Characterization of a new bifunctional endo-1,4-β-xylanase/esterase found in the rumen metagenome

AbstractMetagenomic data mining of the Nellore cattle rumen microbiota identified a new bifunctional enzyme, endo-1,4-β-xylanase/esterase, which was subsequently overexpressed in E. coli BL21 (DE3). This enzyme was stable at pH intervals of 5 to 6.5 and temperatures between 30 and 45 °C, and under the test conditions, it had a Vmax of 30.959 ± 2.334 µmol/min/mg, Km of 3.6 ± 0.6 mM and kcat of 2.323 ± 175 s−1. Additionally, the results showed that the enzyme is tolerant to NaCl and organic solvents and therefore is suitable for industrial environments. Xylanases are widely applicable, and the synergistic activity of endo-1,4-β-xylanase/esterase in a single molecule will improve the degradation efficiency of heteroxylans via the creation of xylanase binding sites. Therefore, this new molecule has the potential for use in lignocellulosic biomass processing and as an animal feed food additive and could improve xylooligosaccharide production efficiency.

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In situ genotyping of a pooled strain library after characterizing complex phenotypes

10.1101/142729 ◽

2017 ◽

Cited By ~ 2

Author(s):

Michael J. Lawson ◽

Daniel Camsund ◽

Jimmy Larsson ◽

Özden Baltekin ◽

David Fange ◽

...

Keyword(s):

Fluorescence Imaging ◽

Single Molecule ◽

Single Molecule Fluorescence ◽

Detailed Characterization ◽

E Coli ◽

Complex Phenotypes

So far, it has not been possible to perform advanced microscopy on pool generated strain libraries and at the same time know each strain’s genotype. We have overcome this barrier by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single molecule fluorescence imaging of E. coli strains harboring barcoded plasmids that express a sgRNA which suppress different genes through dCas9.

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Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking

10.1101/2020.09.30.321232 ◽

2020 ◽

Author(s):

Martin Rieu ◽

Jessica Valle-Orero ◽

Bertrand Ducos ◽

Jean-François Allemand ◽

Vincent Croquette

Keyword(s):

Single Molecule ◽

Functional Characterization ◽

Nucleic Acid Binding ◽

Single Molecule Force Spectroscopy ◽

E Coli ◽

Natural Function ◽

Protein Nucleic Acid ◽

Free Quantification

ABSTRACTFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.

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Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding

Communications Biology ◽

10.1038/s42003-021-02606-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Martin Rieu ◽

Jessica Valle-Orero ◽

Bertrand Ducos ◽

Jean-François Allemand ◽

Vincent Croquette

Keyword(s):

Single Molecule ◽

Functional Characterization ◽

Nucleic Acid Binding ◽

Single Molecule Force Spectroscopy ◽

E Coli ◽

Natural Function ◽

Protein Nucleic Acid ◽

Free Quantification

AbstractFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

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Characterization of cellulase from E. coli BPPTCC-EGRK2

E3S Web of Conferences ◽

10.1051/e3sconf/20185200024 ◽

2018 ◽

Vol 52 ◽

pp. 00024

Author(s):

Mas Gunawan Haryanto ◽

Siswa Setyahadi ◽

Muhammad Sahlan ◽

Masafumi Yohda ◽

Yosuke Fukutani ◽

...

Keyword(s):

Molecular Weight ◽

Enzyme Activity ◽

Animal Feed ◽

Maximum Velocity ◽

Cellulase Production ◽

Luria Bertani ◽

E Coli ◽

Sds Page ◽

Weight Analysis

Cellulase enzymes are widely used in various industries such as detergent industry, bioethanol, animal feed, textile and paper. This research focused on characterization of cellulase produced from Eschericia coli BPPT-CC EgRK2, which is a recombinant that can produce protein enzymes endo- β-1,4-glucanase. Eschericia coli BPPT-CC EgRK2 was cultured in 1 litre liquid medium Luria Bertani. Because the bacteria is intracellular, sonication is needed for cell disruption to get the cellulase enzyme. The enzyme activity was then analyzed by CMC substrate at different concentration. The protein content analysis was carried out using Bradford method; the molecular weight analysis was done using SDS-PAGE; while the enzyme kinetics was plotted using Michaelis-Menten model. Our results showed the highest enzyme activity was 2.403 U/ml and the protein concentration was 5.352 mg/ml. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for CMC substrate hydrolysis were 0.07 μmol/ml and 2.49 μmol/ml/sec, respectively. The cellulase molecular weight was 58 kDa using SDS-PAGE with 7.5% stacking gel. The results indicated that Eschericia coli BPPT-CC EgRK2 is a promising renewable source for cellulase production for industrial application.

