scholarly journals High-Level Extracellular Expression of a New β-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc

2021 ◽  
Vol 12 ◽  
Author(s):  
Congna Li ◽  
Shun Jiang ◽  
Chao Du ◽  
Zhenghui Lu ◽  
Nisha He ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chemical Process ◽  
Specific Activity ◽  
High Specific Activity ◽  
Culture Conditions ◽  
Extracellular Expression ◽  
Secreted Expression ◽  
Encoding Gene ◽  
High Level ◽  
The Cost

N-acetyl-β-D glucosamine (GlcNAc) is wildly used in cosmetics, nutraceuticals and pharmaceuticals. The traditional chemical process for GlcNAc production from chitin causes serious acidic pollution. Therefore, the enzymatic hydrolysis becomes a great promising and alternative strategy to produce GlcNAc. β-N-acetylglucosaminidase (NAGase) can hydrolyze chitin to produce GlcNAc. Here, a GH3 family NAGase encoding gene BlNagZ from Bacillus licheniformis was expressed extracellularly in Escherichia coli guided by signal peptide PelB. The recombinant BlNagZ presented the best activity at 60°C and pH 5.5 with a high specific activity of 13.05 U/mg. The BlNagZ activity in the fermentation supernatant can reach 13.62 U/mL after optimizing the culture conditions, which is 4.25 times higher than optimization before. Finally, combining BlNagZ with chitinase ChiA we identified before, chitin conversion efficiency to GlcNAc can reach 89.2% within 3.5 h. In all, this study provided not only a high active NAGase, and a secreted expression strategy to reduce the cost of production, which is conducive to the industrial application.

scholarly journals Constitutive High Level Expression of an Endoxylanase Gene from the Newly IsolatedBacillus subtilisAQ1 inEscherichia coli

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Is Helianti ◽  
Niknik Nurhayati ◽  
Maria Ulfah ◽  
Budiasih Wahyuntari ◽  
Siswa Setyahadi
Keyword(s):  
Specific Activity ◽  
Biochemical Properties ◽  
Extracellular Expression ◽  
High Level Expression ◽  
Periplasmic Fraction ◽  
16S Rdna Sequence ◽  
E Coli ◽  
Encoding Gene ◽  
High Level ◽  
Very High

A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted asBacillus subtilis(B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed inE. coli. InE. colithe recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely,2173.6 ± 51.4and2745.3 ± 11 U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the nativeB. subtilisAQ1 endoxylanase and that of 95% homologous recombinant one fromB. subtilisDB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression inE. coliand hence made the production inE. colifeasible.

Preparation of Isotope-labeled Dihydroneopterin 3'-Triphosphate with High Specific Activity

Pteridines ◽  
1990 ◽  
Vol 2 (3) ◽  
pp. 169-174 ◽  
Author(s):  
Gerd Katzenmeier ◽  
Bruno Schwarzkopf ◽  
Quang Le Van ◽  
Cornelia Schmid ◽  
Adelbert Bacher
Keyword(s):  
Escherichia Coli ◽  
Adenylate Kinase ◽  
Specific Activity ◽  
High Specific Activity ◽  
Starting Material ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Guanylate Kinase ◽  
Phosphoribosyl Transferase ◽  
Phosphoribosylpyrophosphate Synthetase

SummaryIsotope-labeled dihydroneopterin 3'-triphosphate with 3H at positions C-1' and C-2', respectively, has been prepared from isotope-labeled glucose as starting material. Glucose was first converted enzymatically to ribose 5-phosphate. GMP was subsequently obtained by the action of phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase. It was subsequently phosphorylated to GTP in two steps using adenylate kinase and guanylate kinase. Dihydroneopterin triphosphate was prepared from GTP by the action of recombinant GTP-cyclohydrolase I from Escherichia coli. The method allows the incorporation of 3H and 14C isotope labels into any desired position of dihydroneopterin triphosphate. Rapid purfication procedures for phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase as well as HPLC assays for their determinations are described.

A Mutation That Improves Soluble Recombinant Hemoglobin Accumulation in Escherichia coli in Heme Excess

1999 ◽  
Vol 65 (2) ◽  
pp. 640-647 ◽  
Author(s):  
Michael J. Weickert ◽  
Maria Pagratis ◽  
Christopher B. Glascock ◽  
Richard Blackmore
Keyword(s):  
Escherichia Coli ◽  
Binding Pocket ◽  
Culture Conditions ◽  
Molar Ratio ◽  
Cell Protein ◽  
Wild Type ◽  
E Coli ◽  
Soluble Cell ◽  
Hemoglobin Variants ◽  
High Level

