Research status and development of application fields in enzyme technology

NASA Astrophysics Data System (ADS)

Ji, Y. B.; Wang, S. W.; Yu, M.; Ru, X.; Wei, C.; Zhu, H. J.; Li, Z. Y.; Zhao, H.; Qiao, A. N.; Guo, S. Z.; Lu, L.

2018-01-01

Biological enzymes are catalyzed by living cells, most of which are proteins, and very few are RNA. Biological engineering as a new high-tech has been rapid development, Enzyme manufacturing and application areas are gradually expanding, In this paper, the status and progress of the application of enzyme technology are reviewed by reviewing the literature. and aims to provide reference for the application of enzyme technology and provide scientific basis for its future research and development in new field.

Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prasannan, Priya; Suliman, Huda S.; Robertus, Jon D., E-mail: jrobertus@mail.utexas.edu

2009-05-15

Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sitesmore » were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.« less

Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts.

PubMed

Prier, Christopher K; Arnold, Frances H

2015-11-11

Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.

[Advances on enzymes and enzyme inhibitors research based on microfluidic devices].

PubMed

Hou, Feng-Hua; Ye, Jian-Qing; Chen, Zuan-Guang; Cheng, Zhi-Yi

2010-06-01

With the continuous development in microfluidic fabrication technology, microfluidic analysis has evolved from a concept to one of research frontiers in last twenty years. The research of enzymes and enzyme inhibitors based on microfluidic devices has also made great progress. Microfluidic technology improved greatly the analytical performance of the research of enzymes and enzyme inhibitors by reducing the consumption of reagents, decreasing the analysis time, and developing automation. This review focuses on the development and classification of enzymes and enzyme inhibitors research based on microfluidic devices.

de novo computational enzyme design.

PubMed

Zanghellini, Alexandre

2014-10-01

Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Technological advances and applications of hydrolytic enzymes for valorization of lignocellulosic biomass.

PubMed

Manisha; Yadav, Sudesh Kumar

2017-12-01

Hydrolytic enzymes are indispensable tools in the production of various foodstuffs, drugs, and consumables owing to their applications in almost every industrial process nowadays. One of the foremost areas of interest involving the use of hydrolytic enzymes is in the transformation of lignocellulosic biomass into value added products. However, limitations of the processes due to inadequate enzyme activity and stability with a narrow range of pH and temperature optima often limit their effective usage. The innovative technologies, involving manipulation of enzyme activity and stability through mutagenesis, genetic engineering and metagenomics lead to a major leap in all the fields using hydrolytic enzymes. This article provides recent advancement towards the isolation and use of microbes for lignocellulosic biomass utilisation, microbes producing the hydrolytic enzymes, the modern age technologies used to manipulate and enhance the hydrolytic enzyme activity and the applications of such enzymes in value added products development from lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

Advances in Targeted Pesticides with Environmentally Responsive Controlled Release by Nanotechnology

PubMed Central

Huang, Bingna; Chen, Feifei; Shen, Yue; Wang, Yan; Sun, Changjiao; Zhao, Xiang; Cui, Bo; Gao, Fei; Zeng, Zhanghua; Cui, Haixin

2018-01-01

Pesticides are the basis for defending against major biological disasters and important for ensuring national food security. Biocompatible, biodegradable, intelligent, and responsive materials are currently an emerging area of interest in the field of efficient, safe, and green pesticide formulation. Using nanotechnology to design and prepare targeted pesticides with environmentally responsive controlled release via compound and chemical modifications has also shown great potential in creating novel formulations. In this review, special attention has been paid to intelligent pesticides with precise controlled release modes that can respond to micro-ecological environment changes such as light-sensitivity, thermo-sensitivity, humidity sensitivity, soil pH, and enzyme activity. Moreover, establishing intelligent and controlled pesticide release technologies using nanomaterials are reported. These technologies could increase pesticide-loading, improve the dispersibility and stability of active ingredients, and promote target ability. PMID:29439498

Pre-set extrusion bioprinting for multiscale heterogeneous tissue structure fabrication.

PubMed

Kang, Donggu; Ahn, Geunseon; Kim, Donghwan; Kang, Hyun-Wook; Yun, Seokhwan; Yun, Won-Soo; Shim, Jin-Hyung; Jin, Songwan

2018-06-06

Recent advances in three-dimensional bioprinting technology have led to various attempts in fabricating human tissue-like structures. However, current bioprinting technologies have limitations for creating native tissue-like structures. To resolve these issues, we developed a new pre-set extrusion bioprinting technique that can create heterogeneous, multicellular, and multimaterial structures simultaneously. The key to this ability lies in the use of a precursor cartridge that can stably preserve a multimaterial with a pre-defined configuration that can be simply embedded in a syringe-based printer head. The multimaterial can be printed and miniaturized through a micro-nozzle without conspicuous deformation according to the pre-defined configuration of the precursor cartridge. Using this system, we fabricated heterogeneous tissue-like structures such as spinal cords, hepatic lobule, blood vessels, and capillaries. We further obtained a heterogeneous patterned model that embeds HepG2 cells with endothelial cells in a hepatic lobule-like structure. In comparison with homogeneous and heterogeneous cell printing, the heterogeneous patterned model showed a well-organized hepatic lobule structure and higher enzyme activity of CYP3A4. Therefore, this pre-set extrusion bioprinting method could be widely used in the fabrication of a variety of artificial and functional tissues or organs.

Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Challenges and Opportunities.

PubMed

Heinisch, Tillmann; Ward, Thomas R

2016-09-20

The biotin-streptavidin technology offers an attractive means to engineer artificial metalloenzymes (ArMs). Initiated over 50 years ago by Bayer and Wilchek, the biotin-(strept)avidin techonology relies on the exquisite supramolecular affinity of either avidin or streptavidin for biotin. This versatile tool, commonly referred to as "molecular velcro", allows nearly irreversible anchoring of biotinylated probes within a (strept)avidin host protein. Building upon a visionary publication by Whitesides from 1978, several groups have been exploiting this technology to create artificial metalloenzymes. For this purpose, a biotinylated organometallic catalyst is introduced within (strept)avidin to afford a hybrid catalyst that combines features reminiscent of both enzymes and organometallic catalysts. Importantly, ArMs can be optimized by chemogenetic means. Combining a small collection of biotinylated organometallic catalysts with streptavidin mutants allows generation of significant diversity, thus allowing optimization of the catalytic performance of ArMs. Pursuing this strategy, the following reactions have been implemented: hydrogenation, alcohol oxidation, sulfoxidation, dihydroxylation, allylic alkylation, transfer hydrogenation, Suzuki cross-coupling, C-H activation, and metathesis. In this Account, we summarize our efforts in the latter four reactions. X-ray analysis of various ArMs based on the biotin-streptavidin technology reveals the versatility and commensurability of the biotin-binding vestibule to accommodate and interact with transition states of the scrutinized organometallic transformations. In particular, streptavidin residues at positions 112 and 121 recurrently lie in close proximity to the biotinylated metal cofactor. This observation led us to develop a streamlined 24-well plate streptavidin production and screening platform to optimize the performance of ArMs. To date, most of the efforts in the field of ArMs have focused on the use of purified protein samples. This seriously limits the throughput of the optimization process. With the ultimate goal of complementing natural enzymes in the context of synthetic and chemical biology, we outline the milestones required to ultimately implement ArMs within a cellular environment. Indeed, we believe that ArMs may allow signficant expansion of the natural enzymes' toolbox to access new-to-nature reactivities in vivo. With this ambitious goal in mind, we report on our efforts to (i) activate the biotinylated catalyst precursor upon incorporation within streptavidin, (ii) minimize the effect of the cellular environment on the ArM's performance, and (iii) demonstrate the compatibility of ArMs with natural enzymes in cascade reactions.

Structure of Escherichia coli dGTP Triphosphohydrolase: A Hexameric Enzyme with DNA Effector Molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Deepa; Gawel, Damian; Itsko, Mark

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present paper, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNAmore » with high affinity (K d ~ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Finally, our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.« less

Structure of Escherichia coli dGTP Triphosphohydrolase: A Hexameric Enzyme with DNA Effector Molecules

DOE PAGES

Singh, Deepa; Gawel, Damian; Itsko, Mark; ...

2015-02-18

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present paper, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNAmore » with high affinity (K d ~ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Finally, our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.« less

Printable enzyme-embedded materials for methane to methanol conversion

DOE PAGES

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.; ...

2016-06-15

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scalemore » structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions.« less

Printable enzyme-embedded materials for methane to methanol conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scalemore » structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions.« less

Printable enzyme-embedded materials for methane to methanol conversion

PubMed Central

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.; DeOtte, Joshua R.; Oakdale, James S.; Maiti, Amitesh; Lenhardt, Jeremy M.; Sirajuddin, Sarah; Rosenzweig, Amy C.; Baker, Sarah E.

2016-01-01

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scale structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions. PMID:27301270

Phenylketonuria

MedlinePlus

... inherited disorder that causes an amino acid called phenylalanine to build up in the body. PKU is ... helps create the enzyme needed to break down phenylalanine. Without the enzyme necessary to process phenylalanine, a ...

Accelerating the Rate-Limiting Step in Novel Enzymatic Carbohydrate-to-Hydrogen Technology by Enzyme Engineering

DTIC Science & Technology

2011-10-30

stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low- cost high-capacity celiulosic...NOVEL ENZYMATIC CARBOHYDRATE-TO-HYDROGEN TECHNOLOGY BY ENZYME ENGINEERING Grant/Contract Number: FA9550-08-1-0145 Program Manager: Dr. Walt...bbtransformation (SyPaB) is the implementation of complicated biochemical reactions by in vitro assembly of enzyme and coenzymes. Different from in vivo

Process for preparing multilayer enzyme coating on a fiber

DOEpatents

Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

2009-11-03

A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

Approach to Cultural Asset Preservation "Enzyme Filter Media Has Bactericidal Effect and Bacteriostasis Effect in Air Phase"

NASA Astrophysics Data System (ADS)

Isomae, Kazuro

Enzyme air-filter media according to the bactericidal effect as an environmental green technology acquired the high appraisal and the result in the domestic and foreign clean room and the air conditioning field. The mechanism of this enzyme technology, safety, and the bactericidal effect in the real environment are discussed by using the electron microscopic picture etc. And it proposes to apply these technologies to the cultural asset preservation.

Green polymer chemistry: enzyme catalysis for polymer functionalization.

PubMed

Sen, Sanghamitra; Puskas, Judit E

2015-05-21

Enzyme catalyzed reactions are green alternative approaches to functionalize polymers compared to conventional methods. This technique is especially advantageous due to the high selectivity, high efficiency, milder reaction conditions, and recyclability of enzymes. Selected reactions can be conducted under solventless conditions without the application of metal catalysts. Hence this process is becoming more recognized in the arena of biomedical applications, as the toxicity created by solvents and metal catalyst residues can be completely avoided. In this review we will discuss fundamental aspects of chemical reactions biocatalyzed by Candida antarctica lipase B, and their application to create new functionalized polymers, including the regio- and chemoselectivity of the reactions.

The synthesis of starch from carbon dioxide using isolubilized stabilized enzymes

NASA Technical Reports Server (NTRS)

Bassham, J. A.; Bearden, L.; Wilke, C.; Carroad, P.; Mitra, G.; Ige, R.

1972-01-01

Systems for artificial manufacture of starch and for delineation of technological areas, and the rationale for studying them are considered. A discussion of the enzyme-catalyzed routes of synthesis available and a choice as to the most promising route are presented. A discussion of the enzymes involved, of enzyme insolubilization technology, and of possible engineering approaches, with examples in the form of model calculations for both reactors and separators, are also presented.

An Integrated Circuit for Chip-Based Analysis of Enzyme Kinetics and Metabolite Quantification.

PubMed

Cheah, Boon Chong; Macdonald, Alasdair Iain; Martin, Christopher; Streklas, Angelos J; Campbell, Gordon; Al-Rawhani, Mohammed A; Nemeth, Balazs; Grant, James P; Barrett, Michael P; Cumming, David R S

2016-06-01

We have created a novel chip-based diagnostic tools based upon quantification of metabolites using enzymes specific for their chemical conversion. Using this device we show for the first time that a solid-state circuit can be used to measure enzyme kinetics and calculate the Michaelis-Menten constant. Substrate concentration dependency of enzyme reaction rates is central to this aim. Ion-sensitive field effect transistors (ISFET) are excellent transducers for biosensing applications that are reliant upon enzyme assays, especially since they can be fabricated using mainstream microelectronics technology to ensure low unit cost, mass-manufacture, scaling to make many sensors and straightforward miniaturisation for use in point-of-care devices. Here, we describe an integrated ISFET array comprising 2(16) sensors. The device was fabricated with a complementary metal oxide semiconductor (CMOS) process. Unlike traditional CMOS ISFET sensors that use the Si3N4 passivation of the foundry for ion detection, the device reported here was processed with a layer of Ta2O5 that increased the detection sensitivity to 45 mV/pH unit at the sensor readout. The drift was reduced to 0.8 mV/hour with a linear pH response between pH 2-12. A high-speed instrumentation system capable of acquiring nearly 500 fps was developed to stream out the data. The device was then used to measure glucose concentration through the activity of hexokinase in the range of 0.05 mM-231 mM, encompassing glucose's physiological range in blood. Localised and temporal enzyme kinetics of hexokinase was studied in detail. These results present a roadmap towards a viable personal metabolome machine.

Study on the technology of compound enzymatic hydrolysis of whole passion fruit

NASA Astrophysics Data System (ADS)

Yang, Yu-xia; Duan, Zhen-hua; Kang, Chao; Zhu, Xiang-hao; Li, Ding-jin

2017-12-01

Fresh Whole Passion Fruit was used as raw material, The enzymatic hydrolysis technology of Passion Fruit by Complex enzyme were studied, The effects of enzyme dosage, Enzyme ratio(cellulose: pectinase), pH, temperature and time on the hydrolysis were investigated by single-tests and orthogonal tests, the hydrolysis indicators of single-factor tests and orthogonal tests were juice yield. The optimal hydrolysis conditions of Passion Fruit by Complex enzyme were enzyme dosage 0.12%, Enzyme ratio 5:1, hydrolysis temperature 50°C, pH4.0 and time 3.5 h. Under such conditions, juice yield of Passion Fruit was 92.91%.

Technology Prospecting on Enzymes: Application, Marketing and Engineering

PubMed Central

Li, Shuang; Yang, Xiaofeng; Yang, Shuai; Zhu, Muzi; Wang, Xiaoning

2012-01-01

Enzymes are protein molecules functioning as specialized catalysts for chemical reactions. They have contributed greatly to the traditional and modern chemical industry by improving existing processes. In this article, we first give a survey of representative industrial applications of enzymes, focusing on the technical applications, feed industry, food processing and cosmetic products. The recent important developments and applications of enzymes in industry are reviewed. Then large efforts are dedicated to the worldwide enzyme market from the demand and production perspectives. Special attention is laid on the Chinese enzyme market. Although enzyme applications are being developed in full swing, breakthroughs are needed to overcome their weaknesses in maintaining activities during the catalytic processes. Strategies of metagomic analysis, cell surface display technology and cell-free system might give valuable solutions in novel enzyme exploiting and enzyme engineering. PMID:24688658

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6.

PubMed

Pereira, Jose Henrique; Heins, Richard A; Gall, Daniel L; McAndrew, Ryan P; Deng, Kai; Holland, Keefe C; Donohue, Timothy J; Noguera, Daniel R; Simmons, Blake A; Sale, Kenneth L; Ralph, John; Adams, Paul D

2016-05-06

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50-70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pereira, Jose Henrique; Heins, Richard A.; Gall, Daniel L.

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding tomore » 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.« less

Empirical evaluation of a virtual laboratory approach to teach lactate dehydrogenase enzyme kinetics.

PubMed

Booth, Christine; Cheluvappa, Rajkumar; Bellinson, Zack; Maguire, Danni; Zimitat, Craig; Abraham, Joyce; Eri, Rajaraman

2016-06-01

Personalised instruction is increasingly recognised as crucial for efficacious learning today. Our seminal work delineates and elaborates on the principles, development and implementation of a specially-designed adaptive, virtual laboratory. We strived to teach laboratory skills associated with lactate dehydrogenase (LDH) enzyme kinetics to 2nd-year biochemistry students using our adaptive learning platform. Pertinent specific aims were to:(1)design/implement a web-based lesson to teach lactate dehydrogenase(LDH) enzyme kinetics to 2nd-year biochemistry students(2)determine its efficacious in improving students' comprehension of enzyme kinetics(3)assess their perception of its usefulness/manageability(vLab versus Conventional Tutorial). Our tools were designed using HTML5 technology. We hosted the program on an adaptive e-learning platform (AeLP). Provisions were made to interactively impart informed laboratory skills associated with measuring LDH enzyme kinetics. A series of e-learning methods were created. Tutorials were generated for interactive teaching and assessment. The learning outcomes herein were on par with that from a conventional classroom tutorial. Student feedback showed that the majority of students found the vLab learning experience "valuable"; and the vLab format/interface "well-designed". However, there were a few technical issues with the 1st roll-out of the platform. Our pioneering effort resulted in productive learning with the vLab, with parity with that from a conventional tutorial. Our contingent discussion emphasises not only the cornerstone advantages, but also the shortcomings of the AeLP method utilised. We conclude with an astute analysis of possible extensions and applications of our methodology.

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6

DOE PAGES

Pereira, Jose Henrique; Heins, Richard A.; Gall, Daniel L.; ...

2016-03-03

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding tomore » 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.« less

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6*

PubMed Central

Pereira, Jose Henrique; Heins, Richard A.; Gall, Daniel L.; McAndrew, Ryan P.; Deng, Kai; Holland, Keefe C.; Donohue, Timothy J.; Noguera, Daniel R.; Simmons, Blake A.; Sale, Kenneth L.; Ralph, John; Adams, Paul D.

2016-01-01

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts. PMID:26940872

Applications of Microbial Enzymes in Food Industry.

PubMed

Raveendran, Sindhu; Parameswaran, Binod; Ummalyma, Sabeela Beevi; Abraham, Amith; Mathew, Anil Kuruvilla; Madhavan, Aravind; Rebello, Sharrel; Pandey, Ashok

2018-03-01

The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed.

Applications of Microbial Enzymes in Food Industry

PubMed Central

2018-01-01

Summary The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed. PMID:29795993

Polypyrrole membranes as scaffolds for biomolecule immobilization

NASA Astrophysics Data System (ADS)

Hery, Travis M.; Satagopan, Sriram; Northcutt, Robert G.; Tabita, F. Robert; Sundaresan, Vishnu-Baba

2016-12-01

Enzymes have evolved over hundreds of years through changes in ecosystems (climate, atmosphere, hydrology, etc). The evolutionary changes driven by the need to survive has led to enzymes with diverse functionality such as reduction of carbon dioxide and methane to other forms of carbon, fixation of nitrogen, and high temperature biochemical processes. While these enzymes have useful properties, engineering a scalable cell-free system with these enzymes will be useful for stable production of desired products without involving the vagaries of cellular metabolism. This article presents various approaches to incorporate ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a conducting polymer (polypyrrole (PPy)) doped with a bulky anion (dodecylbenzenesulfonate (DBS)) in an effort to create functional devices for the conversion of carbon dioxide into precursors for high-value chemicals. We demonstrate that the tailored device creates an environment where the enzyme can retain its function while being protected from denaturing conditions. It is envisioned that the 3-PGA produced by RuBisCO will be converted into value-added products.

Quality-related enzymes in plant-based products: effects of novel food-processing technologies part 3: ultrasonic processing.

PubMed

Terefe, Netsanet Shiferaw; Buckow, Roman; Versteeg, Cornelis

2015-01-01

High-power ultrasound is a versatile technology which can potentially be used in many food processing applications including food preservation. This is part 2 of a series of review articles dealing with the effectiveness of nonthermal food processing technologies in food preservation focusing on their effect on enzymes. Typically, ultrasound treatment alone does not efficiently cause microbial or enzyme inactivation sufficient for food preservation. However, combined with mild heat with or without elevated pressure (P ≤ 500 kPa), ultrasound can effectively inactivate enzymes and microorganisms. Synergistic effects between ultrasound and mild heat have been reported for the inactivation of both enzymes and microorganisms. The application of ultrasound has been shown to enhance the rate of inactivation of quality degrading enzymes including pectin methylesterase (PME), polygalacturonase (PG), peroxidase (POD), polyphenol oxidase (PPO), and lipoxygenase (LOX) at mild temperature by up to 400 times. Moreover, ultrasound enables the inactivation of relatively heat-resistant enzymes such as tomato PG1 and thermostable orange PME at mild temperature conditions. The extent to which ultrasound enhances the inactivation rate depends on the type of enzyme, the medium in which the enzyme is suspended, and the processing condition including frequency, ultrasonic intensity, temperature, and pressure. The physical and chemical effects of cavitation are considered to be responsible for the ultrasound-induced inactivation of enzymes, although the dominant mechanism depends on the structure of the enzyme.

Using ultrasound technology for the inactivation and thermal sensitization of peroxidase in green coconut water.

PubMed

Rojas, Meliza Lindsay; Trevilin, Júlia Hellmeister; Funcia, Eduardo Dos Santos; Gut, Jorge Andrey Wilhelms; Augusto, Pedro Esteves Duarte

2017-05-01

Green coconut water has unique nutritional and sensorial qualities. Despite the different technologies already studied, its enzymatic stability is still challenging. This study evaluated the use of ultrasound technology (US) for inactivating/sensitizing coconut water peroxidase (POD). The effect of both US application alone and as a pre-treatment to thermal processing was evaluated. The enzyme activity during US processing was reduced 27% after 30min (286W/L, 20kHz), demonstrating its high resistance. The thermal inactivation was described by the Weibull model under non-isothermal conditions. The enzyme became sensitized to heat after US pre-treatment. Further, the use of US resulted in more uniform heat resistance. The results suggest that US is a good technology for sensitizing enzymes before thermal processing (even for an enzyme with high thermal resistance). Therefore, the use of this technology could decrease the undesirable effects of long times and/or the high temperatures of the conventional thermal processing. Copyright © 2016 Elsevier B.V. All rights reserved.

The amyloid architecture provides a scaffold for enzyme-like catalysts.

PubMed

Al-Garawi, Z S; McIntosh, B A; Neill-Hall, D; Hatimy, A A; Sweet, S M; Bagley, M C; Serpell, L C

2017-08-03

Natural biological enzymes possess catalytic sites that are generally surrounded by a large three-dimensional scaffold. However, the proportion of the protein molecule that participates in the catalytic reaction is relatively small. The generation of artificial or miniature enzymes has long been a focus of research because enzyme mimetics can be produced with high activity at low cost. These enzymes aim to mimic the active sites without the additional architecture contributed by the protein chain. Previous work has shown that amyloidogenic peptides are able to self-assemble to create an active site that is capable of binding zinc and catalysing an esterase reaction. Here, we describe the structural characterisation of a set of designed peptides that form an amyloid-like architecture and reveal that their capability to mimic carbonic anhydrase and serve as enzyme-like catalysts is related to their ability to self-assemble. These amyloid fibril structures can bind the metal ion Zn 2+ via a three-dimensional arrangement of His residues created by the amyloid architecture. Our results suggest that the catalytic efficiency of amyloid-like assembly is not only zinc-dependent but also depends on an active centre created by the peptides which is, in turn, dependent on the ordered architecture. These fibrils have good esterase activity, and they may serve as good models for the evolution of modern-day enzymes. Furthermore, they may be useful in designing self-assembling fibrils for applications as metal ion catalysts. This study also demonstrates that the ligands surrounding the catalytic site affect the affinity of the zinc-binding site to bind the substrate contributing to the enzymatic activity of the assembled peptides.

Recent advances in enzyme extraction strategies: A comprehensive review.

PubMed

Nadar, Shamraja S; Pawar, Rohini G; Rathod, Virendra K

2017-08-01

The increasing interest of industrial enzymes demands for development of new downstream strategies for maximizing enzyme recovery. The significant efforts have been focused on the development of newly adapted technologies to purify enzymes in catalytically active form. Recently, an aqueous two phase system (ATPS) is emerged as powerful tools for efficient extraction and purification of enzymes due to their versatility, lower cost, process integration capability and easy scale-up. The present review gives an overview of effect of parameters such as tie line length, pH, neutral salts, properties of polymer and salt involved in traditional polymer/polymer and polymer/salt ATPS for enzyme recovery. Further, advanced ATPS have been developed based on alcohols, surfactants, micellar compounds to avoid tedious recovery steps for getting desired enzyme. In order to improve the selectivity and efficiency of ATPS, recent approaches of conventional ATPS combined with different techniques like affinity ligands, ionic liquids, thermoseparating polymers and microfluidic device based ATPS have been reviewed. Moreover, three phase partitioning is also highlighted for enzymes enrichment as a blooming technology for efficiently integrated bioseparation techniques. At the end, it includes an overview of CLEAs technology and organic-inorganic nanoflowers preparation as novel strategies for simultaneous extraction, purification and immobilization of enzymes. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel photonic technique creates micrometer resolution protein arrays and provides a new approach to coupling of genes, peptide hormones and drugs to nanoparticle carriers

NASA Astrophysics Data System (ADS)

Duroux, M.; Duroux, L.; Neves-Petersen, M. T.; Skovsen, E.; Petersen, S. B.

2007-07-01

We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either thiolated quartz, gold or silicon. The reaction mechanism behind the reported new technology involves light-induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. In general, the protein molecules retain their function. The size of the immobilization spot is limited to the focal point of illumination being as small as a few micrometers. This new technology allows for dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalised new materials, such as nano-biosensors. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated image processing software has been developed for making quality assessment of the protein arrays. This novel technology is ideal to couple drugs and other bio-molecules to nanoparticles which can be used as carriers into cells for therapeutic purposes.

The CoFactor database: organic cofactors in enzyme catalysis.

PubMed

Fischer, Julia D; Holliday, Gemma L; Thornton, Janet M

2010-10-01

Organic enzyme cofactors are involved in many enzyme reactions. Therefore, the analysis of cofactors is crucial to gain a better understanding of enzyme catalysis. To aid this, we have created the CoFactor database. CoFactor provides a web interface to access hand-curated data extracted from the literature on organic enzyme cofactors in biocatalysis, as well as automatically collected information. CoFactor includes information on the conformational and solvent accessibility variation of the enzyme-bound cofactors, as well as mechanistic and structural information about the hosting enzymes. The database is publicly available and can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/CoFactor.

Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

PubMed

Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

2015-11-15

The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzymes- An Existing and Promising Tool of Food Processing Industry.

PubMed

Ray, Lalitagauri; Pramanik, Sunita; Bera, Debabrata

2016-01-01

The enzyme catalyzed process technology has enormous potential in the food sectors as indicated by the recent patents studies. It is very well realized that the adaptation of the enzyme catalyzed process depends on the availability of enzyme in affordable prices. Enzymes may be used in different food sectors like dairy, fruits & vegetable processing, meat tenderization, fish processing, brewery and wine making, starch processing and many other. Commercially only a small number of enzymes are used because of several factors including instability of enzymes during processing and high cost. More and more enzymes for food technology are now derived from specially selected or genetically modified microorganisms grown in industrial scale fermenters. Enzymes with microbial source have commercial advantages of using microbial fermentation rather than animal and plant extraction to produce food enzymes. At present only a relatively small number of enzymes are used commercially in food processing. But the number is increasing day by day and field of application will be expanded more and more in near future. The purpose of this review is to describe the practical applications of enzymes in the field of food processing.

Bioremediation of Industrial Waste Through Enzyme Producing Marine Microorganisms.

PubMed

Sivaperumal, P; Kamala, K; Rajaram, R

Bioremediation process using microorganisms is a kind of nature-friendly and cost-effective clean green technology. Recently, biodegradation of industrial wastes using enzymes from marine microorganisms has been reported worldwide. The prospectus research activity in remediation area would contribute toward the development of advanced bioprocess technology. To minimize industrial wastes, marine enzymes could constitute a novel alternative in terms of waste treatment. Nowadays, the evidence on the mechanisms of bioremediation-related enzymes from marine microorganisms has been extensively studied. This review also will provide information about enzymes from various marine microorganisms and their complexity in the biodegradation of comprehensive range of industrial wastes. © 2017 Elsevier Inc. All rights reserved.

Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes.

PubMed

Suplatov, D A; Arzhanik, V K; Svedas, V K

2011-01-01

Comparative bioinformatic analysis is the cornerstone of the study of enzymes' structure-function relationship. However, numerous enzymes that derive from a common ancestor and have undergone substantial functional alterations during natural selection appear not to have a sequence similarity acceptable for a statistically reliable comparative analysis. At the same time, their active site structures, in general, can be conserved, while other parts may largely differ. Therefore, it sounds both plausible and appealing to implement a comparative analysis of the most functionally important structural elements - the active site structures; that is, the amino acid residues involved in substrate binding and the catalytic mechanism. A computer algorithm has been developed to create a library of enzyme active site structures based on the use of the PDB database, together with programs of structural analysis and identification of functionally important amino acid residues and cavities in the enzyme structure. The proposed methodology has been used to compare some α,β-hydrolase superfamily enzymes. The insight has revealed a high structural similarity of catalytic site areas, including the conservative organization of a catalytic triad and oxyanion hole residues, despite the wide functional diversity among the remote homologues compared. The methodology can be used to compare the structural organization of the catalytic and substrate binding sites of various classes of enzymes, as well as study enzymes' evolution and to create of a databank of enzyme active site structures.

Using Phage Display to Create Recombinant Antibodies.

PubMed

Dasch, James R; Dasch, Amy L

2017-09-01

A variety of phage display technologies have been developed since the approach was first described for antibodies. The most widely used approaches incorporate antibody sequences into the minor coat protein pIII of the nonlytic filamentous phage fd or M13. Libraries of variable gene sequences, encoding either scFv or Fab fragments, are made by incorporating sequences into phagemid vectors. The phagemid is packaged into phage particles with the assistance of a helper phage to produce the antibody display phage. This protocol describes a method for creating a phagemid library. The multiple cloning site (MCS) of the pBluescript KS(-) phagemid vector is replaced by digestion with the restriction enzyme BssHII, followed by the insertion of four overlapping oligonucleotides to create a new MCS within the vector. Next, the 3' portion of gene III (from M13mp18) is amplified and combined with an antibody sequence using overlap extension PCR. This product is inserted into the phagemid vector to create pPDS. Two helper plasmids are also created from the modified pBluescript vector: pLINK provides the linker between the heavy and light chains, and pFABC provides the CH1 domain of the heavy chain. An antibody cDNA library is constructed from the RNA of interest and ligated into pPDS. The phagemid library is electroporated into Escherichia coli cells along with the VCS-M13 helper phage. © 2017 Cold Spring Harbor Laboratory Press.

Inhibitors of SOD1 Interaction as an Approach to Slow the Progressive Spread of ALS Symptoms

DTIC Science & Technology

2016-07-01

luciferase enzyme can be split into 2 halves. These 2 halves can be forced to reconstitute an active enzyme if they are brought together by some...force. In our assay, this force is the normal interaction that occurs when 2 individual SOD1 proteins come together to form a normal active enzyme ...Using recombinant DNA, we create fusion proteins of SOD1 and each half of the luciferase enzyme . In the past year, we have characterized and optimized

Biomimetic enzyme nanocomplexes and their use as antidotes and preventive measures for alcohol intoxication

NASA Astrophysics Data System (ADS)

Liu, Yang; Du, Juanjuan; Yan, Ming; Lau, Mo Yin; Hu, Jay; Han, Hui; Yang, Otto O.; Liang, Sheng; Wei, Wei; Wang, Hui; Li, Jianmin; Zhu, Xinyuan; Shi, Linqi; Chen, Wei; Ji, Cheng; Lu, Yunfeng

2013-03-01

Organisms have sophisticated subcellular compartments containing enzymes that function in tandem. These confined compartments ensure effective chemical transformation and transport of molecules, and the elimination of toxic metabolic wastes. Creating functional enzyme complexes that are confined in a similar way remains challenging. Here we show that two or more enzymes with complementary functions can be assembled and encapsulated within a thin polymer shell to form enzyme nanocomplexes. These nanocomplexes exhibit improved catalytic efficiency and enhanced stability when compared with free enzymes. Furthermore, the co-localized enzymes display complementary functions, whereby toxic intermediates generated by one enzyme can be promptly eliminated by another enzyme. We show that nanocomplexes containing alcohol oxidase and catalase could reduce blood alcohol levels in intoxicated mice, offering an alternative antidote and prophylactic for alcohol intoxication.

A new paper-based platform technology for point-of-care diagnostics.

PubMed

Gerbers, Roman; Foellscher, Wilke; Chen, Hong; Anagnostopoulos, Constantine; Faghri, Mohammad

2014-10-21

Currently, the Lateral flow Immunoassays (LFIAs) are not able to perform complex multi-step immunodetection tests because of their inability to introduce multiple reagents in a controlled manner to the detection area autonomously. In this research, a point-of-care (POC) paper-based lateral flow immunosensor was developed incorporating a novel microfluidic valve technology. Layers of paper and tape were used to create a three-dimensional structure to form the fluidic network. Unlike the existing LFIAs, multiple directional valves are embedded in the test strip layers to control the order and the timing of mixing for the sample and multiple reagents. In this paper, we report a four-valve device which autonomously directs three different fluids to flow sequentially over the detection area. As proof of concept, a three-step alkaline phosphatase based Enzyme-Linked ImmunoSorbent Assay (ELISA) protocol with Rabbit IgG as the model analyte was conducted to prove the suitability of the device for immunoassays. The detection limit of about 4.8 fm was obtained.

A designed bifunctional laccase/β-1,3-1,4-glucanase enzyme shows synergistic sugar release from milled sugarcane bagasse.

PubMed

Furtado, G P; Ribeiro, L F; Lourenzoni, M R; Ward, R J

2013-01-01

A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the β-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall β-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/β-1,3-1,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75°C that showed a V(max) 30% higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50°C, with a slight reduction in V(max) by ∼10% compared with the parental glucanase. A decreased K(M) resulted in an overall increase in the K(cat)/K(M) value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20% higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function.

Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM Enzymes in Pathogenic Streptococci.

PubMed

Schramma, Kelsey R; Seyedsayamdost, Mohammad R

2017-04-21

Macrocycles represent a common structural framework in many naturally occurring peptides. Several strategies exist for macrocyclization, and the enzymes that incorporate them are of great interest, as they enhance our repertoire for creating complex molecules. We recently discovered a new peptide cyclization reaction involving a crosslink between the side chains of lysine and tryptophan that is installed by a radical SAM enzyme. Herein, we characterize relatives of this metalloenzyme from the pathogens Streptococcus agalactiae and Streptococcus suis. Our results show that the corresponding enzymes, which we call AgaB and SuiB, contain multiple [4Fe-4S] clusters and catalyze Lys-Trp crosslink formation in their respective substrates. Subsequent high-resolution-MS and 2D-NMR analyses located the site of macrocyclization. Moreover, we report that AgaB can accept modified substrates containing natural or unnatural amino acids. Aside from providing insights into the mechanism of this unusual modification, the substrate promiscuity of AgaB may be exploited to create diverse macrocyclic peptides.

DNA-Based Enzyme Reactors and Systems

PubMed Central

Linko, Veikko; Nummelin, Sami; Aarnos, Laura; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.

2016-01-01

During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme) cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications. PMID:28335267

Expanding P450 catalytic reaction space through evolution and engineering

PubMed Central

McIntosh, John A.; Farwell, Christopher C.; Arnold, Frances H.

2014-01-01

Advances in protein and metabolic engineering have led to wider use of enzymes to synthesize important molecules. However, many desirable transformations are not catalyzed by any known enzyme, driving interest in understanding how new enzymes can be created. The cytochrome P450 enzyme family, whose members participate in xenobiotic metabolism and natural products biosynthesis, catalyzes an impressive range of difficult chemical reactions that continues to grow as new enzymes are characterized. Recent work has revealed that P450-derived enzymes can also catalyze useful reactions previously accessible only to synthetic chemistry. The evolution and engineering of these enzymes provides an excellent case study for how to genetically encode new chemistry and expand biology’s reaction space. PMID:24658056

Novel Formulation to Destroy Biothreat Agents

DTIC Science & Technology

2016-02-01

partial disassembly of these structures and/or 2) enzymes , and in particular proteases, can be used to create breaches in these structures. If successful...susceptible to a mild decontaminant. The initial goals of this work involved a broad search among diverse chemistries and enzymes for those that...exosporium. At the start of the work, no such chemistries or enzymes were known, and no obvious candidates were available. Therefore, in practice there

Delivery of Formulated Industrial Enzymes with Acoustic Technology.

