scholarly journals 300-Fold Increase in Production of the Zn2+-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

2014 ◽  
Vol 80 (13) ◽  
pp. 4003-4011 ◽  
Author(s):  
Colin J. Jackson ◽  
Christopher W. Coppin ◽  
Paul D. Carr ◽  
Alexey Aleksandrov ◽  
Matthew Wilding ◽  
...  
Keyword(s):  
Soluble Protein ◽  
Metal Ion ◽  
Large Scale ◽  
Computational Simulation ◽  
Active Form ◽  
Fold Increase ◽  
Soluble Form ◽  
Scale Production ◽  
Content Type ◽  
The Stability

ABSTRACTMicrobial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme fromArthrobacter aurescenswith environmental applications that catalyzes the hydrolysis of triazine herbicides, inEscherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.

scholarly journals Introduction of Surface Loops as a Tool for Encapsulin Functionalization.

2021 ◽  
Author(s):  
Sandra Michel-Souzy ◽  
Naomi M. Hamelmann ◽  
Sara Zarzuela-Pura ◽  
Jos M. J. Paulusse ◽  
Jeroen J. L. M. Cornelissen
Keyword(s):  
Biomedical Applications ◽  
Large Scale ◽  
Thermotoga Maritima ◽  
Scale Production ◽  
Structure Property ◽  
Protein Subunit ◽  
Protein Cages ◽  
Notable Increase ◽  
The Stability ◽  
Surface Loops

Encapsulin based protein cages are nanoparticles with different biomedical applications, such as targeted drug delivery or imaging agents. These particles are biocompatible and can be produced in bacteria, allowing large scale production and protein engineering. In order to use these bacterial nanocages in different applications, it is important to further explore the potential of their surface modification and optimize their production. In this study we design and show new surface modifications of the Thermotoga maritima (Tm) and Brevibacterium linens (Bl) encapsulins. Two new loops on Tm encapsulin with a His-tag insertion after the residue 64 and the residue 127, and the modification of the C-terminal on Bl encapsulin, are reported. The multi-modification of the Tm encapsulin enables up to 240 different functionalities on the cage surface, resulting from 4 potential modifications per protein subunit. We furthermore report an improved protocol giving a better stability and providing a notable increase of the production yield of the cages. Finally, we tested the stability of different encapsulin variants over a year and the results show a difference in stability arising from the tag insertion position. These first insights in the structure-property relationship of encapsulins, with respect to the position of a function loop, allow for further study of the use of these protein nanocages in biomedical applications.

scholarly journals Blakeslea trispora Photoreceptors: Identification and Functional Analysis

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Wei Luo ◽  
Chao Xue ◽  
Yuzheng Zhao ◽  
Huili Zhang ◽  
Zhiming Rao ◽  
...  
Keyword(s):  
Large Scale ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Flavin Mononucleotide ◽  
Blakeslea Trispora ◽  
Scale Production ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Flavin Binding

ABSTRACT Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora. Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa. The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli. The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trispora. IMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides. The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.

scholarly journals AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

2015 ◽  
Vol 197 (24) ◽  
pp. 3788-3796 ◽  
Author(s):  
Takayuki Kuge ◽  
Haruhiko Teramoto ◽  
Masayuki Inui
Keyword(s):  
Corynebacterium Glutamicum ◽  
Binding Sites ◽  
Large Scale ◽  
Transcriptional Regulator ◽  
Scale Production ◽  
Primary Role ◽  
Promoter Regions ◽  
Independent Manner ◽  
Content Type

ABSTRACTInCorynebacterium glutamicumATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression ofl-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDAandgalM-araR) and two (BSE1and BSE2) upstream ofaraE.l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSBmutation resulted in derepression of botharaBDAandgalM-araRoperons. The effects of BSE1and/or BSE2mutation onaraEexpression revealed that the two sites independently function as theciselements, but BSE1plays the primary role. However, AraR was shown to bind to these sites with almost the same affinityin vitro. Taken together, the expression ofaraBDAandaraEis strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of thegalM-araRandaraEpromoters is less effective in repression. Furthermore, downregulation ofaraBDAandaraEdependent onl-arabinose catabolism observed in the BSBmutant and the AraR-independentaraRpromoter identified withingalM-araRadd complexity to regulation of the AraR regulon derepressed byl-arabinose.IMPORTANCECorynebacterium glutamicumhas a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. MostC. glutamicumstrains are unable to use a pentose sugarl-arabinose as a carbon source. However, genes forl-arabinose utilization and its regulation have been recently identified inC. glutamicumATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression byl-arabinose and thereby highlights the complex regulatory feedback loops in combination withl-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

Stability of jets in a shallow water layer

2015 ◽  
Vol 25 (2) ◽  
pp. 358-374 ◽  
Author(s):  
Zhanhong Wan ◽  
Saihua Huang ◽  
Zhilin Sun ◽  
Zhenjiang You
Keyword(s):  
Reynolds Number ◽  
Stability Analysis ◽  
Shallow Water ◽  
Large Scale ◽  
Bottom Topography ◽  
Bottom Friction ◽  
Rossby Number ◽  
Content Type ◽  
The Stability ◽  
Topographic Parameters

