scholarly journals Improvement of a recombinant β-glucosidase activity of a sequence derived from metagenome database of Binh Chau hotspring

2020 ◽  
Vol 17 (3) ◽  
pp. 545-551
Author(s):  
Tran Thanh Thuy ◽  
Lai Thi Hong Nhung ◽  
Le Thi Thanh Xuan ◽  
Nguyen Kim Thoa
Keyword(s):  
Hot Spring ◽  
Biomass Conversion ◽  
New Approach ◽  
E Coli ◽  
Metabolic Products ◽  
Predicted Values ◽  
Metagenome Sequencing ◽  
High Alkali ◽  
Host Strains ◽  
Medium Culture

Metabolic products obtaining from microorganisms of geothermal ecologies often show special characteristics which help their cells to survive, grow and develop under extreme conditions. Exploiting the microbial gene resource of those environments demands a new approach via uncultured methods. Thanks to the development of metagenomics and bioinformatic softwares, we can exploit novel genes from environment directly. Based on Binh Chau hotspring’s DNA metagenome sequencing, ORF [denovogenes]_32768 encoding  for β-glucosidase is selected for expression into pET17b vector because it shown a low similartity of amino acid sequence as compared to others in Genbank, a high alkali and Tm predicted values. To improve the expression efficiency of β-glucosidase, some factors (host strains, medium culture, IPTG concentration, aeration…) are investigated. The results showed that the recombinant E. coli C43(DE3) reached the highest dried biomass at 8.26 g/L and the maximum enzymatic activity at 0.34 U/mL in shaking condition (TB medium plus 0.25 mM IPTG with the ratio of cultured /flask volume is 20%, 42-48 hours, 30°C). This study demonstrates the capacity of mining a novel gene encoded for enzyme from DNA metagenome of Vietnam hot spring as well as produces recombinant enzyme for biomass conversion.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

scholarly journals Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
María-Eugenia DeCastro ◽  
Michael P. Doane ◽  
Elizabeth Ann Dinsdale ◽  
Esther Rodríguez-Belmonte ◽  
María-Isabel González-Siso
Keyword(s):  
Microbial Community ◽  
Hot Spring ◽  
Functional Characterization ◽  
Tca Cycle ◽  
Maximal Activity ◽  
Sulfur Cycle ◽  
E Coli ◽  
Complex Microbial Community ◽  
Relationship Of ◽  
The Relationship

AbstractIn the present study we investigate the microbial community inhabiting As Burgas geothermal spring, located in Ourense (Galicia, Spain). The approximately 23 Gbp of Illumina sequences generated for each replicate revealed a complex microbial community dominated by Bacteria in which Proteobacteria and Aquificae were the two prevalent phyla. An association between the two most prevalent genera, Thermus and Hydrogenobacter, was suggested by the relationship of their metabolism. The high relative abundance of sequences involved in the Calvin–Benson cycle and the reductive TCA cycle unveils the dominance of an autotrophic population. Important pathways from the nitrogen and sulfur cycle are potentially taking place in As Burgas hot spring. In the assembled reads, two complete ORFs matching GH2 beta-galactosidases were found. To assess their functional characterization, the two ORFs were cloned and overexpressed in E. coli. The pTsbg enzyme had activity towards o-Nitrophenyl-β-d-galactopyranoside (ONPG) and p-Nitrophenyl-β-d-fucopyranoside, with high thermal stability and showing maximal activity at 85 °C and pH 6, nevertheless the enzyme failed to hydrolyze lactose. The other enzyme, Tsbg, was unable to hydrolyze even ONPG or lactose. This finding highlights the challenge of finding novel active enzymes based only on their sequence.

Endurance vs. strength training: comparison of cardiac structures using normal predicted values

1988 ◽  
Vol 64 (6) ◽  
pp. 2552-2557 ◽  
Author(s):  
A. Roy ◽  
M. Doyon ◽  
J. G. Dumesnil ◽  
J. Jobin ◽  
F. Landry
Keyword(s):  
Individual Differences ◽  
Strength Training ◽  
Body Surface ◽  
Normal Values ◽  
Volume Overload ◽  
Left Ventricular ◽  
New Approach ◽  
Lv Mass ◽  
Lv Volume ◽  
Predicted Values

There are still disagreements concerning the adaptation of cardiac structures in relation to different training stimuli. To eliminate some of the variance due to individual differences in body surface area, we utilized a new approach based on the calculation of the percentages of each individual's normal predicted values (%NPV). We studied 46 strength (S, bodybuilders) and 57 endurance (E, runners) athletes. Left ventricular (LV) mass was 143.8 +/- 21.9 %NPV (mean +/- SD) in E vs. 134.3 +/- 23.4 %NPV in S (P less than 0.05), and LV volume was 131.0 +/- 24.0 %NPV in E vs. 120.0 +/- 25.5 %NPV in S (P less than 0.05). Moreover, the LV wall thickness-to-radius ratio did not differ from normal values in either group. From these data we conclude that 1) cardiac modifications are greater in E than S, 2) the predominant stimulus is a volume overload type in both groups, and 3) concentric LV hypertrophy may not be as prevalent in S as previously suggested.