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Single-Molecule Characterization of E. Coli Pol III Core Catalytic Activity

Biophysical Journal ◽

10.1016/j.bpj.2016.11.2778 ◽

2017 ◽

Vol 112 (3) ◽

pp. 514a

Author(s):

M. Nabuan Naufer ◽

David A. Murison ◽

Ioulia Rouzina ◽

Penny J. Beuning ◽

Mark C. Williams

Keyword(s):

Catalytic Activity ◽

Single Molecule ◽

E Coli ◽

Pol Iii

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Single-molecule mechanochemical characterization of E. coli pol III core catalytic activity

Protein Science ◽

10.1002/pro.3152 ◽

2017 ◽

Vol 26 (7) ◽

pp. 1413-1426 ◽

Cited By ~ 9

Author(s):

M. Nabuan Naufer ◽

David A. Murison ◽

Ioulia Rouzina ◽

Penny J. Beuning ◽

Mark C. Williams

Keyword(s):

Catalytic Activity ◽

Single Molecule ◽

E Coli ◽

Pol Iii

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Single molecule characterization of proteins involved in replication of E. coli, the LINE1 retrotransposon, and retroviruses

10.17760/d20316396 ◽

2019 ◽

Author(s):

Mohamed Nabuan Naufer

Keyword(s):

Single Molecule ◽

E Coli

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Identification and functional characterization of a β-glucosidase from Bacillus tequelensis BD69 expressed in bacterial and yeast heterologous systems

PeerJ ◽

10.7717/peerj.8792 ◽

2020 ◽

Vol 8 ◽

pp. e8792 ◽

Cited By ~ 1

Author(s):

Ahmad Raza ◽

Ratnasri Pothula ◽

Heba Abdelgaffar ◽

Saira Bashir ◽

Juan Luis Jurat-Fuentes

Keyword(s):

Molecular Docking ◽

Animal Feed ◽

Functional Characterization ◽

Industrial Applications ◽

Biofuel Production ◽

Cloning And Expression ◽

E Coli ◽

Glucosidase Activity ◽

Hydrolysis Of

Background The identification and characterization of novel β-glucosidase genes has attracted considerable attention because of their valuable use in a variety of industrial applications, ranging from biofuel production to improved digestibility of animal feed. We previously isolated a fiber-degrading strain of Bacillus tequelensis from buffalo dung samples, and the goal of the current work was to identify β-glucosidase genes in this strain. We describe the cloning and expression of a new β-glucosidase gene (Bteqβgluc) from Bacillus tequelensis strain BD69 in bacterial and yeast hosts. The recombinant Bteqβgluc were used to characterize specificity and activity parameters, and candidate active residues involved in hydrolysis of different substrates were identified through molecular docking. Methods The full length Bteqβgluc gene was cloned and expressed in Escherichia coli and Pichia pastoris cultures. Recombinant Bteqβgluc proteins were purified by immobilized metal affinity or anion exchange chromatography and used in β-glucosidase activity assays measuring hydrolysis of ρ-nitrophenyl-β-D-glucopyranoside (pNPG). Activity parameters were determined by testing relative β-glucosidase activity after incubation under different temperature and pH conditions. Candidate active residues in Bteqβgluc were identified using molecular operating environment (MOE) software. Results The cloned Bteqβgluc gene belongs to glycoside hydrolase (GH) family 4 and encoded a 54.35 kDa protein. Specific activity of the recombinant β-glucosidase was higher when expressed in P. pastoris (1,462.25 U/mg) than in E. coli (1,445.09 U/mg) hosts using same amount of enzyme. Optimum activity was detected at pH 5 and 50 °C. The activation energy (Ea) was 44.18 and 45.29 kJ/mol for Bteqβgluc produced by P. pastoris and E. coli, respectively. Results from other kinetic parameter determinations, including pKa for the ionizable groups in the active site, Gibbs free energy of activation (ΔG‡), entropy of activation (ΔS‡), Michaelis constant (Km) and maximum reaction velocity (Vmax) for pNPG hydrolysis support unique kinetics and functional characteristics that may be of interest for industrial applications. Molecular docking analysis identified Glu, Asn, Phe, Tyr, Thr and Gln residues as important in protein-ligand catalytic interactions.