Purpose – The present work is devoted to the numerical study of the stability of shallow jet. The effects of important parameters on the stability behavior for large scale shallow jets are considered and investigated. Connections between the stability theory and observed features reported in the literature are emphasized. The paper aims to discuss these issues. Design/methodology/approach – A linear stability analysis of shallow jet incorporating the effects of bottom topography, bed friction and viscosity has been carried out by using the shallow water stability equation derived from the depth averaged shallow water equations in conjunction with both Chézy and Manning resistance formulae. Effects of the following main factors on the stability of shallow water jets are examined: Rossby number, bottom friction number, Reynolds number, topographic parameters, base velocity profile and resistance model. Special attention has been paid to the Coriolis effects on the jet stability by limiting the rotation number in the range of Ro∈[0, 1.0]. Findings – It is found that the Rossby number may either amplify or attenuate the growth of the flow instability depending on the values of the topographic parameters. There is a regime where the near cancellation of Coriolis effects due to other relevant parameters influences is responsible for enhancement of stability. The instability can be suppressed by the bottom friction when the bottom friction number is large enough. The amplification rate may become sensitive to the relatively small Reynolds number. The stability region using the Manning formula is larger than that using the Chézy formula. The combination of these effects may stabilize or destabilize the shallow jet flow. These results of the stability analysis are compared with those from the literature. Originality/value – Results of linear stability analysis on shallow jets along roughness bottom bed are presented. Different from the previous studies, this paper includes the effects of bottom topography, Rossby number, Reynolds number, resistance formula and bed friction. It is found that the influence of Reynolds number on the stability of the jet is notable for relative small value. Therefore, it is important to experimental investigators that the viscosity should be considered with comparison to the results from inviscid assumption. In contrast with the classical analysis, the use of multi-parameters of the base velocity and topographic profile gives an extension to the jet stability analysis. To characterize the large scale motion, besides the bottom friction as proposed in the related literature, the Reynolds number Re, Rossby number Ro, the topographic parameters and parameters controlling base velocity profile may also be important to the stability analysis of shallow jet flows.

scholarly journals Efficient Production of l-Ribose with a Recombinant Escherichia coli Biocatalyst

2008 ◽  
Vol 74 (10) ◽  
pp. 2967-2975 ◽  
Author(s):  
Ryan D. Woodyer ◽  
Nathan J. Wymer ◽  
F. Michael Racine ◽  
Shama N. Khan ◽  
Badal C. Saha
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Active Form ◽  
Apium Graveolens ◽  
Efficient Production ◽  
Scale Production ◽  
Gene Encoding ◽  
Large Scale Production ◽  
One Step ◽  
Rare Sugars

ABSTRACT A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.

scholarly journals Call for papers on special issue “Perovskite photovoltaics and optoelectronic devices”

2020 ◽  
Vol 2 (1) ◽  
pp. 049-049
Keyword(s):  
Solar Cells ◽  
Large Scale ◽  
Perovskite Solar Cells ◽  
Optoelectronic Devices ◽  
Scale Production ◽  
Special Issue ◽  
Device Structure ◽  
Stability Performance ◽  
Large Scale Production ◽  
The Stability

Aim & Scope: Metal halide perovskitehave been regarded as promising classes of materials for photovoltaics and optoelectronic devices, owing to the unique characteristics, such as long charge carrier diffusion lengths, precise tunable bandgaps, high light absorption coefficients, and high defect tolerance. Research on perovskite in the fields including photovoltaics, light-emitting diodes, lasers, X-ray imaging, and photodetectors has been gaining increasingly interest over the past years. Up to now, the efficiency of perovskite solar cells has grown from 3.8% in single-junction solar cells in 2009 to more than 25%, catching up the efficiency level of commercial silicon cells. Up to now, the key issues of perovskite photovoltaics and optoelectronic devices have become the stability, performance and large-scale production. This requires optimization of the film morphology, interface, device structure and the fabrication process. A lot work has been done on this issue and has made remarkable progress. We kindly invite you to submit a manuscript(s) for this Special Issue. Full papers, communications, and reviews are all welcome.

scholarly journals Metal-Organic Frameworks for Metal-Ion Batteries: Towards Scalability

2021 ◽  
Vol 8 (3) ◽  
pp. 20210304
Author(s):  
Semyon Bachinin ◽  
Venera Gilemkhanova ◽  
Maria Timofeeva ◽  
Yuliya Kenzhebayeva ◽  
Andrei Yankin ◽  
...  
Keyword(s):  
Material Science ◽  
Metal Ion ◽  
Large Scale ◽  
Gas Adsorption ◽  
Metal Organic Frameworks ◽  
Scale Production ◽  
Structural Elements ◽  
Large Scale Production ◽  
Metal Organic ◽  
And Storage

Metal-organic frameworks (MOFs), being a family of highly crystalline and porous materials, have attracted particular attention in material science due to their unprecedented chemical and structural tunability. Next to their application in gas adsorption, separation, and storage, MOFs also can be utilized for energy transfer and storage in batteries and supercapacitors. Based on recent studies, this review describes the latest developments about MOFs as structural elements of metal-ion battery with a focus on their industry-oriented and large-scale production.