ABSTRACT High-level expression of soluble recombinant human hemoglobin (rHb) in Escherichia coli was obtained with several hemoglobin variants. Under identical conditions, two rHbs containing the Presbyterian mutation (Asn-108→Lys) in β-globin accumulated to approximately twofold less soluble globin than rHbs containing the corresponding wild-type β-globin subunit accumulated. The β-globin Providence(asp) mutation (Lys-82→Asp) significantly improved soluble rHb accumulation compared to the wild-type β-globin subunit and restored soluble accumulation of rHbs containing the Presbyterian mutation to wild-type levels. The Providenceasp substitution introduced a negatively charged residue into the normally cationic 2,3-bisphosphoglycerate binding pocket, potentially reducing the electrostatic repulsion in the absence of the polyanion. The average soluble globin accumulation when there was coexpression of di-α-globin and β-Lys-82→Asp-globin (rHb9.1) and heme was present in at least a threefold molar excess was 36% ± 3% of the soluble cell protein in E. coli. The average total accumulation (soluble globin plus insoluble globin) was 56% ± 7% of the soluble cell protein. Fermentations yielded 6.0 ± 0.3 g of soluble rHb9.1 per liter 16 h after induction and 6.4 ± 0.2 g/liter 24 h after induction. The average total globin yield was 9.4 g/liter 16 h after induction. High-level accumulation of soluble rHb in E. coli depends on culture conditions, the protein sequence, and the molar ratio of the heme cofactor added.

scholarly journals Extracellular expression of Aerococcus viridans pyruvate oxidase in recombinant Escherichia coli through SecB co-expression

RSC Advances ◽  
2019 ◽  
Vol 9 (45) ◽  
pp. 26291-26301 ◽  
Author(s):  
Junwen Lu ◽  
Jianguo Zhang
Keyword(s):  
Escherichia Coli ◽  
Recombinant Escherichia Coli ◽  
Extracellular Expression ◽  
Pyruvate Oxidase ◽  
E Coli ◽  
Aerococcus Viridans ◽  
High Level

Extracellular pyruvate oxidase was expressed at a high level using E. coli by co-expression of chaperone SecB under bla promoter, and therefore cultivation optimization.

Inhibition of Escherichia coli CTP synthase by glutamate γ-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis

2001 ◽  
Vol 356 (1) ◽  
pp. 223-232 ◽  
Author(s):  
Stephen L. BEARNE ◽  
Omid HEKMAT ◽  
Jennifer E. MacDONNELL
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Specific Activity ◽  
High Specific Activity ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Open Chain ◽  
Tetrahedral Intermediate ◽  
Allosteric Effector ◽  
Ctp Synthase

Cytidine 5′-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate γ-semialdehyde. Glutamate γ-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate γ-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (Kis 0.16±0.03mM; Kii 0.4±0.1mM) and a competitive inhibitor with respect to ammonia (Ki 0.39±0.06mM) in the presence of GTP at pH8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate γ-semialdehyde. Although glutamate γ-semialdehyde exists in solution primarily in its cyclic form, Δ1-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Δ1-pyrroline-5-carboxylate (l-proline, l-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate γ-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.

scholarly journals N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms

1999 ◽  
Vol 55 (7) ◽  
pp. 1350-1352 ◽  
Author(s):  
Fernando Gil ◽  
Santiago Ramón-Maiques ◽  
Alberto Marina ◽  
Ignacio Fita ◽  
Vicente Rubio
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
High Specific Activity ◽  
Unit Cell ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Space Group ◽  
Cell Parameters ◽  
E Coli ◽  
Carbamate Kinase

The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E. coli, allowing enzyme purification in three steps. NAGK exhibits high specific activity (1.1 µmol s−1 mg−1), lacks Met1 and forms dimers (shown by cross-linking). Crystals of unliganded NAGK diffract to 2 Å and belong to space group P6122 or its enantiomorph P6522 (unit-cell parameters a = b = 78.6, c = 278.0 Å) with two monomers in the asymmetric unit. Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 Å and belong to space group C2221 (unit-cell parameters a = 60.0, b = 71.9, c = 107.4 Å), with one monomer in the asymmetric unit. NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase.

scholarly journals Targeted Radionuclide Therapy for Patients with Metastatic Pheochromocytoma and Paraganglioma: From Low-Specific-Activity to High-Specific-Activity Iodine-131 Metaiodobenzylguanidine

Cancers ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 1018 ◽  
Author(s):  
Camilo Jimenez ◽  
William Erwin ◽  
Beth Chasen
Keyword(s):  
Clinical Practice ◽  
Specific Activity ◽  
High Specific Activity ◽  
Novel Therapy ◽  
Pivotal Phase ◽  
The Us ◽  
Disease Stabilization ◽  
Iodine 131 ◽  
High Level ◽  
Collective Information

Low-specific-activity iodine-131–radiolabeled metaiodobenzylguanidine (I-131-MIBG) was introduced last century as a potential systemic therapy for patients with malignant pheochromocytomas and paragangliomas. Collective information derived from mainly retrospective studies has suggested that 30–40% of patients with these tumors benefit from this treatment. A low index of radioactivity, lack of therapeutic standardization, and toxicity associated with intermediate to high activities (absorbed radiation doses) has prevented the implementation of I-131-MIBG’s in clinical practice. High-specific-activity, carrier-free I-131-MIBG has been developed over the past two decades as a novel therapy for patients with metastatic pheochromocytomas and paragangliomas that express the norepinephrine transporter. This drug allows for a high level of radioactivity, and as yet is not associated with cardiovascular toxicity. In a pivotal phase two clinical trial, more than 90% of patients achieved partial responses and disease stabilization with the improvement of hypertension. Furthermore, many patients exhibited long-term persistent antineoplastic effects. Currently, the high-specific-activity I-131-MIBG is the only approved therapy in the US for patients with metastatic pheochromocytomas and paragangliomas. This review will discuss the historical development of high-specific-activity I-131-MIBG, its benefits and adverse events, and future directions for clinical practice applicability and trial development.

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