PubMed

Hwang, Jennifer Dorcas; Ortiz-Maldonado, Mariliz; Paramonov, Sergey

2016-02-01

Industrial enzymes are instrumental in many applications, including carbohydrate processing, fabric and household care, biofuels, food, and animal nutrition, among others. Enzymes have to be active and stable not only in harsh application conditions, but also during shipment and storage. In protein stability studies, formulated concentrated enzyme solutions are frequently diluted gravimetrically prior to enzyme activity measurements, making it challenging to move toward more high-throughput techniques using conventional robotic equipment. Current assay methods pose difficulties when measuring highly concentrated proteins. For example, plastic pipette tips can introduce error because proteins adsorb to the tip surface, despite the presence of detergents, decreasing precision and overall efficiency of protein activity assays. Acoustic liquid handling technology, frequently used for various dilute small-molecule assays, may overcome such problems. Originally shown to effectively deliver dilute solutions of small molecules, this technology is used here as an effective alternative to the aforementioned challenge with viscous concentrated protein solutions. Because the acoustic liquid handler transfers nanoliter quantities of liquids without using pipette tips and without sample loss, it rapidly and uniformly prepares assay plates for enzyme activity measurements within minutes. This increased efficiency transforms the nature of enzyme stability studies toward high precision and throughput. © 2015 Society for Laboratory Automation and Screening.

novPTMenzy: a database for enzymes involved in novel post-translational modifications

PubMed Central

Khater, Shradha; Mohanty, Debasisa

2015-01-01

With the recent discoveries of novel post-translational modifications (PTMs) which play important roles in signaling and biosynthetic pathways, identification of such PTM catalyzing enzymes by genome mining has been an area of major interest. Unlike well-known PTMs like phosphorylation, glycosylation, SUMOylation, no bioinformatics resources are available for enzymes associated with novel and unusual PTMs. Therefore, we have developed the novPTMenzy database which catalogs information on the sequence, structure, active site and genomic neighborhood of experimentally characterized enzymes involved in five novel PTMs, namely AMPylation, Eliminylation, Sulfation, Hydroxylation and Deamidation. Based on a comprehensive analysis of the sequence and structural features of these known PTM catalyzing enzymes, we have created Hidden Markov Model profiles for the identification of similar PTM catalyzing enzymatic domains in genomic sequences. We have also created predictive rules for grouping them into functional subfamilies and deciphering their mechanistic details by structure-based analysis of their active site pockets. These analytical modules have been made available as user friendly search interfaces of novPTMenzy database. It also has a specialized analysis interface for some PTMs like AMPylation and Eliminylation. The novPTMenzy database is a unique resource that can aid in discovery of unusual PTM catalyzing enzymes in newly sequenced genomes. Database URL: http://www.nii.ac.in/novptmenzy.html PMID:25931459

Biotechnological Applications of Marine Enzymes From Algae, Bacteria, Fungi, and Sponges.

PubMed

Parte, S; Sirisha, V L; D'Souza, J S

Diversity is the hallmark of all life forms that inhabit the soil, air, water, and land. All these habitats pose their unique inherent challenges so as to breed the "fittest" creatures. Similarly, the biodiversity from the marine ecosystem has evolved unique properties due to challenging environment. These challenges include permafrost regions to hydrothermal vents, oceanic trenches to abyssal plains, fluctuating saline conditions, pH, temperature, light, atmospheric pressure, and the availability of nutrients. Oceans occupy 75% of the earth's surface and harbor most ancient and diverse forms of organisms (algae, bacteria, fungi, sponges, etc.), serving as an excellent source of natural bioactive molecules, novel therapeutic compounds, and enzymes. In this chapter, we introduce enzyme technology, its current state of the art, unique enzyme properties, and the biocatalytic potential of marine algal, bacterial, fungal, and sponge enzymes that have indeed boosted the Marine Biotechnology Industry. Researchers began exploring marine enzymes, and today they are preferred over the chemical catalysts for biotechnological applications and functions, encompassing various sectors, namely, domestic, industrial, commercial, and healthcare. Next, we summarize the plausible pros and cons: the challenges encountered in the process of discovery of the potent compounds and bioactive metabolites such as biocatalysts/enzymes of biomedical, therapeutic, biotechnological, and industrial significance. The field of Marine Enzyme Technology has recently assumed importance, and if it receives further boost, it could successfully substitute other chemical sources of enzymes useful for industrial and commercial purposes and may prove as a beneficial and ecofriendly option. With appropriate directions and encouragement, marine enzyme technology can sustain the rising demand for enzyme production while maintaining the ecological balance, provided any undesired exploitation of the marine ecosystem is avoided. © 2017 Elsevier Inc. All rights reserved.

Combinatorial enzyme technology: Conversion of pectin to oligo species and its effect on microbial growth

USDA-ARS?s Scientific Manuscript database

Plant cell wall polysaccharides, which consist of polymeric backbones with various types of substitution, were studied using the concept of combinatorial enzyme technology for conversion of agricultural fibers to functional products. Using citrus pectin as the starting substrate, an active oligo spe...

Cross-Linked Enzyme Aggregates for Applications in Aqueous and Nonaqueous Media.

PubMed

Roy, Ipsita; Mukherjee, Joyeeta; Gupta, Munishwar N

2017-01-01

Extensive cross-linking of a precipitate of a protein by a cross-linking reagent (glutaraldehyde has been most commonly used) creates an insoluble enzyme preparation called cross-linked enzyme aggregates (CLEAs). CLEAs show high stability and performance in conventional aqueous as well as nonaqueous media. These are also stable at fairly high temperatures. CLEAs with more than one kind of enzyme activity can be prepared, and such CLEAs are called combi-CLEAs or multipurpose CLEAs. Extent of cross-linking often influences their morphology, stability, activity, and enantioselectivity.

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

PubMed

Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

2017-09-01

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

Printing enzymatic reactions.

PubMed

Tian, Junfei; Shen, Wei

2011-02-07

We used relief and planographic printing methods to print the catalytic effect of an enzyme, but not the enzyme molecules, onto paper. Printing enzymatic reactions have applications in bioactive papers, low-cost diagnostics, anti-counterfeiting devices and advanced packaging materials. These methods can create novel printing effects on commodity surfaces for advanced applications.

Production and characterization of murine models of classic and intermediate maple syrup urine disease

PubMed Central

Homanics, Gregg E; Skvorak, Kristen; Ferguson, Carolyn; Watkins, Simon; Paul, Harbhajan S

2006-01-01

Background Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. Methods To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. Results By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. Conclusion These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. PMID:16579849

Enzyme processes for pulp and paper : a review of recent developments

Treesearch

William R. Kenealy; Thomas W. Jeffries

2003-01-01

The pulp and paper industry is applying new, ecologically sound technology in its manufacturing processes. Many interesting enzymatic applications have been proposed in the literature. Implemented technologies tend to change the existing industrial process as little as possible. Commercial applications include xylanases in prebleaching kraft pulps and various enzymes...

Engineered control of enzyme structural dynamics and function.

PubMed

Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

2018-04-01

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers.

PubMed

Botyanszki, Zsofia; Tay, Pei Kun R; Nguyen, Peter Q; Nussbaumer, Martin G; Joshi, Neel S

2015-10-01

Biocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long-term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on high cellular metabolism, the modified curli-based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces. © 2015 Wiley Periodicals, Inc.

Engineering thermal stability of L-asparaginase by in vitro directed evolution.

PubMed

Kotzia, Georgia A; Labrou, Nikolaos E

2009-03-01

L-asparaginase (EC 3.5.1.1, L-ASNase) catalyses the hydrolysis of l-Asn, producing L-Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukaemia. In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability. A library of enzyme variants was created by a staggered extension process using the genes that code for the L-ASNases from Erwinia chrysanthemi and Erwinia carotovora. The amino acid sequences of the parental L-ASNases show 77% identity, but their half-inactivation temperature (T(m)) differs by 10 degrees C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The T(m) of this variant was 55.8 degrees C, whereas the wild-type enzyme has a T(m) of 46.4 degrees C. At 50 degrees C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respectively. Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability. Screening of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu mutation confers optimal thermostability.

Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics.

PubMed

Güleç, Hacı Ali

2013-04-01

The aim of this study was to investigate the effects of surface characteristics of plain and plasma modified cellulose acetate (CA) membranes on the immobilization yield of β-galactosidases from Kluyveromyces lactis (KLG) and its galacto-oligosaccharide (GOS) yield, respectively. Low pressure plasma treatments involving oxygen plasma activation, plasma polymerization (PlsP) of ethylenediamine (EDA) and PlsP of 2-mercaptoethanol were used to modify plain CA membrane surfaces. KLG enzyme was immobilized onto plain and oxygen plasma treated membrane surfaces by simple adsorption. Oxygen plasma activation increased the hydrophylicity of CA membrane surfaces and it improved the immobilization yield of the enzyme by 42%. KLG enzyme was also immobilized onto CA membrane surfaces through amino groups created by PlsP of EDA via covalent binding. Plasma action at 60W plasma power and 15 min. exposure time improved the amount of membrane bounded enzyme by 3.5-fold. The enrichment of the amount of amino groups via polyethyleneimine (PEI) addition enhanced this increase from 3.5-fold to 4.5-fold. Although high enzyme loading was achived (65-83%), both of the methods dramatically decreased the enzyme activity (11-12%) and GOS yield due to probably negative effects of active amino groups. KLG enzyme was more effectively immobilized onto thiolated CA membrane surface created by PlsP of 2-mercaptoethanol with high immobilization yield (70%) and especially high enzyme activity (46%). Immobilized enzymes on the CA membranes treated by PlsP were successively reutilized for 5-8 cycles at 25°C and enzymatic derivatives retained approximately 75-80% of their initial activites at the end of the reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Enzyme-triggered compound release using functionalized antimicrobial peptide derivatives† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04435b Click here for additional data file.

PubMed Central

Kashibe, Masayoshi; Matsumoto, Kengo; Hori, Yuichiro

2017-01-01

Controlled release is one of the key technologies for medical innovation, and many stimulus-responsive nanocarriers have been developed to utilize this technology. Enzyme activity is one of the most useful stimuli, because many enzymes are specifically activated in diseased tissues. However, controlled release stimulated by enzyme activity has not been frequently reported. One of the reasons for this is the lack of versatility of carriers. Most of the reported stimulus-responsive systems involve a sophisticated design and a complicated process for the synthesis of stimulus-responsive nanocarrier components. The purpose of this study was to develop versatile controlled release systems triggered by various stimuli, including enzyme activity, without modifying the nanocarrier components. We developed two controlled release systems, both of which comprised a liposome as the nanocarrier and a membrane-damaging peptide, temporin L (TL), and its derivatives as the release-controllers. One system utilized branched peptides for proteases, and the other utilized phosphopeptides for phosphatases. In our systems, the target enzymes converted the non-membrane-damaging TL derivatives into membrane-damaging peptides and released the liposome inclusion. We demonstrated the use of our antimicrobial peptide-based controlled release systems for different enzymes and showed the promise of this technology as a novel theranostic tool. PMID:28451373

Innovations in food technology for health.

PubMed

Hsieh, Yun-Hwa Peggy; Ofori, Jack Appiah

2007-01-01

Modern nutritional science is providing ever more information on the functions and mechanisms of specific food components in health promotion and/or disease prevention. In response to demands from increasingly health conscious consumers, the global trend is for food industries to translate nutritional information into consumer reality by developing food products that provide not only superior sensory appeal but also nutritional and health benefits. Today's busy life styles are also driving the development of healthy convenience foods. Recent innovations in food technologies have led to the use of many traditional technologies, such as fermentation, extraction, encapsulation, fat replacement, and enzyme technology, to produce new health food ingredients, reduce or remove undesirable food components, add specific nutrient or functional ingredients, modify food compositions, mask undesirable flavors or stabilize ingredients. Modern biotechnology has even revolutionized the way foods are created. Recent discoveries in gene science are making it possible to manipulate the components in natural foods. In combination with biofermentation, desirable natural compounds can now be produced in large amounts at a low cost and with little environmental impact. Nanotechnology is also beginning to find potential applications in the area of food and agriculture. Although the use of new technologies in the production of health foods is often a cause for concern, the possibility that innovative food technology will allow us to produce a wide variety of food with enhanced flavor and texture, while at the same time conferring multiple health benefits on the consumer, is very exciting.

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium

NASA Astrophysics Data System (ADS)

Freeman, Michael F.; Helf, Maximilian J.; Bhushan, Agneya; Morinaka, Brandon I.; Piel, Jörn

2017-04-01

Uncultivated bacteria represent a massive resource of new enzymes and bioactive metabolites, but such bacteria remain functionally enigmatic. Polytheonamides are potent peptide cytotoxins produced by uncultivated bacteria that exist as symbionts in a marine sponge. Outside glycobiology, polytheonamides represent the most heavily post-translationally modified biomolecules that are derived from amino acids. The biosynthesis of polytheonamides involves up to 50 site-specific modifications to create a membrane-spanning β-helical structure. Here, we provide functional evidence that only seven enzymes are necessary for this process. They iteratively catalyse epimerization, methylation and hydroxylation of diverse amino acids. To reconstitute C-methylation, we employed the rarely used heterologous host Rhizobium leguminosarum to invoke the activities of two cobalamin-dependent C-methyltransferases. We observed 44 of the modifications to systematically unravel the biosynthesis of one of the most densely modified and metabolically obscure ribosome-derived molecules found in nature.

Biochemical Conversion Processes of Lignocellulosic Biomass to Fuels and Chemicals - A Review.

PubMed

Brethauer, Simone; Studer, Michael H

2015-01-01

Lignocellulosic biomass - such as wood, agricultural residues or dedicated energy crops - is a promising renewable feedstock for production of fuels and chemicals that is available at large scale at low cost without direct competition for food usage. Its biochemical conversion in a sugar platform biorefinery includes three main unit operations that are illustrated in this review: the physico-chemical pretreatment of the biomass, the enzymatic hydrolysis of the carbohydrates to a fermentable sugar stream by cellulases and finally the fermentation of the sugars by suitable microorganisms to the target molecules. Special emphasis in this review is put on the technology, commercial status and future prospects of the production of second-generation fuel ethanol, as this process has received most research and development efforts so far. Despite significant advances, high enzyme costs are still a hurdle for large scale competitive lignocellulosic ethanol production. This could be overcome by a strategy termed 'consolidated bioprocessing' (CBP), where enzyme production, enzymatic hydrolysis and fermentation is integrated in one step - either by utilizing one genetically engineered superior microorganism or by creating an artificial co-culture. Insight is provided on both CBP strategies for the production of ethanol as well as of advanced fuels and commodity chemicals.

A Broader View: Microbial Enzymes and Their Relevance in Industries, Medicine, and Beyond

PubMed Central

Bose, Sutapa; Rai, Vivek

2013-01-01

Enzymes are the large biomolecules that are required for the numerous chemical interconversions that sustain life. They accelerate all the metabolic processes in the body and carry out a specific task. Enzymes are highly efficient, which can increase reaction rates by 100 million to 10 billion times faster than any normal chemical reaction. Due to development in recombinant technology and protein engineering, enzymes have evolved as an important molecule that has been widely used in different industrial and therapeutical purposes. Microbial enzymes are currently acquiring much attention with rapid development of enzyme technology. Microbial enzymes are preferred due to their economic feasibility, high yields, consistency, ease of product modification and optimization, regular supply due to absence of seasonal fluctuations, rapid growth of microbes on inexpensive media, stability, and greater catalytic activity. Microbial enzymes play a major role in the diagnosis, treatment, biochemical investigation, and monitoring of various dreaded diseases. Amylase and lipase are two very important enzymes that have been vastly studied and have great importance in different industries and therapeutic industry. In this review, an approach has been made to highlight the importance of different enzymes with special emphasis on amylase and lipase in the different industrial and medical fields. PMID:24106701

Artificial enzymes based on supramolecular scaffolds.

PubMed

Dong, Zeyuan; Luo, Quan; Liu, Junqiu

2012-12-07

Enzymes are nanometer-sized molecules with three-dimensional structures created by the folding and self-assembly of polymeric chain-like components through supramolecular interactions. They are capable of performing catalytic functions usually accompanied by a variety of conformational states. The conformational diversities and complexities of natural enzymes exerted in catalysis seriously restrict the detailed understanding of enzymatic mechanisms in molecular terms. A supramolecular viewpoint is undoubtedly helpful in understanding the principle of enzyme catalysis. The emergence of supramolecular artificial enzymes therefore provides an alternative way to approach the structural complexity and thus to unravel the mystery of enzyme catalysis. This critical review covers the recent development of artificial enzymes designed based on supramolecular scaffolds ranging from the synthetic macrocycles to self-assembled nanometer-sized objects. Such findings are anticipated to facilitate the design of supramolecular artificial enzymes as well as their potential uses in important fields, such as manufacturing and food industries, environmental biosensors, pharmaceutics and so on.

MTA-enriched nanocomposite TiO(2)-polymeric powder coatings support human mesenchymal cell attachment and growth.

PubMed

Shi, Wen; Mozumder, Mohammad Sayem; Zhang, Hui; Zhu, Jesse; Perinpanayagam, Hiran

2012-10-01

The objective of the study described in this paper was the development of novel polymer/ceramic nanocomposite coatings for implants through the application of ultrafine powder coating technology. Polyester resins were combined with µm-sized TiO(2) (25%) as the biocompatibility agent, nTiO(2) (0.5%) as the flow additive and mineral trioxide aggregates (ProRoot® MTA, 5%) as bioactive ceramics. Ultrafine powders were prepared and applied to titanium to create continuous polymeric powder coatings (PPCs) through the application of electrostatic ultrafine powder coating technology. Energy dispersive x-ray analysis confirmed that MTA had been incorporated into the PPCs, and elemental mapping showed that it had formed small clusters that were evenly distributed across the surface. Scanning electron microscopy (SEM) revealed continuous and smooth, but highly textured surface coatings that contrasted with the scalloped appearance of commercially pure titanium (cpTi) controls. Atomic force microscopy revealed intricate nano-topographies with an abundance of submicron-sized pits and nano-projections, evenly dispersed across their surfaces. Inverted fluorescence microscopy, SEM and cell counts showed that human embryonic palatal mesenchymal cells attached and spread out onto PPC and MTA-enriched PPCs within 24 h. Mitochondrial enzyme activity measured viable and metabolically active cells on all of the surfaces. After 72 h of growth, cell counts and metabolic activity were significantly higher (P < 0.05) on the grey-MTA enriched PPC surfaces, than on unmodified PPC and cpTi. The novel polymer/ceramic nanocomposites that were created with ultrafine powder coating technology were continuous, homogenous and nano-rough coatings that enhanced human mesenchymal cell attachment and growth.

Using Technology to Create Safer Schools.

ERIC Educational Resources Information Center

Townley, Arthur J.; Martinez, Kenneth

1995-01-01

Although classes to create student self-esteem and antigang programs are gaining in popularity, most school districts have not used available technology to help create safer campuses. Increased availability of telephones and two-way radios would enhance school security, along with incorporation of newer technologies such as computers, digitized…

Enzyme Analysis to Determine Glucose Content

NASA Astrophysics Data System (ADS)

Carpenter, Charles; Ward, Robert E.

Enzyme analysis is used for many purposes in food science and technology. Enzyme activity is used to indicate adequate processing, to assess enzyme preparations, and to measure constituents of foods that are enzyme substrates. In this experiment, the glucose content of corn syrup solids is determined using the enzymes, glucose oxidase and peroxidase. Glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide (H2O2), which then reacts with a dye in the presence of peroxidase to give a stable colored product.

From immunology to MRI data anlysis: Problems in mathematical biology

NASA Astrophysics Data System (ADS)

Waters, Ryan Samuel

This thesis represents a collection of four distinct biological projects rising from immunology and metabolomics that required unique and creative mathematical approaches. One project focuses on understanding the role IL-2 plays in immune response regulation and exploring how these effects can be altered. We developed several dynamic models of the receptor signaling network which we analyze analytically and numerically. In a second project focused also on MS, we sought to create a system for grading magnetic resonance images (MRI) with good correlation with disability. The goal is for these MRI scores to provide a better standard for large-scale clinical drug trials, which limits the bias associated with differences in available MRI technology and general grader/participant variability. The third project involves the study of the CRISPR adaptive immune system in bacteria. Bacterial cells recognize and acquire snippets of exogenous genetic material, which they incorporate into their DNA. In this project we explore the optimal design for the CRISPR system given a viral distribution to maximize its probability of survival. The final project involves the study of the benefits for colocalization of coupled enzymes in metabolic pathways. The hypothesized kinetic advantage, known as `channeling', of putting coupled enzymes closer together has been used as justification for the colocalization of coupled enzymes in biological systems. We developed and analyzed a simple partial differential equation of the diffusion of the intermediate substrate between coupled enzymes to explore the phenomena of channeling. The four projects of my thesis represent very distinct biological problems that required a variety of techniques from diverse areas of mathematics ranging from dynamical modeling to statistics, Fourier series and calculus of variations. In each case, quantitative techniques were used to address biological questions from a mathematical perspective ultimately providing insight back to the biological problems which motivated them.

Disentangling DNA molecules

NASA Astrophysics Data System (ADS)

Vologodskii, Alexander

2016-09-01

The widespread circular form of DNA molecules inside cells creates very serious topological problems during replication. Due to the helical structure of the double helix the parental strands of circular DNA form a link of very high order, and yet they have to be unlinked before the cell division. DNA topoisomerases, the enzymes that catalyze passing of one DNA segment through another, solve this problem in principle. However, it is very difficult to remove all entanglements between the replicated DNA molecules due to huge length of DNA comparing to the cell size. One strategy that nature uses to overcome this problem is to create the topoisomerases that can dramatically reduce the fraction of linked circular DNA molecules relative to the corresponding fraction at thermodynamic equilibrium. This striking property of the enzymes means that the enzymes that interact with DNA only locally can access their topology, a global property of circular DNA molecules. This review considers the experimental studies of the phenomenon and analyzes the theoretical models that have been suggested in attempts to explain it. We describe here how various models of enzyme action can be investigated computationally. There is no doubt at the moment that we understand basic principles governing enzyme action. Still, there are essential quantitative discrepancies between the experimental data and the theoretical predictions. We consider how these discrepancies can be overcome.

The Enzyme Portal: a case study in applying user-centred design methods in bioinformatics.

PubMed

de Matos, Paula; Cham, Jennifer A; Cao, Hong; Alcántara, Rafael; Rowland, Francis; Lopez, Rodrigo; Steinbeck, Christoph

2013-03-20

User-centred design (UCD) is a type of user interface design in which the needs and desires of users are taken into account at each stage of the design process for a service or product; often for software applications and websites. Its goal is to facilitate the design of software that is both useful and easy to use. To achieve this, you must characterise users' requirements, design suitable interactions to meet their needs, and test your designs using prototypes and real life scenarios.For bioinformatics, there is little practical information available regarding how to carry out UCD in practice. To address this we describe a complete, multi-stage UCD process used for creating a new bioinformatics resource for integrating enzyme information, called the Enzyme Portal (http://www.ebi.ac.uk/enzymeportal). This freely-available service mines and displays data about proteins with enzymatic activity from public repositories via a single search, and includes biochemical reactions, biological pathways, small molecule chemistry, disease information, 3D protein structures and relevant scientific literature.We employed several UCD techniques, including: persona development, interviews, 'canvas sort' card sorting, user workflows, usability testing and others. Our hope is that this case study will motivate the reader to apply similar UCD approaches to their own software design for bioinformatics. Indeed, we found the benefits included more effective decision-making for design ideas and technologies; enhanced team-working and communication; cost effectiveness; and ultimately a service that more closely meets the needs of our target audience.

Microscale Measurements of Michaelis-Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage.

PubMed

Gattu, Srikanth; Crihfield, Cassandra L; Holland, Lisa A

2017-01-03

Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis-Menten constants (K M ) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A K M value of 3.3 ± 0.8 mM (V max , 2100 ± 200 μM/min) was obtained for 3'-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a K M of 2 ± 1 mM (V max , 400 ± 100 μM/min) was obtained for the 6'-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a K M value of 3 ± 2 mM (V max , 900 ± 300 μM/min) for 3'-sialyllactose. With a knowledge of V max , the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3' and 6' sialic acid linkages.

Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage

PubMed Central

2016-01-01

Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis–Menten constants (KM) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A KM value of 3.3 ± 0.8 mM (Vmax, 2100 ± 200 μM/min) was obtained for 3′-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a KM of 2 ± 1 mM (Vmax, 400 ± 100 μM/min) was obtained for the 6′-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a KM value of 3 ± 2 mM (Vmax, 900 ± 300 μM/min) for 3′-sialyllactose. With a knowledge of Vmax, the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3′ and 6′ sialic acid linkages. PMID:27936604

Some Nutritional, Technological and Environmental Advances in the Use of Enzymes in Meat Products

PubMed Central

Marques, Anne y Castro; Maróstica, Mário Roberto; Pastore, Gláucia Maria

2010-01-01

The growing consumer demand for healthier products has stimulated the development of nutritionally enhanced meat products. However, this can result in undesirable sensory consequences to the product, such as texture alterations in low-salt and low-phosphate meat foods. Additionally, in the meat industry, economical aspects have stimulated researchers to use all the animal parts to maximize yields of marketable products. This paper aimed to show some advances in the use of enzymes in meat processing, particularly the application of the proteolytic enzymes transglutaminase and phytases, associated with nutritional, technological, and environmental improvements. PMID:21048865

Fungal biodegradation and enzymatic modification of lignin

PubMed Central

Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng

2010-01-01

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed. PMID:21968746

Biomedical Applications of Enzymes From Marine Actinobacteria.

PubMed

Kamala, K; Sivaperumal, P

Marine microbial enzyme technologies have progressed significantly in the last few decades for different applications. Among the various microorganisms, marine actinobacterial enzymes have significant active properties, which could allow them to be biocatalysts with tremendous bioactive metabolites. Moreover, marine actinobacteria have been considered as biofactories, since their enzymes fulfill biomedical and industrial needs. In this chapter, the marine actinobacteria and their enzymes' uses in biological activities and biomedical applications are described. © 2017 Elsevier Inc. All rights reserved.

Japan Report, Science and Technology.

DTIC Science & Technology

1987-05-06

Figure 17. Microcapsule Type Enzyme Immobilized Membrane Such metabolites as creatinine, uric acid , and vitamin B12 are relatively easily adsorbed by...WEST EUROPE ivory AFRICA (SUB-SAHARA) tan SCIENCE $ TECHNOLOGY gray WORLDWIDES pewter The changes that are of interest to readers of this report...condition and that they are unstable in strong acids , strong bases, organic solvents, and heat. Ordinarily, an enzyme docks with a substrate in an

A survey on faculty perspectives on the transition to a biochemistry course-based undergraduate research experience laboratory.

PubMed

Craig, Paul A

2017-09-01

It will always remain a goal of an undergraduate biochemistry laboratory course to engage students hands-on in a wide range of biochemistry laboratory experiences. In 2006, our research group initiated a project for in silico prediction of enzyme function based only on the 3D coordinates of the more than 3800 proteins "of unknown function" in the Protein Data Bank, many of which resulted from the Protein Structure Initiative. Students have used the ProMOL plugin to the PyMOL molecular graphics environment along with BLAST, Pfam, and Dali to predict protein functions. As young scientists, these undergraduate research students wanted to see if their predictions were correct and so they developed an approach for in vitro testing of predicted enzyme function that included literature exploration, selection of a suitable assay and the search for commercially available substrates. Over the past two years, a team of faculty members from seven different campuses (California Polytechnic San Luis Obispo, Hope College, Oral Roberts University, Rochester Institute of Technology, St. Mary's University, Ursinus College, and Purdue University) have transferred this approach to the undergraduate biochemistry teaching laboratory as a Course-based Undergraduate Research Experience. A series of ten course modules and eight instructional videos have been created (www.promol.org/home/basil-modules-1) and the group is now expanding these resources, creating assessments and evaluating how this approach helps student to grow as scientists. The focus of this manuscript will be the logistical implications of this transition on campuses that have different cultures, expectations, schedules, and student populations. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):426-436, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Protein Hydrolysates as Hypoallergenic, Flavors and Palatants for Companion Animals

NASA Astrophysics Data System (ADS)

Nagodawithana, Tilak W.; Nelles, Lynn; Trivedi, Nayan B.

Early civilizations have relied upon their good sense and experience to develop and improve their food quality. The discovery of soy sauce centuries ago can now be considered one of the earliest protein hydrolysates made by man to improve palatability of foods. Now, it is well known that such savory systems are not just sources for enjoyment but complex semiotic systems that direct the humans to satisfy the body's protein need for their sustenance. Recent developments have resulted in a wide range of cost effective savory flavorings, the best known of which are autolyzed yeast extracts and hydrolyzed vegetable proteins. New technologies have helped researchers to improve the savory characteristics of yeast extracts through the application of Maillard reaction and by generating specific flavor enhancers through the use of enzymes. An interesting parallel exists in the pet food industry, where a similar approach is taken in using animal protein hydrolysates to create palatability enhancers via Maillard reaction scheme. Protein hydrolysates are also utilized extensively as a source of nutrition to the elderly, young children and immuno-compromised patient population. These hydrolysates have an added advantage in having peptides small enough to avoid any chance of an allergenic reaction which sometimes occur with the consumption of larger sized peptides or proteins. Accordingly, protein hydrolysates are required to have an average molecular weight distribution in the range 800-1,500 Da to make them non-allergenic. The technical challenge for scientists involved in food and feed manufacture is to use an appropriate combination of enzymes within the existing economic constraints and other physical factors/limitations, such as heat, pH, and time, to create highly palatable, yet still nutritious and hypoallergenic food formulations.

Converting Enzymes into Tools of Industrial Importance.

PubMed

Prasad, Shivcharan; Roy, Ipsita

2018-01-01

Enzymes have applications in numerous biotechnological products and processes that are commonly used in the production of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes, however, are optimized to function under physiological conditions. Any change in reaction conditions results in their activity as well as stability being compromised. Hence, most of the natural biomolecules are not suitable for industrial applications. Modifications are required to develop efficient and successful reagents as per demand. Protein engineering can be applied to cope up with these situations. This review describes some of the novel uses/unusual properties of enzymes as biological catalysts. It explains the different ways in which enzymes can be and have been used under non-native conditions. Different strategies have been discussed regarding stabilization of enzyme as well optimum conditions of its uses in different industries. The following patents databases were consulted: European Patent Office (EPO), the United States Patent and Trademark Office (USPTO), Patent scope Search International and National Patent Collections (WIPO) and Google Patents. The review illustrates the width of the umbrella of applications covered by biocatalysts. Employing the tools of solvent and protein engineering, viz. non-aqueous media, additives, immobilization, mutagenesis, to name a few; biotechnology has been able to make enzyme catalyzed processes an essential components of the industrialist's armoury. The article lists a number of successful examples, both of patented technology as well as biocatalysts which are currently being used in the industry, to highlight the accomplishments of technologies which have been adopted till now for making enzyme technology industrially viable. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Software-assisted stacking of gene modules using GoldenBraid 2.0 DNA-assembly framework.

PubMed

Vazquez-Vilar, Marta; Sarrion-Perdigones, Alejandro; Ziarsolo, Peio; Blanca, Jose; Granell, Antonio; Orzaez, Diego

2015-01-01

GoldenBraid (GB) is a modular DNA assembly technology for plant multigene engineering based on type IIS restriction enzymes. GB speeds up the assembly of transcriptional units from standard genetic parts and facilitates the stacking of several genes within the same T-DNA in few days. GBcloning is software-assisted with a set of online tools. The GBDomesticator tool assists in the adaptation of DNA parts to the GBstandard. The combination of GB-adapted parts to build new transcriptional units is assisted by the GB TU Assembler tool. Finally, the assembly of multigene modules is simulated by the GB Binary Assembler. All the software tools are available at www.gbcloning.org . Here, we describe in detail the assembly methodology to create a multigene construct with three transcriptional units for polyphenol metabolic engineering in plants.

Synthetic biology: exploring and exploiting genetic modularity through the design of novel biological networks.

PubMed

Agapakis, Christina M; Silver, Pamela A

2009-07-01

Synthetic biology has been used to describe many biological endeavors over the past thirty years--from designing enzymes and in vitro systems, to manipulating existing metabolisms and gene expression, to creating entirely synthetic replicating life forms. What separates the current incarnation of synthetic biology from the recombinant DNA technology or metabolic engineering of the past is an emphasis on principles from engineering such as modularity, standardization, and rigorously predictive models. As such, synthetic biology represents a new paradigm for learning about and using biological molecules and data, with applications in basic science, biotechnology, and medicine. This review covers the canonical examples as well as some recent advances in synthetic biology in terms of what we know and what we can learn about the networks underlying biology, and how this endeavor may shape our understanding of living systems.

Designing a dynamic dissolution method: a review of instrumental options and corresponding physiology of stomach and small intestine.

PubMed

Culen, Martin; Rezacova, Anna; Jampilek, Josef; Dohnal, Jiri

2013-09-01

Development of new pharmaceutical compounds and dosage forms often requires in vitro dissolution testing with the closest similarity to the human gastrointestinal (GI) tract. To create such conditions, one needs a suitable dissolution apparatus and the appropriate data on the human GI physiology. This review discusses technological approaches applicable in biorelevant dissolutions as well as the physiology of stomach and small intestine in both fasted and fed state, that is, volumes of contents, transit times for water/food and various solid oral dosage forms, pH, osmolality, surface tension, buffer capacity, and concentrations of bile salts, phospholipids, enzymes, and Ca(2+) ions. The information is aimed to provide clear suggestions on how these conditions should be set in a dynamic biorelevant dissolution test. Copyright © 2013 Wiley Periodicals, Inc.

Release of enzymes from cells: transport and distribution within the extracellular space.

PubMed

Mattenheimer, H; Friedel, R

1977-01-01

The distribution in the extracellular space of enzymes released from organ cells was investigated using three models: (1) comparison of enzyme activities in blood plasma and lymph of the ductus thoracicus (dog) and plasma and intestinal lymph (rat); (2) i.v. injection of heterologous, homologous and autologous enzymes in order to increase acutely the activities and to measure the rate constants for the distribution and elimination of the enzymes (rat); or (3) plasmapheresis in order to create an enzyme activity gradient from the interstitial space and to determine the rate constants for the reestablishment of the equilibrium between the extra and intravascular compartments (rat). The results suggest that the enzymes are mainly released into the interstitial fluid and transported via the lymph into the intravascular compartment. From there the enzymes diffuse back into the interstitial compartment and are eliminated by a yet unknown mechanism. Transport of enzymes across the capillary membranes in both directions depends on (1) the permeability of the capillary membranes, which varies from region to region and (2) the molecular seizes of the enzymes.

Industrial applications of enzyme biocatalysis: Current status and future aspects.

PubMed

Choi, Jung-Min; Han, Sang-Soo; Kim, Hak-Sung

2015-11-15

Enzymes are the most proficient catalysts, offering much more competitive processes compared to chemical catalysts. The number of industrial applications for enzymes has exploded in recent years, mainly owing to advances in protein engineering technology and environmental and economic necessities. Herein, we review recent progress in enzyme biocatalysis, and discuss the trends and strategies that are leading to broader industrial enzyme applications. The challenges and opportunities in developing biocatalytic processes are also discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

PubMed

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-10

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

A multi-step chromatographic strategy to purify three fungal endo-β-glucanases.

PubMed

McCarthy, Tracey; Tuohy, Maria G

2011-01-01

Fungi and fungal enzymes have traditionally occupied a central role in biotechnology. Understanding the biochemical properties of the variety of enzymes produced by these eukaryotes has been an area of research interest for decades and again more recently due to global interest in greener bio-production technologies. Purification of an individual enzyme allows its unique biochemical and functional properties to be determined, can provide key information as to the role of individual biocatalysts within a complex enzyme system, and can inform both protein engineering and enzyme production strategies in the development of novel green technologies based on fungal biocatalysts. Many enzymes of current biotechnological interest are secreted by fungi into the extracellular culture medium. These crude enzyme mixtures are typically complex, multi-component, and generally also contain other non-enzymatic proteins and secondary metabolites. In this chapter, we describe a multi-step chromatographic strategy required to isolate three new endo-β-glucanases (denoted EG V, EG VI, and EG VII) with activity against cereal mixed-linkage β-glucans from the thermophilic fungus Talaromyces emersonii. This work also illustrates the challenges frequently involved in isolating individual extracellular fungal proteins in general.

