scholarly journals Construction and comprehensive characterization of an EcLDCc-CatIB set—varying linkers and aggregation inducing tags

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Kira Küsters ◽  
Martina Pohl ◽  
Ulrich Krauss ◽  
Gizem Ölçücü ◽  
Sandor Albert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Inclusion Bodies ◽  
Early Stage ◽  
Volumetric Productivity ◽  
Efficient Production ◽  
Wild Type ◽  
Conversion Rates ◽  
Active Inclusion Bodies

Abstract Background In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes. Results Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of l-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L− 1 d− 1 and a specific volumetric productivity of 256 g L− 1 d− 1 gCatIB−1. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L− 1 d− 1, specific volumetric activity: 106 g L− 1 d− 1 gCatIB− 1). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. Conclusions Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.

scholarly journals Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

2021 ◽  
Vol 7 (5) ◽  
pp. 325
Author(s):  
Laura Isabel de de Eugenio ◽  
Rosa Peces-Pérez ◽  
Dolores Linde ◽  
Alicia Prieto ◽  
Jorge Barriuso ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Veratryl Alcohol ◽  
Dye Decolorization ◽  
Irpex Lacteus ◽  
Type D ◽  
Lignin Peroxidases

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

scholarly journals EDL933 Strains of Escherichia coli O157 can Demonstrate Genetic Diversity and Differential Adherence to Bovine Recto-Anal Junction Squamous Epithelial Cells

2021 ◽  
Vol 15 (1) ◽  
pp. 129-138
Author(s):  
Raegan S. Hoefler ◽  
Indira T. Kudva
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
Cell Adherence ◽  
O157 Strain ◽  
Shiga Toxins ◽  
The Us ◽  
Strain Type ◽  
Enterocyte Effacement ◽  
Different Sources

Background: Differences between Escherichia coli O157 (O157) strains are well-established with some of these strains being associated with major outbreaks in the US. EDL933 is one such O157 strain that caused a multistate outbreak in 1982 and has since been used as a prototype in various O157-related experiments. Objective: As O157 can readily acquire genetic mutations, we sought to determine if the genetic and phenotypic profiles of EDL933 strains from different sources would be consistent. Methods: We evaluated wild-type O157 strains stocked as EDL933 from three different laboratories, in the strain typing Polymorphic Amplified Typing Sequence (PATS) and the bovine rectal-anal junction squamous epithelial (RSE) cell- and HEp-2 cell- adherence assays. In addition, we also verified if Shiga toxins (Stx), the Locus of Enterocyte Effacement (LEE) or curli fimbriae contributed to the adherence phenotypes observed using mutant and wild-type EDL933 isolates. Results: Our results showed differences in PATS profiles and RSE cell-adherence phenotype, with no influence from the Stx or LEE genes, between EDL933 from different sources. Interestingly, the EDL933 strain that demonstrated the most contrasting diffuse adherence phenotype on RSE cells, EDL933-T, had decreased curli production that may have contributed to this phenotype. Conclusion: Our observations suggest that a comprehensive characterization of bacterial isolates, even if assigned to the same strain type prior to use in experiments, is warranted to ensure consistency and reproducibility of results.

Isolation and partial characterization of the Asp-B10, Tyr-B25-des-(B26-B30)-proinsulin analog from inclusion bodies in Escherichia coli

1996 ◽  
Vol 10 (10) ◽  
Author(s):  
A.M.A. Nascimento ◽  
H.R.T. Souza ◽  
M.S. Xavier ◽  
J.E. Thiemann ◽  
L. Vilela ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Partial Characterization

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

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