scholarly journals Catechol 1,2-Dioxygenase From Paracoccus sp. MKU1—A Greener and Cleaner Bio-Machinery for cis, cis-Muconic Acid Production by Recombinant E. coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Manikka Kubendran Aravind ◽  
Perumal Varalakshmi ◽  
Swamidoss Abraham John ◽  
Balasubramaniem Ashokkumar
Keyword(s):  
Low Cost ◽  
3D Structure ◽  
Single Step ◽  
Potential Candidate ◽  
Muconic Acid ◽  
Whole Cells ◽  
E Coli ◽  
C Terminus ◽  
Catechol 1,2 Dioxygenase ◽  
Α Helix

Cis, cis-muconic acid (ccMA) is known for its industrial importance as a precursor for the synthesis of several biopolymers. Catechol 1,2-dioxygenase (C12O) is involved in aromatic compounds catabolism and ccMA synthesis in a greener and cleaner way. This is the first study on C12O gene from a metabolically versatile Paracoccus sp. MKU1, which was cloned and expressed in E. coli to produce ccMA from catechol. From the E. coli transformant, recombinant C12O enzyme was purified and found to be a homotrimer with a subunit size of 38.6 kDa. The apparent Km and Vmax for C12O was 12.89 µM and 310.1 U.mg−1, respectively, evidencing high affinity to catechol than previously reported C12Os. The predicted 3D-structure of C12O from MKU1 consisted of five α-helices in N-terminus, one α-helix in C-terminus, and nine β-sheets in C-terminus. Moreover, a unique α-helix signature ‘EESIHAN’ was identified in C-terminus between 271 and 277 amino acids, however the molecular insight of conservative α-helix remains obscure. Further, fed-batch culture was employed using recombinant E. coli expressing C12O gene from Paracoccus sp. MKU1 to produce ccMA by whole-cells catalyzed bioconversion of catechol. With the successive supply of 120 mM catechol, the transformant produced 91.4 mM (12.99 g/L) of ccMA in 6 h with the purity of 95.7%. This single step conversion of catechol to ccMA using whole-cells reactions of recombinants did not generate any by-products in the reaction mixtures. Thus, the recombinant E. coli expressing high activity C12O from Paracoccus sp. MKU1 holds promise as a potential candidate for yielding high concentrations of ccMA at faster rates in low cost settings.

scholarly journals The Extended C-Terminal α-Helix of the HypC Chaperone Restricts Recognition of Large Subunit Precursors by the Hyp-Scaffold Machinery during [NiFe]-Hydrogenase Maturation in Escherichia coli

2018 ◽  
Vol 28 (2) ◽  
pp. 87-97
Author(s):  
Claudia Thomas ◽  
Mandy Waclawek ◽  
Kerstin Nutschan ◽  
Constanze Pinske ◽  
R. Gary Sawers
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Large Subunit ◽  
Protein Family ◽  
Variable Regions ◽  
E Coli ◽  
Hydrogenase Maturation ◽  
C Terminus ◽  
Α Helix

Members of the HypC protein family are chaperone-like proteins that play a central role in the maturation of [NiFe]-hydrogenases (Hyd). <i>Escherichia coli</i> has a second copy of HypC, called HybG, and, as a component of the HypDEF maturation scaffold, these proteins help synthesize the NiFe-cofactor and guide the scaffold to its designated hydrogenase large subunit precursor. HypC is required to synthesize active Hyd-1 and Hyd-3, while HybG facilitates Hyd-2 and Hyd-1 synthesis. To identify determinants on HypC that allow it to discriminate against Hyd-2, we made amino acid exchanges in 3 variable regions, termed VR1, VR2, and VR3, of HypC, that make it more similar to HybG. Region VR3 includes a HypC-specific C-terminal α-helical extension, and this proved particularly important in preventing the maturation of Hyd-2 by HypC. Truncation of this extension on HypC increased Hyd-2 activity in the absence of HybG, while retaining maturation of Hyd-3 and Hyd-1. Combining this truncation with amino acid exchanges in VR1 and VR2 of HypC negatively affected the synthesis of active Hyd-1. The C-terminus of <i>E. coli</i> HypC is thus a key determinant in hindering Hyd-2 maturation, while VR1 and VR2 appear more important for Hyd-1 matu­ration.

scholarly journals Chitosan Stabilized Silver Nanoparticles for the Electrochemical Detection of Lipopolysaccharide: A Facile Biosensing Approach for Gram-Negative Bacteria

Micromachines ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 413 ◽  
Author(s):  
Muhammad Imran ◽  
Christopher J. Ehrhardt ◽  
Massimo F. Bertino ◽  
Muhammad R. Shah ◽  
Vamsi K. Yadavalli
Keyword(s):  
Silver Nanoparticles ◽  
Low Cost ◽  
Age Groups ◽  
Single Step ◽  
Indirect Detection ◽  
Detection Methods ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Strong Electrostatic Interaction