A Qualitative Study of Technology-Based Training in Organizations that Hire Agriculture and Life Sciences Students

ERIC Educational Resources Information Center

Bedgood, Leslie; Murphrey, Theresa Pesl; Dooley, Kim E.

2008-01-01

Technological advances have created unlimited opportunities in education. Training and technology have merged to create new methods referred to as technology-based training. The purpose of this study was to identify organizations that hire agriculture and life sciences students for positions involving technology-based training and identify…

Immobilized enzyme reactors in HPLC and its application in inhibitor screening: A review

PubMed Central

Fang, Si-Meng; Wang, Hai-Na; Zhao, Zhong-Xi; Wang, Wei-Hong

2011-01-01

This paper sets out to summarize the literatures based on immobilized enzyme bio-chromatography and its application in inhibitors screening in the last decade. In order to screen enzyme inhibitors from a mass of compounds in preliminary screening, multi-pore materials with good biocompatibility are used for the supports of immobilizing enzymes, and then the immobilized enzyme reactor applied as the immobilized enzyme stationary phase in HPLC. Therefore, a technology platform of high throughput screening is gradually established to screen the enzyme inhibitors as new anti-tumor drugs. Here, we briefly summarize the selective methods of supports, immobilization techniques, co-immobilized enzymes system and the screening model. PMID:29403726

Ballistic Motion of Enzymes that Catalyze Highly Exothermic Reactions

NASA Astrophysics Data System (ADS)

Tsekouras, Konstantinos; Pressé, Steve

Recently we proposed that the experimentally observed enhanced diffusion of enzymes catalyzing highly exothermic reactions is a consequence of their mechanism for dissipating reaction energy. More specifically, we proposed that reaction energy spreads out from the reaction site in the form of an acoustic wave which causes the enzyme to asymmetrically deform into the solvent. The solvent reaction propels the enzyme. However, it has been noted that in water, high viscosity should reduce enzyme momentum to zero within a few ps, so any diffusion increase should not be observable. Here we provide a model explaining how small volumetric expansions of biomolecules inside water may cause fluid compression that in turn creates regions of low fluid density around the biomolecule. We then investigate the dynamics of the biomolecule in the presence of these perturbations.

Molecular engineering of industrial enzymes: recent advances and future prospects.

PubMed

Yang, Haiquan; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Liu, Long; Chen, Jian

2014-01-01

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.

Development of rapid bioconversion with integrated recycle technology for ethanol production from extractive ammonia pretreated corn stover.

PubMed

Jin, Mingjie; Liu, Yanping; da Costa Sousa, Leonardo; Dale, Bruce E; Balan, Venkatesh

2017-08-01

High enzyme loading and low productivity are two major issues impeding low cost ethanol production from lignocellulosic biomass. This work applied rapid bioconversion with integrated recycle technology (RaBIT) and extractive ammonia (EA) pretreatment for conversion of corn stover (CS) to ethanol at high solids loading. Enzymes were recycled via recycling unhydrolyzed solids. Enzymatic hydrolysis with recycled enzymes and fermentation with recycled yeast cells were studied. Both enzymatic hydrolysis time and fermentation time were shortened to 24 h. Ethanol productivity was enhanced by two times and enzyme loading was reduced by 30%. Glucan and xylan conversions reached as high as 98% with an enzyme loading of as low as 8.4 mg protein per g glucan. The overall ethanol yield was 227 g ethanol/kg EA-CS (191 g ethanol/kg untreated CS). Biotechnol. Bioeng. 2017;114: 1713-1720. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Supramolecular catalysis beyond enzyme mimics.

PubMed

Meeuwissen, Jurjen; Reek, Joost N H

2010-08-01

Supramolecular catalysis - the assembly of catalyst species by harnessing multiple weak intramolecular interactions - has, until recently, been dominated by enzyme-inspired approaches. Such approaches often attempt to create an enzyme-like 'active site' and have concentrated on reactions similar to those catalysed by enzymes themselves. Here, we discuss the application of supramolecular assembly to the more traditional transition metal catalysis and to small-molecule organocatalysis. The modularity of self-assembled multicomponent catalysts means that a relatively small pool of catalyst components can provide rapid access to a large number of catalysts that can be evaluated for industrially relevant reactions. In addition, we discuss how catalyst-substrate interactions can be tailored to direct substrates along particular reaction paths and selectivities.

Artificial enzymes with protein scaffolds: structural design and modification.

PubMed

Matsuo, Takashi; Hirota, Shun

2014-10-15

Recent development in biochemical experiment techniques and bioinformatics has enabled us to create a variety of artificial biocatalysts with protein scaffolds (namely 'artificial enzymes'). The construction methods of these catalysts include genetic mutation, chemical modification using synthetic molecules and/or a combination of these methods. Designed evolution strategy based on the structural information of host proteins has become more and more popular as an effective approach to construct artificial protein-based biocatalysts with desired reactivities. From the viewpoint of application of artificial enzymes for organic synthesis, recently constructed artificial enzymes mediating oxidation, reduction and C-C bond formation/cleavage are introduced in this review article. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mesoporous silica-encapsulated gold nanoparticles as artificial enzymes for self-activated cascade catalysis.

PubMed

Lin, Youhui; Li, Zhenhua; Chen, Zhaowei; Ren, Jinsong; Qu, Xiaogang

2013-04-01

A significant challenge in chemistry is to create synthetic structures that mimic the complexity and function of natural systems. Here, a self-activated, enzyme-mimetic catalytic cascade has been realized by utilizing expanded mesoporous silica-encapsulated gold nanoparticles (EMSN-AuNPs) as both glucose oxidase- and peroxidase-like artificial enzymes. Specifically, EMSN helps the formation of a high degree of very small and well-dispersed AuNPs, which exhibit an extraordinarily stability and dual enzyme-like activities. Inspired by these unique and attractive properties, we further piece them together into a self-organized artificial cascade reaction, which is usually completed by the oxidase-peroxidase coupled enzyme system. Our finding may pave the way to use matrix as the structural component for the design and development of biomimetic catalysts and to apply enzyme mimics for realizing higher functions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biotechnological Processes in Microbial Amylase Production

PubMed Central

Arshad, M. K. Md; Lakshmipriya, Thangavel; Hashim, Uda; Chinni, Suresh V.

2017-01-01

Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology. It is produced mainly from microbial sources and is used in many industries. Industrial sectors with top-down and bottom-up approaches are currently focusing on improving microbial amylase production levels by implementing bioengineering technologies. The further support of energy consumption studies, such as those on thermodynamics, pinch technology, and environment-friendly technologies, has hastened the large-scale production of the enzyme. Herein, the importance of microbial (bacteria and fungi) amylase is discussed along with its production methods from the laboratory to industrial scales. PMID:28280725

Biotechnological Processes in Microbial Amylase Production.

PubMed

Gopinath, Subash C B; Anbu, Periasamy; Arshad, M K Md; Lakshmipriya, Thangavel; Voon, Chun Hong; Hashim, Uda; Chinni, Suresh V

2017-01-01

Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology. It is produced mainly from microbial sources and is used in many industries. Industrial sectors with top-down and bottom-up approaches are currently focusing on improving microbial amylase production levels by implementing bioengineering technologies. The further support of energy consumption studies, such as those on thermodynamics, pinch technology, and environment-friendly technologies, has hastened the large-scale production of the enzyme. Herein, the importance of microbial (bacteria and fungi) amylase is discussed along with its production methods from the laboratory to industrial scales.

Creating a Technologically Literate Classroom: Professional's Guide. Teacher Created Materials No. 887.

ERIC Educational Resources Information Center

Garfield, Gary M.; McDonough, Suzanne

This book discusses how to effectively integrate technology into the classroom. It examines the benefits of curriculum development utilizing technology and presents sample learning activities. Highlights include: technology's past and present role in education; access to computers; the roles of teacher and learner; professional development;…

JPRS Report, Science & Technology: China.

DTIC Science & Technology

1989-01-31

et al.; GUANGXUE XUEBAO, No 8, Aug 88] 130 Hardening Treatment for the Surface of 50 SiMnP Steel for High Power Laser [Yu Hongtao , Cao Tianshun...and Qian Bin [6929 2430]; helped with the technical work; the tool enzyme unit provided the enzymes; and the Shanghai Biological Products Institute...154 [Article by Yu Hongtao ]0060 3163 3447] and Cao Tianshun [2580 1131 7311] of Angang College of Technology] [Abstract] The article proposes a

The Enzyme Portal: a case study in applying user-centred design methods in bioinformatics

PubMed Central

2013-01-01

User-centred design (UCD) is a type of user interface design in which the needs and desires of users are taken into account at each stage of the design process for a service or product; often for software applications and websites. Its goal is to facilitate the design of software that is both useful and easy to use. To achieve this, you must characterise users’ requirements, design suitable interactions to meet their needs, and test your designs using prototypes and real life scenarios. For bioinformatics, there is little practical information available regarding how to carry out UCD in practice. To address this we describe a complete, multi-stage UCD process used for creating a new bioinformatics resource for integrating enzyme information, called the Enzyme Portal (http://www.ebi.ac.uk/enzymeportal). This freely-available service mines and displays data about proteins with enzymatic activity from public repositories via a single search, and includes biochemical reactions, biological pathways, small molecule chemistry, disease information, 3D protein structures and relevant scientific literature. We employed several UCD techniques, including: persona development, interviews, ‘canvas sort’ card sorting, user workflows, usability testing and others. Our hope is that this case study will motivate the reader to apply similar UCD approaches to their own software design for bioinformatics. Indeed, we found the benefits included more effective decision-making for design ideas and technologies; enhanced team-working and communication; cost effectiveness; and ultimately a service that more closely meets the needs of our target audience. PMID:23514033

Zinc finger nuclease technology: advances and obstacles in modelling and treating genetic disorders.

PubMed

Jabalameli, Hamid Reza; Zahednasab, Hamid; Karimi-Moghaddam, Amin; Jabalameli, Mohammad Reza

2015-03-01

Zinc finger nucleases (ZFNs) are engineered restriction enzymes designed to target specific DNA sequences within the genome. Assembly of zinc finger DNA-binding domain to a DNA-cleavage domain enables the enzyme machinery to target unique locus in the genome and invoke endogenous DNA repair mechanisms. This machinery offers a versatile approach in allele editing and gene therapy. Here we discuss the architecture of ZFNs and strategies for generating targeted modifications within the genome. We review advances in gene therapy and modelling of the disease using these enzymes and finally, discuss the practical obstacles in using this technology. Copyright © 2014 Elsevier B.V. All rights reserved.

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

PubMed Central

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-01

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

Enzymes as modular catalysts for redox half-reactions in H2-powered chemical synthesis: from biology to technology.

PubMed

Reeve, Holly A; Ash, Philip A; Park, HyunSeo; Huang, Ailun; Posidias, Michalis; Tomlinson, Chloe; Lenz, Oliver; Vincent, Kylie A

2017-01-15

The present study considers the ways in which redox enzyme modules are coupled in living cells for linking reductive and oxidative half-reactions, and then reviews examples in which this concept can be exploited technologically in applications of coupled enzyme pairs. We discuss many examples in which enzymes are interfaced with electronically conductive particles to build up heterogeneous catalytic systems in an approach which could be termed synthetic biochemistry We focus on reactions involving the H + /H 2 redox couple catalysed by NiFe hydrogenase moieties in conjunction with other biocatalysed reactions to assemble systems directed towards synthesis of specialised chemicals, chemical building blocks or bio-derived fuel molecules. We review our work in which this approach is applied in designing enzyme-modified particles for H 2 -driven recycling of the nicotinamide cofactor NADH to provide a clean cofactor source for applications of NADH-dependent enzymes in chemical synthesis, presenting a combination of published and new work on these systems. We also consider related photobiocatalytic approaches for light-driven production of chemicals or H 2 as a fuel. We emphasise the techniques available for understanding detailed catalytic properties of the enzymes responsible for individual redox half-reactions, and the importance of a fundamental understanding of the enzyme characteristics in enabling effective applications of redox biocatalysis. © 2017 The Author(s).

Leveraging Simulation Against the F-16 Flying Training Gap

DTIC Science & Technology

2005-11-01

must leverage emerging simulation technology into combined flight training to counter mission employment complexity created by technology itself...two or more of these stand-alone simulators creates a mission training center (MTC), which when further networked create distributed mission...operations (DMO). Ultimately, the grand operational vision of DMO is to interconnect non-collocated users creating a “virtual” joint training environment

iAB-RBC-283: A proteomically derived knowledge-base of erythrocyte metabolism that can be used to simulate its physiological and patho-physiological states.

PubMed

Bordbar, Aarash; Jamshidi, Neema; Palsson, Bernhard O

2011-07-12

The development of high-throughput technologies capable of whole cell measurements of genes, proteins, and metabolites has led to the emergence of systems biology. Integrated analysis of the resulting omic data sets has proved to be hard to achieve. Metabolic network reconstructions enable complex relationships amongst molecular components to be represented formally in a biologically relevant manner while respecting physical constraints. In silico models derived from such reconstructions can then be queried or interrogated through mathematical simulations. Proteomic profiling studies of the mature human erythrocyte have shown more proteins present related to metabolic function than previously thought; however the significance and the causal consequences of these findings have not been explored. Erythrocyte proteomic data was used to reconstruct the most expansive description of erythrocyte metabolism to date, following extensive manual curation, assessment of the literature, and functional testing. The reconstruction contains 281 enzymes representing functions from glycolysis to cofactor and amino acid metabolism. Such a comprehensive view of erythrocyte metabolism implicates the erythrocyte as a potential biomarker for different diseases as well as a 'cell-based' drug-screening tool. The analysis shows that 94 erythrocyte enzymes are implicated in morbid single nucleotide polymorphisms, representing 142 pathologies. In addition, over 230 FDA-approved and experimental pharmaceuticals have enzymatic targets in the erythrocyte. The advancement of proteomic technologies and increased generation of high-throughput proteomic data have created the need for a means to analyze these data in a coherent manner. Network reconstructions provide a systematic means to integrate and analyze proteomic data in a biologically meaning manner. Analysis of the red cell proteome has revealed an unexpected level of complexity in the functional capabilities of human erythrocyte metabolism.

PCP IMMUMOASSAY TECHNOLOGIES - INNOVATIVE TECHNOLOGY EVALUATION REPORT

EPA Science Inventory

Three enzyme-linked immunosorbent assay technologies for pentachlorophenol (PCP) testing in soil and water were evaluated. Penta RISc Test Systems (formerly ENSYS, Inc.), EnviroGard™ PCP Immunoassay Test Kit (Millipore Corp.), and Pentachlorophenol RaPID Assay (formerly Ohmicron ...

Spatial and temporal control of microwave triggered chemiluminescence: a protein detection platform.

PubMed

Previte, Michael J R; Aslan, Kadir; Geddes, Chris D

2007-09-15

We have combined the principles of microwave circuitry and antenna design and our recent work in microwave-triggered metal-enhanced chemiluminescence to now "trigger" chemically and enzyme-catalyzed chemiluminescent reactions with spatial and temporal control. With this technology platform, we achieve spatial and temporal control of enzyme and chemically catalyzed chemiluminescence reactions to achieve more than 500-fold increases in "on-demand" photon flux from chemically catalyzed chemiluminescent reactions. We also report a 6-fold increase in photon flux from HRP-catalyzed assays on disposable coverslips functionalized with HRP and placed proximal to the substrates modified with thin-film aluminum triangle disjointed "bow-tie" structures. In addition, we demonstrate the applicability of this technology to develop multiplexed or high-throughput chemiluminescent assays. We also demonstrate the clinical and biological relevance of this technology platform by affixing aluminum structures in proximity to HRP protein immobilized on nitrocellulose to improve the sensitivity for this model Western blot scheme by 50-fold. We believe analytical applications that rely on enzyme-catalyzed chemiluminescence, such as immunoassays, may greatly benefit from this new platform technology.

Engineered Antibodies for Monitoring of Polynuclear Aromatic Hydrocarbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alexander E. Karu Ph.D; Victoria A. Roberts Ph.D.; Qing X. Li, Ph.D.

2002-01-17

This project was undertaken to fill needs in ODE's human and ecosystem health effects research, site remediation, rapid emergency response, and regulatory compliance monitoring programs. Doe has greatly stimulated development and validation of antibody-based, rapid, field-portable detection systems for small hazardous compounds. These range from simple dipsticks, microplate enzyme-linked immunosorbent assays (ELISAs), and hand-held colorimeters, to ultrasensitive microfluidic reactors, fiber-optic sensors and microarrays that can identify multiple analytes from patterns of cross-reactivity. Unfortunately, the technology to produce antibodies with the most desirable properties did not keep pace. Lack of antibodies remains a limiting factor in production and practical use ofmore » such devices. The goals of our project were to determine the chemical and structural bases for the antibody-analyte binding interactions using advanced computational chemistry, and to use this information to create useful new binding properties through in vitro genetic engineering and combinatorial library methods.« less

Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

PubMed

Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

2015-08-10

The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

The role of adenosine monophosphate kinase in remodeling white adipose tissue metabolism.

PubMed

Gaidhu, Mandeep Pinky; Ceddia, Rolando Bacis

2011-04-01

Recent evidence indicates that the enzyme adenosine monophosphate (AMP) kinase exerts important fat-reducing effects in the adipose tissue, which has created great interest in this enzyme as a potential target for obesity treatment. This review summarizes our findings that chronic AMP kinase activation remodels adipocyte glucose and lipid metabolism and enhances the ability of adipose tissue to dissipate energy within itself and reduce adiposity.

Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions

PubMed Central

Maréchal, J-D; Kemp, C A; Roberts, G C K; Paine, M J I; Wolf, C R; Sutcliffe, M J

2008-01-01

The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity—including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes. PMID:18026129

Effects of combined pressure and temperature on enzymes related to quality of fruits and vegetables: from kinetic information to process engineering aspects.

PubMed

Ludikhuyze, L; Van Loey, A; Indrawati; Smout, C; Hendrickx, M

2003-01-01

Throughout the last decade, high pressure technology has been shown to offer great potential to the food processing and preservation industry in delivering safe and high quality products. Implementation of this new technology will be largely facilitated when a scientific basis to assess quantitatively the impact of high pressure processes on food safety and quality becomes available. Besides, quantitative data on the effects of pressure and temperature on safety and quality aspects of foods are indispensable for design and evaluation of optimal high pressure processes, i.e., processes resulting in maximal quality retention within the constraints of the required reduction of microbial load and enzyme activity. Indeed it has to be stressed that new technologies should deliver, apart from the promised quality improvement, an equivalent or preferably enhanced level of safety. The present paper will give an overview from a quantitative point of view of the combined effects of pressure and temperature on enzymes related to quality of fruits and vegetables. Complete kinetic characterization of the inactivation of the individual enzymes will be discussed, as well as the use of integrated kinetic information in process engineering.

Engineering Technology Of Fish Farming Floating Nets Cages On Polka Dot Grouper (Cromileptes Altivelis) Used Artificial Feed Enriched Phytase Enzyme

NASA Astrophysics Data System (ADS)

Samidjan, Istiyanto; Rachmawati, Diana

2018-02-01

One solution is to utilize engineering technology cultivation floating cage net polka dot grouper (ducker grouper), which is given artificial feed enriched with phytase enzymes. The objectives of this study was to examine the use of technology engineering floating net on ducker grouper on artificial feed that is enriched with different dose phytase enzymes to accelerate growth and survival. The research method used ducker grouper fish size 15,5 ± 0,5 cm in the net cages unit (1 m x 1 m x 1 m), 250 fish per cage, using 12 cages. Each net-cages was made of polyethylens netting, mesh size 12.5 mm. with complete randomized design (CRD) 4 treatment and 3 replication were feed Artificial enriched of phytase enzyme with the doses of A (0 FTU · kg-1 diet), B (200 FTU · kg-1 diet), C (500 FTU · kg-1 diet), and D (800 FTU · kg-1 diet) phytase enzyme. Feed was given 2 times a day in the morning and afternoon with 5% biomass per day. Data includes the growth of absolute weight polka dot grouper, FCR, and survival rate analyzed variety and Test Tukey.The result of the research showed that the difference of artificial feeding enriched phytase enzyme significantly (P <0,05) to growth, food conversion ratio (FCR), survival rete of polka dot grouper. The best treatment at C (500 mg / kg of feed) increase growth of absolute weight of 128.75 g, 1.75 (FCR), and a survival rate of 93.5%.

Designing proteins for therapeutic applications.

PubMed

Lazar, Greg A; Marshall, Shannon A; Plecs, Joseph J; Mayo, Stephen L; Desjarlais, John R

2003-08-01

Protein design is becoming an increasingly useful tool for optimizing protein drugs and creating novel biotherapeutics. Recent progress includes the engineering of monoclonal antibodies, cytokines, enzymes and viral fusion inhibitors.

Towards efficient chemical synthesis via engineering enzyme catalysis in biomimetic nanoreactors.

PubMed

Liu, Jia; Yang, Qihua; Li, Can

2015-09-18

Biocatalysis with immobilized enzymes as catalysts holds enormous promise in developing more efficient and sustainable processes for the synthesis of fine chemicals, chiral pharmaceuticals and biomass feedstocks. Despite the appealing potentials, nowadays the industrial-scale application of biocatalysts is still quite modest in comparison with that of traditional chemical catalysts. A critical issue is that the catalytic performance of enzymes, the sophisticated and vulnerable catalytic machineries, strongly depends on their intracellular working environment; however the working circumstances provided by the support matrix are radically different from those in cells. This often leads to various adverse consequences on enzyme conformation and dynamic properties, consequently decreasing the overall performance of immobilized enzymes with regard to their activity, selectivity and stability. Engineering enzyme catalysis in support nanopores by mimicking the physiological milieu of enzymes in vivo and investigating how the interior microenvironment of nanopores imposes an influence on enzyme behaviors in vitro are of paramount significance to modify and improve the catalytic functions of immobilized enzymes. In this feature article, we have summarized the recent advances in mimicking the working environment and working patterns of intracellular enzymes in nanopores of mesoporous silica-based supports. Especially, we have demonstrated that incorporation of polymers into silica nanopores could be a valuable approach to create the biomimetic microenvironment for enzymes in the immobilized state.

Biochemical Platform Processing Integration

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

The objective of this project is to facilitate deployment of enzyme-based biomass conversion technology. The immediate goal is to explore integration issues that impact process performance and to demonstrate improved performance of the lower-cost enzymes being developed by Genencor and Novozymes.

Creating an X Window Terminal-Based Information Technology Center.

ERIC Educational Resources Information Center

Klassen, Tim W.

1997-01-01

The creation of an information technology center at the University of Oregon Science Library is described. Goals included providing access to Internet-based resources and multimedia software, platforms for running science-oriented software, and resources so students can create multimedia materials. A mixed-lab platform was created with Unix-based…

Finding Sequences for over 270 Orphan Enzymes

PubMed Central

Shearer, Alexander G.; Altman, Tomer; Rhee, Christine D.

2014-01-01

Despite advances in sequencing technology, there are still significant numbers of well-characterized enzymatic activities for which there are no known associated sequences. These ‘orphan enzymes’ represent glaring holes in our biological understanding, and it is a top priority to reunite them with their coding sequences. Here we report a methodology for resolving orphan enzymes through a combination of database search and literature review. Using this method we were able to reconnect over 270 orphan enzymes with their corresponding sequence. This success points toward how we can systematically eliminate the remaining orphan enzymes and prevent the introduction of future orphan enzymes. PMID:24826896

Computational Enzymology and Organophosphorus Degrading Enzymes: Promising Approaches Toward Remediation Technologies of Warfare Agents and Pesticides.

PubMed

Ramalho, Teodorico C; de Castro, Alexandre A; Silva, Daniela R; Silva, Maria Cristina; Franca, Tanos C C; Bennion, Brian J; Kuca, Kamil

2016-01-01

The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and help in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.

Self-assembly of protein-based biomaterials initiated by titania nanotubes.

PubMed

Forstater, Jacob H; Kleinhammes, Alfred; Wu, Yue

2013-12-03

Protein-based biomaterials are a promising strategy for creating robust highly selective biocatalysts. The assembled biomaterials must sufficiently retain the near-native structure of proteins and provide molecular access to catalytically active sites. These requirements often exclude the use of conventional assembly techniques, which rely on covalent cross-linking of proteins or entrapment within a scaffold. Here we demonstrate that titania nanotubes can initiate and template the self-assembly of enzymes, such as ribonuclease A, while maintaining their catalytic activity. Initially, the enzymes form multilayer thick ellipsoidal aggregates centered on the nanotube surface; subsequently, these nanosized entities assemble into a micrometer-sized enzyme material that has enhanced enzymatic activity and contains as little as 0.1 wt % TiO2 nanotubes. This phenomenon is uniquely associated with the active anatase (001)-like surface of titania nanotubes and does not occur on other anatase nanomaterials, which contain significantly fewer undercoordinated Ti surface sites. These findings present a nanotechnology-enabled mechanism of biomaterial growth and open a new route for creating stable protein-based biomaterials and biocatalysts without the need for chemical modification.

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

PubMed Central

Fischbach, Michael A.; Lai, Jonathan R.; Roche, Eric D.; Walsh, Christopher T.; Liu, David R.

2007-01-01

Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with ≈10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (103 to 104 clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts. PMID:17620609

Fungal Enzymes for Bio-Products from Sustainable and Waste Biomass.

PubMed

Gupta, Vijai K; Kubicek, Christian P; Berrin, Jean-Guy; Wilson, David W; Couturier, Marie; Berlin, Alex; Filho, Edivaldo X F; Ezeji, Thaddeus

2016-07-01

Lignocellulose, the most abundant renewable carbon source on earth, is the logical candidate to replace fossil carbon as the major biofuel raw material. Nevertheless, the technologies needed to convert lignocellulose into soluble products that can then be utilized by the chemical or fuel industries face several challenges. Enzymatic hydrolysis is of major importance, and we review the progress made in fungal enzyme technology over the past few years with major emphasis on (i) the enzymes needed for the conversion of polysaccharides (cellulose and hemicellulose) into soluble products, (ii) the potential uses of lignin degradation products, and (iii) current progress and bottlenecks for the use of the soluble lignocellulose derivatives in emerging biorefineries. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stalking The Silent Killers.

ERIC Educational Resources Information Center

DeYoung, H. Garrett

1983-01-01

New technologies have been developed to diagnose such disorders as cancer, heart disease, and defects of the newborn in presymptomatic stages. Several technologies, including amniocentesis, radioimmunoassay, enzyme immunoassay, multichannel analysis and others are discussed. Development of these technologies and issues related to their use are…

Using Internet Technologies To Enhance Training.

ERIC Educational Resources Information Center

Pollock, Carl; Masters, Robert

1997-01-01

Describes how to use Internet technologies to create an intranet, or an online training database system, for improving company communications, effectiveness, and job performance. Topics include technology and performance; educating managers and key decision makers; creating a graphic model of the training system; and fitting into the existing…

Creating Standards-Based Technology Education Facilities

ERIC Educational Resources Information Center

Daugherty, Michael K.; Klenke, Andrew M.; Neden, Michael

2008-01-01

One of the most intimidating tasks faced by new or practicing technology education teachers is the challenge of creating new facilities or renovating current facilities for a new purpose. While the fourth program standard in "Advancing Excellence in Technological Literacy: Student Assessment, Professional Development, and Program Standards (AETL)"…

Pilot study of a novel vacuum-assisted method for decellularization of tracheae for clinical tissue engineering applications.

PubMed

Lange, P; Greco, K; Partington, L; Carvalho, C; Oliani, S; Birchall, M A; Sibbons, P D; Lowdell, M W; Ansari, T

2017-03-01

Tissue engineered tracheae have been successfully implanted to treat a small number of patients on compassionate grounds. The treatment has not become mainstream due to the time taken to produce the scaffold and the resultant financial costs. We have developed a method for decellularization (DC) based on vacuum technology, which when combined with an enzyme/detergent protocol significantly reduces the time required to create clinically suitable scaffolds. We have applied this technology to prepare porcine tracheal scaffolds and compared the results to scaffolds produced under normal atmospheric pressures. The principal outcome measures were the reduction in time (9 days to prepare the scaffold) followed by a reduction in residual DNA levels (DC no-vac: 137.8±48.82 ng/mg vs. DC vac 36.83±18.45 ng/mg, p<0.05.). Our approach did not impact on the collagen or glycosaminoglycan content or on the biomechanical properties of the scaffolds. We applied the vacuum technology to human tracheae, which, when implanted in vivo showed no significant adverse immunological response. The addition of a vacuum to a conventional decellularization protocol significantly reduces production time, whilst providing a suitable scaffold. This increases clinical utility and lowers production costs. To our knowledge this is the first time that vacuum assisted decellularization has been explored. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade

NASA Astrophysics Data System (ADS)

Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry

2016-12-01

A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

Fungal Beta-Glucosidases: A Bottleneck in Industrial Use of Lignocellulosic Materials

PubMed Central

Sørensen, Annette; Lübeck, Mette; Lübeck, Peter S.; Ahring, Birgitte K.

2013-01-01

Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, beta-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose accumulation. In this review, we discuss the important role beta-glucosidases play in complex biomass hydrolysis and how they create a bottleneck in industrial use of lignocellulosic materials. An efficient beta-glucosidase facilitates hydrolysis at specified process conditions, and key points to consider in this respect are hydrolysis rate, inhibitors, and stability. Product inhibition impairing yields, thermal inactivation of enzymes, and the high cost of enzyme production are the main obstacles to commercial cellulose hydrolysis. Therefore, this sets the stage in the search for better alternatives to the currently available enzyme preparations either by improving known or screening for new beta-glucosidases. PMID:24970184

Entrapment of subtilisin in ceramic sol-gel coating for antifouling applications.

PubMed

Regina, Viduthalai Rasheedkhan; Søhoel, Helmer; Lokanathan, Arcot Raghupathi; Bischoff, Claus; Kingshott, Peter; Revsbech, Niels Peter; Meyer, Rikke Louise

2012-11-01

Enzymes with antifouling properties are of great interest in developing nontoxic antifouling coatings. A bottleneck in developing enzyme-based antifouling coatings is to immobilize the enzyme in a suitable coating matrix without compromising its activity and stability. Entrapment of enzymes in ceramics using the sol-gel method is known to have several advantages over other immobilization methods. The sol-gel method can be used to make robust coatings, and the aim of this study was to explore if sol-gel technology can be used to develop robust coatings harboring active enzymes for antifouling applications. We successfully entrapped a protease, subtilisin (Savinase, Novozymes), in a ceramic coating using a sol-gel method. The sol-gel formulation, when coated on a stainless steel surface, adhered strongly and cured at room temperature in less than 8 h. The resultant coating was smoother and less hydrophobic than stainless steel. Changes in the coating's surface structure, thickness and chemistry indicate that the coating undergoes gradual erosion in aqueous medium, which results in release of subtilisin. Subtilisin activity in the coating increased initially, and then gradually decreased. After 9 months, 13% of the initial enzyme activity remained. Compared to stainless steel, the sol-gel-coated surfaces with active subtilisin were able to reduce bacterial attachment of both Gram positive and Gram negative bacteria by 2 orders of magnitude. Together, our results demonstrate that the sol-gel method is a promising coating technology for entrapping active enzymes, presenting an interesting avenue for enzyme-based antifouling solutions.

Enzyme assisted extraction of biomolecules as an approach to novel extraction technology: A review.

PubMed

Nadar, Shamraja S; Rao, Priyanka; Rathod, Virendra K

2018-06-01

An interest in the development of extraction techniques of biomolecules from various natural sources has increased in recent years due to their potential applications particularly for food and nutraceutical purposes. The presence of polysaccharides such as hemicelluloses, starch, pectin inside the cell wall, reduces the extraction efficiency of conventional extraction techniques. Conventional techniques also suffer from low extraction yields, time inefficiency and inferior extract quality due to traces of organic solvents present in them. Hence, there is a need of the green and novel extraction methods to recover biomolecules. The present review provides a holistic insight to various aspects related to enzyme aided extraction. Applications of enzymes in the recovery of various biomolecules such as polyphenols, oils, polysaccharides, flavours and colorants have been highlighted. Additionally, the employment of hyphenated extraction technologies can overcome some of the major drawbacks of enzyme based extraction such as longer extraction time and immoderate use of solvents. This review also includes hyphenated intensification techniques by coupling conventional methods with ultrasound, microwave, high pressure and supercritical carbon dioxide. The last section gives an insight on application of enzyme immobilization as a strategy for large scale extraction. Immobilization of enzymes on magnetic nanoparticles can be employed to enhance the operational performance of the system by multiple use of expensive enzymes making them industrially and economically feasible. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Biosensor development in clinical analysis].

PubMed

Boitieux, J L; Desmet, G; Thomas, D

1985-01-01

The use of enzymes immobilized or as markers formed the subject of more than thousand publications in the field of industry or biomedical applications, during the last five years. Recently, some authors published works concerning immobilization of total microorganisms for catalytic purposes, others use the enzymatic activity for marking molecules involved in immunological analysis processes. Together industrial biotechnology and medical analysis laboratory are interested with the evolution of these procedures involving the activity of immobilized enzymes. Enzyme immobilization allowed the lowering of analysis costs for, in this case, the enzyme can be used several times. We take account of the two main cases which are encountered during utilization of immobilized enzymes of analytical purposes. The enzyme is used directly for the catalysed reaction or it is used as enzymatic marker. These both aspects are developed mainly for the elaboration of enzymatic and immunoenzymatic electrodes and the realization of automatic computerized devices allowing continuous estimation of numerous biological blood parameters. From these two precise examples, glucose and antigen determination, the authors show the evolution of these technologies in the field of immobilized enzymes or captors and the analysis of signals given by these electrodes requiring a computerized treatment. This new technology opens to important potentialities in the analytical field. The automatization of these devices allowing the control in real time, will probably make easier the optimization steps of procedures actually used in the biomedical sphere.

Online Experiential Education for Technological Entrepreneurs

ERIC Educational Resources Information Center

Ermolovich, Thomas R.

2011-01-01

Technological Entrepreneurship is both an art and a science. As such, the education of a technological entrepreneur requires both an academic and an experiential component. One form of experiential education is creating real new ventures with student teams. When these ventures are created in an online modality, students work in virtual teams and…

Podcasting as Transformative Work

ERIC Educational Resources Information Center

Vasquez, Vivian Maria

2015-01-01

Although technology is most often heralded an intrinsic efficiency, the process of using technology to create a text is full of opportunities to learn new information, create and distribute knowledge, and engage in identity work. This article explores how the production of 1 such technological text, a podcast, offered students opportunities to…

Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

PubMed Central

Jung, Sang-Kyu; Parisutham, Vinuselvi; Jeong, Seong Hun; Lee, Sung Kuk

2012-01-01

A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. PMID:22911272

Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms.

PubMed

Thallinger, Barbara; Prasetyo, Endry N; Nyanhongo, Gibson S; Guebitz, Georg M

2013-01-01

With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme-based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi-enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum-sensing molecules. The inclusion of anti-quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti-biofilm enzymes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Binding Database: data management and interface design.

PubMed

Chen, Xi; Lin, Yuhmei; Liu, Ming; Gilson, Michael K

2002-01-01

The large and growing body of experimental data on biomolecular binding is of enormous value in developing a deeper understanding of molecular biology, in developing new therapeutics, and in various molecular design applications. However, most of these data are found only in the published literature and are therefore difficult to access and use. No existing public database has focused on measured binding affinities and has provided query capabilities that include chemical structure and sequence homology searches. We have created Binding DataBase (BindingDB), a public, web-accessible database of measured binding affinities. BindingDB is based upon a relational data specification for describing binding measurements via Isothermal Titration Calorimetry (ITC) and enzyme inhibition. A corresponding XML Document Type Definition (DTD) is used to create and parse intermediate files during the on-line deposition process and will also be used for data interchange, including collection of data from other sources. The on-line query interface, which is constructed with Java Servlet technology, supports standard SQL queries as well as searches for molecules by chemical structure and sequence homology. The on-line deposition interface uses Java Server Pages and JavaBean objects to generate dynamic HTML and to store intermediate results. The resulting data resource provides a range of functionality with brisk response-times, and lends itself well to continued development and enhancement.