scholarly journals Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem

2019 ◽  
Vol 20 (17) ◽  
pp. 4268
Author(s):  
Chetna Tyagi ◽  
Tamás Marik ◽  
Csaba Vágvölgyi ◽  
László Kredics ◽  
Ferenc Ötvös
Keyword(s):  
Molecular Dynamics ◽  
Enhanced Sampling ◽  
New Approach ◽  
E Coli ◽  
Accelerated Molecular Dynamics ◽  
Simulation Techniques ◽  
Unfolded State ◽  
Folding Dynamics ◽  
Transmembrane Channels ◽  
Dynamics Simulations

The use of enhanced sampling molecular dynamics simulations to facilitate the folding of proteins is a relatively new approach which has quickly gained momentum in recent years. Accelerated molecular dynamics (aMD) can elucidate the dynamic path from the unfolded state to the near-native state, “flattened” by introducing a non-negative boost to the potential. Alamethicin F30/3 (Alm F30/3), chosen in this study, belongs to the class of peptaibols that are 7–20 residue long, non-ribosomally synthesized, amphipathic molecules that show interesting membrane perturbing activity. The recent studies undertaken on the Alm molecules and their transmembrane channels have been reviewed. Three consecutive simulations of ~900 ns each were carried out where N-terminal folding could be observed within the first 100 ns, while C-terminal folding could only be achieved almost after 800 ns. It took ~1 μs to attain the near-native conformation with stronger potential boost which may take several μs worth of classical MD to produce the same results. The Alm F30/3 hexamer channel was also simulated in an E. coli mimicking membrane under an external electric field that correlates with previous experiments. It can be concluded that aMD simulation techniques are suited to elucidate peptaibol structures and to understand their folding dynamics.

scholarly journals Hybrid Atmosphere Processing of Lead-Free Piezoelectric Sodium Potassium Niobate-Based Ceramics

Ceramics ◽  
2019 ◽  
Vol 2 (3) ◽  
pp. 460-471 ◽  
Author(s):  
Astri Bjørnetun Haugen
Keyword(s):  
Lead Free ◽  
Piezoelectric Response ◽  
Potassium Niobate ◽  
Sodium Potassium ◽  
New Approach ◽  
Sodium Potassium Niobate ◽  
Phase Pure ◽  
High Alkali ◽  
Surface Decomposition ◽  
Air Processing

K0.5Na0.5NbO3-based ceramics, a promising group of lead-free piezoelectrics, are challenging to sinter dense while avoiding alkali evaporation. This work explores hybrid atmosphere processing, a new approach where reducing atmospheres is used during heating to avoid coarsening from alkali carbonates and hydroxides, and oxidizing atmospheres is used during sintering to avoid alkali evaporation. Discs of Li0.06(K0.52Na0.48)0.94Nb0.71Ta0.29O3 with 0.25 mol% Mn (KNNLTM) were sintered in air, N2, 9% H2 in N2, or 9% H2 in N2 during heating and air during sintering (hybrid atmosphere processing). The highest density was obtained by sintering in 9% H2 in N2, but resulted in high alkali loss and decomposition of the surface, followed by low piezoelectric response. However, with the hybrid H2/air processing it was possible to both avoid surface decomposition and leakage currently associated with alkali evaporation during sintering in H2, and to obtain a denser, more phase-pure and small-grained KNNLTM ceramic with a higher piezoelectric response than obtained by sintering in air or N2.

scholarly journals TheEscherichia coli-Derived Thymosinβ4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Xiaolei Wang ◽  
Guihua Yang ◽  
Shanshuang Li ◽  
Meifeng Gao ◽  
Pangfeng Zhao ◽  
...  
Keyword(s):  
Spleen Cells ◽  
New Approach ◽  
E Coli ◽  
Sds Page ◽  
Strain Bl21 ◽  
Healing Wound ◽  
Production Strategy ◽  
Better Than

Thymosinβ4 (Tβ4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tβ4 will increase. In order to develop a new approach to produce recombinant Tβ4, a 168 bp DNA (termedTβ4) was designed based on the Tβ4 protein sequence and used to express a 4 ×Tβ4 concatemer (four tandem copies of Tβ4, termed 4 ×Tβ4) together with a histidine tag (6 × His) inE. coli(strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tβ4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropylβ-D-thiogalactoside (IPTG). TheE. coli-derived 4 ×Tβ4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cellsin vitroandin vivowound healing. The results demonstrate that these activities of theE. coli-derived recombinant 4 × Tβ4 were similar or even better than existing commercially obtained Tβ4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tβ4.