Negatively charged lipopolysaccharide (LPS), a major endotoxin and component of the outer membrane of several Gram-negative bacteria, provides a useful biomarker for the indirect detection of these pathogens. For instance, Escherichia coli (E. coli) is a pathogenic bacterium that causes infections in almost all age groups, and has been implicated in food and water contamination. Current diagnostic and detection methods tend to be labor-intensive or expensive, necessitating the need for an easy, sensitive, rapid, and low-cost method. We report on the synthesis and use of positively charged chitosan stabilized silver nanoparticles (Chi-AgNPs) as a sensitive electrochemical nanobiosensor for the detection of LPS. Chi-AgNPs were synthesized through a facile, single step protocol, and characterized for size, charge, and morphology. Glassy carbon electrodes modified with Chi-AgNPs resulted in an enhancement of signal in the presence of both LPS and E. coli. Detection was accomplished over a large concentration range (several orders of magnitude) of 0.001–100 ng/mL and 10–107 CFU/mL. The biosensors can reliably detect LPS and E. coli at very low concentrations. Chi-AgNPs have potential as low cost, sensitive nanobiosensors for Gram-negative bacteria due to strong electrostatic interaction with LPS present in their outer membranes.

scholarly journals Purification and Properties of NADH-Dependent 5,10-Methylenetetrahydrofolate Reductase (MetF) fromEscherichia coli

1999 ◽  
Vol 181 (3) ◽  
pp. 718-725 ◽  
Author(s):  
Christal A. Sheppard ◽  
Elizabeth E. Trimmer ◽  
Rowena G. Matthews
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Specific Activity ◽  
Single Step ◽  
Turnover Number ◽  
E Coli ◽  
Apparent Homogeneity ◽  
C Terminus ◽  
Purification And Properties ◽  
K 12 ◽  
Peptostreptococcus Productus

ABSTRACT A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 μM, the Km for CH2-H4folate is 0.8 μM, and the turnover number under V max conditions estimated for the reaction is 1,800 mol of NADH oxidized min−1 (mol of enzyme-bound FAD)−1. NADPH also serves as a reductant, but exhibits a much higher Km . MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme fromPeptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.

scholarly journals Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens

2021 ◽  
Vol 9 (11) ◽  
pp. 2367
Author(s):  
Angela Sun ◽  
Jo-Ann L. Stanton ◽  
Peter L. Bergquist ◽  
Anwar Sunna
Keyword(s):  
Water Samples ◽  
Sampling Method ◽  
Tap Water ◽  
Low Cost ◽  
Scale Up ◽  
Single Step ◽  
E Coli ◽  
Conventional Culture ◽  
Insoluble Particles ◽  
Acid Wash

A universal filtration and enzyme-based workflow has been established to allow for the rapid and sensitive quantification of leading pathogens Cryptosporidium parvum, Giardia gamblia, Campylobacter jejuni, and Escherichia coli from tap water samples with volumes up to 100 mL, and the potential to scale up to larger volumes. qPCR limits of quantification as low as four oocysts for Cryptosporidium, twelve cysts for Giardia, two cells for C. jejuni, and nineteen cells for E. coli per reaction were achieved. A polycarbonate filter-based sampling method coupled with the prepGEM enzyme-based DNA extraction system created a single-step transfer workflow that required as little as 20 min of incubation time and a 100 µL reaction mix. The quantification via qPCR was performed directly on the prepGEM extract, bypassing time-consuming, labour-intensive conventional culture-based methods. The tap water samples were shown to contain insoluble particles that inhibited detection by reducing the quantification efficiency of a representative pathogen (C. jejuni) to 30–60%. This sample inhibition was effectively removed by an on-filter treatment of 20% (v/v) phosphoric acid wash. Overall, the established workflow was able to achieve quantification efficiencies of 92% and higher for all four leading water pathogens, forming the basis of a rapid, portable, and low-cost solution to water monitoring.

scholarly journals Biochemical Properties of an Alkaline Xylanase From Bacillus Agaradhaerens C9 and Its Application in Producing Xylo-oligosaccharides With High Degree of Polymerization From Wheat Bran

2021 ◽  
Author(s):  
Jiawen Liu ◽  
Di Sun ◽  
Zhen Dong ◽  
Cong Liu ◽  
Jingrong Zhu ◽  
...  
Keyword(s):  
Wheat Bran ◽  
Low Cost ◽  
Maximal Activity ◽  
Biochemical Properties ◽  
Degree Of Polymerization ◽  
Single Step ◽  
Transition Elements ◽  
E Coli ◽  
High Degree ◽  
Bacillus Agaradhaerens