High-throughput selection for cellulase catalysts using chemical complementation.

PubMed

Peralta-Yahya, Pamela; Carter, Brian T; Lin, Hening; Tao, Haiyan; Cornish, Virginia W

2008-12-24

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation

PubMed Central

Peralta-Yahya, Pamela; Carter, Brian T.; Lin, Hening; Tao, Haiyan; Cornish, Virginia W.

2010-01-01

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases however is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Due to the large number of enzyme variants selections can test compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity. PMID:19053460

Acquisition Modernization: Transitioning Technology Into Warfighter Capability

DTIC Science & Technology

2011-08-01

to test and evaluate the technology and integrate the new capability into operational weapon systems (Figure 4). This funding model creates stove...misalignment between missions, TRLs, and the RDT&E funding model is a major 11 contributor to the valley of death. Technologies become obsolete on... funding model of the acquisition system. Create an individual budget account to fund the development of promising technologies. The Acquisition

A Laboratory Exercise to Understand the Importance of Enzyme Technology in the Fruit-Processing Industry: Viscosity Decrease and Phenols Release from Apple Mash

ERIC Educational Resources Information Center

Pinelo, Manuel; Nielsen, Michael K.; Meyer, Anne S.

2011-01-01

In a 4-h laboratory exercise, students accomplish a series of enzymatic macerations of apple mash, assess the viscosity of the mash during the maceration, extract the juice by centrifugation, and measure the levels of antioxidant phenols extracted into the juice after different enzyme treatments. The exercise shows the impact of enzyme-catalyzed…

Identification of Metabolic Routes and Catabolic Enzymes Involved in Phytoremediation of the Nitro-Substituted Explosives TNT, RDX, and HMX

DTIC Science & Technology

2006-07-31

Identification of Metabolic Routes and Catabolic Enzymes Involved in Phytoremediation of the Nitro- Substituted Explosives TNT, RDX...Routes and Catabolic Enzymes Involved in Phytoremediation of the Nitro-Substituted Explosives TNT, RDX, and HMX Final Technical Report 5a. CONTRACT NUMBER... Phytoremediation has been shown to provide a cost-effective alternative to classical technologies for cleaning up nitro-substituted explosive

Production of Enzymes from Marine Actinobacteria.

PubMed

Zhao, X Q; Xu, X N; Chen, L Y

Marine actinobacteria are well recognized for their capabilities to produce valuable natural products, which have great potential for applications in medical, agricultural, and fine chemical industries. In addition to producing unique enzymes responsible for biosynthesis of natural products, many marine actinobacteria also produce hydrolytic enzymes which are able to degrade various biopolymers, such as cellulose, xylan, and chitin. These enzymes are important to produce biofuels and biochemicals of interest from renewable biomass. In this chapter, the recent reports of novel enzymes produced by marine actinobacteria are reviewed, and advanced technologies that can be applied to search for novel marine enzymes as well as for improved enzyme production by marine actinobacteria are summarized, which include ribosome engineering, genome mining, as well as synthetic biology studies. © 2016 Elsevier Inc. All rights reserved.

Can enzyme engineering benefit from the modulation of protein motions? Lessons learned from NMR relaxation dispersion experiments.

PubMed

Doucet, Nicolas

2011-04-01

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant 'invisible states' adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing 'structure-function' analyses with 'flexibility-function' investigations. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

An Examination of Faculty Innovativeness in Relation to Inductive Teaching and the Use of Technology

ERIC Educational Resources Information Center

Forrer, Donald A.; Wyant, Nancey A.; Gordin, Patricia C.

2014-01-01

The conceptual framework for this research describes the processes faculty use to create an online course that meets learning outcomes while creating a positive learning experience for the online student. This involves acceptance of technology to create a course structured for inductive learning in addition to traditional deductive learning. The…

Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

PubMed

Tóth, Eszter; Huszár, Krisztina; Bencsura, Petra; Kulcsár, Péter István; Vodicska, Barbara; Nyeste, Antal; Welker, Zsombor; Tóth, Szilvia; Welker, Ervin

2014-01-01

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease--a Body Double of the Type IIP enzyme--is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

Creating Games as Authentic Learning in the Information Technology Classroom

ERIC Educational Resources Information Center

Frydenberg, Mark

2015-01-01

Teaching students to create computer games has become a common practice in both K-12 and tertiary education to introducing programming concepts, increasing student engagement, and recruiting majors and minors in technology fields. This study describes a project where first-year college students in an introductory technology concepts course use a…

The Tools of Teacher Education: Preservice Teachers' Use of Technology To Create Instructional Materials.

ERIC Educational Resources Information Center

Roberts, Sherron Killingsworth; Hsu, Ying-Shao

2000-01-01

Examines the effectiveness/efficiency of preservice teachers' use of technology to create instructional materials developed in an undergraduate reading/language arts course. Results showed no significant difference between measures of overall quality of the technology assistance as compared to handmade prompts. Eighty-five percent of the teachers…

Comic Relief: Graduate Students Address Multiple Meanings for Technology Integration with Digital Comic Creation

ERIC Educational Resources Information Center

Sockman, Beth Rajan; Sutton, Rhonda; Herrmann, Michele

2016-01-01

This study determined the usefulness of digital comic creation with 77 graduate students in a teacher technology course. Students completed an assigned reading and created digital comics that addressed technology integration concerns in the schools and society. Using practical action research, 77 student-created comics were analyzed. The findings…

Directed evolution: an approach to engineer enzymes.

PubMed

Kaur, Jasjeet; Sharma, Rohit

2006-01-01

Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.

A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA).

PubMed

Kai, Junhai; Puntambekar, Aniruddha; Santiago, Nelson; Lee, Se Hwan; Sehy, David W; Moore, Victor; Han, Jungyoup; Ahn, Chong H

2012-11-07

In this work we introduce a novel microfluidic enzyme linked immunoassays (ELISA) microplate as the next generation assay platform for unparalleled assay performances. A combination of microfluidic technology with standard SBS-configured 96-well microplate architecture, in the form of microfluidic microplate technology, allows for the improvement of ELISA workflows, conservation of samples and reagents, improved reaction kinetics, and the ability to improve the sensitivity of the assay by multiple analyte loading. This paper presents the design and characterization of the microfluidic microplate, and its application in ELISA.

The synthesis and host-guest applications of synthetic receptor molecules

NASA Astrophysics Data System (ADS)

Osner, Zachary R.

2011-12-01

Host-guest chemistry involves the complimentary binding between two molecules. Host molecules have been synthesized to bind negative, positive, and neutral molecules such as proteins and enzymes, and have been used as optical sensors, electrochemical sensors, supramolecular catalysts, and in the pharmaceutical industry as anti-cancer agents.1 The field of nanoscience has exploited guest-host interactions to create optical sensors with colloidal gold and Dip-Pen nanolithography technologies. Gold nanoparticles, have been functionalized with DNA, and have been developed as a selective colorimetric detection system, that upon binding turns the solution from a red to blue in color.2 Cyclotriveratrylene (CTV) 1 is a common supramolecular scaffold that has been previously employed in guest-host chemistry, and the construction of CTV involves the cyclic trimerization of veratryl alcohol via the veratryl cation.3 Due to the rigid bowl shaped structure of CTV, CTV has been shown to act as a host molecule for fullerene-C60.4 Lectin binding receptor proteins are a specific class of proteins found in bacteria, viruses, plants, and animals that can bind to complimentary carbohydrates. It is these lectins that are believed to be responsible for cell-cell interactions and the formation of biofilms in pathenogenic bacteria.5 P. aeruginosa is a pathenogenic bacterium, shown to have a high resistance to many antibiotics, which can form biofilms in human lung tissue, causing respiratory tract infections in patients with compromised immune systems. 5 I will exploit guest-host interactions to create synthetic supramolecular and carbohydrate receptor molecules to that will be of use as biological sensing devices via self-assembled monolayers on solid surfaces and nanoparticle technologies. *Please refer to dissertation for references/footnotes.

Industrial Competitiveness and Technological Advancement: Debate Over Government Policy

DTIC Science & Technology

2007-03-19

private - sector technological development. Legislative activity over the past two decades has created a policy for technology development, albeit an ad hoc one. Because of the lack of consensus on the scope and direction of a national policy, Congress has taken an incremental approach aimed at creating new mechanisms to facilitate technological advancement in particular areas and making changes and improvements as necessary. Congressional action has mandated specific technology development programs and obligations in federal agencies that did not initially support such

Enzyme-Responsive Delivery of Multiple Proteins with Spatiotemporal Control.

PubMed

Zhu, Suwei; Nih, Lina; Carmichael, S Thomas; Lu, Yunfeng; Segura, Tatiana

2015-06-24

Orchestrated biological materials such as enzymes and growth factors regulate the growth of tissues and organs. A chirality-controlled, single-protein technology is devised to tailor the spatiotemporally defined delivery of therapeutic proteins in response to natural enzymes present at wound sites. Sustained delivery of one protein and sequential delivery of two proteins are demonstrated for stroke and skin wound healing. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization.

PubMed

Cowan, Don A; Fernandez-Lafuente, Roberto

2011-09-10

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process. Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications. Copyright © 2011 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jia, Feng

The main objective of this project is to design novel nano-structured carriers and strategies to co-localize multiple enzymes to mimic the functionalities of MECs. In order to achieve this goal, distinct approaches for enzyme co-localization were developed and evaluated. Specifically, we investigated different polymeric nano-carriers, both flexible and rigid, as platforms for co-localization, as well as distinct enzyme attachment techniques using model enzyme systems using glucose oxidase and horseradish peroxidase to control the spatial arrangement of the multiple enzymes on the nanocarriers. This platform technology can be potentially used to co-localize various enzyme systems and its broad applicability will bemore » tested using the sclareol biosynthesis process to control the formation of products through the formation of MECs with multiple enzymes NgCPS and sSsSS to regulate the pathway of reactive intermediate to enhance the final product conversion rate.« less

77 FR 48157 - Office of the National Coordinator for Health Information Technology; Announcement of...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-13

... on YouTube.com or Vimeo.com (the Video should be no longer than 2 minutes); A text description of... open call for the public to create short, inspiring videos sharing how you use technology to manage... Challenge invites you to create short, inspiring videos sharing how you use technology to manage your...

Fusing Technology into the School Design Process for Today and Tomorrow

ERIC Educational Resources Information Center

Strube, Marilyn K.; Thompson, Ann L.

2012-01-01

Students are creating content in a variety of ways and using numerous technologies to share that content that did not exist when their teachers were students. Students are interacting with the new technologies they see on TV, at the movies and through their social networks. They are creating and sharing websites, posting self made videos and…

Technological Innovation: Higher Education, Small Manufacturing Enterprises Growth and the Five (I) Technological Development Model in Kenya

ERIC Educational Resources Information Center

Ng'ang'a, S. I.; Kabethi, J. M.; Kiumbe, P. M.; Otii, Leonard

2014-01-01

In Less Developed Countries (LDCs), most graduates from higher institutions of learning are absorbed in the informal sector and/or micro and small enterprises. Knowledge development through training, research and experiential learning may lead to creating or discovering new knowledge/technology or creating new value, by applying…

What Is Mitochondrial DNA?

MedlinePlus

... to create adenosine triphosphate (ATP), the cell’s main energy source. A set of enzyme complexes, designated as complexes I-V, carry out oxidative phosphorylation within mitochondria. In addition to energy production, mitochondria play a role in several other ...

A Novel Technology to Investigate Students' Understandings of Enzyme Representations

ERIC Educational Resources Information Center

Linenberger, Kimberly J.; Bretz, Stacey Lowery

2012-01-01

Digital pen-and-paper technology, although marketed commercially as a bridge between old and new note-taking capabilities, synchronizes the collection of both written and audio data. This manuscript describes how this technology was used to improve data collection in research regarding students' learning, specifically their understanding of…

"America COMPETES Reauthorization Act of 2010" or the "America Creating Opportunities to Meaningfully Promote Excellence in Technology, Education, and Science Reauthorization Act of 2010." House of Representatives, One Hundred Eleventh Congress, Second Session. H.R. 5116

ERIC Educational Resources Information Center

US House of Representatives, 2010

2010-01-01

The America COMPETES (Creating Opportunities to Meaningfully Promote Excellence in Technology, Education, and Science) Reauthorization Act of 2010 documented here is divided into the following titles: (1) Office of Science and Technology Policy (Coordination of Federal STEM [science, technology, engineering, and mathematics] education;…

The Impact of Enzyme Characteristics on Corn Stover Fiber Degradation and Acid Production During Ensiled Storage

NASA Astrophysics Data System (ADS)

Ren, Haiyu; Richard, Tom L.; Moore, Kenneth J.

Ensilage can be used to store lignocellulosic biomass before industrial bioprocessing. This study investigated the impacts of seven commerical enzyme mixtures derived from Aspergillus niger, Trichoderma reesei, and T. longibrachiatum. Treatments included three size grades of corn stover, two enzyme levels (1.67 and 5 IU/g dry matter based on hemicellulase), and various ratios of cellulase to hemicellulase (C ∶ H). The highest C ∶ H ratio tested, 2.38, derived from T. reesei, resulted in the most effective fermentation, with lactic acid as the dominant product. Enzymatic activity during storage may complement industrial pretreatment; creating synergies that could reduce total bioconversion costs.

AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) TESTING OF ENZYMATIC TEST KITS FOR WARFARE AGENTS AND PESTICIDES IN DRINKING WATER

EPA Science Inventory

Enzymatic test kits, generally designed to be handheld and portable, detect the presence of chemical agents, carbamate pesticides, and/or organophosphate pesticides by relying on the reaction of the cholinesterase enzyme. Under normal conditions, the enzyme reacts as expected wi...

Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

EPA Science Inventory

This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

Embedded enzymes catalyse capture

NASA Astrophysics Data System (ADS)

Kentish, Sandra

2018-05-01

Membrane technologies for carbon capture can offer economic and environmental advantages over conventional amine-based absorption, but can suffer from limited gas flux and selectivity to CO2. Now, a membrane based on enzymes embedded in hydrophilic pores is shown to exhibit combined flux and selectivity that challenges the state of the art.

Computational Enzymology and Organophosphorus Degrading Enzymes: Promising Approaches Toward Remediation Technologies of Warfare Agents and Pesticides

DOE PAGES

Ramalho, Teodorico C.; DeCastro, Alexandre A.; Silva, Daniela R.; ...

2015-08-26

The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and helpmore » in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and help in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.« less

Computational Enzymology and Organophosphorus Degrading Enzymes: Promising Approaches Toward Remediation Technologies of Warfare Agents and Pesticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramalho, Teodorico C.; DeCastro, Alexandre A.; Silva, Daniela R.

The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and helpmore » in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and help in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.« less

Ready to use bioinformatics analysis as a tool to predict immobilisation strategies for protein direct electron transfer (DET).

PubMed

Cazelles, R; Lalaoui, N; Hartmann, T; Leimkühler, S; Wollenberger, U; Antonietti, M; Cosnier, S

2016-11-15

Direct electron transfer (DET) to proteins is of considerable interest for the development of biosensors and bioelectrocatalysts. While protein structure is mainly used as a method of attaching the protein to the electrode surface, we employed bioinformatics analysis to predict the suitable orientation of the enzymes to promote DET. Structure similarity and secondary structure prediction were combined underlying localized amino-acids able to direct one of the enzyme's electron relays toward the electrode surface by creating a suitable bioelectrocatalytic nanostructure. The electro-polymerization of pyrene pyrrole onto a fluorine-doped tin oxide (FTO) electrode allowed the targeted orientation of the formate dehydrogenase enzyme from Rhodobacter capsulatus (RcFDH) by means of hydrophobic interactions. Its electron relays were directed to the FTO surface, thus promoting DET. The reduction of nicotinamide adenine dinucleotide (NAD(+)) generating a maximum current density of 1μAcm(-2) with 10mM NAD(+) leads to a turnover number of 0.09electron/s/molRcFDH. This work represents a practical approach to evaluate electrode surface modification strategies in order to create valuable bioelectrocatalysts. Copyright © 2016 Elsevier B.V. All rights reserved.

In-vitro engineering of novel bioactivity in the natural enzymes

NASA Astrophysics Data System (ADS)

Tiwari, Vishvanath

2016-10-01

Enzymes catalyze various biochemical functions with high efficiency and specificity. In-vitro design of the enzyme leads to novel bioactivity in this natural biomolecule that give answers of some vital questions like crucial residues in binding with substrate, molecular evolution, cofactor specificity etc. Enzyme engineering technology involves directed evolution, rational designing, semi-rational designing and structure-based designing using chemical modifications. Similarly, combined computational and in-vitro evolution approaches together help in artificial designing of novel bioactivity in the natural enzyme. DNA shuffling, error prone PCR and staggered extension process are used to artificially redesign active site of enzyme, which can alter its efficiency and specificity. Modifications of the enzyme can lead to the discovery of new path of molecular evolution, designing of efficient enzymes, locating active sites and crucial residues, shift in substrate and cofactor specificity. The methods and thermodynamics of in-vitro designing of the enzyme are also discussed. Similarly, engineered thermophilic and psychrophilic enzymes attain substrate specificity and activity of mesophilic enzymes that may also be beneficial for industry and therapeutics.

[Complex enzyme combined with ultrasound extraction technology, physicochemical properties and antioxidant activity of Hedysarum polysaccharides].

PubMed

Yang, Xiu-Yan; Xue, Zhi-Yuan; Yang, Ya-Fei; Fang, Yao-Yao; Zhou, Xiang-Lin; Zhao, Liang-Gong; Feng, Shi-Lan

2018-06-01

In this study, complex enzymes combined with ultrasonic extraction technology（MC） were used, to select optimal extraction combinations by single factor and orthogonal test, with Hedysarum polysaccharides yield and content as the comprehensive indexes. The components, physicochemical properties and antioxidant activity of Hedysarum polysaccharides from complex enzyme combined with ultrasonic extraction（HPS-MC）and the Hedysarum polysaccharides from hot water extraction（HPS-R）were analyzed. The results showed that：complex enzymes had significant effect on the yield and content of Hedysarum polysaccharides, and the ultrasonic power could significantly improve the content of Hedysarum polysaccharides. The optimum technological parameters were as follows: complex enzyme ratio 1:1, ultrasonic power 105 W, ultrasonic time 60 min, and enzymatic hydrolysis pH 5, achieving （14.01±0.64）% and （92.45±1.47）% respectively for the yield and content of Polysaccharides. As compared with HPS-R, the molecular weight, absolute viscosity and protein content of HPS-MC were decreased, while the content of uronic acid was increased. In the antioxidant system, the concentration of polysaccharide was within the range of 1-7 g·L⁻¹; the antioxidant activity of HPS-MC was higher than that of HPS-R, and HPS-MC (80%) with the lowest molecular weight showed a significant dose effect relationship with the increase of the experimental concentration. In conclusion, MC is a simple, convenient, economical and environmentally friendly extraction technology, and the Hedysarum polysaccharides extracted by this method have obvious antioxidant activity. Copyright© by the Chinese Pharmaceutical Association.

Dissecting enzyme function with microfluidic-based deep mutational scanning.

PubMed

Romero, Philip A; Tran, Tuan M; Abate, Adam R

2015-06-09

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.

Directed evolution: tailoring biocatalysts for industrial applications.

PubMed

Kumar, Ashwani; Singh, Suren

2013-12-01

Current challenges and promises of white biotechnology encourage protein engineers to use a directed evolution approach to generate novel and useful biocatalysts for various sets of applications. Different methods of enzyme engineering have been used in the past in an attempt to produce enzymes with improved functions and properties. Recent advancement in the field of random mutagenesis, screening, selection and computational design increased the versatility and the rapid development of enzymes under strong selection pressure with directed evolution experiments. Techniques of directed evolution improve enzymes fitness without understanding them in great detail and clearly demonstrate its future role in adapting enzymes for use in industry. Despite significant advances to date regarding biocatalyst improvement, there still remains a need to improve mutagenesis strategies and development of easy screening and selection tools without significant human intervention. This review covers fundamental and major development of directed evolution techniques, and highlights the advances in mutagenesis, screening and selection methods with examples of enzymes developed by using these approaches. Several commonly used methods for creating molecular diversity with their advantages and disadvantages including some recently used strategies are also discussed.

Microgravity

NASA Image and Video Library

2004-04-15

The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Pharmaceutical companies have been developing drugs that can inhibit the function of neuraminidase hoping to create an effective weapon against the flu. Researchers from the pharmaceutical industry and from the Center for Macromolecular Crystallography have grown crystals of neuraminidase in space. These improved, space-grown crystals have provided information that have helped design drugs which form a stronger interaction with the enzyme. These drugs inhibit neuraminidase by attaching themselves to the enzyme. Since the drugs are less likely to detach from the enzyme, they are more effective, require smaller dosages, and have fewer side effects. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

Neuraminidase

NASA Technical Reports Server (NTRS)

2004-01-01

The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Pharmaceutical companies have been developing drugs that can inhibit the function of neuraminidase hoping to create an effective weapon against the flu. Researchers from the pharmaceutical industry and from the Center for Macromolecular Crystallography have grown crystals of neuraminidase in space. These improved, space-grown crystals have provided information that have helped design drugs which form a stronger interaction with the enzyme. These drugs inhibit neuraminidase by attaching themselves to the enzyme. Since the drugs are less likely to detach from the enzyme, they are more effective, require smaller dosages, and have fewer side effects. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

NASA Astrophysics Data System (ADS)

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

PubMed

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

Enzyme-polymer composites with high biocatalytic activity and stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jungbae; Kosto, Timothy J.; Manimala, Joseph C.

2004-08-22

We have applied vacuum-spraying and electrospinning to incorporate an enzyme into a polymer matrix, creating a novel and highly active biocatalytic composite. As a unique technical approach, enzymes were co-dissolved in toluene with polymers, and the solvent was then rapidly removed by injecting the mixture into a vacuum chamber or by electrospinning. Subsequent crosslinking of the enzyme with glutaraldehyde resulted in stable entrapped enzyme within the polymeric matrices. For example, an amorphous composite of alpha-chymotrypsin and polyethylene showed no significant loss of enzymatic activity in aqueous buffer for one month. Nanofibers of alpha-chymotrypsin and polystyrene also showed no decrease inmore » activity for more than two weeks. The normalized activity of amorphous composite in organic solvents was 3-13 times higher than that of native alpha-chymotrypsin. The activity of nanofibers was 5-7 times higher than that of amorphous composite in aqueous buffer solution. The composites of alpha-chymotrypsin and polymers demonstrate the feasibility of obtaining a wide variety of active and stable biocatalytic materials with many combinations of enzymes and polymers.« less

Phage display: concept, innovations, applications and future.

PubMed

Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

2010-01-01

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field. Copyright © 2010 Elsevier Inc. All rights reserved.

Synthesis and Characterization of Magnetic Carriers Based on Immobilized Enzyme

NASA Astrophysics Data System (ADS)

Li, F. H.; Tang, N.; Wang, Y. Q.; Zhang, L.; Du, W.; Xiang, J.; Cheng, P. G.

2018-05-01

Several new types of carriers and technologies have been implemented to improve traditional enzyme immobilization in industrial biotechnology. The magnetic immobilized enzyme is a kind of new method of enzyme immobilization developed in recent years. An external magnetic field can be used to control the motion mode and direction of immobilized enzyme, and to improve the catalytic efficiency of immobilized enzyme. In this paper, Fe3O4-CaCO3-PDA complex and CaCO3/Fe3O4 composite modified by PEI were prepared. The results show that the morphology of Fe3O4-CaCO3-PDA complex formation is irregular, while the morphology of CaCO3/Fe3O4 composite modified by PEI is regular and has a porous structure.

Synthetic metabolism: metabolic engineering meets enzyme design.

PubMed

Erb, Tobias J; Jones, Patrik R; Bar-Even, Arren

2017-04-01

Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Programmable DNA-Guided Artificial Restriction Enzymes.

PubMed

Enghiad, Behnam; Zhao, Huimin

2017-05-19

Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research. However, they have limited sequence specificity and availability. Here we report a Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by reannealing of the cleaved single stranded DNAs. We used this platform to generate over 18 AREs for DNA fingerprinting and molecular cloning of PCR-amplified or genomic DNAs. These AREs work as efficiently as their naturally occurring counterparts, and some of them even do not have any naturally occurring counterparts, demonstrating easy programmability, generality, versatility, and high efficiency for this new technology.

Translational Science by Public Biotechnology Companies in the IPO“Class of 2000”: The Impact of Technological Maturity

PubMed Central

McNamee, Laura; Ledley, Fred

2013-01-01

The biotechnology industry plays a central role in the translation of nascent biomedical science into both products that offer material health benefits and creating capital growth. This study examines the relationship between the maturity of technologies in a characteristic life cycle and value creation by biotechnology companies. We examined the core technology, product development pipelines, and capitalization for a cohort of biotechnology companies that completed an IPO in 2000. Each of these companies was well financed and had core technologies on the leading edge of biological science. We found that companies with the least mature technologies had significantly higher valuations at IPO, but failed to develop products based on these technologies over the ensuing decade, and created less capital growth than companies with more mature technologies at IPO. The observation that this cohort of recently public biotechnology companies was not effective in creating value from nascent science suggests the need for new, evidence-based business strategies for translational science. PMID:24358154

Translational science by public biotechnology companies in the IPO "class of 2000": the impact of technological maturity.

PubMed

McNamee, Laura; Ledley, Fred

2013-01-01

The biotechnology industry plays a central role in the translation of nascent biomedical science into both products that offer material health benefits and creating capital growth. This study examines the relationship between the maturity of technologies in a characteristic life cycle and value creation by biotechnology companies. We examined the core technology, product development pipelines, and capitalization for a cohort of biotechnology companies that completed an IPO in 2000. Each of these companies was well financed and had core technologies on the leading edge of biological science. We found that companies with the least mature technologies had significantly higher valuations at IPO, but failed to develop products based on these technologies over the ensuing decade, and created less capital growth than companies with more mature technologies at IPO. The observation that this cohort of recently public biotechnology companies was not effective in creating value from nascent science suggests the need for new, evidence-based business strategies for translational science.

Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.

PubMed

Renata, Hans; Wang, Z Jane; Arnold, Frances H

2015-03-09

High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature's known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression and Regulation of Drug Transporters and Metabolizing Enzymes in the Human Gastrointestinal Tract.

PubMed

Drozdzik, M; Oswald, S

2016-01-01

Orally administered drugs must pass through the intestinal wall and then through the liver before reaching systemic circulation. During this process drugs are subjected to different processes that may determine the therapeutic value. The intestinal barrier with active drug metabolizing enzymes and drug transporters in enterocytes plays an important role in the determination of drug bioavailability. Accumulating information demonstrates variable distribution of drug metabolizing enzymes and transporters along the human gastrointestinal tract (GI), that creates specific barrier characteristics in different segments of the GI. In this review, expression of drug metabolizing enzymes and transporters in the healthy and diseased human GI as well as their regulatory aspects: genetic, miRNA, DNA methylation are outlined. The knowledge of unique interplay between drug metabolizing enzymes and transporters in specific segments of the GI tract allows more precise definition of drug release sites within the GI in order to assure more complete bioavailability and prediction of drug interactions.

Toxin detection using a tyrosinase-coupled oxygen electrode.

PubMed

Smit, M H; Rechnitz, G A

1993-02-15

An enzyme-based "electrochemical canary" is described for the detection of cyanide. The sensing system imitates cyanide's site of toxicity in the mitochondria. The terminal sequence of electron transfer in aerobic respiration is mimicked by mediator coupling of tyrosinase catalysis to an electro-chemical system. An enzyme-coupled oxygen electrode is created which is sensitive to selective poisoning. Biocatalytic reduction of oxygen is promoted by electrochemically supplying tyrosinase with electrons. Thus, ferrocyanide is generated at a cathode and mediates the enzymatic reduction of oxygen to water. An enzyme-dependent reductive current can be monitored which is inhibited by cyanide in a concentration-dependent manner. Oxygen depletion in the reaction layer can be minimized by addressing enzyme activity using a potential pulsing routine. Enzyme activity is electrochemically initiated and terminated and the sensor becomes capable of continuous monitoring. Cyanide poisoning of the biological component is reversible, and it can be reused after rinsing. The resulting sensor detects cyanide based on its biological activity rather than its physical or chemical properties.

Inserting new technology into small missions

NASA Technical Reports Server (NTRS)

Deutsch, L. J.

2001-01-01

Part of what makes small missions small is that they have less money. Executing missions at low cost implies extensive use of cost sharing with other missions or use of existing solutions. However, in order to create many small missions, new technology must be developed, applied, and assimilated. Luckily, there are methods for creating new technology and inserting it into faster-better-cheaper (FBC) missions.

Applications of Protein Hydrolysates in Biotechnology

NASA Astrophysics Data System (ADS)

Pasupuleti, Vijai K.; Holmes, Chris; Demain, Arnold L.

By definition, protein hydrolysates are the products that are obtained after the hydrolysis of proteins and this can be achieved by enzymes, acid or alkali. This broad definition encompasses all the products of protein hydrolysis - peptides, amino acids and minerals present in the protein and acid/alkali used to adjust pH (Pasupuleti 2006). Protein hydrolysates contain variable side chains depending on the enzymes used. These side chains could be carboxyl, amino, imidazole, sulfhydryl, etc. and they may exert specific physiological roles in animal, microbial, insect and plant cells. This introductory chapter reviews the applications of protein hydrolysates in biotechnology. The word biotechnology is so broad and for the purpose of this book, we define it as a set of technologies such as cell culture technology, bioprocessing technology that includes fermentations, genetic engineering technology, microbiology, and so on. This chapter provides introduction and leads to other chapters on manufacturing and applications of protein hydrolysates in biotechnology.

Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Adam J.; Brown, Melissa N.; Black, Margaret E., E-mail: blackm@vetmed.wsu.edu

2011-12-09

Highlights: Black-Right-Pointing-Pointer Goal was to enhance dFdC cytotoxicity by the creation of a UCMK/dCK fusion enzyme. Black-Right-Pointing-Pointer The UCMK/dCK fusion enzyme possesses both native activities. Black-Right-Pointing-Pointer The fusion renders cells equally sensitive to dFdC relative to dCK expression alone. Black-Right-Pointing-Pointer Dual activities of fusion not sufficient to augment cell dFdC sensitivity in vitro. Black-Right-Pointing-Pointer Data may warrant the implementation of UCMK mutagenesis studies. -- Abstract: While gemcitabine (2 Prime -2 Prime -difluoro-2 Prime -deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhancemore » dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.« less

Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study.

PubMed

Németh, Attila; Kamondi, Szilárd; Szilágyi, András; Magyar, Csaba; Kovári, Zoltán; Závodszky, Péter

2002-05-02

Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects.

PubMed

Wang, Yan; Song, Qinghao; Zhang, Xiao-Hua

2016-09-22

Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes.

Laser Technology.

ERIC Educational Resources Information Center

Gauger, Robert

1993-01-01

Describes lasers and indicates that learning about laser technology and creating laser technology activities are among the teacher enhancement processes needed to strengthen technology education. (JOW)

3D-Printing Technologies for Craniofacial Rehabilitation, Reconstruction, and Regeneration.

PubMed

Nyberg, Ethan L; Farris, Ashley L; Hung, Ben P; Dias, Miguel; Garcia, Juan R; Dorafshar, Amir H; Grayson, Warren L

2017-01-01

The treatment of craniofacial defects can present many challenges due to the variety of tissue-specific requirements and the complexity of anatomical structures in that region. 3D-printing technologies provide clinicians, engineers and scientists with the ability to create patient-specific solutions for craniofacial defects. Currently, there are three key strategies that utilize these technologies to restore both appearance and function to patients: rehabilitation, reconstruction and regeneration. In rehabilitation, 3D-printing can be used to create prostheses to replace or cover damaged tissues. Reconstruction, through plastic surgery, can also leverage 3D-printing technologies to create custom cutting guides, fixation devices, practice models and implanted medical devices to improve patient outcomes. Regeneration of tissue attempts to replace defects with biological materials. 3D-printing can be used to create either scaffolds or living, cellular constructs to signal tissue-forming cells to regenerate defect regions. By integrating these three approaches, 3D-printing technologies afford the opportunity to develop personalized treatment plans and design-driven manufacturing solutions to improve aesthetic and functional outcomes for patients with craniofacial defects.

3D-Printing Technologies for Craniofacial Rehabilitation, Reconstruction, and Regeneration

PubMed Central

Nyberg, Ethan L.; Farris, Ashley L.; Hung, Ben P.; Dias, Miguel; Garcia, Juan R.; Dorafshar, Amir H.; Grayson, Warren L.

2016-01-01

The treatment of craniofacial defects can present many challenges due to the variety of tissue-specific requirements and the complexity of anatomical structures in that region. 3D-printing technologies provide clinicians, engineers and scientists with the ability to create patient-specific solutions for craniofacial defects. Currently, there are 3 key strategies that utilize these technologies to restore both appearance and function to patients: rehabilitation, reconstruction and regeneration. In rehabilitation, 3D-printing can be used to create prostheses to replace or cover damaged tissues. Reconstruction, through plastic surgery, can also leverage 3D-printing technologies to create custom cutting guides, fixation devices, practice models and implanted medical devices to improve patient outcomes. Regeneration of tissue attempts to replace defects with biological materials. 3D-printing can be used to create either scaffolds or living, cellular constructs to signal tissue-forming cells to regenerate defect regions. By integrating these three approaches, 3D-printing technologies afford the opportunity to develop personalized treatment plans and design-driven manufacturing solutions to improve aesthetic and functional outcomes for patients with craniofacial defects. PMID:27295184

White biotechnology: State of the art strategies for the development of biocatalysts for biorefining.

PubMed

Heux, S; Meynial-Salles, I; O'Donohue, M J; Dumon, C

2015-12-01

White biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers. This review proposes an original and global overview of highly complementary fields of biotechnology at both enzyme and microorganism level. A certain number of state of the art approaches that are now being used to improve the industrial fitness of biocatalysts particularly focused on the biorefinery sector are presented. The first part deals with the technologies that underpin the development of industrial biocatalysts, notably the discovery of new enzymes and enzyme improvement using directed evolution techniques. The second part describes the toolbox available by the cell engineer to shape the metabolism of microorganisms. And finally the last part focuses on the 'omic' technologies that are vital for understanding and guide microbial engineering toward more efficient microbial biocatalysts. Altogether, these techniques and strategies will undoubtedly help to achieve the challenging task of developing consolidated bioprocessing (i.e. CBP) readily available for industrial purpose. Copyright © 2015 Elsevier Inc. All rights reserved.

Enzyme-modified starch as an oil delivery system for bake-only chicken nuggets.

PubMed

Purcell, Sarah; Wang, Ya-Jane; Seo, Han-Seok

2014-05-01

This study investigated the effects of enzyme modification on starch as an effective oil delivery system for bake-only chicken nuggets. Various native starches were hydrolyzed by amyloglucosidase to a hydrolysis degree of 20% to 25% and plated with 50% (w/w, starch dry basis) with canola oil to create a starch-oil matrix. This matrix was then blended into a dry ingredient blend for batter and breader components. Nuggets were prepared by coated with predust, hydrated batter, and breader, and the coated nuggets were steam-baked until fully cooked and then frozen until texture and sensory analyses. The enzyme-modified starches showed a significant decrease in pasting viscosities for all starch types. For textural properties of nuggets, no clear relationship was found between peak force and starch source or amylose content. Sensory attributes related to fried foods (for example, crispness and mouth-coating) did not significantly differ between bake-only nuggets formulated using the enzyme-modified starches and the partially fried and baked ones. The present findings suggest that enzyme-modified starches can deliver sufficient quantity of oil to create sensory attributes similar to those of partially fried chicken nuggets. Further study is needed to optimize the coating formulation of bake-only chicken nugget to become close to the fried one in sensory aspects. The food industry has become increasingly focused on healthier items. Frying imparts several critical and desirable product functionalities, such as developing texture and color, and providing mouth-feel and flavor. The food industry has yet to duplicate all of the unique characteristics of fried chicken nuggets with a baking process. This study investigated the application of enzyme-modified starch as an oil delivery system in bake-only chicken nugget formulation in attempts to provide characteristics of fried items. This information is useful to improve the nutritional value of fried food by eliminating the frying process while preserving the desired characteristics of fried products. © 2014 Institute of Food Technologists®

Aroma Release in Wine Using Co-Immobilized Enzyme Aggregates.