Improving Forecasting Performance by Exploiting Expert Knowledge: Evidence from Guangzhou Port

2016 ◽  
Vol 15 (02) ◽  
pp. 387-401 ◽  
Author(s):  
Anqiang Huang ◽  
Han Qiao ◽  
Shouyang Wang ◽  
John Liu
Keyword(s):  
Expert Knowledge ◽  
Constraint Equation ◽  
Positive Contribution ◽  
New Approach ◽  
Subjective Judgment ◽  
Forecasting Performance ◽  
Predicted Values

Expert knowledge has been proved by substantial studies to be contributory to higher forecasting performance; meanwhile, its application is criticized and opposed by some groups for biases and inconsistency inherent in experts’ subjective judgment. This paper proposes a new approach to improving forecasting performance, which takes advantage of expert knowledge by constructing a constraint equation rather than directly adjusting the predicted values by experts. For the comparison purpose, the proposed approach, together with several widely used models including ARIMA, BP-ANN and the judgment model (JM), is applied to forecasting the container throughput of Guangzhou Port, which is one of the most important ports of China. Forecasting performances of the above models are compared and the results clearly show superiority of the proposed approach over its rivals, which implies that expert knowledge will make positive contribution as long as it is used in a right way.

Field observations and management strategy for hot spring wastewater in Wulai area, Taiwan

2010 ◽  
Vol 61 (9) ◽  
pp. 2309-2316
Author(s):  
J. Y. Lin ◽  
C. F. Chen ◽  
F. R. Lei ◽  
C. D. Hsieh
Keyword(s):  
Hot Springs ◽  
Hot Spring ◽  
Management Strategies ◽  
Water Bodies ◽  
E Coli ◽  
Untreated Wastewater ◽  
Quality Of Water ◽  
Treatment Equipment ◽  
Different Seasons

Hot springs are important centers for recreation and tourism. However, the pollution that may potentially be caused by hot spring wastewater has rarely been discussed. More than half of Taiwan's hot springs are located in areas where the water quality of water bodies is to be protected, and untreated wastewater could pollute the receiving water bodies. In this study, we investigate hot spring wastewater in the Wulai area, one of Taiwan's famous hot spring resorts. Used water from five hot spring hotels was sampled and ten sampling events were carried out to evaluate the changes in the quality of used water in different seasons, at different periods of the week, and from different types of hotels. The concentrations of different pollutants in hot spring wastewater were found to exhibit wide variations, as follows: COD, 10–250 mg/L; SS, N.D.−93 mg/L; NH3-N, 0.01–1.93 mg/L; TP, 0.01–0.45 mg/L; and E. coli, 10–27,500 CFU/100 mL. The quality of hot spring wastewater depends on the operation of public pools, because this affects the frequency of supplementary fresh water and the outflow volume. Two management strategies, namely, onsite treatment systems and individually packaged treatment equipment, are considered, and a multi-objective optimization model is used to determine the optimal strategy.

scholarly journals Different impacts of naturally occurring variants of the globally disseminated carbapenemase-encoding plasmid pKpQIL on the biology of host strains

2018 ◽  
Author(s):  
Howard T. H. Saw ◽  
Mark A. Webber ◽  
Neil Woodford ◽  
Laura J. V. Piddock
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Host Range ◽  
Generation Time ◽  
Enterobacter Cloacae ◽  
Bacterial Host ◽  
E Coli ◽  
Naturally Occurring ◽  
Filter Mating ◽  
Host Strains

SynopsisKlebsiella-associated plasmid pKpQIL and its variant have been isolated globally. Our study aimed to determine whether a naturally occurring variant has altered host range and impacts on the fitness of different bacterial host strains. The plasmids pKpQIL-UK and pKpQIL-D2 were transferred from the original clinical isolate host strains of Klebsiella pneumoniae into Escherichia coli, Salmonella Typhimurium, Enterobacter cloacae and Serratia marcescens strains by filter-mating and conjugation frequencies determined and compared. The fitness of the resulting transconjugants was assessed by determining growth kinetics, ability to form a biofilm and persistence of the plasmids in each host was also measured. Transfer of either plasmid into Salmonella and S. marcescens was similar. However, pKpQIL-UK transferred into E. coli at a higher rate than did pKpQIL-D2; the reverse was found for E. cloacae. Both plasmids were rapidly lost from the E. coli population. Plasmid pKpQIL-UK, but not -D2, was able to persist in Salmonella. Although pKpQIL-UK imposed a greater fitness cost (inferred from an increased generation time) than -D2 on E. cloacae, it was able to persist as well as pKpQIL-D2 in this host. The pKpQIL-D2 plasmid did not confer any fitness benefit on any of the hosts under the conditions tested. Variants of the globally important pKpQIL plasmid have arisen in patients due to recombination. The impacts of the pKpQIL-UK plasmid and the -D2 variant in various Enterobacteriaceae are host-dependent. Continuing evolution of pKpQIL may alter its host range in the future.

Sign in / Sign up

Export Citation Format

Share Document