dolores Gutiérrez ◽  
Beatriz Martínez ◽  
Ana Rodríguez ◽  
Pilar García
Keyword(s):  
Staphylococcus Aureus ◽  
Lactococcus Lactis ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Content Type ◽  
Inducible Promoters ◽  
Secretion Signal ◽  
Cell Extracts ◽  
Culture Supernatants ◽  
Secretion Signal Sequence

ABSTRACTBacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded byStaphylococcus aureusphage vB_SauS-phi-IPLA88, was expressed and secreted inLactococcus lactisusing the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts.

scholarly journals Improved Production of Recombinant Myrosinase in Pichia pastoris

2021 ◽  
Vol 22 (21) ◽  
pp. 11889
Author(s):  
Zuzana Rosenbergová ◽  
Zuzana Hegyi ◽  
Miroslav Ferko ◽  
Natália Andelová ◽  
Martin Rebroš
Keyword(s):  
Pichia Pastoris ◽  
Signal Sequence ◽  
Specific Productivity ◽  
Endoplasmic Reticulum Membrane ◽  
Volumetric Activity ◽  
Secretion Signal ◽  
Plant Enzymes ◽  
Optimum Ph ◽  
Mating Factor ◽  
Native Signal Sequence

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

scholarly journals Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus

1998 ◽  
Vol 64 (12) ◽  
pp. 4857-4861 ◽  
Author(s):  
Toshiyuki Murai ◽  
Mitsuyoshi Ueda ◽  
Takashi Kawaguchi ◽  
Motoo Arai ◽  
Atsuo Tanaka
Keyword(s):  
Cell Surface ◽  
Rhizopus Oryzae ◽  
Signal Sequence ◽  
Hydrolytic Enzymes ◽  
Water Soluble ◽  
Aspergillus Aculeatus ◽  
Cellulosic Materials ◽  
Secretion Signal ◽  
Engineered Yeast ◽  
Secretion Signal Sequence

ABSTRACT Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and β-glucosidase from the fungusAspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and β-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for β-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and β-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and β-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.

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