• Title/Summary/Keyword: ATP regeneration

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In-vitro Production of Glutathione Using Yeast ATP Regeneration System and Recombinant Synthetic Enzymes from Escherichia coli. (효모의 ATP 재생산계와 대장균 유래의 재조합 생산효소를 이용한 in vitro 글루타치온 생산)

  • 고성영;구윤모
    • Microbiology and Biotechnology Letters
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    • v.26 no.3
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    • pp.213-220
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    • 1998
  • An ATP regeneration system was used for the production of glutathione which was synthesized by a sequential action of ${\gamma}$-glutamyl-cysteine synthetase and glutathione synthetase. The synthetases above were produced in the recombinant E. coli (TG1/pDG7) with the highest specific production yield of 31 mg glutathione/g wet cell. Bakers yeast was considered to have economically a better ATP regeneration system although the glutathione production yield was lower than that of acetate kinase. It was also observed that the ATP regeneration system of bakers yeast was superior to that of Saccharomyces cerevisiae ATCC24858. The yield of glutathione production with bakers yeast was 36% with the ATP concentration of 5 mM. To avoid the cysteine limitation during the early phase of glutatione production, an extra cysteine was added at 2 hours after reaction and the production yield increased 1.91 times. The effectiveness of bakers yeast as an ATP regeneration system was proved by several sets of extra feeding experiments. The product inhibition by glutathione above 14 mM was also observed.

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Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

  • Lee, Sung-Ju;Kwon, Chae-Hwa;Kim, Yong-Keun
    • The Korean Journal of Physiology and Pharmacology
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    • v.13 no.1
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    • pp.15-22
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    • 2009
  • This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide(KCN)/0.1 mM iodoacetic acid(IAA), and membrane transport function and cell viability were evaluated by measuring $Na^+$-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in $Na^+$-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited $Na^+$-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a $Na^+$ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in $Na^+$-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers(dimethylthiourea and thiourea), and amino acids(glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase $A_2(cPLA_2)$. The ATP depletion-dependent arachidonic acid release was inhibited by $cPLA_2$ specific inhibitor $AACOCF_3$. ATP depletion-induced alterations in $Na^+$-dependent phosphate uptake and cell viability were prevented by $AACOCF_3$. Inhibition of $Na^+$-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and $cPLA_2$ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

One-pot Enzymatic Synthesis of UDP-D-glucose from UMP and Glucose-1-phosphate Using an ATP Regeneration System

  • Lee, Hei-Chan;Lee, Seung-Don;Sohng, Jae-Kyung;Liou, Kwang-Kyoung
    • BMB Reports
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    • v.37 no.4
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    • pp.503-506
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    • 2004
  • Glucose-1-phosphate uridylyltransferase from E. coli K12 was used to convert uridine-5'-triphosphate and glucose-1-phosphate to UDP-D-glucose. The conversion was efficient and completed within 5 minutes under the employed conditions. In addition, thymidine-5'-monophosphate kinase and acetate kinase were proven to be non-specific, converting udridine-5'-monophosphate to uridine-5'-triphosphate with 55% conversion after 6 h, which was much slower than the production of TTP under the same conditions (complete conversion within one hour). Since these two reactions could proceed under the same conditions, a one-pot synthesis of UDP-D-glucose with ATP regeneration was designed from easily available starting materials, and conversion up to 40% by HPLC peak integration was achieved given a reaction time of 4 h.

Regeneration of ATP through an Activated Glycolytic Pathway in a Cell-free Extract and its Application for Protein Expression (해당과정의 활성화를 통한 무세포 단백질 발현 시스템에서의 ATP 재생)

  • Kim Dong Myung;Keum Jeong Won;Kim Tae Wan;Oh In Seok;Choi Cha-yong
    • KSBB Journal
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    • v.19 no.6 s.89
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    • pp.467-470
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    • 2004
  • We have investigated the key parameters affecting ATP regeneration in a cell-free protein synthesis system derived from Escherichia coli. When glucose-6-phosphate was used as an energy source, the efficiency of ATP regeneration sharply responded to pH change of reaction mixture. In addition, both productivity and reproducibility of protein synthesis was substantially enhanced by introducing appropriate amount of NAD into the reaction mixture. As a result, through the activation of glycolytic pathway under an optimal pH, the batch cell-free system produced over $300\;{\mu}g$ of protein in a 1 mL reaction.

