scholarly journals Simultaneous optimization of donor and acceptor substrate specificity for transketolase by a small but smart library

2019 ◽  
Author(s):  
Haoran Yu ◽  
Roberto Icken Hernández López ◽  
David Steadman ◽  
Daniel Méndez-Sánchez ◽  
Sally Higson ◽  
...  
Keyword(s):  
Industrial Applications ◽  
Rapid Expansion ◽  
Simultaneous Optimization ◽  
Library Screening ◽  
Diverse Range ◽  
Substrate Specificities ◽  
E Coli ◽  
Donor Substrate ◽  
Donor And Acceptor ◽  
Reversible Transfer

AbstractA narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimization of both substrate specificities, is also required for the rapid expansion of accepted substrates. Transketolase catalyses the reversible transfer of a C2-ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for widely industrial applications. Herein, transketolase from E. coli was engineered to simultaneously accept both pyruvate as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid. Twenty single-mutant variants were firstly designed and characterized experimentally. Beneficial mutations were then recombined to construct a small but smart library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to WT. Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% ee for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-formylbenzoic acid. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor acceptances, that would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

scholarly journals Current Strategies for Noble Metal Nanoparticle Synthesis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Giyaullah Habibullah ◽  
Jitka Viktorova ◽  
Tomas Ruml
Keyword(s):  
Noble Metal ◽  
Noble Metals ◽  
Drug Carriers ◽  
Industrial Applications ◽  
Metal Nanoparticle ◽  
Noble Metal Nanoparticles ◽  
Synthesis Methods ◽  
Gold And Silver Nanoparticles ◽  
Diverse Range ◽  
The Past

AbstractNoble metals have played an integral part in human history for centuries; however, their integration with recent advances in nanotechnology and material sciences have provided new research opportunities in both academia and industry, which has resulted in a new array of advanced applications, including medical ones. Noble metal nanoparticles (NMNPs) have been of great importance in the field of biomedicine over the past few decades due to their importance in personalized healthcare and diagnostics. In particular, platinum, gold and silver nanoparticles have achieved the most dominant spot in the list, thanks to a very diverse range of industrial applications, including biomedical ones such as antimicrobial and antiviral agents, diagnostics, drug carriers and imaging probes. In particular, their superior resistance to extreme conditions of corrosion and oxidation is highly appreciated. Notably, in the past two decades there has been a tremendous advancement in the development of new strategies of more cost-effective and robust NMNP synthesis methods that provide materials with highly tunable physicochemical, optical and thermal properties, and biochemical functionalities. As a result, new advanced hybrid NMNPs with polymer, graphene, carbon nanotubes, quantum dots and core–shell systems have been developed with even more enhanced physicochemical characteristics that has led to exceptional diagnostic and therapeutic applications. In this review, we aim to summarize current advances in the synthesis of NMNPs (Au, Ag and Pt).

P. vortex-mediated strategies for polysaccharides decomposition

TECHNOLOGY ◽  
2015 ◽  
Vol 03 (02n03) ◽  
pp. 80-83
Author(s):  
Mark Polikovsky ◽  
Eshel Ben-Jacob ◽  
Alin Finkelshtein
Keyword(s):  
Degradation Products ◽  
Cellulose Degradation ◽  
Cellulose Hydrolysis ◽  
Industrial Applications ◽  
Biofuel Production ◽  
E Coli ◽  
Novel Approach ◽  
Textile Manufacture ◽  
Hydrolysis Of Cellulose ◽  
The Relationship

Cellulose hydrolysis has many industrial applications such as biofuel production, food, paper and textile manufacture. Here, we present a novel approach to cellulose hydrolysis using a consortium of motile bacteria, Paenibacillus vortex, that can swarm on solid medium carrying a non-motile recombinant E. coli cargo strain expressing the β-glucosidase and cellulase genes that facilitate the hydrolysis of cellulose. These two species cooperate; the relationship is mutually beneficial: the E. coli is dispersed over long distances, while the P. vortex bacteria gain from the supply of cellulose degradation products. This enables the use of such consortia in this area of biotechnology.