Abstract A xylanase of Bacillus agaradhaerens C9 was heterologously expressed and was then investigated. The recombinant xylanase (rBaxyl11) showed maximal activity at 60°C and pH 8.0-9.0. Under optimal conditions, Kcat of rBaxyl11 for arabinoxylan and glucuronoxylan were 599 s-1 and 330 s-1, respectively. rBaxyl11 showed a good stability at pH ranging from 5.0 to 9.0, and retained 50% of activity after 6-hour incubation at 70°C. However, it was markedly inactivated by transition elements including Fe3+, Ni2+, Mn2+, Co2+, Zn2+, Cu2+ and Fe2+. rBaxyl11 generated xylo-oligosaccharides (XOS) whose degree of polymerization (DP) is greater than 3 when hydrolyzing arabinoxylan, while the DP of XOS ranged from 2 to 6 when acting on glucuronoxylan. Simultaneously producing xylanase and XOS by recombinant E. coli containing rBaxyl11 were then carried out. Results showed that the engineering E. coli generated xylanase and high-DP XOS extracellularly using wheat bran as substrate, and concentration of XOS reached 73 mg/g substrate after 12-hour fermentation. This study indicates the feasibility of producing XOS by a single-step fermentation approach with low cost using rBaxyl11.

scholarly journals Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

2015 ◽  
Vol 81 (23) ◽  
pp. 8037-8043 ◽  
Author(s):  
Sudeshna Sengupta ◽  
Sudhakar Jonnalagadda ◽  
Lakshani Goonewardena ◽  
Veeresh Juturu
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Carbon Sources ◽  
Raw Material ◽  
Hydroxybenzoic Acid ◽  
Muconic Acid ◽  
Content Type ◽  
E Coli ◽  
Catechol 1,2 Dioxygenase ◽  
K 12

ABSTRACTcis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain ofEscherichia coliK-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes,pobA,aroY, andcatA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed inE. colito establish the MA biosynthetic pathway.E. colinative genesubiC,aroFFBR,aroE, andaroLwere overexpressed and the genesptsH,ptsI,crr, andpykFwere deleted from theE. coligenome in order to increase the precursors of the proposed MA pathway. The final engineeredE. colistrain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.

scholarly journals Direct purification of CRISPR/Cas ribonucleoprotein from E. coli in a single step

2018 ◽  
Author(s):  
Siyu Lin ◽  
Jie Qiao ◽  
Lixin Ma ◽  
Yi Liu
Keyword(s):  
Large Scale ◽  
Low Cost ◽  
Cost Effective ◽  
Nuclease Activity ◽  
Single Step ◽  
Simple Method ◽  
E Coli ◽  
Purification System

AbstractCRISPR/Cas ribonucleoprotein (RNP) complexes have been recently used as promising biological tools with plenty of applications, however, there are by far no efficient methods to prepare them at large scale and low cost. Here, we present a simple method to directly produce and purify Cas RNP, including the widely used Cas9 and Cas12a nuclease, from E.coli in a single step using an ultra-high-affinity CL7/Im7 purification system. The prepared Cas RNP shows high stability, solid nuclease activity in vitro, and profound genome editing efficiency in vivo. Our method is convenient, cost-effective, and applicable to prepare other CRISPR associated nucleases.

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals Synthesis and characterization of Mg–Zn ferrite based flexible microwave composites and its application as SNG metamaterial

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Md Atiqur Rahman ◽  
Mohammad Tariqul Islam ◽  
Mandeep Singh Jit Singh ◽  
Md Samsuzzaman ◽  
Muhammad E. H. Chowdhury
Keyword(s):  
Low Cost ◽  
Sol Gel ◽  
Microwave Frequency ◽  
Dielectric Substrate ◽  
Dielectric Constants ◽  
Potential Candidate ◽  
Flexible Composites ◽  
Average Grain Size ◽  
Zn Ferrite ◽  
High Flexibility

AbstractIn this article, we propose SNG (single negative) metamaterial fabricated on Mg–Zn ferrite-based flexible microwave composites. Firstly, the flexible composites are synthesized by the sol-gel method having four different molecular compositions of MgxZn(1−x)Fe2O4, which are denoted as Mg20, Mg40, Mg60, and Mg80. The structural, morphological, and microwave properties of the synthesized flexible composites are analyzed using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), and conventional dielectric assessment kit (DAK) to justify their possible application as dielectric substrate at microwave frequency regime. Thus the average grain size is found from 20 to 24 nm, and the dielectric constants are 6.01, 5.10, 4.19, and 3.28, as well as loss tangents, are 0.002, 0.004, 0.006, and 0.008 for the prepared Mg–Zn ferrites, i.e., Mg20, Mg40, Mg60, and Mg80 respectively. Besides, the prepared low-cost Mg–Zn ferrite composites exhibit high flexibility and lightweight, which makes them a potential candidate as a metamaterial substrate. Furthermore, a single negative (SNG) metamaterial unit cell is fabricated on the prepared, flexible microwave composites, and their essential electromagnetic behaviors are observed. Very good effective medium ratios (EMR) vales are obtained from 14.65 to 18.47, which ensure the compactness of the fabricated prototypes with a physical dimension of 8 × 6.5 mm2. Also, the proposed materials have shown better performances comparing with conventional FR4 and RO4533 materials, and they have covered S-, C-, X-, Ku-, and K-band of microwave frequency region. Thus, the prepared, flexible SNG metamaterials on MgxZn(1−x)Fe2O4 composites are suitable for microwave and flexible technologies.

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