Enhancement of Glucose-Fueled Cell-Free Protein Synthesis by the Addition of Lipids (지질의 첨가를 통한 포도당 기반 무세포 단백질 합성 시스템의 단백질 발현 효율 향상)

  • Lee, So Jeong;Kim, Ho-Cheol;Kim, Dong-Myung
    • Korean Chemical Engineering Research
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    • v.57 no.1
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    • pp.85-89
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    • 2019
  • Cell-free protein synthesis utilizes the translational machinery in a cell extract. Unlike the conventional cell-based expression methods, not being affected by the conditions for cell growth, cell-free protein synthesis enables flexible manipulation of individual factors affecting the efficiency protein biosynthesis. However, the high cost and low stability of the energy sources to regenerate ATP have limited the use of cell-free synthesis for large-scale production of recombinant proteins. One of the approaches to address this problem is to use glucose as an alternative energy source to regenerate ATP through the glucose-metabolizing pathways in a cell extract. In this study, in an attempt to improve the efficiency of ATP regeneration by reinforcing oxidative phosphorylation process, we supplemented with cellular lipids to a glucose-fueled reaction mixture for cell-free protein synthesis. As a result of the lipid supplementation, the productivity of chloramphenicol acetyltransferase in a cell-free synthesis system using glucose increased more than 6 fold compared to when the lipid was not supplemented.

Proposed Mechanisms of Photobiomodulation (PBM) Mediated via the Stimulation of Mitochondrial Activity in Peripheral Nerve Injuries

  • Choi, Ji Eun
    • Medical Lasers
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    • v.10 no.4
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    • pp.195-200
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    • 2021
  • Evidence shows that nerve injury triggers mitochondrial dysfunction during axonal degeneration. Mitochondria play a pivotal role in axonal regeneration. Therefore, normalizing mitochondrial energy metabolism may represent an elective therapeutic strategy contributing to nerve recovery after damage. Photobiomodulation (PBM) induces a photobiological effect by stimulating mitochondrial activity. An increasing body of evidence demonstrates that PBM improves ATP generation and modulates many of the secondary mediators [reactive oxygen species (ROS), nitric oxide (NO), cyclic adenosine monophosphate (cAMP), and calcium ions (Ca2+)], which in turn activate multiple pathways involved in axonal regeneration.

Transformation of Glutamic Acid to Glutamine by E. coli Glutamine Synthetase (E. coli Glutamine Synthetase에 의한 Glutamic Acid로부터 Glutamine 으로의 전환)

  • 전복환;신형순;이왕식;방원기
    • Microbiology and Biotechnology Letters
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    • v.17 no.4
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    • pp.379-384
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    • 1989
  • Glutamine production from glutamate was carried out using glutamine synthetase from E. coli K-12 pgln 6 and baker's yeast, which supplies ATP into the reaction system through alcohol fermentation, simultaneously. With whole cells of E. coli K-12 pgln 6 as an enzyme source of glutamine synthetase, 11.8 g/ι of glutamine produced after 18-h incubation (60% yield based on a substrate, glutamate). Using the partially purified glutamine synthetase, 19.8 git of glutamine was produced after 5-h incubation. This amount of glutamine was correspond to 90% yield, based on substrate, glutamate.

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Fermentative Production of 5'-GMP from 5'-XMP by XMP aminase and ATP-generation System of Saccharomyces cerevisiae (효모 Saccharomyces cevevisiae의 ATP 생성계와 XMP aminase에 의한 5'-XMP로부터 5'-GMP 발효생산)

  • Cho, Jung-Il
    • The Korean Journal of Mycology
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    • v.21 no.4
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    • pp.285-292
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    • 1993
  • For the enzymatic conversion of 5'-XMP to 5'-GMP, partially purified XMP aminase from Escherichia coli was coupled with the yeast, Saccharomycrs cerevisiae, capable of ATP regeneration through glycolytic pathway. In order to elevate the level of XMP aminase in E. coli, $guaB^{-}(IMP\;dehydrogenase-less)$ mutant were introduced, and the yeast used as ATP supplier was treated by some method to increase its membrane permeability. The optimum conditions for efficient conversion reaction by energy-coupled system were investigated. As the results, a CH 41, $guaB^-$ mutant of E. coli K-12, showed 2.75 fold increase in the level of XMP aminase, compared with its parent cell. And the lyophylized yeast was the most effective at the ATP supplier. The optimum temperature and pH of conversion reaction were $40{\circ]C$ and pH 7.4, and the highest conversion ratio was shown under the reaction condition of 100 mM glucose, 100 mM inorganic phosphate and 6 mM AMP. When 36 units/ml XMP aminase used under the above conditions, the amount of 60 mg/ml yeast was sufficient to be used. Under the optimum condition, 71% of 1.8 mM(65.6 mg/100 ml) 5'-XMP was converted to 5'-GMP within 8 hr.