PubMed

Ahumada, Katherine; Martínez-Gil, Ana; Moreno-Simunovic, Yerko; Illanes, Andrés; Wilson, Lorena

2016-11-08

Aroma is a remarkable factor of quality and consumer preference in wine, representing a distinctive feature of the product. Most aromatic compounds in varietals are in the form of glycosidic precursors, which are constituted by a volatile aglycone moiety linked to a glucose residue by an O -glycosidic bond; glucose is often linked to another sugar (arabinose, rhamnose or apiose). The use of soluble β-glycosidases for aroma liberation implies the addition of a precipitating agent to remove it from the product and precludes its reuse after one batch. An attractive option from a technological perspective that will aid in removing such constraints is the use of immobilized glycosidases. Immobilization by aggregation and crosslinking is a simple strategy producing enzyme catalysts of very high specific activity, being an attractive option to conventional immobilization to solid inert supports. The purpose of this work was the evaluation of co-immobilized β-glycosidases crosslinked aggregates produced from the commercial preparation AR2000, which contains the enzymes involved in the release of aromatic terpenes in Muscat wine (α-l-arabinofuranosidase and β-d-glucopyranosidase). To do so, experiments were conducted with co-immobilized crosslinked enzyme aggregates (combi-CLEAs), and with the soluble enzymes, using an experiment without enzyme addition as control. Stability of the enzymes at the conditions of winemaking was assessed and the volatiles composition of wine was determined by SPE-GC-MS. Stability of enzymes in combi-CLEAs was much higher than in soluble form, 80% of the initial activity remaining after 60 days in contact with the wine; at the same conditions, the soluble enzymes had lost 80% of their initial activities after 20 days. Such higher stabilities will allow prolonged use of the enzyme catalyst reducing its impact in the cost of winemaking. Wine treated with combi-CLEAs was the one exhibiting the highest concentration of total terpenes (18% higher than the control) and the highest concentrations of linalool (20% higher), nerol (20% higher) and geraniol (100% higher), which are the most important terpenes in determining Muscat typicity. Co-immobilized enzymes were highly stable at winemaking conditions, so their reutilization is possible and technologically attractive by reducing the impact of enzyme cost on winemaking cost.

Adopting new technologies in stroke rehabilitation: the influence of the US health care system.

PubMed

Stein, J

2009-06-01

Stroke rehabilitation is entering a new era of technological innovation, including the development of robotic aids for therapy, peripheral electrical stimulation devices, and brain stimulation systems. These technologies have the potential to significantly improve the efficiency and efficacy of stroke rehabilitation. The United States health care system creates both opportunities for new technologies to be created and adopted, as well as important barriers. Inadequate support of clinical trials of the efficacy of new non-invasive devices is a particular concern for practitioners seeking to determine if new devices are clinically useful. Government support of clinical trials of efficacy, coupled with reform of FDA approval processes for novel therapies, is needed to create an evidence-based approach to improving stroke rehabilitation.

A Self-Assisting Protein Folding Model for Teaching Structural Molecular Biology.

PubMed

Davenport, Jodi; Pique, Michael; Getzoff, Elizabeth; Huntoon, Jon; Gardner, Adam; Olson, Arthur

2017-04-04

Structural molecular biology is now becoming part of high school science curriculum thus posing a challenge for teachers who need to convey three-dimensional (3D) structures with conventional text and pictures. In many cases even interactive computer graphics does not go far enough to address these challenges. We have developed a flexible model of the polypeptide backbone using 3D printing technology. With this model we have produced a polypeptide assembly kit to create an idealized model of the Triosephosphate isomerase mutase enzyme (TIM), which forms a structure known as TIM barrel. This kit has been used in a laboratory practical where students perform a step-by-step investigation into the nature of protein folding, starting with the handedness of amino acids to the formation of secondary and tertiary structure. Based on the classroom evidence we collected, we conclude that these models are valuable and inexpensive resource for teaching structural molecular biology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bioproduction of Chitooligosaccharides: Present and Perspectives

PubMed Central

Jung, Woo-Jin; Park, Ro-Dong

2014-01-01

Chitin and chitosan oligosaccharides (COS) have been traditionally obtained by chemical digestion with strong acids. In light of the difficulties associated with these traditional production processes, environmentally compatible and reproducible production alternatives are desirable. Unlike chemical digestion, biodegradation of chitin and chitosan by enzymes or microorganisms does not require the use of toxic chemicals or excessive amounts of wastewater. Enzyme preparations with chitinase, chitosanase, and lysozymeare primarily used to hydrolyze chitin and chitosan. Commercial preparations of cellulase, protease, lipase, and pepsin provide another opportunity for oligosaccharide production. In addition to their hydrolytic activities, the transglycosylation activity of chitinolytic enzymes might be exploited for the synthesis of desired chitin oligomers and their derivatives. Chitin deacetylase is also potentially useful for the preparation of oligosaccharides. Recently, direct production of oligosaccharides from chitin and crab shells by a combination of mechanochemical grinding and enzymatic hydrolysis has been reported. Together with these, other emerging technologies such as direct degradation of chitin from crustacean shells and microbial cell walls, enzymatic synthesis of COS from small building blocks, and protein engineering technology for chitin-related enzymes have been discussed as the most significant challenge for industrial application. PMID:25353253

The production, properties, and applications of thermostable steryl glucosidases.

PubMed

Aguirre, Andres; Eberhardt, Florencia; Hails, Guillermo; Cerminati, Sebastian; Castelli, María Eugenia; Rasia, Rodolfo M; Paoletti, Luciana; Menzella, Hugo G; Peiru, Salvador

2018-02-21

Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable β-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.

Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant E. coli.

PubMed

Fakruddin, Md; Mohammad Mazumdar, Reaz; Bin Mannan, Khanjada Shahnewaj; Chowdhury, Abhijit; Hossain, Md Nur

2013-01-01

E. coli is the most frequently used host for production of enzymes and other proteins by recombinant DNA technology. E. coli is preferable for its relative simplicity, inexpensive and fast high-density cultivation, well-known genetics, and large number of compatible molecular tools available. Despite all these advantages, expression and production of recombinant enzymes are not always successful and often result in insoluble and nonfunctional proteins. There are many factors that affect the success of cloning, expression, and mass production of enzymes by recombinant E. coli. In this paper, these critical factors and approaches to overcome these obstacles are summarized focusing controlled expression of target protein/enzyme in an unmodified form at industrial level.

Bioengineering recombinant tung tree diacylglycerol acyltransferases

USDA-ARS?s Scientific Manuscript database

Understanding plant oil biosynthesis will help to create new oilseed crops with value-added properties to replace petroleum-based compounds. Diacylglycerol acyltransferases (DGATs) are key enzymes catalyzing the last step of triacylglycerol (TAG) biosynthesis in eukaryotes. Plants and animals defici...

Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone

USDA-ARS?s Scientific Manuscript database

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone derivatives with modified ring structure. In one chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates ...

Vault Nanoparticles Packaged with Enzymes as an Efficient Pollutant Biodegradation Technology.

PubMed

Wang, Meng; Abad, Danny; Kickhoefer, Valerie A; Rome, Leonard H; Mahendra, Shaily

2015-11-24

Vault nanoparticles packaged with enzymes were synthesized as agents for efficiently degrading environmental contaminants. Enzymatic biodegradation is an attractive technology for in situ cleanup of contaminated environments because enzyme-catalyzed reactions are not constrained by nutrient requirements for microbial growth and often have higher biodegradation rates. However, the limited stability of extracellular enzymes remains a major challenge for practical applications. Encapsulation is a recognized method to enhance enzymatic stability, but it can increase substrate diffusion resistance, lower catalytic rates, and increase the apparent half-saturation constants. Here, we report an effective approach for boosting enzymatic stability by single-step packaging into vault nanoparticles. With hollow core structures, assembled vault nanoparticles can simultaneously contain multiple enzymes. Manganese peroxidase (MnP), which is widely used in biodegradation of organic contaminants, was chosen as a model enzyme in the present study. MnP was incorporated into vaults via fusion to a packaging domain called INT, which strongly interacts with vaults' interior surface. MnP fused to INT and vaults packaged with the MnP-INT fusion protein maintained peroxidase activity. Furthermore, MnP-INT packaged in vaults displayed stability significantly higher than that of free MnP-INT, with slightly increased Km value. Additionally, vault-packaged MnP-INT exhibited 3 times higher phenol biodegradation in 24 h than did unpackaged MnP-INT. These results indicate that the packaging of MnP enzymes in vault nanoparticles extends their stability without compromising catalytic activity. This research will serve as the foundation for the development of efficient and sustainable vault-based bioremediation approaches for removing multiple contaminants from drinking water and groundwater.

Commercialization of a novel fermentation concept.

PubMed

Mazumdar-Shaw, Kiran; Suryanarayan, Shrikumar

2003-01-01

Fermentation is the core of biotechnology where current methodologies span across technologies based on the use of either solid or liquid substrates. Traditionally, solid substrate fermentation technologies have been the widely practiced in the Far East to manufacture fermented foods such as soya sauce, sake etc. The Western World briefly used solid substrate fermentation for the manufacture of antibiotics and enzymes but rapidly replaced this technology with submerged fermentation which proved to be a superior technology in terms of automation, containment and large volume fermentation. Biocon India developed its enzyme technology based on solid substrate fermentation as a low-cost, low-energy option for the production of specialty enzymes. However, the limitations of applying solid substrate fermentation to more sophisticated biotechnology products as well as large volume fermentations were recognized by Biocon India as early as 1990 and the company embarked on a 8 year research and development program to develop a novel bioreactor capable of conducting solid substrate fermentation with comparable levels of automation and containment as those practiced by submerged fermentation. In addition, the novel technology enabled fed-batch fermentation, in situ extraction and other enabling features that will be discussed in this article. The novel bioreactor was christened the "PlaFractor" (pronounced play-fractor). The next level of research on this novel technology is now focused on addressing large volume fermentation. This article traces the evolution of Biocon India's original solid substrate fermentation to the PlaFractor technology and provides details of the scale-up and commercialization processes that were involved therein. What is also apparent in the article is Biocon India's commercially focused research programs and the perceived need to be globally competitive through low costs of innovation that address, at all times, processes and technologies that exhibit high degrees of conformance to the international standards of regulatory and good manufacturing practice.

Education, Technology and Health Literacy.

PubMed

Lindgren, Kurt; Koldkjær Sølling, Ina; Carøe, Per; Siggaard Mathiesen, Kirsten

2015-01-01

The purpose of this study is to develop an interdisciplinary learning environment between education in technology, business, and nursing. This collaboration creates natural interest and motivation for welfare technology. The aim of establishing an interaction between these three areas of expertise is to create an understanding of skills and cultural differences in each area. Futhermore, the aim is to enable future talents to gain knowledge and skills to improve health literacy among senior citizens. Based on a holistic view of welfare technology, a Student Academy was created as a theoretically- and practically-oriented learning center. The mission of the Student Academy is to support and facilitate education in order to maintain and upgrade knowledge and skills in information technology and information management related to e-health and health literacy. The Student Academy inspires students, stakeholders, politicians, DanAge Association members, companies, and professionals to participate in training, projects, workshops, and company visits.

Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

NASA Astrophysics Data System (ADS)

Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo

An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.

Creating a Pilot Educational Psychiatry Website: Opportunities, Barriers, and Next Steps.

PubMed

Torous, John; O'Connor, Ryan; Franzen, Jamie; Snow, Caitlin; Boland, Robert; Kitts, Robert

2015-11-05

While medical students and residents may be utilizing websites as online learning resources, medical trainees and educators now have the opportunity to create such educational websites and digital tools on their own. However, the process and theory of building educational websites for medical education have not yet been fully explored. To understand the opportunities, barriers, and process of creating a novel medical educational website. We created a pilot psychiatric educational website to better understand the options, opportunities, challenges, and processes involved in the creation of a psychiatric educational website. We sought to integrate visual and interactive Web design elements to underscore the potential of such Web technology. A pilot website (PsychOnCall) was created to demonstrate the potential of Web technology in medical and psychiatric education. Creating an educational website is now technically easier than ever before, and the primary challenge no longer is technology but rather the creation, validation, and maintenance of information for such websites as well as translating text-based didactics into visual and interactive tools. Medical educators can influence the design and implementation of online educational resources through creating their own websites and engaging medical students and residents in the process.

Creating a Pilot Educational Psychiatry Website: Opportunities, Barriers, and Next Steps

PubMed Central

O'Connor, Ryan; Franzen, Jamie; Snow, Caitlin; Boland, Robert; Kitts, Robert

2015-01-01

Background While medical students and residents may be utilizing websites as online learning resources, medical trainees and educators now have the opportunity to create such educational websites and digital tools on their own. However, the process and theory of building educational websites for medical education have not yet been fully explored. Objective To understand the opportunities, barriers, and process of creating a novel medical educational website. Methods We created a pilot psychiatric educational website to better understand the options, opportunities, challenges, and processes involved in the creation of a psychiatric educational website. We sought to integrate visual and interactive Web design elements to underscore the potential of such Web technology. Results A pilot website (PsychOnCall) was created to demonstrate the potential of Web technology in medical and psychiatric education. Conclusions Creating an educational website is now technically easier than ever before, and the primary challenge no longer is technology but rather the creation, validation, and maintenance of information for such websites as well as translating text-based didactics into visual and interactive tools. Medical educators can influence the design and implementation of online educational resources through creating their own websites and engaging medical students and residents in the process. PMID:27731837

Distribution of enzyme activity hotspots induced by earthworms in top- and subsoil

NASA Astrophysics Data System (ADS)

Hoang, D. T. T.

2016-12-01

Earthworms (Lumbricus terrestris L.) not only affect soil physics, but they also boost microbial activities and consequently create important hotspots of microbial mediated carbon and nutrient turnover through their burrowing activity. However, it is still unknown to which extend earthworms change the enzyme distribution and activity inside their burrows in top- and subsoil horizons. We hypothesized that earthworm burrows, which are enriched in available substrates, have higher percentage of enzyme activity hotspots than soil without earthworms, and that enzyme activities decreased with increasing depth because of the increasing recalcitrance of organic matter in subsoil. We visualized enzyme distribution inside and outside of worm burrows (biopores) by in situ soil zymography and measured enzyme kinetics of 6 enzymes - β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) - in pore and bulk soil material up to 105 cm. Zymography showed a heterogeneous distribution of hotspots in worm burrows. The hotspot areas was 2.4 to 14 times larger in the burrows than in soil without earthworms. However, the dispersion index of hotspot distribution showed more aggregated hotspots in soil without earthworms than in soil with earthworms and burrow wall. Enzyme activities decreased with depth, by a factor of 2 to 8 due to fresh C input from the soil surface. Compared to bulk soil, enzyme activities in topsoil biopores were up to 11 times higher for all enzymes, but in the subsoil activities of XYL, NAG and APT were lower in earthworm biopores than bulk soil. In conclusion, hotspots were twice as concentrated close to earthworm burrows as in surrounding soil. Earthworms exerted stronger effects on enzyme activities in biopores in the topsoil than in subsoil. Keywords: Earthworms, hotspots, enzyme activities, enzyme distribution, subsoil

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

PubMed

Nadar, Shamraja S; Rathod, Virendra K

2017-08-22

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (k m and V max ) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

Creation of a federated database of blood proteins: a powerful new tool for finding and characterizing biomarkers in serum

PubMed Central

2014-01-01

Protein biomarkers offer major benefits for diagnosis and monitoring of disease processes. Recent advances in protein mass spectrometry make it feasible to use this very sensitive technology to detect and quantify proteins in blood. To explore the potential of blood biomarkers, we conducted a thorough review to evaluate the reliability of data in the literature and to determine the spectrum of proteins reported to exist in blood with a goal of creating a Federated Database of Blood Proteins (FDBP). A unique feature of our approach is the use of a SQL database for all of the peptide data; the power of the SQL database combined with standard informatic algorithms such as BLAST and the statistical analysis system (SAS) allowed the rapid annotation and analysis of the database without the need to create special programs to manage the data. Our mathematical analysis and review shows that in addition to the usual secreted proteins found in blood, there are many reports of intracellular proteins and good agreement on transcription factors, DNA remodelling factors in addition to cellular receptors and their signal transduction enzymes. Overall, we have catalogued about 12,130 proteins identified by at least one unique peptide, and of these 3858 have 3 or more peptide correlations. The FDBP with annotations should facilitate testing blood for specific disease biomarkers. PMID:24476026

Technology in the Learning Sanctuary.

ERIC Educational Resources Information Center

Williams, Robert L.

1990-01-01

Adult residential programs can use educational technology to (1) create information icons that allow the learner to focus on and distill information; (2) remove barriers of time and space and create a measured reality within the learning sanctuary; and (3) develop open learning systems that are interactive and nonlinear. (SK)

Detection of Antibodies to the Biofilm Exopolysaccharide of Histophilus somni following Infection in Cattle by Enzyme-Linked Immunosorbent Assay

PubMed Central

Pan, Yu; Fisher, Taylor; Olk, Christina

2014-01-01

An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibodies to Histophilus somni exopolysaccharide (EPS), which is created during biofilm formation. When an index value of 0.268 was used, the sensitivity of the assay for infected calves was 90.5% at 3 weeks postinfection, but the number of positive animals increased by week 4. The specificity of the assay for healthy calves was 92.5%. The EPS ELISA may aid in identifying calves with H. somni diseases. PMID:25143338

Production of ethanol from winter barley by the EDGE (enhanced dry grind enzymatic) process

PubMed Central

2010-01-01

Background US legislation requires the use of advanced biofuels to be made from non-food feedstocks. However, commercialization of lignocellulosic ethanol technology is more complex than expected and is therefore running behind schedule. This is creating a demand for non-food, but more easily converted, starch-based feedstocks other than corn that can fill the gap until the second generation technologies are commercially viable. Winter barley is such a feedstock but its mash has very high viscosity due to its high content of β-glucans. This fact, along with a lower starch content than corn, makes ethanol production at the commercial scale a real challenge. Results A new fermentation process for ethanol production from Thoroughbred, a winter barley variety with a high starch content, was developed. The new process was designated the EDGE (enhanced dry grind enzymatic) process. In this process, in addition to the normal starch-converting enzymes, two accessory enzymes were used to solve the β-glucan problem. First, β-glucanases were used to hydrolyze the β-glucans to oligomeric fractions, thus significantly reducing the viscosity to allow good mixing for the distribution of the yeast and nutrients. Next, β-glucosidase was used to complete the β-glucan hydrolysis and to generate glucose, which was subsequently fermented in order to produce additional ethanol. While β-glucanases have been previously used to improve barley ethanol production by lowering viscosity, this is the first full report on the benefits of adding β-glucosidases to increase the ethanol yield. Conclusions In the EDGE process, 30% of total dry solids could be used to produce 15% v/v ethanol. Under optimum conditions an ethanol yield of 402 L/MT (dry basis) or 2.17 gallons/53 lb bushel of barley with 15% moisture was achieved. The distillers dried grains with solubles (DDGS) co-product had extremely low β-glucan (below 0.2%) making it suitable for use in both ruminant and mono-gastric animal feeds. PMID:20426816

National transportation technology plan

DOT National Transportation Integrated Search

2000-05-01

The National Science and Technology Council (NSTC) Committee on Technology, Subcommittee on Transportation Research and Development (R&D), has created a National Transportation Technology Plan that builds on the initial Technology Plan released in 19...

DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody

DTIC Science & Technology

2016-03-01

performance in an enzyme-linked immunosorbent assay ( ELISA ), with little regard for quantification of the full spectrum of variables affecting antibody...Program (ATP) Quality MS2 coat protein (MS2CP) Enzyme-linked immunosorbent assay ( ELISA ) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT...5 2.7 ELISA ................................................................................................................5

Federal Technology Transfer Act Success Stories

EPA Pesticide Factsheets

Successful Federal Technology Transfer Act (FTTA) partnerships demonstrate the many advantages of technology transfer and collaboration. EPA and partner organizations create valuable and applicable technologies for the marketplace.

Diffusing Supply Chain Innovations at Hewlett-Packard Company: Applications of Performance Technology.

ERIC Educational Resources Information Center

Cargille, Brian; Branvold, Dwight

2000-01-01

Explains how Hewlett-Packard creates supply chain management innovations and effectively diffuses new technologies. Outlines how performance technologists help accelerate the diffusion and adoption of innovations by modifying innovations, define the client adoption path, create resources to lead clients through adoption, and improve the diffusion…

Learning through Creating Robotic Models of Biological Systems

ERIC Educational Resources Information Center

Cuperman, Dan; Verner, Igor M.

2013-01-01

This paper considers an approach to studying issues in technology and science, which integrates design and inquiry activities towards creating and exploring technological models of scientific phenomena. We implemented this approach in a context where the learner inquires into a biological phenomenon and develops its representation in the form of a…

Bioinspired catalytic materials for energy-relevant conversions

NASA Astrophysics Data System (ADS)

Artero, Vincent

2017-09-01

The structure of active sites of enzymes involved in bioenergetic processes can inspire design of active, stable and cost-effective catalysts for renewable-energy technologies. For these materials to reach maturity, the benefits of bioinspired systems must be combined with practical technological requirements.

Stereoselective hydrogenation of olefins using rhodium-substituted carbonic anhydrase--a new reductase.

PubMed

Jing, Qing; Okrasa, Krzysztof; Kazlauskas, Romas J

2009-01-01

One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA-[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site-directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA-[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis- over trans-stilbene by about 20:1. This enzyme is the first cofactor-independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme-catalyzed reactions.

Fabrication of Protein Microparticles and Microcapsules with Biomolecular Tools

NASA Astrophysics Data System (ADS)

Cheung, Kwan Yee; Lai, Kwok Kei; Mak, Wing Cheung

2018-05-01

Microparticles have attracted much attention for medical, analytical and biological applications. Calcium carbonate (CaCO3) templating method with the advantages of having narrow size distribution, controlled morphology and good biocompatibility that has been widely used for the synthesis of various protein-based microparticles. Despite CaCO3 template is biocompatible, most of the conventional methods to create stable protein microparticles are mainly driven by chemical crosslink reagents which may induce potential harmful effect and remains undesirable especially for biomedical or clinical applications. In this article, we demonstrate the fabrication of protein microparticles and microcapsules with an innovative method using biomolecular tools such as enzymes and affinity molecules to trigger the assembling of protein molecules within a porous CaCO3 template followed by a template removal step. We demonstrated the enzyme-assisted fabrication of collagen microparticles triggered by transglutaminase, as well as the affinity-assisted fabrication of BSA-biotin avidin microcapsules triggered by biotin-avidin affinity interaction, respectively. Based on the different protein assemble mechanisms, the collagen microparticles appeared as a solid-structured particles, while the BSA-biotin avidin microcapsules appeared as hollow-structured morphology. The fabrication procedures are simple and robust that allows producing protein microparticles or microcapsules under mild conditions at physiological pH and temperature. In addition, the microparticle morphologies, protein compositions and the assemble mechanisms were studied. Our technology provides a facile approach to design and fabricate protein microparticles and microcapsules that are useful in the area of biomaterials, pharmaceuticals and analytical chemistry.

Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates

NASA Astrophysics Data System (ADS)

Fuhrmann, Gregor; Grotzky, Andrea; Lukić, Ružica; Matoori, Simon; Luciani, Paola; Yu, Hao; Zhang, Baozhong; Walde, Peter; Schlüter, A. Dieter; Gauthier, Marc A.; Leroux, Jean-Christophe

2013-07-01

Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

Modification of enzymes by use of high-pressure homogenization.

PubMed

Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi

2018-07-01

High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.

Health and Wellness Technology Use by Historically Underserved Health Consumers: Systematic Review

PubMed Central

Perchonok, Jennifer

2012-01-01

Background The implementation of health technology is a national priority in the United States and widely discussed in the literature. However, literature about the use of this technology by historically underserved populations is limited. Information on culturally informed health and wellness technology and the use of these technologies to reduce health disparities facing historically underserved populations in the United States is sparse in the literature. Objective To examine ways in which technology is being used by historically underserved populations to decrease health disparities through facilitating or improving health care access and health and wellness outcomes. Methods We conducted a systematic review in four library databases (PubMed, PsycINFO, Web of Science, and Engineering Village) to investigate the use of technology by historically underserved populations. Search strings consisted of three topics (eg, technology, historically underserved populations, and health). Results A total of 424 search phrases applied in the four databases returned 16,108 papers. After review, 125 papers met the selection criteria. Within the selected papers, 30 types of technology, 19 historically underserved groups, and 23 health issues were discussed. Further, almost half of the papers (62 papers) examined the use of technology to create effective and culturally informed interventions or educational tools. Finally, 12 evaluation techniques were used to assess the technology. Conclusions While the reviewed studies show how technology can be used to positively affect the health of historically underserved populations, the technology must be tailored toward the intended population, as personally relevant and contextually situated health technology is more likely than broader technology to create behavior changes. Social media, cell phones, and videotapes are types of technology that should be used more often in the future. Further, culturally informed health information technology should be used more for chronic diseases and disease management, as it is an innovative way to provide holistic care and reminders to otherwise underserved populations. Additionally, design processes should be stated regularly so that best practices can be created. Finally, the evaluation process should be standardized to create a benchmark for culturally informed health information technology. PMID:22652979

Enzyme reactor design under thermal inactivation.

PubMed

Illanes, Andrés; Wilson, Lorena

2003-01-01

Temperature is a very relevant variable for any bioprocess. Temperature optimization of bioreactor operation is a key aspect for process economics. This is especially true for enzyme-catalyzed processes, because enzymes are complex, unstable catalysts whose technological potential relies on their operational stability. Enzyme reactor design is presented with a special emphasis on the effect of thermal inactivation. Enzyme thermal inactivation is a very complex process from a mechanistic point of view. However, for the purpose of enzyme reactor design, it has been oversimplified frequently, considering one-stage first-order kinetics of inactivation and data gathered under nonreactive conditions that poorly represent the actual conditions within the reactor. More complex mechanisms are frequent, especially in the case of immobilized enzymes, and most important is the effect of catalytic modulators (substrates and products) on enzyme stability under operation conditions. This review focuses primarily on reactor design and operation under modulated thermal inactivation. It also presents a scheme for bioreactor temperature optimization, based on validated temperature-explicit functions for all the kinetic and inactivation parameters involved. More conventional enzyme reactor design is presented merely as a background for the purpose of highlighting the need for a deeper insight into enzyme inactivation for proper bioreactor design.

Translations on Eastern Europe, Political, Sociological, and Military Affairs, Number 1404-A

DTIC Science & Technology

1977-06-22

Personality Development in Light of Technological Progress (Harry Nick; EINHEIT, Apr 77) 128 Significance of National Culture in Socialism...industrialization in such a way as to create "labor intensive" technologies in the Hungarian regions, preferably those which require low levels of training... technology , as well as the large number of well-paid personnel create excellent con- ditions for the implementation of the plans of the ideological

High pressure processing of fresh seafoods.

PubMed

Simpson, B K

1998-01-01

Crude proteolytic enzyme extracts were prepared from the muscle tissues of two fish species, bluefish and sheephead, and subjected to high hydrostatic pressure treatments (from 1,000-3,000 atm), and monitored for residual activity for cathepsin C, collagenase, chymotrypsin-like and trypsin-like enzymes versus homologous enzymes from bovine. The fish enzymes were more sensitive to hydrostatic pressure than the mammalian enzymes. The extent of enzyme inactivation achieved depended on both the amount of pressure applied, the duration of pressurization, and on the source material. Pressure treatment of fresh fish flesh formed products whose color deteriorated (cooked appearance) with increasing pressure as well as holding time. Application of pressure also improved tissue firmness or strength of fresh fish up to 2,000 atm and a holding time of 10 min, beyond which texture generally deteriorated. The combined use of pressure in combination with the broad spectrum protease inhibitor, alpha 2-macroglobulin, enhanced the capacity of the hydrostatic pressure technology to achieve a more lasting inactivation of endogenous enzymes to form stable fish gels.

Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Trygg, Johan; Vivier, Melané A

2015-03-18

Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.

Purification of recombinant tung tree diacylglycerol acyltransferases from E. coli

USDA-ARS?s Scientific Manuscript database

Understanding plant oil biosynthesis will help to create new oilseed crops with value-added properties to replace petroleum-based compounds. Diacylglycerol acyltransferases (DGATs) are key enzymes catalyzing the last step of triacylglycerol (TAG) biosynthesis in eukaryotes. Over-expression of DGATs ...

Biocatalysis and biomimetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burrington, J.D.; Clark, D.S.

1989-01-01

This book presents recent advances in catalytic science and biotechnology. The chapters illustrate how many of the key challenges in biotechnology can be addressed by bringing together traditionally separate disciplines within chemistry and biology. The authors focus on emerging enabling technologies at the interfaces of catalysis and biology that will provide new opportunities for the chemicals industries. Key aspects to be presented within this major theme of catalysis and biotechnology are biomimetics and hybrid catalysts, biocatalytic applications of computers and expert systems, enzyme solid-state structure and immobilization, enzyme structure-activity relationships, and the use of enzymes under novel conditions.

Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.

PubMed

Cravatt, Benjamin F; Wright, Aaron T; Kozarich, John W

2008-01-01

Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.

Protein Engineering Towards Natural Product Synthesis and Diversification

PubMed Central

Zabala, Angelica O.; Cacho, Ralph A.; Tang, Yi

2014-01-01

A dazzling array of enzymes is used by nature in making structurally complex natural products. These enzymes constitute a molecular toolbox that may be used in the construction and fine-tuning of pharmaceutically active molecules. Aided by technological advancements in protein engineering, it is now possible to tailor the activities and specificities of these enzymes as biocatalysts in the production of both natural products and their unnatural derivatives. These efforts are crucial in drug discovery and development, where there is a continuous quest for more potent agents. Both rational and random evolution techniques have been utilized in engineering these enzymes. This review will highlight some examples from several large families of natural products. PMID:22006344

Profiling microbial lignocellulose degradation and utilization by emergent omics technologies.

PubMed

Rosnow, Joshua J; Anderson, Lindsey N; Nair, Reji N; Baker, Erin S; Wright, Aaron T

2017-08-01

The use of plant materials to generate renewable biofuels and other high-value chemicals is the sustainable and preferable option, but will require considerable improvements to increase the rate and efficiency of lignocellulose depolymerization. This review highlights novel and emerging technologies that are being developed and deployed to characterize the process of lignocellulose degradation. The review will also illustrate how microbial communities deconstruct and metabolize lignocellulose by identifying the necessary genes and enzyme activities along with the reaction products. These technologies include multi-omic measurements, cell sorting and isolation, nuclear magnetic resonance spectroscopy (NMR), activity-based protein profiling, and direct measurement of enzyme activity. The recalcitrant nature of lignocellulose necessitates the need to characterize the methods microbes employ to deconstruct lignocellulose to inform new strategies on how to greatly improve biofuel conversion processes. New technologies are yielding important insights into microbial functions and strategies employed to degrade lignocellulose, providing a mechanistic blueprint in order to advance biofuel production.

"Trojan Horse" strategy for deconstruction of biomass for biofuels production.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinclair, Michael B.; Hadi, Masood Z.; Timlin, Jerilyn Ann

2008-08-01

Production of renewable biofuels to displace fossil fuels currently consumed in the transportation sector is a pressing multi-agency national priority. Currently, nearly all fuel ethanol is produced from corn-derived starch. Dedicated 'energy crops' and agricultural waste are preferred long-term solutions for renewable, cheap, and globally available biofuels as they avoid some of the market pressures and secondary greenhouse gas emission challenges currently facing corn ethanol. These sources of lignocellulosic biomass are converted to fermentable sugars using a variety of chemical and thermochemical pretreatments, which disrupt cellulose and lignin cross-links, allowing exogenously added recombinant microbial enzymes to more efficiently hydrolyze themore » cellulose for 'deconstruction' into glucose. This process is plagued with inefficiencies, primarily due to the recalcitrance of cellulosic biomass, mass transfer issues during deconstruction, and low activity of recombinant deconstruction enzymes. Costs are also high due to the requirement for enzymes and reagents, and energy-intensive and cumbersome pretreatment steps. One potential solution to these problems is found in synthetic biology; they propose to engineer plants that self-produce a suite of cellulase enzymes targeted to the apoplast for cleaving the linkages between lignin and cellulosic fibers; the genes encoding the degradation enzymes, also known as cellulases, are obtained from extremophilic organisms that grow at high temperatures (60-100 C) and acidic pH levels (<5). These enzymes will remain inactive during the life cycle of the plant but become active during hydrothermal pretreatment i.e., elevated temperatures. Deconstruction can be integrated into a one-step process, thereby increasing efficiency (cellulose-cellulase mass-transfer rates) and reducing costs. The proposed disruptive technologies address biomass deconstruction processes by developing transgenic plants encoding a suite of enzymes used in cellulosic deconstruction. The unique aspects of this technology are the rationally engineered, highly productive extremophilic enzymes, targeted to specific cellular locations (apoplast) and their dormancy during normal plant proliferation, which become Trojan horses during pretreatment conditions. They have been leveraging established Sandia's enzyme-engineering and imaging capabilities. Their technical approach not only targets the recalcitrance and mass-transfer problem during biomass degradation but also eliminates the costs associated with industrial-scale production of microbial enzymes added during processing.« less

Characterization of lignocellulolytic enzymes from white-rot fungi.

PubMed

Manavalan, Tamilvendan; Manavalan, Arulmani; Heese, Klaus

2015-04-01

The development of alternative energy sources by applying lignocellulose-based biofuel technology is critically important because of the depletion of fossil fuel resources, rising fossil fuel prices, security issues regarding the fossil fuel supply, and environmental issues. White-rot fungi have received much attention in recent years for their valuable enzyme systems that effectively degrade lignocellulosic biomasses. These fungi have powerful extracellular oxidative and hydrolytic enzymes that degrade lignin and cellulose biopolymers, respectively. Lignocellulosic biomasses from either agricultural or forestry wastes are abundant, low-cost feedstock alternatives in nature but require hydrolysis into simple sugars for biofuel production. This review provides a complete overview of the different lignocellulose biomasses and their chemical compositions. In addition, a complete list of the white-rot fungi-derived lignocellulolytic enzymes that have been identified and their molecular structures, mechanism of action in lignocellulose hydrolysis, and biochemical properties is summarized in detail. These enzymes include ligninolytic enzymes (laccase, manganese peroxidase, lignin peroxidase, and versatile peroxidase) and cellulolytic enzymes (endo-glucanase, cellobiohydrolase, and beta-glucosidase). The use of these fungi for low-cost lignocellulolytic enzyme production might be attractive for biofuel production.

Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes.

PubMed

Sirisha, V L; Jain, Ankita; Jain, Amita

Immobilized enzymes can be used in a wide range of processes. In recent years, a variety of new approaches have emerged for the immobilization of enzymes that have greater efficiency and wider usage. During the course of the last two decades, this area has rapidly expanded into a multidisciplinary field. This current study is a comprehensive review of a variety of literature produced on the different enzymes that have been immobilized on various supporting materials. These immobilized enzymes have a wide range of applications. These include applications in the sugar, fish, and wine industries, where they are used for removing organic compounds from waste water. This study also reviews their use in sophisticated biosensors for metabolite control and in situ measurements of environmental pollutants. Immobilized enzymes also find significant application in drug metabolism, biodiesel and antibiotic production, bioremediation, and the food industry. The widespread usage of immobilized enzymes is largely due to the fact that they are cheaper, environment friendly, and much easier to use when compared to equivalent technologies. © 2016 Elsevier Inc. All rights reserved.

Immobilization, stabilization and patterning techniques for enzyme based sensor systems.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flounders, A.W.; Carichner, S.C.; Singh, A.K.

