scholarly journals Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

2020 ◽  
Vol 47 (8) ◽  
pp. 563-571
Author(s):  
Hongmei Han ◽  
Weizhu Zeng ◽  
Guoqiang Zhang ◽  
Jingwen Zhou
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Industrial Application ◽  
Enzyme Activities ◽  
Inclusion Bodies ◽  
Carboxyl Terminus ◽  
Tyrosine Phenol Lyase ◽  
Functional Peptides ◽  
Important Enzyme ◽  
Aggregate Particles

Abstract The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-l-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA.

scholarly journals Function of the N-terminal propeptide of an aminopeptidase from Vibrio proteolyticus

2000 ◽  
Vol 350 (3) ◽  
pp. 671-676 ◽  
Author(s):  
Zhen-Zhong ZHANG ◽  
Satoru NIRASAWA ◽  
Yoshiaki NAKAJIMA ◽  
Michiteru YOSHIDA ◽  
Kiyoshi HAYASHI
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Active Region ◽  
Protein Expression ◽  
Inclusion Bodies ◽  
Active Enzyme ◽  
Vibrio Proteolyticus ◽  
Show Evidence ◽  
Inhibited Enzyme

An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli. Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro. Using l-Leu-p-nitroanilide as the substrate, the kinetic parameters (kcat and Km) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.

scholarly journals Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli

2009 ◽  
Vol 74 (6) ◽  
pp. 607-618 ◽  
Author(s):  
Mira Milisavljevic ◽  
Drazen Papic ◽  
Gordana Timotijevic ◽  
Vesna Maksimovic
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Inclusion Bodies ◽  
Bond Formation ◽  
Aspartic Protease ◽  
The Other ◽  
Fusion Partner ◽  
Disulfide Bond Formation ◽  
Insoluble Protein ◽  
Successful Production

Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality.

scholarly journals PHYSIOLOGICAL CHARACTERIZATION OF POLAR Tn5-INDUCED ISOLEUCINE-VALINE AUXOTROPHS IN ESCHERICHIA COLI K-12: EVIDENCE FOR AN INTERNAL PROMOTER IN THE ilvOGEDA OPERON

Genetics ◽  
1979 ◽  
Vol 93 (2) ◽  
pp. 309-319
Author(s):  
Claire M Berg ◽  
Karen J Shaw ◽  
Joyce Vender ◽  
Maryla Borucka-Mankiewicz
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Transposable Element ◽  
Feeding Behavior ◽  
Enzyme Activities ◽  
Internal Promoter ◽  
Physiological Characterization ◽  
Growth Requirements ◽  
K 12

ABSTRACT The properties of 22 isoleucine-valine auxotrophs induced in Escherichia coli K-12 by the transposable element, Tn5, were characterized on the basis of growth requirements, cross-feeding behavior, and enzyme activity. Mutants defective in ilvA, ilvC, ilvD and ilvE were found. Mutations in ilvE were not completely polar on ilvD and ilvA enzyme activities (that is, ilvE mutants possessed a low constitutive level of expression of the enzymes coded by ilvD and ilvA), while mutations in ilvD were completely polar on ilvA enzyme activity. The data suggest that there is an internal promoter between the sites of Tn5 insertion in ilvE and ilvD.

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

2020 ◽  
Vol 14 (4) ◽  
pp. 269-282
Author(s):  
Sadra S. Tehrani ◽  
Golnaz Goodarzi ◽  
Mohsen Naghizadeh ◽  
Seyyed H. Khatami ◽  
Ahmad Movahedpour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Clinical Aspects ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
E Coli ◽  
Secretory Production ◽  
Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

Conditions Resulting in Formation of Properly Assembled Retroviral Capsids within Inclusion Bodies of Escherichia coli

1999 ◽  
Vol 64 (8) ◽  
pp. 1348-1356 ◽  
Author(s):  
Michaela Rumlová-Kliková ◽  
Iva Pichová ◽  
Eric Hunter ◽  
Tomáš Ruml
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Amorphous Material ◽  
Bacterial Expression ◽  
Biologically Active ◽  
Capsid Assembly ◽  
Cultivation Medium ◽  
Viral Particles ◽  
Induction Temperature

It has been generally accepted that inclusion bodies (IBs) formed in Escherichia coli consist of non-biologically active aggregated proteins, which are stabilized by non-productive interactions. We show here that bacterial expression of a retroviral capsid polyprotein results in formation of insoluble IBs that contain fully assembled viral particles connected with amorphous material. The efficiency of IBs formation and capsid assembly was not significantly affected by changes in induction temperature, pH of cultivation medium or the level of expression.

Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application

Biologia ◽  
2009 ◽  
Vol 64 (6) ◽  
Author(s):  
Yue-Hong Wang ◽  
Yu Jiang ◽  
Zuo-Ying Duan ◽  
Wei-Lan Shao ◽  
Hua-Zhong Li
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Optimal Condition ◽  
Industrial Application ◽  
Conversion Rate ◽  
Hydrolytic Activity ◽  
Thermoanaerobacter Ethanolicus ◽  
Transglycosylation Activity

AbstractIn this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K m and V max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).

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.

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