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Continuous Cell-Free Protein Synthesis Using Glycolytic Intermediates as Energy Sources

  • Kim, Ho-Cheol;Kim, Tae-Wan;Park, Chang-Gil;Oh, In-Seok;Park, Kyung-Moon;Kim, Dong-Myung
    • Journal of Microbiology and Biotechnology
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    • v.18 no.5
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    • pp.885-888
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    • 2008
  • In this work, we demonstrate that glycolytic intermediates can serve as efficient energy sources to regenerate ATP during continuous-exchange cell-free (CECF) protein synthesis reactions. Through the use of an optimal energy source, approximately 10 mg/ml of protein was generated from a CECF protein synthesis reaction at greatly reduced reagent costs. Compared with the conventional reactions utilizing phosphoenol pyruvate as an energy source, the described method yields 10-fold higher productivity per unit reagent cost, making the techniques of CECF protein synthesis a more realistic alternative for rapid protein production.

Development of Intelligent ATP System Using Genetic Algorithm (유전 알고리듬을 적용한 지능형 ATP 시스템 개발)

  • Kim, Tai-Young
    • Journal of Intelligence and Information Systems
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    • v.16 no.4
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    • pp.131-145
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    • 2010
  • The framework for making a coordinated decision for large-scale facilities has become an important issue in supply chain(SC) management research. The competitive business environment requires companies to continuously search for the ways to achieve high efficiency and lower operational costs. In the areas of production/distribution planning, many researchers and practitioners have developedand evaluated the deterministic models to coordinate important and interrelated logistic decisions such as capacity management, inventory allocation, and vehicle routing. They initially have investigated the various process of SC separately and later become more interested in such problems encompassing the whole SC system. The accurate quotation of ATP(Available-To-Promise) plays a very important role in enhancing customer satisfaction and fill rate maximization. The complexity for intelligent manufacturing system, which includes all the linkages among procurement, production, and distribution, makes the accurate quotation of ATP be a quite difficult job. In addition to, many researchers assumed ATP model with integer time. However, in industry practices, integer times are very rare and the model developed using integer times is therefore approximating the real system. Various alternative models for an ATP system with time lags have been developed and evaluated. In most cases, these models have assumed that the time lags are integer multiples of a unit time grid. However, integer time lags are very rare in practices, and therefore models developed using integer time lags only approximate real systems. The differences occurring by this approximation frequently result in significant accuracy degradations. To introduce the ATP model with time lags, we first introduce the dynamic production function. Hackman and Leachman's dynamic production function in initiated research directly related to the topic of this paper. They propose a modeling framework for a system with non-integer time lags and show how to apply the framework to a variety of systems including continues time series, manufacturing resource planning and critical path method. Their formulation requires no additional variables or constraints and is capable of representing real world systems more accurately. Previously, to cope with non-integer time lags, they usually model a concerned system either by rounding lags to the nearest integers or by subdividing the time grid to make the lags become integer multiples of the grid. But each approach has a critical weakness: the first approach underestimates, potentially leading to infeasibilities or overestimates lead times, potentially resulting in excessive work-inprocesses. The second approach drastically inflates the problem size. We consider an optimized ATP system with non-integer time lag in supply chain management. We focus on a worldwide headquarter, distribution centers, and manufacturing facilities are globally networked. We develop a mixed integer programming(MIP) model for ATP process, which has the definition of required data flow. The illustrative ATP module shows the proposed system is largely affected inSCM. The system we are concerned is composed of a multiple production facility with multiple products, multiple distribution centers and multiple customers. For the system, we consider an ATP scheduling and capacity allocationproblem. In this study, we proposed the model for the ATP system in SCM using the dynamic production function considering the non-integer time lags. The model is developed under the framework suitable for the non-integer lags and, therefore, is more accurate than the models we usually encounter. We developed intelligent ATP System for this model using genetic algorithm. We focus on a capacitated production planning and capacity allocation problem, develop a mixed integer programming model, and propose an efficient heuristic procedure using an evolutionary system to solve it efficiently. This method makes it possible for the population to reach the approximate solution easily. Moreover, we designed and utilized a representation scheme that allows the proposed models to represent real variables. The proposed regeneration procedures, which evaluate each infeasible chromosome, makes the solutions converge to the optimum quickly.