Barriers to intergenerational learning: a case of a workplace in Turkey

2020 ◽  
Vol 41 (3) ◽  
pp. 431-447
Author(s):  
Soner Polat ◽  
Yılmazer Yılmaz
Keyword(s):  
Large Scale ◽  
Study Data ◽  
Intergenerational Learning ◽  
Scale Production ◽  
Personal Factors ◽  
Structured Interviews ◽  
Content Type ◽  
Organization And Management ◽  
Relational Factors ◽  
The Hierarchical Structure

PurposeThe purpose of this study is to determine barriers to intergenerational learning.Design/methodology/approachThe research design is phenomenological, which is a qualitative research method. The study was carried out in a large-scale production facility in the Central District of Kocaeli, Turkey, and the study group of the research consists of 61 participants determined using the maximum diversity technique, one of the purposeful sampling techniques. Significant attention was paid to the examination of employees from all levels and every generation within the hierarchical structure of the company while selecting the working groups for the study. Data were collected through semi-structured interviews, and content analysis method was applied in the analysis of the data. Participants were questioned about barriers to their prejudices.FindingsAs a result of the research, it was observed that barriers “to being a leader for intergenerational learning” were gathered under three distinct themes: personal factors, relational factors and managerial factors. The most frequently stated subthemes of these were: “seeing knowledge and experience as a power” and “lack of job commitment and motivation” under personal factors, “communication problems” under relational factors and “insufficient support or not being supported or encouraged by management” under managerial factors.Originality/valueThis study should be of interest to researchers interested in educational organization use of barriers to intergenerational learning: a case of a workplace in Turkey, use of intergenerational learning by organization and management.

Decentralized fuzzy C-means robust algorithm for continuous systems

2019 ◽  
Vol 92 (2) ◽  
pp. 222-228
Author(s):  
Tim Chen ◽  
J.C.Y. Chen
Keyword(s):  
Stability Criterion ◽  
Large Scale ◽  
Clustering Algorithm ◽  
Closed Loop ◽  
Linear Matrix ◽  
Model Description ◽  
Continuous Systems ◽  
Content Type ◽  
Fuzzy C Means ◽  
The Stability

Purpose This paper aims to address the robust controller design problem for a class of fuzzy C-means clustering algorithm that is robust against both the plant parameter perturbations and controller gain variations. Based on Takagi–Sugeno (T-S) fuzzy model description, the stability and control problems of nonlinear systems are studied. Design/methodology/approach A recently proposed integral inequality is selected based on the free-weight matrix, and the less conservative stability criterion is given in the form of linear matrix inequalities (LMIs). Findings Under the premise that the controller and the system share the same, the method does not require the number of membership functions and rules. Practical implications Furthermore, the modified controller in a large-scale nonlinear system is utilized as a stability criterion for a closed-loop T-S fuzzy system obtained by LMI, and is rearranged by a machine learning membership function. Originality/value The closed-loop controller criterion is derived by energy functions to guarantee the stability of systems. Finally, an example is given to demonstrate the results.

Heterologous Expression, Characterization and Evolution Prediction of a Diaphorase From Geobacillus sp. Y4.1MC1

2021 ◽  
Author(s):  
Jinzhao Shang ◽  
Shuohao Yue ◽  
Fang Zeng ◽  
Yun Chen ◽  
Longgang Jia
Keyword(s):  
Large Scale ◽  
Ketone Bodies ◽  
Scale Production ◽  
Hydroxybutyric Acid ◽  
E Coli ◽  
Ph Values ◽  
Structure Changes ◽  
Large Scale Production ◽  
Expression Characterization ◽  
The Stability

Abstract β-hydroxybutyric acid is the most sensitive indicator in ketoacidosis detection, and accounts for nearly 78% of the ketone bodies. Diaphorase is commonly used to detect the β-hydroxybutyric acid in clinical diagnosis. However, the extraction of diaphorase from animal myocardium is complex and low-yield, which is not convenient for large-scale production. In this study, a diaphorase from Geobacillus sp. Y4.1MC1 was efficiently heterologous expressed and purified in E. coli a yield of 110 mg/L culture. The optimal temperature and pH of this recombinant diaphorase (rDIA) were 55 °C and 6.5, respectively. It was proved that rDIA was a dual acid- and thermo-stable enzyme, and which showed much more accurate detection of β-hydroxybutyric acid than the commercial enzyme. Additionally, we also investigated the molecular interaction of rDIA with the substrate, and the conformation transition in different pH values by using homology modeling and molecular dynamics (MD) simulation. The results showed that 141-161 domain of rDIA played important role in the structure changes and conformations transmission at different pH values. Moreover, it was predicted that F105W, F105R and M186R mutants were able to improve the binding affinity of rDIA, and A2Y, P35F, Q36D, N210L, F211Y mutants were benefit for the stability of rDIA.

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