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Production and Characterization of Novel Board-spectrum Antimicrobial 5-butyl-2-pyridine carboxylic acid from Aspergillus fumigatus nHF-01

10.21203/rs.3.rs-1162299/v1 ◽

2022 ◽

Author(s):

Vivekananda Mandal ◽

Narendra Nath Ghosh ◽

Prashanta Kumar Mitra ◽

Sukhendu Mandal ◽

Vivekananda Mandal

Keyword(s):

Carboxylic Acid ◽

Broad Spectrum ◽

Pathogenic Bacteria ◽

Synergistic Activity ◽

Antimicrobial Compound ◽

Gram Negative ◽

E Coli ◽

Pyridine Carboxylic Acid ◽

Pyridine Carboxylic

Abstract Objectives: The present study aims to report on the production optimization, purification, and characterization of structural and functional attributes of a novel broad-spectrum antibacterial compound produced by Aspergillus fumigatus nHF-01 (GenBank Ac. No. MN190286).Materials and Methods: The culture conditions were optimized by using rigorous culture-set preparation considering various abiotic and biotic factors for a higher amount of antimicrobial production. The produced antimicrobial was solvent extracted and purified by preparative TLC and HPLC methods followed by characterization using UV-Vis, FT-IR, ESI-MS, and 1H-NMR spectroscopy. The MIC and MBC of the antimicrobials were determined against a set of Gram-positive and Gram-negative human pathogenic bacteria. The mode of action on cellular morphology and integrity were determined by LDH and SEM studies. Its biofilm-inhibition properties and synergistic activity with antibiotics were studied. The possible cytotoxic effect on human cell lines was also tested by MTT assay. The putative target site of action was evaluated through in silico molecular docking study. Results: The micro-fungus A. fumigatus nHF-01 produced the maximum antibacterial compound while grown in a combination of 2% MEB (w/v) and 4% YE (w/v) at pH 6.0 and 20 °C temperature with 100 rpm agitation for ten days. The DCM extractable crude compound has a potent growth inhibition against the target human food and topical pathogenic bacteria at a 15 mg/ml concentration and is stable up to 100 °C. The spectroscopic studies confirmed the antimicrobial compound as 5-butyl-2-pyridine carboxylic acid with MIC values from 0.069±0.0034 to 1.12±0.052 mg/ml and from 8.925±0.39 to 17.85±0.78 mg/ml; and MBC values from 8.925±0.40 to 17.85±0.776 mg/ml and from 0.069±0.0034 to 0.139±0.0065 mg/ml against human pathogenic Gram-positive and Gram-negative bacteria, respectively. A concentration of 0.139 and 17.85 mg/ml decreased the viability sharply within 15 min of the incubation period with the gradual increase in LDH activity, indicating a robust bactericidal and lytic mode of action. The time-kill kinetics study shows that at a 17.85 mg/ml dose (i.e. MBC), the compound caused zero viability of E. coli and S. epidermidis cells from the initial log CFU/ml 5.78 after 15 h of treatment. It caused a remarkable change in morphology like the formation of blebbing, notch, rupture of the entire cell walls, and entire dissolution of cell integrity at a concentration of 4 µg/ml and 129 µg/ml. It had cytotoxicity against the tested human lung carcinoma A549 cell line. It showed a notable antibiofilm activity at 20 µg/ml and 4 µg/ml comparable to the standard antibiofilm drug usnic acid 10 µg/ml and 64 µg/ml against E. coli and B. cereus. It had a synergistic activity with streptomycin, whereas ciprofloxacin and vancomycin showed additive effects. It showed the highest binding affinities with Quinol-Fumarate Reductase (1l0v), a respiratory enzyme. Conclusion: Thus, the above findings can be concluded that the strain A. fumigatus nHF-01 produces a novel broad-spectrum antimicrobial compound 5-butyl-2-pyridine carboxylic acid with potent bactericidal activity against human food and topical pathogenic bacteria. This is the first report of such a compound from the A. fumigatus.

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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