scholarly journals A New Clone Sweeps Clean: the Enigmatic Emergence of Escherichia coli Sequence Type 131

2014 ◽  
Vol 58 (9) ◽  
pp. 4997-5004 ◽  
Author(s):  
Ritu Banerjee ◽  
James R. Johnson
Keyword(s):  
Escherichia Coli ◽  
Sequence Type ◽  
Rapid Expansion ◽  
Future Research ◽  
Content Type ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Phylogenetic Studies ◽  
Ecological Success

ABSTRACTEscherichia colisequence type 131 (ST131) is an extensively antimicrobial-resistantE. coliclonal group that has spread explosively throughout the world. Recent molecular epidemiologic and whole-genome phylogenetic studies have elucidated the fine clonal structure of ST131, which comprises multiple ST131 subclones with distinctive resistance profiles, including the (nested) H30, H30-R, and H30-Rx subclones. The most prevalent ST131 subclone, H30, arose from a single common fluoroquinolone (FQ)-susceptible ancestor containing allele 30 offimH(type 1 fimbrial adhesin gene). An early H30 subclone member acquired FQ resistance and launched the rapid expansion of the resulting FQ-resistant subclone, H30-R. Subsequently, a member of H30-R acquired the CTX-M-15 extended-spectrum beta-lactamase and launched the rapid expansion of the CTX-M-15-containing subclone within H30-R, H30-Rx. Clonal expansion clearly is now the dominant mechanism for the rising prevalence of both FQ resistance and CTX-M-15 production in ST131 and inE. coligenerally. Reasons for the successful dissemination and expansion of the key ST131 subclones remain undefined but may include increased transmissibility, greater ability to colonize and/or persist in the intestine or urinary tract, enhanced virulence, and more-extensive antimicrobial resistance compared to otherE. coli. Here we discuss the epidemiology and molecular phylogeny of ST131 and its key subclones, possible mechanisms for their ecological success, implications of their widespread dissemination, and future research needs.

An Architecture to Implement the Internet-of-Things using the Prometheus Methodology

2020 ◽  
pp. 609-629
Author(s):  
Bogdan Manațe ◽  
Florin Fortiş ◽  
Philip Moore
Keyword(s):  
Internet Of Things ◽  
Rapid Expansion ◽  
The Internet ◽  
Data Types ◽  
Diverse Range ◽  
Complex Agent ◽  
Multi Agent ◽  
Complex Scenario ◽  
The Internet Of Things ◽  
Prometheus Methodology

The rapid expansion of the Internet of Things (IoT) will generate a diverse range of data types that needs to be handled, processed and stored. This paper aims to create a multi-agent system that suits the needs introduced by the IoT expansion, thus being able to oversee the Big Data collection and processing and also to maintain the semantic links between the data sources and data consumers. In order to build a complex agent oriented architecture, we have assessed the existing agent oriented methodologies searching for the best solution that is not bound to a specific programming language of framework, and it is flexible enough to be applied in such a divers domain like IoT. As complex scenario, the proposed approach has been applied to medical diagnosis and motoring of mental disorders.

A kinetic colorimetric assay of gamma-glutamyltransferase.

1986 ◽  
Vol 32 (8) ◽  
pp. 1581-1584 ◽  
Author(s):  
P Fossati ◽  
G V Melzi d'Eril ◽  
G Tarenghi ◽  
L Prencipe ◽  
G Berti
Keyword(s):  
Dynamic Range ◽  
Colorimetric Assay ◽  
Colorimetric Method ◽  
Volume Ratio ◽  
Ascorbate Oxidase ◽  
Good Precision ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Donor Substrate ◽  
Donor And Acceptor