1997-01-01

Sandia National Laboratories has recently opened the Chemical and Radiation Detection Laboratory (CRDL) in Livermore CA to address the detection needs of a variety of government agencies (e.g., Department of Energy, Environmental Protection Agency, Department of Agriculture) as well as provide a fertile environment for the cooperative development of new industrial technologies. This laboratory consolidates a variety of existing chemical and radiation detection efforts and enables Sandia to expand into the novel area of biochemically based sensors. One aspect of this biosensor effort is further development and optimization of enzyme modified field effect transistors (EnFETs). Recent work has focused uponmore » covalent attachment of enzymes to silicon dioxide and silicon nitride surfaces for EnFET fabrication. They are also investigating methods to pattern immobilized proteins; a critical component for development of array-based sensor systems. Novel enzyme stabilization procedures are key to patterning immobilized enzyme layers while maintaining enzyme activity. Results related to maximized enzyme loading, optimized enzyme activity and fluorescent imaging of patterned surfaces will be presented.« less

Integrative computational approach for genome-based study of microbial lipid-degrading enzymes.

PubMed

Vorapreeda, Tayvich; Thammarongtham, Chinae; Laoteng, Kobkul

2016-07-01

Lipid-degrading or lipolytic enzymes have gained enormous attention in academic and industrial sectors. Several efforts are underway to discover new lipase enzymes from a variety of microorganisms with particular catalytic properties to be used for extensive applications. In addition, various tools and strategies have been implemented to unravel the functional relevance of the versatile lipid-degrading enzymes for special purposes. This review highlights the study of microbial lipid-degrading enzymes through an integrative computational approach. The identification of putative lipase genes from microbial genomes and metagenomic libraries using homology-based mining is discussed, with an emphasis on sequence analysis of conserved motifs and enzyme topology. Molecular modelling of three-dimensional structure on the basis of sequence similarity is shown to be a potential approach for exploring the structural and functional relationships of candidate lipase enzymes. The perspectives on a discriminative framework of cutting-edge tools and technologies, including bioinformatics, computational biology, functional genomics and functional proteomics, intended to facilitate rapid progress in understanding lipolysis mechanism and to discover novel lipid-degrading enzymes of microorganisms are discussed.

Europe Report, Science and Technology.

DTIC Science & Technology

1986-06-18

amylase, heat stable alpha-amylase and glucoamylase for processing starch as a substrate for 71 glucose and its isomerization to fructose using an...continuous column process under laboratory conditions. We have demonstrated that these preparations isomerize glucose syrups up to 42 percent, converting...food industry is the leading consumer of microbial enzymes devouring about 80 percent of the world production of enzymes -- glucose isomerase, alpha

Suitable technological conditions for enzymatic hydrolysis of waste paper by Novozymes® enzymes NS50013 and NS50010.

PubMed

Brummer, Vladimir; Skryja, Pavel; Jurena, Tomas; Hlavacek, Viliam; Stehlik, Petr

2014-10-01

Waste paper belongs to a group of quantitatively the most produced waste types. Enzymatic hydrolysis is becoming a suitable way to treat this type of waste and at the same time, to produce a valuable liquid biofuel, because reducing sugars solutions that are formed during the process of saccharification can be a precursor for following or simultaneous fermentation. If it will be possible to make the enzymatic hydrolysis of the waste paper economically viable, it could serve as one of the new ways to lower the dependence of the transport sector on oil in the future. Only several studies comparing the enzymatic hydrolysis of different waste papers were performed in the past; they are summarized in this manuscript. In our experimental trials, suitable technological conditions for waste paper enzymatic hydrolysis using enzymes from Novozymes® biomass kit: enzymes NS50013 and NS50010 were investigated. The following enzymatic hydrolysis parameters in laboratory scale trials were verified on high cellulose content substrates-filter paper and cellulose pulp: type of buffer, pH, temperature, concentration of the substrate, loading of the enzyme and rate of stirring.

The End of Mass Society?: A Preface to Telecommunications Politics.

ERIC Educational Resources Information Center

Triebwasser, Marc A.

This paper suggests that throughout human history technological revolutions have often created the potential for social revolutions as these new ways of doing things were applied to society. Often, it has been these changes in technology, more than ideology, that have created changes in the way human society is organized. The document contends…

A review of an attempt to create shatter cones with magnetic flyer plate technology

NASA Technical Reports Server (NTRS)

Linnerud, H. J.

1981-01-01

The feasibility of creating shatter cones in a controlled laboratory environment is discussed. Magnetic flyer plate technology, which generates high amplitude shock waves in test materials is discribed. Considerable sample shear and break up was observed, however, no shatter cones are found in the tested samples.

The Academy Is Flat: Using Technology to Create Authentic Multicultural Education

ERIC Educational Resources Information Center

Roberts, Gary O.; Monroe-Baillargeon, Ann

2012-01-01

Thomas Freidman (2005) asserts that technology has created a transnational platform for communication, collaboration, and competition that is stimulating global opportunity for all individuals. After years of robust study abroad programs and the recent "bricks and mortar" expansion of U.S. higher education overseas, we are now seeing the true…

A Wiki Collaboration to Create National Guidelines: Tips for Professional Practice

ERIC Educational Resources Information Center

Moser, Patt

2009-01-01

In 2006, technology directors from independent schools across the country had banded together and created "Principles of Good Practice" for technology in independent schools. These principles "define high standards and ethical behavior in key areas of independent school operations" (NAIS 2006). This author decided that it was time for the National…

Leveraging 21st Century Learning & Technology to Create Caring Diverse Classroom Cultures

ERIC Educational Resources Information Center

Tarbutton, Tanya

2018-01-01

Creating diverse caring classroom environments, for all students, using innovative technology, is the impetus of this article. Administrators and teachers in many states have worked to integrate 21st Century Learning Outcomes and Local Control and Accountability Plans (LCAP) into daily teaching and learning. These initiatives are designed to…

Student Adoption & Development of Digital Learning Media: Action Research and Recommended Practices

ERIC Educational Resources Information Center

Tabor, Sharon W.; Minch, Robert P.

2013-01-01

Digital technologies offer many opportunities for creating engaging course content. In this study we captured student perceptions and adoption choices related to creating and using digital media as learning tools. Podcasts, video and other media were integrated in a variety of contexts and tasks in two undergraduate information technology (IT)…

Creating Micro-Videos to Demonstrate Technology Learning

ERIC Educational Resources Information Center

Frydenberg, Mark; Andone, Diana

2016-01-01

Short videos, also known as micro-videos, have emerged as a platform for sharing ideas, experiences, and life events on online social networks. This paper shares preliminary results of a study involving students from two universities who created six-second videos using the Vine mobile app to explain or illustrate technology concepts. An analysis…

How To Create Effective Information and Communication Technology Learning Programmes. A Guide.

ERIC Educational Resources Information Center

Clarke, Alan

This guide, which is intended for information and communication technology (ICT) tutors and tutor managers in Great Britain's further education sector, explains how to create effective ICT learning programs for adults. The guide emphasizes developing students' confidence and providing them with a sound foundation for further study. The following…

Higher Education Technology and Research: Creating Excellence through State Investments

ERIC Educational Resources Information Center

New Jersey Commission on Higher Education, 2004

2004-01-01

Colleges and universities in New Jersey play a critical role in building and sustaining economic prosperity and quality of life in the state and beyond. Through advancements in technology and research, higher education helps to create new jobs, improve the workforce, develop new knowledge, and boost the overall economy. This report summarizes…

The "Day in the Life of a Teenage Hobo" Project: Integrating Technology with Shneiderman's Collect-Relate-Create- Donate Framework

ERIC Educational Resources Information Center

Reich, Justin; Daccord, Thomas

2009-01-01

Used wisely, academic technology empowers students to take responsibility for their own learning. "In Leonardo's Laptop," Ben Shneiderman provides teachers with a powerful framework, "Collect-Relate-Create-Donate" (CRCD), for designing student-centered learning opportunities using computers. Shneiderman developed his model by…

Genetic technologies and ethics.

PubMed

Ardekani, Ali M

2009-01-01

In the past decade, the human genome has been completely sequenced and the knowledge from it has begun to influence the fields of biological and social sciences in fundamental ways. Identification of about 25000 genes in the human genome is expected to create great benefits in diagnosis and treatment of diseases in the coming years. However, Genetic technologies have also created many interesting and difficult ethical issues which can affect the human societies now and in the future. Application of genetic technologies in the areas of stem cells, cloning, gene therapy, genetic manipulation, gene selection, sex selection and preimplantation diagnosis has created a great potential for the human race to influence and change human life on earth as we know it today. Therefore, it is important for leaders of societies in the modern world to pay attention to the advances in genetic technologies and prepare themselves and those institutions under their command to face the challenges which these new technologies induce in the areas of ethics, law and social policies.

Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

PubMed

Gupta, Sanjeev K; Shukla, Pratyoosh

2016-12-01

Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

Phytoremediation of hazardous wastes. Technical report, 23--26 July 1995

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCutcheon, S.C.; Wolfe, N.L.; Carreria, L.H.

1995-07-26

A new and innovative approach to phytoremediation (the use of plants to degrade hazardous contaminants) was developed. The new approach to phytoremediation involves rigorous pathway analyses, mass balance determinations, and identification of specific enzymes that break down trinitrotoluene (TNT), other explosives (RDX and HMX), nitrobenzene, and chlorinated solvents (e.g., TCE and PCE) (EPA 1994). As a good example, TNT is completely and rapidly degraded by nitroreductase and laccase enzymes. The aromatic ring is broken and the carbon in the ring fragments is incorporated into new plant fiber, as part of the natural lignification process. Half lives for TNT degradation approachmore » 1 hr or less under ideal laboratory conditions. Continuous-flow pilot studies indicate that scale up residence times in created wetlands may be two to three times longer than in laboratory batch studies. The use of created wetlands and land farming techniques guided by rigorous field biochemistry and ecology promises to be a vital part of a newly evolving field, ecological engineering.« less

Phytoremediation of hazardous wastes

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCutcheon, S.C.; Wolfe, N.L.; Carreria, L.H.

1995-11-01

A new and innovative approach to phytoremediation (the use of plants to degrade hazardous contaminants) was developed. The new approach to phytoremediation involves rigorous pathway analyses, mass balance determinations, and identification of specific enzymes that break down trinitrotoluene (TNT), other explosives (RDX and HMX), nitrobenzene, and chlorinated solvents (e.g., TCE and PCE) (EPA 1994). As a good example, TNT is completely and rapidly degraded by nitroreductase and laccase enzymes. The aromatic ring is broken and the carbon in the ring fragments is incorporated into new plant fiber, as part of the natural lignification process. Half lives for TNT degradation approachmore » 1 hr or less under ideal laboratory conditions. Continuous-flow pilot studies indicate that scale up residence times in created wetlands may be two to three times longer than in laboratory batch studies. The use of created wetlands and land farming techniques guided by rigorous field biochemistry and ecology promises to be a vital part of a newly evolving field, ecological engineering.« less

The impact of podcasts, screencasts, and vodcasts on student achievement in the science classroom

NASA Astrophysics Data System (ADS)

Pena, Ruben, Jr.

Educators in today's society are in search for different ways to reach their students in order to keep them engaged and active in the learning process. There are several strategies that teachers have utilized in the classroom in order to reach all students. Now seen more in the classroom is the use of technology in one form or another. There are several types of technologies that one may employ while in the classroom, but seen more recently is the use of podcasts, screencasts, and vodcasts. The major purpose of the study was to investigate the impact of using podcasts, screencasts, and vodcasts in conjunction with science curriculum on student academic achievement. Two intermediate schools from the south Texas region were chosen as a convenience sample for the study because one school utilized the technology of podcasts, screencasts, and vodcasts at the student created level while the other school did not utilize podcasts, screencasts, and vodcasts at the student created level. The researcher collected scores from curriculum based assessments that were aligned with the Texas Essential Knowledge and Skills (TEKS) for comparison between the two different groups, while controlling grade five science TAKS scores for group equalization. Once all data was collected, scores were entered into the Statistical Package for the Social Sciences (SPSS) and were analyzed using an analysis of covariance. The ANCOVA allowed the researcher to see that differences among curriculum based assessments scores existed between the two different schools. Scores were higher for the students who utilized podcasts, screencasts, and vodcasts at the student created level when compared to those scores for students who did not utilize podcasts, screencasts, and vodcasts at the student created level. This study showed the benefits reaped of having students create their own podcasts, screencasts, and vodcasts. Having students create their own technology has them actively engaged in the learning process and this is the goal of every educator. Looking at this notion shows that having students create their own technology projects, any type of technology, enhances academic achievement which is expected in today's classroom.

Genetic engineering approach to toxic waste management: case study for organophosphate waste treatment.

PubMed

Coppella, S J; DelaCruz, N; Payne, G F; Pogell, B M; Speedie, M K; Karns, J S; Sybert, E M; Connor, M A

1990-01-01

Currently, there has been limited use of genetic engineering for waste treatment. In this work, we are developing a procedure for the in situ treatment of toxic organophosphate wastes using the enzyme parathion hydrolase. Since this strategy is based on the use of an enzyme and not viable microorganisms, recombinant DNA technology could be used without the problems associated with releasing genetically altered microorganisms into the environment. The gene coding for parathion hydrolase was cloned into a Streptomyces lividans, and this transformed bacterium was observed to express and excrete this enzyme. Subsequently, fermentation conditions were developed to enhance enzyme production, and this fermentation was scaled-up to the pilot scale. The cell-free culture fluid (i.e., a nonpurified enzyme solution) was observed to be capable of effectively hydrolyzing organophosphate compounds under laboratory and simulated in situ conditions.

National Transportation Science and Technology Strategy.

DOT National Transportation Integrated Search

1999-04-01

The National Science and Technology Council (NSTC) Committee on Technology, Subcommittee on Transportation Research and Development (R&D), has created a National Transportation Science and Technology Strategy that builds on the earlier strategy publi...

The National Network forTechnology Entrepreneurship and Commercialization (N2TEC): Bringing New Technologies to Market

NASA Astrophysics Data System (ADS)

Allen, Kathleen

2003-03-01

N2TEC, the National Network for Technology Entrepreneurship and Commercialization, is a National Science Foundation "Partnerships for Innovation" initiative designed to raise the level of innovation and technology commercialization in colleges, universities, and communities across the nation. N2TEC is creating a network of people and institutions, and a set of technology tools that will facilitate the pooling of resources and knowledge and enable faculty and students to share those resources and collaborate without regard to geographic boundaries. N2TEC will become the backbone by which educational institutions across the nation can move their technologies into new venture startups. The ultimate goal is to create new wealth and strengthen local, regional and national economies.

Robotic surgery update.

PubMed

Jacobsen, G; Elli, F; Horgan, S

2004-08-01

Minimally invasive surgical techniques have revolutionized the field of surgery. Telesurgical manipulators (robots) and new information technologies strive to improve upon currently available minimally invasive techniques and create new possibilities. A retrospective review of all robotic cases at a single academic medical center from August 2000 until November 2002 was conducted. A comprehensive literature evaluation on robotic surgical technology was also performed. Robotic technology is safely and effectively being applied at our institution. Robotic and information technologies have improved upon minimally invasive surgical techniques and created new opportunities not attainable in open surgery. Robotic technology offers many benefits over traditional minimal access techniques and has been proven safe and effective. Further research is needed to better define the optimal application of this technology. Credentialing and educational requirements also need to be delineated.

Atomic-Level Quality Assessment of Enzymes Encapsulated in Bioinspired Silica.

PubMed

Martelli, Tommaso; Ravera, Enrico; Louka, Alexandra; Cerofolini, Linda; Hafner, Manuel; Fragai, Marco; Becker, Christian F W; Luchinat, Claudio

2016-01-04

Among protein immobilization strategies, encapsulation in bioinspired silica is increasingly popular. Encapsulation offers high yields and the solid support is created through a protein-catalyzed polycondensation reaction that occurs under mild conditions. An integrated strategy is reported for the characterization of both the protein and bioinspired silica scaffold generated by the encapsulation of enzymes with an external silica-forming promoter or with the promoter expressed as a fusion to the enzyme. This strategy is applied to the catalytic domain of matrix metalloproteinase 12. Analysis reveals that the structure of the protein encapsulated by either method is not significantly altered with respect to the native form. The structural features of silica obtained by either strategy are also similar, but differ from those obtained by other approaches. In case of the covalently linked R5-enzyme construct, immobilization yields are higher. Encapsulation through a fusion protein, therefore, appears to be the method of choice. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using virtual reality technology and hand tracking technology to create software for training surgical skills in 3D game

NASA Astrophysics Data System (ADS)

Zakirova, A. A.; Ganiev, B. A.; Mullin, R. I.

2015-11-01

The lack of visible and approachable ways of training surgical skills is one of the main problems in medical education. Existing simulation training devices are not designed to teach students, and are not available due to the high cost of the equipment. Using modern technologies such as virtual reality and hands movements fixation technology we want to create innovative method of learning the technics of conducting operations in 3D game format, which can make education process interesting and effective. Creating of 3D format virtual simulator will allow to solve several conceptual problems at once: opportunity of practical skills improvement unlimited by the time without the risk for patient, high realism of environment in operational and anatomic body structures, using of game mechanics for information perception relief and memorization of methods acceleration, accessibility of this program.

The Graphics Tablet - A Valuable Tool for the Digital STEM Teacher

NASA Astrophysics Data System (ADS)

Stephens, Jeff

2018-04-01

I am inspired to write this article after coming across some publications in The Physics Teacher that all hit on topics of personal interest and experience. Similarly to Christensen my goal in writing this is to encourage other physics educators to take advantage of modern technology in delivering content to students and to feel comfortable doing so. There are numerous ways in which to create screencasts and lecture videos, some of which have been addressed in other articles. I invite those interested in learning how to create these videos to contact their educational technology staff or perform some internet searches on the topic. I will focus this article on the technology that enhanced the content I was delivering to my students. I will share a bit of my journey towards creating video materials and introduce a vital piece of technology, the graphics tablet, which changed the way I communicate with my students.

Structural characterization of O- and C-glycosylating variants of the landomycin glycosyltransferase LanGT2.

PubMed

Tam, Heng Keat; Härle, Johannes; Gerhardt, Stefan; Rohr, Jürgen; Wang, Guojun; Thorson, Jon S; Bigot, Aurélien; Lutterbeck, Monika; Seiche, Wolfgang; Breit, Bernhard; Bechthold, Andreas; Einsle, Oliver

2015-02-23

The structures of the O-glycosyltransferase LanGT2 and the engineered, C-C bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed that there is a conformational transition to create the binding sites for the aglycon substrate. This induced-fit transition was explored by molecular docking experiments with various aglycon substrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Technology in Science and Mathematics Education.

ERIC Educational Resources Information Center

Buccino, Alphonse

Provided are several perspectives on technology, addressing changes in learners related to technology, changes in contemporary life related to technology, and changes in subject areas related to technology (indicating that technology has created such new tools for inquiry as computer programming, word processing, online database searches, and…

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT: ECR TECHNOLOGIES, INC., EARTHLINKED GROUND-SOURCE HEAT PUMP WATER HEATING SYSTEM

EPA Science Inventory

EPA has created the Environmental Technology Verification program to provide high quality, peer reviewed data on technology performance. This data is expected to accelerate the acceptance and use of improved environmental protection technologies. The Greenhouse Gas Technology C...

Leveraging technology: creating and sustaining changes for health.

PubMed

Teyhen, Deydre S; Aldag, Matt; Edinborough, Elton; Ghannadian, Jason D; Haught, Andrea; Kinn, Julie; Kunkler, Kevin J; Levine, Betty; McClain, James; Neal, David; Stewart, Tiffany; Thorndike, Frances P; Trabosh, Valerie; Wesensten, Nancy; Parramore, David J

2014-09-01

The rapid growth and evolution of health-related technology capabilities are driving an established presence in the marketplace and are opening up tremendous potential to minimize and/or mitigate barriers associated with achieving optimal health, performance, and readiness. This article summarizes technology-based strategies that promote healthy habits related to physical activity, nutrition, and sleep. The Telemedicine and Advanced Technology Research Center convened a workshop titled "Leveraging Technology: Creating & Sustaining Changes for Health" (May 29-30, 2013, Fort Detrick, MD). Participants included experts from academia (n=3), government (n=33), and industry (n=16). A modified Delphi method was used to establish expert consensus in six topic areas: (1) physical activity, (2) nutrition, (3) sleep, (4) incentives for behavior change, (5) usability/interoperability, and (6) mobile health/open platform. Overall, 162 technology features, constructs, and best practices were reviewed and prioritized for physical activity monitors (n=29), nutrition monitors (n=35), sleep monitors (n=24), incentives for change (n=36), usability and interoperability (n=25), and open data (n=13). Leading practices, gaps, and research needs for technology-based strategies were identified and prioritized. This information can be used to provide a research and development road map for (1) leveraging technology to minimize barriers to enhancing health and (2) facilitating evidence-based techniques to create and sustain healthy behaviors.

Biomimetic structural engineering of P22 virus-like particles for catalysis and immune modulation

NASA Astrophysics Data System (ADS)

Schwarz, Benjamin

Within biology molecules are arranged in hierarchical structures that coordinate and control the many processes that allow for complex organisms to exist. Proteins and other functional macromolecules are often studied outside their natural nanostructural context because it remains difficult to create controlled arrangements of proteins at this size scale. Viruses are elegantly simple nano-systems that exist at the interface of living organisms and non-living biological machines. Studied and viewed primarily as pathogens to be combatted, viruses have emerged as models of structural efficiency at the nanoscale and have spurred the development of biomimetic nanoparticle systems. Virus-like particles (VLPs) are noninfectious protein cages derived from viruses or other cage-forming systems. VLPs provide incredibly regular scaffolds for building at the nanoscale. In this work I have utilized the VLP derived from the bacteriophage P22 as a platform for the organization of enzymes, antigens, and immune-stimulating proteins inside and outside the capsid through purely genetic means. In the case of enzymes, encapsulation of a two-enzyme pathway has led to the development of metabolic nanoparticle catalysts and an expanded understanding of the control that structure exerts on metabolic flux. These same structural elements applied to the delivery of protein subunit antigens directed at cytotoxic T cell immunity result in drastically enhanced antigen processing and lasting immunological memory. Lastly, presentation of immune-stimulating proteins from the Tumor Necrosis Factor Super Family on the surface of the P22 VLP enhances the cell signaling efficiency of these compounds 50-fold and provides strategies for the application of these proteins as immune modulatory oncology therapeutics. In all of these cases, the reintroduction of nanostructure to these protein systems, reminiscent of their natural environment, has led to both new technologies and a better understanding of the role of structure in biological processes.

Biocatalysis for the application of CO2 as a chemical feedstock.

PubMed

Alissandratos, Apostolos; Easton, Christopher J

2015-01-01

Biocatalysts, capable of efficiently transforming CO2 into other more reduced forms of carbon, offer sustainable alternatives to current oxidative technologies that rely on diminishing natural fossil-fuel deposits. Enzymes that catalyse CO2 fixation steps in carbon assimilation pathways are promising catalysts for the sustainable transformation of this safe and renewable feedstock into central metabolites. These may be further converted into a wide range of fuels and commodity chemicals, through the multitude of known enzymatic reactions. The required reducing equivalents for the net carbon reductions may be drawn from solar energy, electricity or chemical oxidation, and delivered in vitro or through cellular mechanisms, while enzyme catalysis lowers the activation barriers of the CO2 transformations to make them more energy efficient. The development of technologies that treat CO2-transforming enzymes and other cellular components as modules that may be assembled into synthetic reaction circuits will facilitate the use of CO2 as a renewable chemical feedstock, greatly enabling a sustainable carbon bio-economy.

The challenges of sequencing by synthesis.

PubMed

Fuller, Carl W; Middendorf, Lyle R; Benner, Steven A; Church, George M; Harris, Timothy; Huang, Xiaohua; Jovanovich, Stevan B; Nelson, John R; Schloss, Jeffery A; Schwartz, David C; Vezenov, Dmitri V

2009-11-01

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to $100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.

Biochemical and Thermodynamical Characterization of Glucose Oxidase, Invertase, and Alkaline Phosphatase Secreted by Antarctic Yeasts.

PubMed

Yuivar, Yassef; Barahona, Salvador; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

2017-01-01

The use of enzymes in diverse industries has increased substantially over past decades, creating a well-established and growing global market. Currently, the use of enzymes that work better at ambient or lower temperatures in order to decrease the temperatures of production processes is desirable. There is thus a continuous search for enzymes in cold environments, especially from microbial sources, with amylases, proteases, lipases and, cellulases being the most studied. Other enzymes, such as glucose oxidase (GOD), invertase (Inv), and alkaline phosphatase (ALP), also have a high potential for application, but have been much less studied in microorganisms living in cold-environments. In this work, secretion of these three enzymes by Antarctic yeast species was analyzed, and five, three, and five species were found to produce extracellular GOD, Inv, and ALP, respectively. The major producers of GOD, Inv, and ALP were Goffeauzyma gastrica, Wickerhamomyces anomalus , and Dioszegia sp., respectively, from which the enzymes were purified and characterized. Contrary to what was expected, the highest GOD and Inv activities were found at 64°C and 60°C, respectively, and at 47°C for ALP. However, the three enzymes maintained a significant percentage of activity at lower temperatures, especially ALP that kept a 67 and 43% of activity at 10°C and 4°C, respectively.

Biochemical and Thermodynamical Characterization of Glucose Oxidase, Invertase, and Alkaline Phosphatase Secreted by Antarctic Yeasts

PubMed Central

Yuivar, Yassef; Barahona, Salvador; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

2017-01-01

The use of enzymes in diverse industries has increased substantially over past decades, creating a well-established and growing global market. Currently, the use of enzymes that work better at ambient or lower temperatures in order to decrease the temperatures of production processes is desirable. There is thus a continuous search for enzymes in cold environments, especially from microbial sources, with amylases, proteases, lipases and, cellulases being the most studied. Other enzymes, such as glucose oxidase (GOD), invertase (Inv), and alkaline phosphatase (ALP), also have a high potential for application, but have been much less studied in microorganisms living in cold-environments. In this work, secretion of these three enzymes by Antarctic yeast species was analyzed, and five, three, and five species were found to produce extracellular GOD, Inv, and ALP, respectively. The major producers of GOD, Inv, and ALP were Goffeauzyma gastrica, Wickerhamomyces anomalus, and Dioszegia sp., respectively, from which the enzymes were purified and characterized. Contrary to what was expected, the highest GOD and Inv activities were found at 64°C and 60°C, respectively, and at 47°C for ALP. However, the three enzymes maintained a significant percentage of activity at lower temperatures, especially ALP that kept a 67 and 43% of activity at 10°C and 4°C, respectively. PMID:29312954

Multicultural Media Authorship: Using Technology to Create Children's Literature Texts

ERIC Educational Resources Information Center

Byker, Erik Jon; Good, Amy J.; Miller, Erin; Kissel, Brian

2018-01-01

The purpose of this article is to describe and report on how a group of teacher candidates used technology to create multicultural texts which integrated literacy and social studies. To meet this purpose, the authors briefly describe the objectives of the assignment, examine 41 teacher candidates' perceptions of the assignment, and discuss media…

Pre-Service Teachers' Perception of and Technology Competency at Creating and Using E-Picture Books

ERIC Educational Resources Information Center

Park, Yong Joon; Yang, Youjin

2013-01-01

This study investigated pre-service teachers' perception of and technology competency in creating and using e-picture books in their future classrooms. Participants were 114 pre-service teachers in a required Early Childhood Education undergraduate course at a mid-western university in the United States. As part of the course assignments,…

Creating a Video Tour to Market Your Center

ERIC Educational Resources Information Center

Gellens, Suzanne; Mathews, Bobbie; Young, Shari

2012-01-01

Today, technology can be used to engage and communicate with others in ever-expanding ways. Many directors are embracing these technological advances to market their child care programs. In addition to printing up brochures and taking out ads in newspapers, directors can create a tour of their child care program that will attract new customers, as…

Digital Photography and Journals in a Kindergarten-First-Grade Classroom: Toward Meaningful Technology Integration in Early Childhood Education

ERIC Educational Resources Information Center

Ching, Cynthia Carter; Wang, X. Christine; Shih, Mei-Li; Kedem, Yore

2006-01-01

To explore meaningful and effective technology integration in early childhood education, we investigated how kindergarten-first-grade students created and employed digital photography journals to support social and cognitive reflection. These students used a digital camera to document their daily school activities and created digital photo…

SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION PROGRAM - TECHNOLOGY PROFILES 4th Edition

EPA Science Inventory

The Superfund Innovative Technology Evaluation (SITE) Program evaluates new and promising treatment technologies for cleanup of hazardous waste sites. The program was created to encourage the development and routine use of innovative treatment technologies. As a result, the SI...

Enzyme immobilisation in biocatalysis: why, what and how.

PubMed

Sheldon, Roger A; van Pelt, Sander

2013-08-07

In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

DTIC Science & Technology

2016-08-01

platforms. 15. SUBJECT TERMS Antibody Antibody Technology Program (ATP) Quality Enzyme-linked immunosorbent assay ( ELISA ) Biosurveillance Single-chain...2.6 Thermal Stress Test............................................................................................4 2.7 ELISA ...3.5 ELISA Results .................................................................................................11 3.6 SPR Results

Elucidation of Genetic Backgrounds Necessary for Chlorophyll a Biosynthesis Toward Artificial Creation of Oxygenic Photosynthesis

NASA Astrophysics Data System (ADS)

Tsukatani, Yusuke; Masuda, Shinji

2015-09-01

We succeeded to create the genetically modified purple photosynthetic bacterium capable of synthesizing chlorophyll a. The results indicate that not only chlorophyll synthase, but also an enzyme for galactolipid synthesis and reaction center proteins are required for accumulating chlorophyll a.

Prostate Cancer Prevention Through Induction of Phase 2 Enzymes

DTIC Science & Technology

2001-04-01

enzymes. During our Phase I Award, we identified sulforaphane as the most potent inducer of carcinogen defenses in the prostate cell. We have...characterized global effects of sulforaphane in prostate cancer cell lines using cDNA microarray technology that allows large-scale determination of changes...of sulforaphane ) and decreased risk of prostate cancer. These findings argue strongly for a preventive intervention trial involving supplementation

Probing Enzyme-Surface Interactions via Protein Engineering and Single-Molecule Techniques

DTIC Science & Technology

2017-06-26

SECURITY CLASSIFICATION OF: The overall objective of this research was to exploit protein engineering and fluorescence single-molecule methods to... Engineering and Single-Molecule Techniques The views, opinions and/or findings contained in this report are those of the author(s) and should not...Status: Technology Transfer: Report Date: 1 FINAL REPORT Project Title: Probing Enzyme-Surface Interactions via Protein Engineering and

The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function

PubMed Central

Johnson, Troy A.; Holyoak, Todd

2012-01-01

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that transitions between an open/disorded conformation to a closed/ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies show that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. In order to more fully investigate the roles of the lid domain in PEPCK function we created three mutations that replaced the 11-residue lid domain with one, two or three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity resulting in a decrease in the catalytic parameters by at least 106. Structural characterization of the mutants in complexes representing the catalytic cycle suggest that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all elements required for chemical conversion of substrates to products remaining intact. PMID:23127136

Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

PubMed

Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

2014-01-01

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT). © 2014 American Institute of Chemical Engineers.

A review of biological delignification and detoxification methods for lignocellulosic bioethanol production.

PubMed

Moreno, Antonio D; Ibarra, David; Alvira, Pablo; Tomás-Pejó, Elia; Ballesteros, Mercedes

2015-01-01

Future biorefineries will integrate biomass conversion processes to produce fuels, power, heat and value-added chemicals. Due to its low price and wide distribution, lignocellulosic biomass is expected to play an important role toward this goal. Regarding renewable biofuel production, bioethanol from lignocellulosic feedstocks is considered the most feasible option for fossil fuels replacement since these raw materials do not compete with food or feed crops. In the overall process, lignin, the natural barrier of the lignocellulosic biomass, represents an important limiting factor in biomass digestibility. In order to reduce the recalcitrant structure of lignocellulose, biological pretreatments have been promoted as sustainable and environmentally friendly alternatives to traditional physico-chemical technologies, which are expensive and pollute the environment. These approaches include the use of diverse white-rot fungi and/or ligninolytic enzymes, which disrupt lignin polymers and facilitate the bioconversion of the sugar fraction into ethanol. As there is still no suitable biological pretreatment technology ready to scale up in an industrial context, white-rot fungi and/or ligninolytic enzymes have also been proposed to overcome, in a separated or in situ biodetoxification step, the effect of the inhibitors produced by non-biological pretreatments. The present work reviews the latest studies regarding the application of different microorganisms or enzymes as useful and environmentally friendly delignification and detoxification technologies for lignocellulosic biofuel production. This review also points out the main challenges and possible ways to make these technologies a reality for the bioethanol industry.

Connexin-Based Therapeutics and Tissue Engineering Approaches to the Amelioration of Chronic Pancreatitis and Type I Diabetes: Construction and Characterization of a Novel Prevascularized Bioartificial Pancreas.

PubMed

Rhett, J Matthew; Wang, Hongjun; Bainbridge, Heather; Song, Lili; Yost, Michael J

2016-01-01

Total pancreatectomy and islet autotransplantation is a cutting-edge technique to treat chronic pancreatitis and postoperative diabetes. A major obstacle has been low islet cell survival due largely to the innate inflammatory response. Connexin43 (Cx43) channels play a key role in early inflammation and have proven to be viable therapeutic targets. Even if cell death due to early inflammation is avoided, insufficient vascularization is a primary obstacle to maintaining the viability of implanted cells. We have invented technologies targeting the inflammatory response and poor vascularization: a Cx43 mimetic peptide that inhibits inflammation and a novel prevascularized tissue engineered construct. We combined these technologies with isolated islets to create a prevascularized bioartificial pancreas that is resistant to the innate inflammatory response. Immunoconfocal microscopy showed that constructs containing islets express insulin and possess a vascular network similar to constructs without islets. Glucose stimulated islet-containing constructs displayed reduced insulin secretion compared to islets alone. However, labeling for insulin post-glucose stimulation revealed that the constructs expressed abundant levels of insulin. This discrepancy was found to be due to the expression of insulin degrading enzyme. These results suggest that the prevascularized bioartificial pancreas is potentially a tool for improving long-term islet cell survival in vivo.

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase*

PubMed Central

Chen, Yaozong; Sun, Yueru; Song, Haigang; Guo, Zhihong

2015-01-01

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes. PMID:26276389

Structure-function mapping of key determinants for hydrocarbon biosynthesis by squalene and squalene synthase-like enzymes from the green alga Botryococcus braunii race B.

PubMed

Bell, Stephen A; Niehaus, Thomas D; Nybo, S Eric; Chappell, Joseph

2014-12-09

Squalene and botryococcene are branched-chain, triterpene compounds that arise from the head-to-head condensation of two molecules of farnesyl diphosphate to yield 1'-1 and 1'-3 linkages, respectively. The enzymes that catalyze their formation have attracted considerable interest from the medical field as potential drug targets and the renewable energy sector for metabolic engineering efforts. Recently, the enzymes responsible for botryococcene and squalene biosynthesis in the green alga Botryococcus braunii race B were characterized. To better understand how the specificity for the 1'-1 and 1'-3 linkages was controlled, we attempted to identify the functional residues and/or domains responsible for this step in the catalytic cascade. Existing crystal structures for the mammalian squalene synthase and Staphylococcus dehydrosqualene synthase enzymes were exploited to develop molecular models for the B. braunii botryococcene and squalene synthase enzymes. Residues within the active sites that could mediate catalytic specificity were identified, and reciprocal mutants were created in an attempt to interconvert the reaction product specificity of the enzymes. We report here the identification of several amino acid positions contributing to the rearrangement of the cyclopropyl intermediate to squalene, but these same positions do not appear to be sufficient to account for the cyclopropyl rearrangement to give botryococcene.

Production and Use of Lipases in Bioenergy: A Review from the Feedstocks to Biodiesel Production

PubMed Central

Ribeiro, Bernardo Dias; de Castro, Aline Machado; Coelho, Maria Alice Zarur; Freire, Denise Maria Guimarães

2011-01-01

Lipases represent one of the most reported groups of enzymes for the production of biofuels. They are used for the processing of glycerides and fatty acids for biodiesel (fatty acid alkyl esters) production. This paper presents the main topics of the enzyme-based production of biodiesel, from the feedstocks to the production of enzymes and their application in esterification and transesterification reactions. Growing technologies, such as the use of whole cells as catalysts, are addressed, and as concluding remarks, the advantages, concerns, and future prospects of enzymatic biodiesel are presented. PMID:21785707

Applications of micellar enzymology to clean coal technology. [Laccase from Polyporus versicolor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walsh, C.T.