Abstract We have explored a kinetic colorimetric method for measuring gamma-glutamyltransferase (EC 2.3.2.2) activity in serum, using L-gamma-glutamyl-3,5-dibromo-4-hydroxyanilide and glycylglycine as donor and acceptor substrates. The released product, 3,5-dibromo-4-hydroxyaniline, reacts with 2,5-dimethylphenol to produce a blue quinone monoimine in the presence of ascorbate oxidase (EC 1.10.3.3). This dye has peak absorption at 610 nm, whereas the donor substrate shows negligible absorption throughout the visible spectrum. The reaction can be run with all the reagents in a single working solution with serum as starter, or with the substrate solution as starting reagent. The sample/reagent volume ratio is 1:24. Adaptation of the method to several automated instruments gave good precision in all cases. Comparison with a method in which L-gamma-glutamyl-3-carboxy-4-nitroanilide is the donor substrate showed good correlation of results (r greater than or equal to 0.987). The dynamic range of the method exceeds the upper limits of the reference intervals for men (9-33 U/L) and women (8-25 U/L) by at least 18-fold.

scholarly journals Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain

Bioimpacts ◽  
2018 ◽  
Vol 9 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Azam Safary ◽  
Rezvan Moniri ◽  
Maryam Hamzeh-Mivehroud ◽  
Siavoush Dastmalchi
Keyword(s):  
Lymphoblastic Leukemia ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Substrate Affinity ◽  
Bacillus Sp ◽  
Recombinant Enzymes ◽  
E Coli ◽  
Bacillus Strain ◽  
Antineoplastic Effect ◽  
Wide Range

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.

scholarly journals Clonal Populations of ThermotolerantEnterobacteriaceae in Recreational Water and Their Potential Interference with Fecal Escherichia coli Counts

2001 ◽  
Vol 67 (10) ◽  
pp. 4934-4938 ◽  
Author(s):  
Sandra L. McLellan ◽  
Annette D. Daniels ◽  
Alissa K. Salmore
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Environmental Protection Agency ◽  
16S Rrna Gene Sequencing ◽  
Fecal Coliforms ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Diverse Range ◽  
E Coli ◽  
Glucuronidase Activity

ABSTRACT Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of β-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. β-Glucuronidase activity was critical in distinguishingE. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.

scholarly journals Dual sequentially addressable dielectrophoretic array for high-throughput, scalable, multiplexed droplet sorting

2021 ◽  
Vol 25 (4) ◽  
Author(s):  
Akihiro Isozaki ◽  
Dunhou Huang ◽  
Yuta Nakagawa ◽  
Keisuke Goda
Keyword(s):  
High Throughput ◽  
Cell Biology ◽  
Digital Pcr ◽  
Industrial Applications ◽  
Droplet Microfluidics ◽  
Diverse Range ◽  
Droplet Sorting ◽  
On Chip ◽  
Single Droplets

AbstractDroplet microfluidics is a powerful tool for a diverse range of biomedical and industrial applications such as single-cell biology, synthetic biology, digital PCR, biosafety monitoring, drug screening, and food, feed, and cosmetic industries. As an integral part of droplet microfluidics, on-chip multiplexed droplet sorting has recently gained enthusiasm, since it enables real-time sorting of single droplets containing cells with different phenotypes into multiple bins. However, conventional sorting methods are limited in throughput and scalability. Here, we present high-throughput, scalable, multiplexed droplet sorting by employing a pair of sequentially addressable dielectrophoretic arrays (SADAs) across a microchannel on a microfluidic chip. A SADA is an on-chip array of electrodes, each of which is sequentially activated and deactivated in synchronization to the position and speed of a flowing droplet of interest. The dual-SADA (dSADA) structure enables high-throughput deflection of droplets in multiple directions in a well-controlled manner. For proof-of-concept demonstration and characterization of the dSADA, we performed fluorescence-activated droplet sorting (FADS) with a 3-way dSADA at a high throughput of 2450 droplets/s. Furthermore, to show the scalability of the dSADA, we also performed FADS with a 5-way dSADA at a high throughput of 473 droplets/s.

scholarly journals Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

2020 ◽  
Vol 477 (15) ◽  
pp. 2791-2805
Author(s):  
Aishat Akere ◽  
Serena H. Chen ◽  
Xiaohan Liu ◽  
Yanger Chen ◽  
Sarath Chandra Dantu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Biochemical Characterization ◽  
Structural Information ◽  
Substrate Recognition ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Donor Substrate ◽  
Substrate Preferences ◽  
Dynamics Simulations ◽  
Experimental Crystal

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

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