1990-07-24

This project is designed to develop methods for pre-combustion coal remediation by implementing recent advances in enzyme biochemistry. The novel approach of this study is incorporation of hydrophilic oxidative enzymes in reverse micelles in an organic solvent. Enzymes from commercial sources or microbial extracts are being investigated for their capacity to remove organic sulfur from coal by oxidation of the sulfur groups, splitting of C-S bonds and loss of sulfur as sulfuric acid. Dibenzothiophen (DBT) and ethylphenylsulfide (EPS) are serving as models of organic sulfur-containing components of coal in initial studies.

Enzymatic desulfurization of coal: Third quarterly report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marquis, Judith K.; Kitchell, Judith P.

Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of ''model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix. In this quarter we obtained important results both with the development of our understanding of the enzyme reaction systems and also with the microbial work at Woods Hole. 12 figs., 11 tabs.

Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger An-76 Induced by Food Processing Byproducts and Based on Integrated Functional Omics.

PubMed

Liu, Lin; Gong, Weili; Sun, Xiaomeng; Chen, Guanjun; Wang, Lushan

2018-02-07

Byproducts of food processing can be utilized for the production of high-value-added enzyme cocktails. In this study, we utilized integrated functional omics technology to analyze composition and functional characteristics of extracellular enzymes produced by Aspergillus niger grown on food processing byproducts. The results showed that oligosaccharides constituted by arabinose, xylose, and glucose in wheat bran were able to efficiently induce the production of extracellular enzymes of A. niger. Compared with other substrates, wheat bran was more effective at inducing the secretion of β-glucosidases from GH1 and GH3 families, as well as >50% of proteases from A1-family aspartic proteases. Compared with proteins induced by single wheat bran or soybean dregs, the protein yield induced by their mixture was doubled, and the time required to reach peak enzyme activity was shortened by 25%. This study provided a technical platform for the complex formulation of various substrates and functional analysis of extracellular enzymes.

Carbonic anhydrase enzymes regulate mast cell–mediated inflammation

PubMed Central

Soteropoulos, Patricia

2016-01-01

Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine–mediated inflammation through their enhanced ability to develop into mast cells. In this study, we show that carbonic anhydrase (Car) enzymes are up-regulated in type 2–associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent mouse mast cell responses and inflammation after Trichinella spiralis infection or the induction of food allergy–like disease. Further, we used CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively, these experiments identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be used to treat mast cell–mediated inflammation. PMID:27526715

SUSTAINABILITY PERSPECTIVE AND CHEMISTRY-BASED TECHNOLOGIES

EPA Science Inventory

Inefficient technologies create adverse and societal impacts while consuming material and energy resources. Yet technology enterprises are the strongest enabler of sustainability. Technologies that will address the complex concerns of these impacts and the consequences of the u...

(Hyper)thermophilic enzymes: production and purification.

PubMed

Falcicchio, Pierpaolo; Levisson, Mark; Kengen, Servé W M; Koutsopoulos, Sotirios

2014-01-01

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

Lignocellulosic ethanol: Technology design and its impact on process efficiency.

PubMed

Paulova, Leona; Patakova, Petra; Branska, Barbora; Rychtera, Mojmir; Melzoch, Karel

2015-11-01

This review provides current information on the production of ethanol from lignocellulosic biomass, with the main focus on relationships between process design and efficiency, expressed as ethanol concentration, yield and productivity. In spite of unquestionable advantages of lignocellulosic biomass as a feedstock for ethanol production (availability, price, non-competitiveness with food, waste material), many technological bottlenecks hinder its wide industrial application and competitiveness with 1st generation ethanol production. Among the main technological challenges are the recalcitrant structure of the material, and thus the need for extensive pretreatment (usually physico-chemical followed by enzymatic hydrolysis) to yield fermentable sugars, and a relatively low concentration of monosaccharides in the medium that hinder the achievement of ethanol concentrations comparable with those obtained using 1st generation feedstocks (e.g. corn or molasses). The presence of both pentose and hexose sugars in the fermentation broth, the price of cellulolytic enzymes, and the presence of toxic compounds that can inhibit cellulolytic enzymes and microbial producers of ethanol are major issues. In this review, different process configurations of the main technological steps (enzymatic hydrolysis, fermentation of hexose/and or pentose sugars) are discussed and their efficiencies are compared. The main features, benefits and drawbacks of simultaneous saccharification and fermentation (SSF), simultaneous saccharification and fermentation with delayed inoculation (dSSF), consolidated bioprocesses (CBP) combining production of cellulolytic enzymes, hydrolysis of biomass and fermentation into one step, together with an approach combining utilization of both pentose and hexose sugars are discussed and compared with separate hydrolysis and fermentation (SHF) processes. The impact of individual technological steps on final process efficiency is emphasized and the potential for use of immobilized biocatalysts is considered. Copyright © 2014 Elsevier Inc. All rights reserved.

Discovery of digestive enzymes in carnivorous plants with focus on proteases.

PubMed

Ravee, Rishiesvari; Mohd Salleh, Faris 'Imadi; Goh, Hoe-Han

2018-01-01

Carnivorous plants have been fascinating researchers with their unique characters and bioinspired applications. These include medicinal trait of some carnivorous plants with potentials for pharmaceutical industry. This review will cover recent progress based on current studies on digestive enzymes secreted by different genera of carnivorous plants: Drosera (sundews), Dionaea (Venus flytrap) , Nepenthes (tropical pitcher plants), Sarracenia (North American pitcher plants) , Cephalotus (Australian pitcher plants) , Genlisea (corkscrew plants) , and Utricularia (bladderworts). Since the discovery of secreted protease nepenthesin in Nepenthes pitcher, digestive enzymes from carnivorous plants have been the focus of many studies. Recent genomics approaches have accelerated digestive enzyme discovery. Furthermore, the advancement in recombinant technology and protein purification helped in the identification and characterisation of enzymes in carnivorous plants. These different aspects will be described and discussed in this review with focus on the role of secreted plant proteases and their potential industrial applications.

Restriction enzyme cutting site distribution regularity for DNA looping technology.

PubMed

Shang, Ying; Zhang, Nan; Zhu, Pengyu; Luo, Yunbo; Huang, Kunlun; Tian, Wenying; Xu, Wentao

2014-01-25

The restriction enzyme cutting site distribution regularity and looping conditions were studied systematically. We obtained the restriction enzyme cutting site distributions of 13 commonly used restriction enzymes in 5 model organism genomes through two novel self-compiled software programs. All of the average distances between two adjacent restriction sites fell sharply with increasing statistic intervals, and most fragments were 0-499 bp. A shorter DNA fragment resulted in a lower looping rate, which was also directly proportional to the DNA concentration. When the length was more than 500 bp, the concentration did not affect the looping rate. Therefore, the best known fragment length was longer than 500 bp, and did not contain the restriction enzyme cutting sites which would be used for digestion. In order to make the looping efficiencies reach nearly 100%, 4-5 single cohesive end systems were recommended to digest the genome separately. Copyright © 2013 Elsevier B.V. All rights reserved.

Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis.

PubMed

Xing, Mei-Ning; Zhang, Xue-Zhu; Huang, He

2012-01-01

Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks. Copyright © 2012 Elsevier Inc. All rights reserved.

A MIDGUT DIGESTIVE PHOSPHOLIPASE A2 IN LARVAL MOSQUITOES, AEDES ALBOPICTUS AND CULEX QUINQUEFASCIATUS

USDA-ARS?s Scientific Manuscript database

Phospholipase A2 (PLA2) is a secretory digestive enzyme that hydrolyzes ester bond at sn-2 position of dietary phospholipids, creating free fatty acid and lysophopholipid. The free fatty acids (arachidonic acid) are absorbed into midgut cells. Aedes albopictus and Culex quinquefasciatus digestive PL...

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells

USDA-ARS?s Scientific Manuscript database

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high ...

Some like it hot, some like it cold: Temperature dependent biotechnological applications and improvements in extremophilic enzymes.

PubMed

Siddiqui, Khawar Sohail

2015-12-01

The full biotechnological exploitation of enzymes is still hampered by their low activity, low stability and high cost. Temperature-dependent catalytic properties of enzymes are a key to efficient and cost-effective translation to commercial applications. Organisms adapted to temperature extremes are a rich source of enzymes with broad ranging thermal properties which, if isolated, characterized and their structure-function-stability relationship elucidated, could underpin a variety of technologies. Enzymes from thermally-adapted organisms such as psychrophiles (low-temperature) and thermophiles (high-temperature) are a vast natural resource that is already under scrutiny for their biotechnological potential. However, psychrophilic and thermophilic enzymes show an activity-stability trade-off that necessitates the use of various genetic and chemical modifications to further improve their properties to suit various industrial applications. This review describes in detail the properties and biotechnological applications of both cold-adapted and thermophilic enzymes. Furthermore, the review critically examines ways to improve their value for biotechnology, concluding by proposing an integrated approach involving thermally-adapted, genetically and magnetically modified enzymes to make biocatalysis more efficient and cost-effective. Copyright © 2015 Elsevier Inc. All rights reserved.

Media, Information Technology, and Language Planning: What Can Endangered Language Communities Learn from Created Language Communities?

ERIC Educational Resources Information Center

Schreyer, Christine

2011-01-01

The languages of Klingon and Na'vi, both created for media, are also languages that have garnered much media attention throughout the course of their existence. Speakers of these languages also utilize social media and information technologies, specifically websites, in order to learn the languages and then put them into practice. While teaching a…

Factors Predicting the Use of Technology: Findings From the Center for Research and Education on Aging and Technology Enhancement (CREATE)

PubMed Central

Czaja, Sara J.; Charness, Neil; Fisk, Arthur D.; Hertzog, Christopher; Nair, Sankaran N.; Rogers, Wendy A.; Sharit, Joseph

2006-01-01

The successful adoption of technology is becoming increasingly important to functional independence. The present article reports findings from the Center for Research and Education on Aging and Technology Enhancement (CREATE) on the use of technology among community-dwelling adults. The sample included 1,204 individuals ranging in age from 18–91 years. All participants completed a battery that included measures of demographic characteristics, self-rated health, experience with technology, attitudes toward computers, and component cognitive abilities. Findings indicate that the older adults were less likely than younger adults to use technology in general, computers, and the World Wide Web. The results also indicate that computer anxiety, fluid intelligence, and crystallized intelligence were important predictors of the use of technology. The relationship between age and adoption of technology was mediated by cognitive abilities, computer self-efficacy, and computer anxiety. These findings are discussed in terms of training strategies to promote technology adoption. PMID:16768579

Accelerating innovation in information and communication technology for health.

PubMed

Crean, Kevin W

2010-02-01

Around the world, inventors are creating novel information and communication technology applications and systems that can improve health for people in disparate settings. However, it is very difficult to find investment funding needed to create business models to expand and develop the prototype technologies. A comprehensive, long-term investment strategy for e-health and m-health is needed. The field of social entrepreneurship offers an integrated approach to develop needed investment models, so that innovations can reach more patients, more effectively. Specialized financing techniques and sustained support from investors can spur the expansion of mature technologies to larger markets, accelerating global health impacts.

Infographic Development by Accelerated Bachelor of Science in Nursing Students: An Innovative Technology-Based Approach to Public Health Education.

PubMed

Falk, Nancy L

Health communications and baccalaureate nursing education are increasingly impacted by new technological tools. This article describes how an Accelerated Bachelor of Science in Nursing program incorporates an infographic assignment into a graduate-level online health information and technology course. Students create colorful, engaging infographics using words and visuals to communicate public health information. The assignment, which incorporates the use of data and evidence, provides students the opportunity to acquire new research and technology skills while gaining confidence creating and innovating. The finished products may be disseminated, serving as vehicles to influence public health and well-being.

Incentives to create and sustain healthy behaviors: technology solutions and research needs.

PubMed

Teyhen, Deydre S; Aldag, Matt; Centola, Damon; Edinborough, Elton; Ghannadian, Jason D; Haught, Andrea; Jackson, Theresa; Kinn, Julie; Kunkler, Kevin J; Levine, Betty; Martindale, Valerie E; Neal, David; Snyder, Leslie B; Styn, Mindi A; Thorndike, Frances; Trabosh, Valerie; Parramore, David J

2014-12-01

Health-related technology, its relevance, and its availability are rapidly evolving. Technology offers great potential to minimize and/or mitigate barriers associated with achieving optimal health, performance, and readiness. In support of the U.S. Army Surgeon General's vision for a "System for Health" and its Performance Triad initiative, the U.S. Army Telemedicine and Advanced Technology Research Center hosted a workshop in April 2013 titled "Incentives to Create and Sustain Change for Health." Members of government and academia participated to identify and define the opportunities, gain clarity in leading practices and research gaps, and articulate the characteristics of future technology solutions to create and sustain real change in the health of individuals, the Army, and the nation. The key factors discussed included (1) public health messaging, (2) changing health habits and the environmental influence on health, (3) goal setting and tracking, (4) the role of incentives in behavior change intervention, and (5) the role of peer and social networks in change. This report summarizes the recommendations on how technology solutions could be employed to leverage evidence-based best practices and identifies gaps in research where further investigation is needed. Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.

Creating a pandemic of health: What is the role of digital technologies?

PubMed

Jadad, Alejandro R

2016-11-01

Imagine a world in which every human being is healthy until the last breath. Thanks to the fast penetration of digital technologies in every region of the planet, this seemingly utopian scenario is not only feasible but also potentially viable. Now that digital technologies have provided almost full interconnectivity among all humans, they should be used to meet key challenges to ensure that health is created and that it spreads to reach every person on earth. The objective of this article is to describe and trigger a serious discussion of such challenges, which include: adopting a new concept of health; positioning self-rated health as the main outcome of the system; creating a health-oriented model to guide service provision; facilitating the identification, scaling up, and sustaining of innovations that can create and spread health; promoting a culture of health promotion; and encouraging the emergence of Precision Health. Once these challenges are met, and health becomes pandemic, public health would have fulfilled its vision, a healthy life for all, at last.

PERFORMANCE VERIFICATION OF ANIMAL WATER TREATMENT TECHNOLOGIES THROUGH EPA'S ENVIRONMENTAL TECHNOLOGY VERIFICATION PROGRAM

EPA Science Inventory

The U.S. Environmental Protection Agency created the Environmental Technology Verification Program (ETV) to further environmental protection by accelerating the commercialization of new and innovative technology through independent performance verification and dissemination of in...

SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION PROGRAM TECHNOLOGY PROFILES: SIXTH EDITION

EPA Science Inventory

The Superfund Innovative Technology Evaluation (SITE) Program evaluates new and promising treatment and monitoring and measurement technologies for cleanup of hazardous waste sites. The program was created to encourage the development and routine use of innovative treatment techn...

SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION PROGRAM - TECHNOLOGY PROFILES - SEVENTH EDITION

EPA Science Inventory

The Superfund Innovative Technology Evaluation (SITE) Program evaluates new and promising treatment and monitoring and measurement technologies for cleanup of hazardous waste sites. The program was created to encourage the development and routine use of innovative treatment techn...

In vivo imaging of inducible tyrosinase gene expression with an ultrasound array-based photoacoustic system

NASA Astrophysics Data System (ADS)

Harrison, Tyler; Paproski, Robert J.; Zemp, Roger J.

2012-02-01

Tyrosinase, a key enzyme in the production of melanin, has shown promise as a reporter of genetic activity. While green fluorescent protein has been used extensively in this capacity, it is limited in its ability to provide information deep in tissue at a reasonable resolution. As melanin is a strong absorber of light, it is possible to image gene expression using tyrosinase with photoacoustic imaging technologies, resulting in excellent resolutions at multiple-centimeter depths. While our previous work has focused on creating and imaging MCF-7 cells with doxycycline-controlled tyrosinase expression, we have now established the viability of these cells in a murine model. Using an array-based photoacoustic imaging system with 5 MHz center frequency, we capture interleaved ultrasound and photoacoustic images of tyrosinase-expressing MCF-7 tumors both in a tissue mimicking phantom, and in vivo. Images of both the tyrosinase-expressing tumor and a control tumor are presented as both coregistered ultrasound-photoacoustic B-scan images and 3-dimensional photoacoustic volumes created by mechanically scanning the transducer. We find that the tyrosinase-expressing tumor is visible with a signal level 12dB greater than that of the control tumor in vivo. Phantom studies with excised tumors show that the tyrosinase-expressing tumor is visible at depths in excess of 2cm, and have suggested that our imaging system is sensitive to a transfection rate of less than 1%.

Optoelectronics technologies for Virtual Reality systems

NASA Astrophysics Data System (ADS)

Piszczek, Marek; Maciejewski, Marcin; Pomianek, Mateusz; Szustakowski, Mieczysław

2017-08-01

Solutions in the field of virtual reality are very strongly associated with optoelectronic technologies. This applies to both process design and operation of VR applications. Technologies such as 360 cameras and 3D scanners significantly improve the design work. What is more, HMD displays with high field of view or optoelectronic Motion Capture systems and 3D cameras guarantee an extraordinary experience in immersive VR applications. This article reviews selected technologies from the perspective of their use in a broadly defined process of creating and implementing solutions for virtual reality. There is also the ability to create, modify and adapt new approaches that show team own work (SteamVR tracker). Most of the introduced examples are effectively used by authors to create different VR applications. The use of optoelectronic technology in virtual reality is presented in terms of design and operation of the system as well as referring to specific applications. Designers and users of VR systems should take a close look on new optoelectronics solutions, as they can significantly contribute to increased work efficiency and offer completely new opportunities for virtual world reception.

Patterns Cancer Prevention Through Induction of Phase 2 Enzymes

DTIC Science & Technology

2003-04-01

2) enzymes. During our Phase I Award, we identified sulforaphane as the most potent inducer of carcinogen defenses in the prostate cell. We have...characterized global effects of sulforaphane in prostate cancer cell lines using cDNA microarray technology that allows large-scale determination of...changes in gene expression. These findings argue strongly for a preventive intervention trial involving with sulforaphane . During our Phase 2 Award, we used

Nanosizing a Metal-Organic Framework Enzyme Carrier for Accelerating Nerve Agent Hydrolysis

DTIC Science & Technology

2016-10-05

Previously, biodegradable liposome nano- carriers have been shown to be effective at providing functionally significant amounts of highly purified enzymes in...AlexaFluor-647 dye was purchased from Life Technologies (Thermo Fisher Scientific). Methyl 6-(pinacolboryl)-2-naphthoate was synthesized using a published...Hitachi) and PXRD (Smartlab, Rigaku). Labeling OPAA with Fluorescent Dye . AlexaFluor-647-labeled OPAA (OPAA647) was prepared by reacting OPAA (0.5

Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes.

PubMed

Lin, Sansan; Fischl, Anthony S; Bi, Xiahui; Parce, Wally

2003-03-01

Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A(2) on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.

A new strategy for genome assembly using short sequence reads and reduced representation libraries.

PubMed

Young, Andrew L; Abaan, Hatice Ozel; Zerbino, Daniel; Mullikin, James C; Birney, Ewan; Margulies, Elliott H

2010-02-01

We have developed a novel approach for using massively parallel short-read sequencing to generate fast and inexpensive de novo genomic assemblies comparable to those generated by capillary-based methods. The ultrashort (<100 base) sequences generated by this technology pose specific biological and computational challenges for de novo assembly of large genomes. To account for this, we devised a method for experimentally partitioning the genome using reduced representation (RR) libraries prior to assembly. We use two restriction enzymes independently to create a series of overlapping fragment libraries, each containing a tractable subset of the genome. Together, these libraries allow us to reassemble the entire genome without the need of a reference sequence. As proof of concept, we applied this approach to sequence and assembled the majority of the 125-Mb Drosophila melanogaster genome. We subsequently demonstrate the accuracy of our assembly method with meaningful comparisons against the current available D. melanogaster reference genome (dm3). The ease of assembly and accuracy for comparative genomics suggest that our approach will scale to future mammalian genome-sequencing efforts, saving both time and money without sacrificing quality.

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

PubMed

Limsakul, Praopim; Peng, Qin; Wu, Yiqian; Allen, Molly E; Liang, Jing; Remacle, Albert G; Lopez, Tyler; Ge, Xin; Kay, Brian K; Zhao, Huimin; Strongin, Alex Y; Yang, Xiang-Lei; Lu, Shaoying; Wang, Yingxiao

2018-04-19

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

PubMed

Hill, Ryan C; Oman, Trent J; Shan, Guomin; Schafer, Barry; Eble, Julie; Chen, Cynthia

2015-08-26

Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

Targeted enzyme prodrug therapies.

PubMed

Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

2010-09-01

The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

JPRS Report, Science & Technology Europe

DTIC Science & Technology

1988-10-13

material, for instance, the single-crystal matrix of a superalloy is reinforced by homogeneously distributed, very fine granules of ceramic...Products/research on new cheese development and cheese flavour problems Enzymes, flavourings , and natural colours for food and drink, pharmaceutical...and diag- nostic industries Dairy products/cheese production and flavour technology Dairy products, including alcohol produced from whey

JPRS Report, Science & Technology, Europe & Latin America

DTIC Science & Technology

1988-04-06

courses and in polytechnics a growing number of undergraduate research theses [ tesi di laurea] are increasingly coming to resemble authentic feasibility...Information Science Eleven Priorities Research Priority Actions — Microbiological engineering —Enzyme engineering —Biotechnological engineering —Food...Foodstuffs Medicine Human and social sciences Technology, computer-integrated manufacturing Electronics, data processing Microbiological

The "Food Polymer Science" approach to the practice of industrial R&D, leading to patent estates based on fundamental starch science and technology.

PubMed

Slade, Louise; Levine, Harry

2018-04-13

This article reviews the application of the "Food Polymer Science" approach to the practice of industrial R&D, leading to patent estates based on fundamental starch science and technology. The areas of patents and patented technologies reviewed here include: (a) soft-from-the-freezer ice creams and freezer-storage-stable frozen bread dough products, based on "cryostabilization technology" of frozen foods, utilizing commercial starch hydrolysis products (SHPs); (b) glassy-matrix encapsulation technology for flavors and other volatiles, based on structure-function relationships for commercial SHPs; (c) production of stabilized whole-grain wheat flours for biscuit products, based on the application of "solvent retention capacity" technology to develop flours with reduced damaged starch; (d) production of improved-quality, low-moisture cookies and crackers, based on pentosanase enzyme technology; (e) production of "baked-not-fried," chip-like, starch-based snack products, based on the use of commercial modified-starch ingredients with selected functionality; (f) accelerated staling of a starch-based food product from baked bread crumb, based on the kinetics of starch retrogradation, treated as a crystallization process for a partially crystalline glassy polymer system; and (g) a process for producing an enzyme-resistant starch, for use as a reduced-calorie flour replacer in a wide range of grain-based food products, including cookies, extruded expanded snacks, and breakfast cereals.

The Technological Dimensions of International Competitiveness.

ERIC Educational Resources Information Center

National Academy of Engineering, Washington, DC. Committee on Technology Issues That Impact International Competitiveness.

This report focuses primarily on the technological dimensions of competitiveness in the United States with its capability to: create and effectively use technology; increase the value added to goods and services through the application of technology; manage technology in industry for more effective relationships among companies, universities, and…

Research challange

DOE Office of Scientific and Technical Information (OSTI.GOV)

Staebler, G.R.

The technological forecast can be used as a research tool by describing the optimum forest from every plausible viewpoint and then charting a course to create it. We must make usable the things we already know, and define, plan, and execute a research attack on those we don't. The three main areas we must consider are full use of the land, maximization of production, and research management and administration. In all these considerations we must keep in mind that technology creates the need for more technology, and that it is biologically necessary to preserve the long look.

Technology-driven dietary assessment: a software developer’s perspective

PubMed Central

Buday, Richard; Tapia, Ramsey; Maze, Gary R.

2015-01-01

Dietary researchers need new software to improve nutrition data collection and analysis, but creating information technology is difficult. Software development projects may be unsuccessful due to inadequate understanding of needs, management problems, technology barriers or legal hurdles. Cost overruns and schedule delays are common. Barriers facing scientific researchers developing software include workflow, cost, schedule, and team issues. Different methods of software development and the role that intellectual property rights play are discussed. A dietary researcher must carefully consider multiple issues to maximize the likelihood of success when creating new software. PMID:22591224

News: Good chemical manufacturing process criteria

EPA Science Inventory

This news column covers topics relating to manufacturing criteria, machine to machine technology, novel process windows, green chemistry indices, business resilience, immobilized enzymes, and Bt crops.

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, TNT DETECTION TECHNOLOGY, TEXAS INSTRUMENTS, SPREETA TM SENSOR

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has created the Environmental Technology Verification Program (ETV) to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the ...

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, GROUNDWATER SAMPLING TECHNOLOGIES, CLEAN ENVIRONMENT EQUIPMENT, SAMPLEASE BLADDER PUMP

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has created the Environmental Technology Verification Program (ETV) to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the...

The SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION program - Technology Profiles

EPA Science Inventory

The Superfund Innovative Technology Evaluation (SITE) program was created to evaluate new and promising treatment technologies for cleanup at hazardous waste sites. The mission of the SITE program is to encourage the development and routine use of innovative treatment technologie...

Environmental Technology Verification Report - NATCO Group, Inc. – Paques THIOPAQ Gas Purification Technology

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has created the Environmental Technology Verification (ETV) program to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the ...

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, GROUNDWATER SAMPLING TECHNOLOGIES, BURGE ENVIRONMENTAL INC. MULTIPROBE 100

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has created the Environmental Technology Verification Program (ETV) to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the...

A Unified Information System for Appropriate Technology.

ERIC Educational Resources Information Center

Unamboowe, Ira

1980-01-01

Considers problems and solutions for transfer of technological information for developing nations. Imbalances created by industrial growth have brought the concept of choice of technologies to the forefront of national objectives. (RAA)

What Is Educational Technology? An Inquiry into the Meaning, Use, and Reciprocity of Technology

ERIC Educational Resources Information Center

Lakhana, Arun

2014-01-01

This position paper explores the ambiguity of technology, toward refined understanding of Educational Technology. The purpose of education is described by John Dewey as growing, or habitual learning. Two philosophical conceptions of technology are reviewed. Dewey positions inquiry as a technology that creates knowledge. Val Dusek offers a…

Developing Indicators for a Classroom Observation Tool on Pedagogy and Technology Integration: A Delphi Study

ERIC Educational Resources Information Center

Elmendorf, Douglas C.; Song, Liyan

2015-01-01

Rapid advances in technology and increased access to technology tools have created new instructional demands and expectations on teachers. Due to the ubiquitous presence of technology in K-12 schools, teachers are being observed on both their pedagogical and technology integration practices. Applying the technological pedagogical and content…

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

PubMed

Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

2015-12-07

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

Unleashing the Future: Educators "Speak Up" about the Use of Emerging Technologies for Learning. Speak Up 2009 National Findings: Teachers, Aspiring Teachers & Administrators

ERIC Educational Resources Information Center

Project Tomorrow, 2010

2010-01-01

Technology has enabled students to have greater access to vast array of resources, classes and experts; empowering students to become "Free Agent Learners" who are creating meaningful personalized learning experiences 24/7 outside of the traditional classroom and school structure. In the report "Creating our Future: Students Speak Up about their…

A Multi-Case Study of Research Using Mobile Imaging, Sensing and Tracking Technologies to Objectively Measure Behavior: Ethical Issues and Insights to Guide Responsible Research Practice

ERIC Educational Resources Information Center

Nebeker, Camille; Linares-Orozco, Rubi; Crist, Katie

2015-01-01

Introduction: The increased availability of mobile sensing technologies is creating a paradigm shift for health research by creating new opportunities for measuring and monitoring behavior. For example, researchers can now collect objective information about a participant's daily activity using wearable devices that have: 1- Global Positioning…

Sol-gel encapsulation of pullulanase in the presence of hybrid magnetic (Fe3O4-chitosan) nanoparticles improves thermal and operational stability.

PubMed

Long, Jie; Li, Xingfei; Zhan, Xiaobei; Xu, Xueming; Tian, Yaoqi; Xie, Zhengjun; Jin, Zhengyu

2017-06-01

Pullulanase was sol-gel encapsulated in the presence of magnetic chitosan/Fe 3 O 4 nanoparticles. The resulting immobilized pullulanase was characterized by scanning electron microscopy, vibrating sample magnetometry, Fourier transform infrared spectroscopy and thermogravimetric analysis. The results showed that the addition of pullulanase created a more regular surface on the sol-gel matrix and an enhanced magnetic response to an applied magnetic field. The maximal activity retention (83.9%) and specific activity (291.7 U/mg) of the immobilized pullulanase were observed under optimized conditions including an octyltriethoxysilane:tetraethoxysilane (OTES:TEOS) ratio of 1:2 and enzyme concentration of 0.484 mg/mL sol. The immobilized enzyme exhibited good thermal stability. When the temperature was above 60 °C, the immobilized pullulanase showed significantly higher activity than the free enzyme (p < 0.01); enzyme immobilized by simple sol-gel encapsulation and co-immobilized by crosslinking-encapsulation retained 52 and 69% of their initial activity after 5 h at 62 °C, respectively, compared to 11% for the free enzyme. Moreover, the stability of the pullulanase was improved by crosslinking-encapsulation, as the enzyme retained more than 85 and 81% of its original activity after 5 and 6 consecutive reuses, respectively, compared to 80 and 72% of its original activity for simple sol-gel encapsulated enzymes. This indicated the leakage of enzyme molecules through the pores of the gel was substantially abated by cross-linking. Such immobilized pullulanase provides high stability and ease of enzyme recovery, characteristics that are advantageous for applications in the food industry that involve continuous starch processing.

Creating Digital Elevation Model Using a Mobile Device

NASA Astrophysics Data System (ADS)

Durmaz, A. İ.

2017-11-01

DEM (Digital Elevation Models) is the best way to interpret topography on the ground. In recent years, lidar technology allows to create more accurate elevation models. However, the problem is this technology is not common all over the world. Also if Lidar data are not provided by government agencies freely, people have to pay lots of money to reach these point clouds. In this article, we will discuss how we can create digital elevation model from less accurate mobile devices' GPS data. Moreover, we will evaluate these data on the same mobile device which we collected data to reduce cost of this modeling.

Marketing NASA Langley Polymeric Materials

NASA Technical Reports Server (NTRS)

Flynn, Diane M.

1995-01-01

A marketing tool was created to expand the knowledge of LaRC developed polymeric materials, in order to facilitate the technology transfer process and increase technology commercialization awareness among a non-technical audience. The created brochure features four materials, LaRC-CP, LaRC-RP46, LaRC-SI, and LaRC-IA, and highlights their competitive strengths in potential commercial applications. Excellent opportunities exist in the $40 million per year microelectronics market and the $6 billion adhesives market. It is hoped that the created brochure will generate inquiries regarding the use of the above materials in markets such as these.

Techno-Human Mesh: The Growing Power of Information Technologies.

ERIC Educational Resources Information Center

West, Cynthia K.

This book examines the intersection of information technologies, power, people, and bodies. It explores how information technologies are on a path of creating efficiency, productivity, profitability, surveillance, and control, and looks at the ways in which human-machine interface technologies, such as wearable computers, biometric technologies,…

Converging technologies in higher education: paradigm for the "new" liberal arts?

PubMed

Balmer, Robert T

2006-12-01

This article discusses the historic relationship between the practical arts (technology) and the mental (liberal) arts, suggesting that Converging Technologies is a new higher education paradigm that integrates the arts, humanities, and sciences with modern technology. It explains that the paradigm really includes all fields in higher education from philosophy to art to music to modern languages and beyond. To implement a transformation of this magnitude, it is necessary to understand the psychology of change in academia. Union College in Schenectady, New York, implemented a Converging Technologies Educational Paradigm in five steps: (1) create a compelling vision, (2) communicate the vision, (3) empower the faculty, (4) create short-term successes, and (5) institutionalize the results. This case study of Union College demonstrates it is possible to build a pillar of educational excellence based on Converging Technologies.

Multi-criteria development and incorporation into decision tools for health technology adoption.

PubMed

Poulin, Paule; Austen, Lea; Scott, Catherine M; Waddell, Cameron D; Dixon, Elijah; Poulin, Michelle; Lafrenière, René

2013-01-01

When introducing new health technologies, decision makers must integrate research evidence with local operational management information to guide decisions about whether and under what conditions the technology will be used. Multi-criteria decision analysis can support the adoption or prioritization of health interventions by using criteria to explicitly articulate the health organization's needs, limitations, and values in addition to evaluating evidence for safety and effectiveness. This paper seeks to describe the development of a framework to create agreed-upon criteria and decision tools to enhance a pre-existing local health technology assessment (HTA) decision support program. The authors compiled a list of published criteria from the literature, consulted with experts to refine the criteria list, and used a modified Delphi process with a group of key stakeholders to review, modify, and validate each criterion. In a workshop setting, the criteria were used to create decision tools. A set of user-validated criteria for new health technology evaluation and adoption was developed and integrated into the local HTA decision support program. Technology evaluation and decision guideline tools were created using these criteria to ensure that the decision process is systematic, consistent, and transparent. This framework can be used by others to develop decision-making criteria and tools to enhance similar technology adoption programs. The development of clear, user-validated criteria for evaluating new technologies adds a critical element to improve decision-making on technology adoption, and the decision tools ensure consistency, transparency, and real-world relevance.

Application of radiation technology in biomedical materials; Fundamentals and applied

NASA Astrophysics Data System (ADS)

Hayashi, K.

1) IMMOBILIZATION OF HEMOGLOBIN Hemoglobin has been immobilized into Poly HEMA Matrix. To increase, Mechanical resistance, at first, CO was coodinated, after immobilization CO was eliminated by photo illumination by visible light from a W lamp and then O 2 was introduced. Oxygencoordiation ability was not damaged by immobilization. 2) REDUCTION MECHANISM OF ENZYME BY THE USE OF PULSE RADIOLYSIS Elementary process of Reduction Mechanism of Myoglobin, Hemoglobin, HRP and Cytochrome Oxidase were investigated in the time range of μsec≈nsec. In the of Cytochrome Oxidase, these are 4 metal ions inside of the Enzyme. The exact step of reduction of this enzyme was elucidated .

Case Study of a Small Scale Polytechnic Entrepreneurship Capstone Course Sequence

ERIC Educational Resources Information Center

Webster, Rustin D.; Kopp, Richard

2017-01-01

A multidisciplinary entrepreneurial senior capstone has been created for engineering technology students at a research I land-grant university statewide extension. The two semester course sequence welcomes students from Mechanical Engineering Technology, Electrical Engineering Technology, Computer Graphics Technology, and Organizational…

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT: BAGHOUSE FILTRATION PRODUCTS--DONALDSON COMPANY, INC. 6277 FILTRATION MEDIA

EPA Science Inventory

The U.S. Environmental Protection Agency has created the Environmental Technology Verification Program to facilitate the deployment of innovative or improved environmental technologies through high quality, peer reviewed data on technology performance to those involved in the des...

Environmental Technology Verification Report -- Baghouse filtration products, GE Energy QG061 filtration media ( tested May 2007)

EPA Science Inventory

EPA has created the Environmental Technology Verification Program to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The Air Pollution Control Technology Verification Center, a cente...

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, GROUNDWATER SAMPLING TECHNOLOGIES, GEOLOG, INC., MICRO-FLO BLADDER PUMP MODEL 57400

EPA Science Inventory

The U.S. Environmental Protection Agency has created the Environmental Technology Verification Program to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the ETV Program...

Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection.

PubMed

Marden, James H

2013-12-01

Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes. © 2013 John Wiley & Sons Ltd.

Self-assembly of amphiphilic janus particles into monolayer capsules for enhanced enzyme catalysis in organic media.

PubMed

Cao, Wei; Huang, Renliang; Qi, Wei; Su, Rongxin; He, Zhimin

2015-01-14

Encapsulation of enzymes during the creation of an emulsion is a simple and efficient route for enhancing enzyme catalysis in organic media. Herein, we report a capsule with a shell comprising a monolayer of silica Janus particles (JPs) (referred to as a monolayer capsule) and a Pickering emulsion for the encapsulation of enzyme molecules for catalysis purposes in organic media using amphiphilic silica JPs as building blocks. We demonstrate that the JP capsules had a monolayer shell consisting of closely packed silica JPs (270 nm). The capsules were on average 5-50 μm in diameter. The stability of the JP capsules (Pickering emulsion) was investigated with the use of homogeneous silica nanoparticles as a control. The results show that the emulsion stabilized via amphiphilic silica JPs presented no obvious changes in physical appearance after 15 days, indicating the high stability of the emulsions and JP capsules. Furthermore, the lipase from Candida sp. was chosen as a model enzyme for encapsulation within the JP capsules during their formation. The catalytic performance of lipase was evaluated according to the esterification of 1-hexanol with hexanoic acid. It was found that the specific activity of the encapsulated enzymes (28.7 U mL(-1)) was more than 5.6 times higher than that of free enzymes in a biphasic system (5.1 U mL(-1)). The enzyme activity was further increased by varying the volume ratio of water to oil and the JPs loadings. The enzyme-loaded capsule also exhibited high stability during the reaction process and good recyclability. In particular, the jellification of agarose in the JP capsules further enhanced their operating stability. We believe that the monolayer structure of the JP capsules, together with their high stability, rendered the capsules to be ideal enzyme carriers and microreactors for enzyme catalysis in organic media because they created a large interfacial area and had low mass transfer resistance through the monolayer shell.

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning.

PubMed

Wan, Haisu; Li, Yongwen; Fan, Yu; Meng, Fanrong; Chen, Chen; Zhou, Qinghua

2012-01-15

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function. Copyright © 2011 Elsevier Inc. All rights reserved.

Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site.

PubMed

Wang, Lu; Fried, Stephen D; Boxer, Steven G; Markland, Thomas E

2014-12-30

Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds.

Effect of ionic liquid on activity, stability, and structure of enzymes: a review.

PubMed

Naushad, Mu; Alothman, Zied Abdullah; Khan, Abbul Bashar; Ali, Maroof

2012-11-01

Ionic liquids have shown their potential as a solvent media for many enzymatic reactions as well as protein preservation, because of their unusual characteristics. It is also observed that change in cation or anion alters the physiochemical properties of the ionic liquids, which in turn influence the enzymatic reactions by altering the structure, activity, enatioselectivity, and stability of the enzymes. Thus, it is utmost need of the researchers to have full understanding of these influences created by ionic liquids before choosing or developing an ionic liquid to serve as solvent media for enzymatic reaction or protein preservation. So, in the present review, we try to shed light on effects of ionic liquids chemistry on structure, stability, and activity of enzymes, which will be helpful for the researchers in various biocatalytic applications. Copyright © 2012. Published by Elsevier B.V.

Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

PubMed Central

Wang, Lu; Fried, Stephen D.; Boxer, Steven G.; Markland, Thomas E.

2014-01-01

Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds. PMID:25503367

Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type.

PubMed

Chabbert, Brigitte; Habrant, Anouck; Herbaut, Mickaël; Foulon, Laurence; Aguié-Béghin, Véronique; Garajova, Sona; Grisel, Sacha; Bennati-Granier, Chloé; Gimbert-Herpoël, Isabelle; Jamme, Frédéric; Réfrégiers, Matthieu; Sandt, Christophe; Berrin, Jean-Guy; Paës, Gabriel

2017-12-19

Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery.

Evolution of a designed retro-aldolase leads to complete active site remodeling

PubMed Central

Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

2013-01-01

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

Biocatalysts for the pharmaceutical industry created by structure-guided directed evolution of stereoselective enzymes.

PubMed

Li, Guangyue; Wang, Jian-Bo; Reetz, Manfred T

2018-04-01

Enzymes have been used for a long time as catalysts in the asymmetric synthesis of chiral intermediates needed in the production of therapeutic drugs. However, this alternative to man-made catalysts has suffered traditionally from distinct limitations, namely the often observed wrong or insufficient enantio- and/or regioselectivity, low activity, narrow substrate range, and insufficient thermostability. With the advent of directed evolution, these problems can be generally solved. The challenge is to develop and apply the most efficient mutagenesis methods which lead to highest-quality mutant libraries requiring minimal screening. Structure-guided saturation mutagenesis and its iterative form have emerged as the method of choice for evolving stereo- and regioselective mutant enzymes needed in the asymmetric synthesis of chiral intermediates. The number of (industrial) applications in the preparation of chiral pharmaceuticals is rapidly increasing. This review features and analyzes typical case studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Arbuscular Mycorrhizal Fungi Can Benefit Heavy Metal Tolerance and Phytoremediation

ERIC Educational Resources Information Center

Forgy, David

2012-01-01

Sites contaminated by heavy metals, such as industrial waste sites, create unwelcoming environments for plant growth. Heavy metals can have a wide range of toxic effects such as replacing essential elements or disrupting enzyme function. While some heavy metals are essential to plant nutrition at low concentrations, high concentrations of any…

A thermophilic phage endolysin fusion to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

Fusion of a thermophilic phage endolysin to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

Microbial Production of Xylitol from L-arabinose by Metabolically Engineered Escherichia coli

USDA-ARS?s Scientific Manuscript database

An Escherichia coli strain, ZUC99(pATX210), which can produce xylitol from L-arabinose at a high yield has been created by introducing a new bioconversion pathway into cells. This pathway consists of three enzymes: L-arabinose isomerase, which converts L-arabinose to L-ribulose; D-psicose 3-epimer...

Dihydroquinazolines as a novel class of Trypanosoma brucei trypanothione reductase inhibitors: discovery, synthesis, and characterization of their binding mode by protein crystallography.

PubMed

Patterson, Stephen; Alphey, Magnus S; Jones, Deuan C; Shanks, Emma J; Street, Ian P; Frearson, Julie A; Wyatt, Paul G; Gilbert, Ian H; Fairlamb, Alan H

2011-10-13

Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei , the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR-ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.

C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products.

PubMed

Yokoyama, Kenichi; Lilla, Edward A

2018-04-10

Covering: up to the end of 2017C-C bond formations are frequently the key steps in cofactor and natural product biosynthesis. Historically, C-C bond formations were thought to proceed by two electron mechanisms, represented by Claisen condensation in fatty acids and polyketide biosynthesis. These types of mechanisms require activated substrates to create a nucleophile and an electrophile. More recently, increasing number of C-C bond formations catalyzed by radical SAM enzymes are being identified. These free radical mediated reactions can proceed between almost any sp3 and sp2 carbon centers, allowing introduction of C-C bonds at unconventional positions in metabolites. Therefore, free radical mediated C-C bond formations are frequently found in the construction of structurally unique and complex metabolites. This review discusses our current understanding of the functions and mechanisms of C-C bond forming radical SAM enzymes and highlights their important roles in the biosynthesis of structurally complex, naturally occurring organic molecules. Mechanistic consideration of C-C bond formation by radical SAM enzymes identifies the significance of three key mechanistic factors: radical initiation, acceptor substrate activation and radical quenching. Understanding the functions and mechanisms of these characteristic enzymes will be important not only in promoting our understanding of radical SAM enzymes, but also for understanding natural product and cofactor biosynthesis.

Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution

PubMed Central

Renata, Hans; Wang, Z. Jane

2015-01-01

High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe. PMID:25649694

Disconnect of microbial structure and function: enzyme activities and bacterial communities in nascent stream corridors.

PubMed

Frossard, Aline; Gerull, Linda; Mutz, Michael; Gessner, Mark O

2012-03-01

A fundamental issue in microbial and general ecology is the question to what extent environmental conditions dictate the structure of communities and the linkages with functional properties of ecosystems (that is, ecosystem function). We approached this question by taking advantage of environmental gradients established in soil and sediments of small stream corridors in a recently created, early successional catchment. Specifically, we determined spatial and temporal patterns of bacterial community structure and their linkages with potential microbial enzyme activities along the hydrological flow paths of the catchment. Soil and sediments were sampled in a total of 15 sites on four occasions spread throughout a year. Denaturing gradient gel electrophoresis (DGGE) was used to characterize bacterial communities, and substrate analogs linked to fluorescent molecules served to track 10 different enzymes as specific measures of ecosystem function. Potential enzyme activities varied little among sites, despite contrasting environmental conditions, especially in terms of water availability. Temporal changes, in contrast, were pronounced and remarkably variable among the enzymes tested. This suggests much greater importance of temporal dynamics than spatial heterogeneity in affecting specific ecosystem functions. Most strikingly, bacterial community structure revealed neither temporal nor spatial patterns. The resulting disconnect between bacterial community structure and potential enzyme activities indicates high functional redundancy within microbial communities even in the physically and biologically simplified stream corridors of early successional landscapes.

Technological Literacy for All: A Course Designed to Raise the Technological Literacy of College Students

ERIC Educational Resources Information Center

Baskette, Kimberly G.; Fantz, Todd D.

2013-01-01

Understanding what technology is, and is not, is the first step in becoming technologically literate. One should also understand how technology is created, how it works, how it shapes society, and how society shapes technology. This study was designed to gauge the ability of a single-semester course to raise students' technological literacy as…

Commercializing Defense Technologies and Helping Defense Firms Succeed in Commercial Markets: A Report on the Objectives, Activities, and Accomplishments of the TAP-IN Program

NASA Technical Reports Server (NTRS)

1997-01-01

Technology Access for Product Innovation (TAP-IN), the largest technology deployment project funded by TRP, was competitively selected through a national solicitation for proposals. TAP-IN was created to help companies access and apply defense technologies and help defense-dependent companies enter new commercial markets. Defense technologies included technologies developed by DoD, DOE, NASA, and their contractors. TAP-IN was structured to provide region-based technology access services that were able to draw on technology resources nationwide. TAP-IN provided expert assistance in all stages of the commercialization process from concept through prototype design to capital sourcing and marketing strategy. TAP-IN helped companies locate new technology, identify business partners, secure financing, develop ideas for new products, identify new markets, license technology, solve technical problems, and develop company-specific applications of federal technology. TAP-IN leveraged NASA's existing commercial technology network to create an integrated national network of organizations that assisted companies in every state. In addition to NASA's six regional technology transfer centers (RTTCs), TAP-IN included business and technology development organizations in every state, the Industrial Designers Society of America, and the Federal Laboratory Consortium (FLC).

Nanosized zeolites as a perspective material for conductometric biosensors creation

NASA Astrophysics Data System (ADS)

Kucherenko, Ivan; Soldatkin, Oleksandr; Kasap, Berna Ozansoy; Kirdeciler, Salih Kaan; Kurc, Burcu Akata; Jaffrezic-Renault, Nicole; Soldatkin, Alexei; Lagarde, Florence; Dzyadevych, Sergei

2015-05-01

In this work, the method of enzyme adsorption on different zeolites and mesoporous silica spheres (MSS) was investigated for the creation of conductometric biosensors. The conductometric transducers consisted of gold interdigitated electrodes were placed on the ceramic support. The transducers were modified with zeolites and MSS, and then the enzymes were adsorbed on the transducer surface. Different methods of zeolite attachment to the transducer surface were used; drop coating with heating to 200°C turned out to be the best one. Nanozeolites beta and L, zeolite L, MSS, and silicalite-1 (80 to 450 nm) were tested as the adsorbents for enzyme urease. The biosensors with all tested particles except zeolite L had good analytical characteristics. Silicalite-1 (450 nm) was also used for adsorption of glucose oxidase, acetylcholinesterase, and butyrylcholinesterase. The glucose and acetylcholine biosensors were successfully created, whereas butyrylcholinesterase was not adsorbed on silicalite-1. The enzyme adsorption on zeolites and MSS is simple, quick, well reproducible, does not require use of toxic compounds, and therefore can be recommended for the development of biosensors when these advantages are especially important.

Mesoporous CLEAs-silica composite microparticles with high activity and enhanced stability

PubMed Central

Cui, Jiandong; Jia, Shiru; Liang, Longhao; Zhao, Yamin; Feng, Yuxiao

2015-01-01

A novel enzyme immobilization approach was used to generate mesoporous enzymes-silica composite microparticles by co-entrapping gelatinized starch and cross-linked phenylalanine ammonia lyase (PAL) aggregates (CLEAs) containing gelatinized starch into biomemitic silica and subsequently removing the starch by α-amylase treatment. During the preparation process, the gelatinzed starch served as a pore-forming agent to create pores in CLEAs and biomimetic silica. The resulting mesoporous CLEAs-silica composite microparticles exhibited higher activity and stability than native PAL, conventional CLEAs, and PAL encapsulated in biomimetic silica. Furthermore, the mesoporous CLEAs-silica composite microparticles displayed good reusability due to its suitable size and mechanical properties, and had excellent stability for storage. The superior catalytic performances were attributed to the combinational unique structure from the intra-cross-linking among enzyme aggregates and hard mesoporous silica shell, which not only decreased the enzyme-support negative interaction and mass-transfer limitations, but also improved the mechanical properties and monodispersity. This approach will be highly beneficial for preparing various bioactive mesoporous composites with excellent catalytic performance. PMID:26374188

Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone.

PubMed

Rich, Joseph O; Budde, Cheryl L; McConeghey, Luke D; Cotterill, Ian C; Mozhaev, Vadim V; Singh, Sheo B; Goetz, Michael A; Zhao, Annie; Michels, Peter C; Khmelnitsky, Yuri L

2009-06-01

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.

Puncturing the pipeline: Do technology companies alienate women in recruiting sessions?

PubMed

Wynn, Alison T; Correll, Shelley J

2018-02-01

A 'chilly' environment limits women's advancement through the educational pipeline leading to jobs in science and technology. However, we know relatively little about the environment women encounter after making it through the educational pipeline. Do technology companies create environments that may dampen women's interest at the juncture when they are launching their careers? Using original observational data from 84 recruiting sessions hosted by technology companies at a prominent university on the US West Coast, we find that company representatives often engage in behaviors that are known to create a chilly environment for women. Through gender-imbalanced presenter roles, geek culture references, overt use of gender stereotypes, and other gendered speech and actions, representatives may puncture the pipeline, lessening the interest of women at the point of recruitment into technology careers.

The Impact of a Technology Coordinator's Belief System upon Using Technology to Create a Community's History

ERIC Educational Resources Information Center

Waring, Scott M.

2010-01-01

As it has been shown that teachers of social studies content are less likely than teachers of other content areas to utilize technology in their classroom, this study focuses on one instructional technology coordinators' beliefs towards technology, instruction, and students and how these beliefs impacted how technology was utilized during a…

ECUT (Energy Conversion and Utilization Technologies Program). Biocatalysis Project

NASA Technical Reports Server (NTRS)

1986-01-01

Presented are the FY 1985 accomplishments, activities, and planned research efforts of the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Program. The Project's technical activities were organized as follows: In the Molecular Modeling and Applied Genetics work element, research focused on (1) modeling and simulation studies to establish the physiological basis of high temperature tolerance in a selected enzyme and the catalytic mechanisms of three species of another enzyme, and (2) determining the degree of plasmid amplification and stability of several DNA bacterial strains. In the Bioprocess Engineering work element, research focused on (1) studies of plasmid propagation and the generation of models, (2) developing methods for preparing immobilized biocatalyst beads, and (3) developing an enzyme encapsulation method. In the Process Design and Analysis work element, research focused on (1) further refinement of a test case simulation of the economics and energy efficiency of alternative biocatalyzed production processes, (2) developing a candidate bioprocess to determine the potential for reduced energy consumption and facility/operating costs, and (3) a techno-economic assessment of potential advancements in microbial ammonia production.

Recent advances in exploiting ionic liquids for biomolecules: Solubility, stability and applications.

PubMed

Sivapragasam, Magaret; Moniruzzaman, Muhammad; Goto, Masahiro

2016-08-01

The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) - potentially attractive "green" and "designer" solvents - rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule-based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein-, enzyme-, and DNA-based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule-friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL-based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule-based applications. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT - GROUNDWATER SAMPLING TECHNOLOGIES - QED ENVIRONMENTAL SYSTEMS INC. WELL WIZARD DEDICATED SAMPLING SYSTEM

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has created the Environmental Technology Verification Program (ETV) to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the ...

Sandia National Labs: Manufacturing Science and Technology

Science.gov Websites

Additional Resources R&D Projects Current Partnerships Creating Partnerships Welcome to the Manufacturing Science and Technology home page Manufacturing Science and Technology Showcase The Manufacturing Science & Technology Center develops and applies advanced manufacturing processes for realization of

Innovative eLearning: Technology Shaping Contemporary Problem Based Learning: A Cross-Case Analysis

ERIC Educational Resources Information Center

Blackburn, Greg

2015-01-01

Preparing students to be critical thinkers and effective communicators is essential in today's multinational and technologically sophisticated environment. New electronic technologies provide opportunities for creating learning environments that extend the possibilities of "old" but still essential technologies: books, blackboards, and…

Environmental Technology Verification: Baghouse Filtration Products--Sinoma Science & Technology Co. Ltd FT-806 Filtration Media

EPA Science Inventory

EPA created the Environmental Technology Verification (ETV) Program to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. It seeks to achieve this goal by providing high-quality, peer r...

NewsWire, 2000-2001.

ERIC Educational Resources Information Center

Richey, Nancy, Ed.; Byrom, Elizabeth, Ed.; Bingham, Margaret, Ed.; Guerrero, Jeanne, Ed.; Thrift, Beth, Ed.; Holton, Brook, Ed.

2001-01-01

This document contains five issues of "NewsWire," a newsletter created for the SouthEast and Islands Regional Technology in Education Consortium (SEIR-TEC). Topics addressed in these issues include: leadership and educational technology; technology program development; resources for teaching and learning with technology; U.S. Department of…

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, GROUNDWATER SAMPLING TECHNOLOGIES, GEOPROBE INC., PNEUMATIC BLADDER PUMP GW 1400 SERIES

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) design efficient processes for conducting has created the Environmental Technology perfofl1lance tests of innovative technologies. Verification Program (E TV) to facilitate the deployment of innovative or improved environmental techn...

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, GROUNDWATER SAMPLING TECHNOLOGIES, GEOPROBE INC, MECHANICAL BLADDER PUMP MODEL MP470

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) design efficient processes for conducting has created the Environmental Technology perfofl1lance tests of innovative technologies. Verification Program (E TV) to facilitate the deployment of innovative or improved environmental techn...

Strategic enzyme patterning for microfluidic biofuel cells

NASA Astrophysics Data System (ADS)

Kjeang, E.; Sinton, D.; Harrington, D. A.

The specific character of biological enzyme catalysts enables combined fuel and oxidant channels and simplified non-compartmentalized fuel cell assemblies. In this work, a microstructured enzymatic biofuel cell architecture is proposed, and species transport phenomena combined with consecutive chemical reactions are studied computationally in order to provide guidelines for optimization. This is the first computational study of this technology, and a 2D CFD model for species transport coupled with laminar fluid flow and Michaelis-Menten enzyme kinetics is established. It is shown that the system is reaction rate limited, indicating that enzyme specific turnover numbers are key parameters for biofuel cell performance. Separated and mixed enzyme patterns in different proportions are analyzed for various Peclet numbers. High fuel utilization is achieved in the diffusion dominated and mixed species transport regimes with separated enzymes arranged in relation to individual turnover rates. However, the Peclet number has to be above a certain threshold value to obtain satisfying current densities. The mixed transport regime is particularly attractive while current densities are maintained close to maximum levels. Optimum performance is achieved by mixed enzyme patterning tailored with respect to individual turnover rates, enabling high current densities combined with nearly complete fuel utilization.

Microbial Enzyme Production Using Lignocellulosic Food Industry Wastes as Feedstock: A Review

PubMed Central

Ravindran, Rajeev; Jaiswal, Amit K.

2016-01-01

Enzymes are of great importance in the industry due to their substrate and product specificity, moderate reaction conditions, minimal by-product formation and high yield. They are important ingredients in several products and production processes. Up to 30% of the total production cost of enzymes is attributed to the raw materials costs. The food industry expels copious amounts of processing waste annually, which is mostly lignocellulosic in nature. Upon proper treatment, lignocellulose can replace conventional carbon sources in media preparations for industrial microbial processes, such as enzyme production. However, wild strains of microorganisms that produce industrially important enzymes show low yield and cannot thrive on artificial substrates. The application of recombinant DNA technology and metabolic engineering has enabled researchers to develop superior strains that can not only withstand harsh environmental conditions within a bioreactor but also ensure timely delivery of optimal results. This article gives an overview of the current complications encountered in enzyme production and how accumulating food processing waste can emerge as an environment-friendly and economically feasible solution for a choice of raw material. It also substantiates the latest techniques that have emerged in enzyme purification and recovery over the past four years. PMID:28952592

Mini review on role of β-galactosidase in lactose intolerance

NASA Astrophysics Data System (ADS)

A, Nivetha; V, Mohanasrinivasan

2017-11-01

This review mainly focuses on the role and properties of β-galactosidase in lactose intolerance and its industrial application. β-Galactosidase, hydrolyses the lactose into glucose and galactose and it is most commonly used in food based technology, particularly in the dairy manufacturing industry. This catalyst mainly focus for the improvement of new and novel products with hydrolyzed lactose, which can be appropriate for the lactose-intolerant persons, to improve the technological, texture and scientific properties of non-fermented dairy products. β-Galactosidase derived from the group of saccharides which is a converting enzymes in the family of hydrolases. They are broadly distributed in the several biological living systems. The enzymatic hydrolysis of lactose is also preferred in food based technology due to the low soluble range of lactose. The concentration lactose was found to be high in fermented dairy products such as ice cream, butter, cheese curd, yogurt, etc., can prompt extreme lactose crystallization bringing about items through a coarse, abrasive surface. Lactose hydrolysis in dairy products enhances adaptability also, richness altogether. These products are extra edible. Also for this purpose, the utilization of β-galactosidase enzyme prior to the condensing operation can reduce the lactose content to a point where lactose was no longer a problem industrial application of β-galactosidase. In Industries, due to the positive and constructive effect on intestinal bacterial microflora, different types of applications are possible in β-galactosidase enzyme.

Using Game Development to Engage Students in Science and Technology

NASA Technical Reports Server (NTRS)

Wiacek, John

2011-01-01

Game design workshops, camps and activities engage K-12 students In STEM disciplines that use game engine and development tools. Game development will have students create games and simulations that Will inspire them to love technology while learning math, physics, and,logic. By using tools such as Gamemaker, Alice, Unity, Gamesalad and others, students will get a sense of confidence and accomplishment creating games and simulations.

3D printing of MRI compatible components: why every MRI research group should have a low-budget 3D printer.

PubMed

Herrmann, Karl-Heinz; Gärtner, Clemens; Güllmar, Daniel; Krämer, Martin; Reichenbach, Jürgen R

2014-10-01

To evaluate low budget 3D printing technology to create MRI compatible components. A 3D printer is used to create customized MRI compatible components, a loop-coil platform and a multipart mouse fixation. The mouse fixation is custom fit for a dedicated coil and facilitates head fixation with bite bar, anesthetic gas supply and biomonitoring sensors. The mouse fixation was tested in a clinical 3T scanner. All parts were successfully printed and proved MR compatible. Both design and printing were accomplished within a few days and the final print results were functional with well defined details and accurate dimensions (Δ<0.4mm). MR images of the mouse head clearly showed reduced motion artifacts, ghosting and signal loss when using the fixation. We have demonstrated that a low budget 3D printer can be used to quickly progress from a concept to a functional device at very low production cost. While 3D printing technology does impose some restrictions on model geometry, additive printing technology can create objects with complex internal structures that can otherwise not be created by using lathe technology. Thus, we consider a 3D printer a valuable asset for MRI research groups. Copyright © 2014 IPEM. Published by Elsevier Ltd. All rights reserved.

Statistical optimization of arsenic biosorption by microbial enzyme via Ca-alginate beads.

PubMed

Banerjee, Suchetana; Banerjee, Anindita; Sarkar, Priyabrata

2018-04-16

Bioremediation of arsenic using green technology via microbial enzymes has attracted scientists due to its simplicity and cost effectiveness. Statistical optimization of arsenate bioremediation was conducted by the enzyme arsenate reductase extracted from arsenic tolerant bacterium Pseudomonas alcaligenes. Response surface methodology based on Box-Behnken design matrix was performed to determine the optimal operational conditions of a multivariable system and their interactive effects on the bioremediation process. The highest biosorptive activity of 96.2 µg gm -1 of beads was achieved under optimized conditions (pH = 7.0; As (V) concentration = 1000 ppb; time = 2 h). SEM analysis showed the morphological changes on the surface of enzyme immobilized gluteraldehyde crosslinked Ca-alginate beads. The immobilized enzyme retained its activity for 8 cycles. ANOVA with a high correlation coefficient (R 2 > 0.99) and lower "Prob > F"value (<0.0001) corroborated the second-order polynomial model for the biosorption process. This study on the adsorptive removal of As (V) by enzyme-loaded biosorbent revealed a possible way of its application in large scale treatment of As (V)-contaminated water bodies.

Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter.

PubMed

van Schie, Morten M C H; Ebrahimi, Kourosh Honarmand; Hagen, Wilfred R; Hagedoorn, Peter-Leon

2018-03-01

Recent developments in microfluidic and nanofluidic technologies have resulted in development of new chip-based microfluidic calorimeters with potential use in different fields. One application would be the accurate high-throughput measurement of enzyme activity. Calorimetry is a generic way to measure activity of enzymes, but unlike conventional calorimeters, chip-based calorimeters can be easily automated and implemented in high-throughput screening platforms. However, application of chip-based microfluidic calorimeters to measure enzyme activity has been limited due to problems associated with miniaturization such as incomplete mixing and a decrease in volumetric heat generated. To address these problems we introduced a calibration method and devised a convenient protocol for using a chip-based microfluidic calorimeter. Using the new calibration method, the progress curve of alkaline phosphatase, which has product inhibition for phosphate, measured by the calorimeter was the same as that recorded by UV-visible spectroscopy. Our results may enable use of current chip-based microfluidic calorimeters in a simple manner as a tool for high-throughput screening of enzyme activity with potential applications in drug discovery and enzyme engineering. Copyright © 2017. Published by Elsevier Inc.

Technology and Terrorism in the Movie Brazil

ERIC Educational Resources Information Center

Stivers, Richard

2006-01-01

The movie "Brazil" calls attention to the relationship between technology and terrorism. Terrorism appears to be a threat to the order that technology creates. But terrorism forces technology to adapt and change so that technology perfects itself as a system. In the movie, terrorism is equated with any form of bureaucratic deviance so that…

Creating a Resource Center for Homeschoolers: The Impact of Technology.

ERIC Educational Resources Information Center

Javid, Mahnaz A.

1998-01-01

Summarizes the findings of a two-month case study of Edmonds Cyberschool (Washington), a resource center for homeschoolers. The study focused on the impact of technology on students' learning as indicated in three areas: attitude toward technology, the use of technology, and value of technology versus other available resources. (Author/LRW)

Instructional Technologists at Public Schools: A Study of the Role and Effectiveness of Technology Resource Teachers.

ERIC Educational Resources Information Center

Moallem, Mahnaz; Micallef, Suzanne

Human resource positions that are becoming known as Technology Coordinator (for district-level personnel) and Technology Resource Teacher (for school-level personnel) have been created to help integrate technology into classroom instruction. This study assessed the progress and effectiveness of Technology Resource Teachers, or TRTs, as technical…

Middle School Mathematics Teachers' Experiences with School-Based and District-Based Support and Its Relationship to Integrating Technology

ERIC Educational Resources Information Center

Hampshire, Onequa N.

2014-01-01

Technological advances play a major role in educating students' in mathematics. Research indicates that technology could create learning environments that support innovativeness and assist teachers in developing a positive attitude toward integrating technology. Unfortunately, teachers are not utilizing technology on a regular basis in mathematics…

S5: Information Technology for Science Missions

NASA Technical Reports Server (NTRS)

Coughlan, Joe

2017-01-01

NASA Missions and Programs create a wealth of science data and information that are essential to understanding our earth, our solar system and the universe. Advancements in information technology will allow many people within and beyond the Agency to more effectively analyze and apply these data and information to create knowledge. The desired end result is to see that NASA data and science information are used to generate the maximum possible impact to the nation: to advance scientific knowledge and technological capabilities, to inspire and motivate the nation's students and teachers, and to engage and educate the public.

Diabetes Information Technology: Designing Informatics Systems to Catalyze Change in Clinical Care

PubMed Central

Lester, William T.; Zai, Adrian H.; Chueh, Henry C.; Grant, Richard W.

2008-01-01

Current computerized reminder and decision support systems intended to improve diabetes care have had a limited effect on clinical outcomes. Increasing pressures on health care networks to meet standards of diabetes care have created an environment where information technology systems for diabetes management are often created under duress, appended to existing clinical systems, and poorly integrated into the existing workflow. After defining the components of diabetes disease management, the authors present an eight-step conceptual framework to guide the development of more effective diabetes information technology systems for translating clinical information into clinical action. PMID:19885355

Competence formation of engineering directions students in the field of energy saving as a way to create new generation technologies

NASA Astrophysics Data System (ADS)

Gilmanshin, I. R.; Gilmanshina, S. I.

2017-09-01

The urgency of the formation of competence in the field of energy saving in the process of studying engineering and technical disciplines at the university is substantiated. The author’s definition of the competence in the field of energy saving is given, allowing to consider the necessity of its formation among students - future engineers as a way to create technologies of a new generation. The essence of this competence is revealed. The system of work, pedagogical conditions and technologies of its formation in the conditions of the federal university is substantiated.

Manufacturing Innovation and Technological Superiority

DTIC Science & Technology

2016-09-01

Defense AT&L: September-October 2016 2 From the Under Secretary of Defense for Acquisit ion, Technology, and Logist ics Manufacturing Innovation ...program to establish Manufacturing Innovation Institutes (MIIs) that would create incubators for advanced manufacturing technology in key

A Mapping of Drug Space from the Viewpoint of Small Molecule Metabolism

PubMed Central

Basuino, Li; Chambers, Henry F.; Lee, Deok-Sun; Wiest, Olaf G.; Babbitt, Patricia C.

2009-01-01

Small molecule drugs target many core metabolic enzymes in humans and pathogens, often mimicking endogenous ligands. The effects may be therapeutic or toxic, but are frequently unexpected. A large-scale mapping of the intersection between drugs and metabolism is needed to better guide drug discovery. To map the intersection between drugs and metabolism, we have grouped drugs and metabolites by their associated targets and enzymes using ligand-based set signatures created to quantify their degree of similarity in chemical space. The results reveal the chemical space that has been explored for metabolic targets, where successful drugs have been found, and what novel territory remains. To aid other researchers in their drug discovery efforts, we have created an online resource of interactive maps linking drugs to metabolism. These maps predict the “effect space” comprising likely target enzymes for each of the 246 MDDR drug classes in humans. The online resource also provides species-specific interactive drug-metabolism maps for each of the 385 model organisms and pathogens in the BioCyc database collection. Chemical similarity links between drugs and metabolites predict potential toxicity, suggest routes of metabolism, and reveal drug polypharmacology. The metabolic maps enable interactive navigation of the vast biological data on potential metabolic drug targets and the drug chemistry currently available to prosecute those targets. Thus, this work provides a large-scale approach to ligand-based prediction of drug action in small molecule metabolism. PMID:19701464

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT; GROUNDWATER SAMPLING TECHNOLOGIES; W.L. GORE AND ASSOCIATES, INC., GORE-SORBER WATER QUALITY MONITORING

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has created the Environmental Technology Verification Program (ETV) to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the...

45 CFR 170.210 - Standards for health information technology to protect electronic health information created...

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Standards for health information technology to... Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES HEALTH INFORMATION TECHNOLOGY HEALTH INFORMATION... FOR HEALTH INFORMATION TECHNOLOGY Standards and Implementation Specifications for Health Information...

45 CFR 170.210 - Standards for health information technology to protect electronic health information created...

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Standards for health information technology to... Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES HEALTH INFORMATION TECHNOLOGY HEALTH INFORMATION... FOR HEALTH INFORMATION TECHNOLOGY Standards and Implementation Specifications for Health Information...

45 CFR 170.210 - Standards for health information technology to protect electronic health information created...

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Standards for health information technology to... Welfare Department of Health and Human Services HEALTH INFORMATION TECHNOLOGY HEALTH INFORMATION... FOR HEALTH INFORMATION TECHNOLOGY Standards and Implementation Specifications for Health Information...

45 CFR 170.210 - Standards for health information technology to protect electronic health information created...

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Standards for health information technology to... Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES HEALTH INFORMATION TECHNOLOGY HEALTH INFORMATION... FOR HEALTH INFORMATION TECHNOLOGY Standards and Implementation Specifications for Health Information...

45 CFR 170.210 - Standards for health information technology to protect electronic health information created...

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Standards for health information technology to... Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES HEALTH INFORMATION TECHNOLOGY HEALTH INFORMATION... FOR HEALTH INFORMATION TECHNOLOGY Standards and Implementation Specifications for Health Information...

Teaching 'How To' Technologies in Context.

ERIC Educational Resources Information Center

Leigh, Patricia Randolph

The introductory instructional technology course at Iowa State University is a survey course covering various technologies. In this case, the instructor chose to create a situated learning environment using low-technology everyday surroundings to teach the fundamentals of photographic and video production, linking the photography, audio, and video…

Carbon prices and incentives for technological development.

PubMed

Lundgren, Tommy; Marklund, Per-Olov; Samakovlis, Eva; Zhou, Wenchao

2015-03-01

There is concern that the carbon prices generated through climate policies are too low to create the incentives necessary to stimulate technological development. This paper empirically analyzes how the Swedish carbon dioxide (CO2) tax and the European Union emission trading system (EU ETS) have affected productivity development in the Swedish pulp and paper industry 1998-2008. A Luenberger total factor productivity (TFP) indicator is computed using data envelopment analysis. The results show that climate policy had a modest impact on technological development in the pulp and paper industry, and if significant it was negative. The price of fossil fuels, on the contrary, seems to have created important incentives for technological development. Hence, the results suggest that the carbon prices faced by the industry through EU ETS and the CO2 tax have been too low. Even though the data for this study is specific for Sweden, the models and results are applicable internationally. When designing policy to mitigate CO2 emissions, it is vital that the policy creates a carbon price that is high enough - otherwise the pressure on technological development will not be sufficiently strong. Copyright © 2014 Elsevier Ltd. All rights reserved.

Designing Improved Enzymes of industrial application from marine microorganisms using Protein Engineering

NASA Astrophysics Data System (ADS)

Prajapati, A. S.; Panchal, K.; Subramanian, R. B.; Patel, D. H.; Sudhir, A. P.; Dave, B. R.

2015-12-01

Global demand for energy has grown with the development of new industries, requiring constant improvement and search for new sources of energy. One of the challenges today is releasing the energy of glucose that nature has cleverly locked into lignocellulosic biomass. Potent and efficient enzyme preparations need to be developed for the enzymatic saccharification process to be more economical. Approaches like enzyme engineering, reconstitution of enzyme mixtures and bioprospecting for superior enzymes are gaining importance. The ocean is considered to be a great reservoir of biodiversity. Because enzymes have unequalled advantages, many industries are keenly interested in adapting enzymatic methods for their processes. Microbial communities in marine environments are ecologically relevant as intermediaries of energy and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. The exploitation of marine bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. Several industrial enzymes are derived from terrestrial sources, whereas, marine environment which encompasses about 71 percent of the earth's surface and a vast resources for useful enzymes, remain unexplored. Marine microorganisms take active part in the mineralization of complex organic matter through degradative pathways of their metabolism. Bacteria from marine environments secrete different enzymes based on their habitat and their ecological functions. Therefore marine microbial enzymes have become the focal point of interest. Even though many of these enzymes are being isolated, the efficiency of hydrolysis is very poor. This could be overcome by altering the substrate specificity of lignocellulases. Protein engineering could prove to be useful to improve the catalytic function these enzymes.

Amperometric IFN-γ immunosensors with commercially fabricated PCB sensing electrodes.

PubMed

Moschou, Despina; Greathead, Louise; Pantelidis, Panagiotis; Kelleher, Peter; Morgan, Hywel; Prodromakis, Themistoklis

2016-12-15

Lab-on-a-Chip (LoC) technology has the potential to revolutionize medical Point-of-Care diagnostics. Currently, considerable research efforts are focused on innovative production technologies that will make commercial upscaling of lab-on-chip products financially viable. Printed circuit board (PCB) manufacturing techniques have several advantages in this field. In this paper we focus on transferring a complete IFN-γ enzyme-linked immune-sorbent assay (ELISA) onto a commercial PCB electrochemical biosensing platform, We adapted a commercially available ELISA to detect the enzyme product TMB/H2O2 using amperometry, successfully reproducing the colorimetry-obtained ELISA standard curve. The results demonstrate the potential for the integration of these components into an automated, disposable, electronic ELISA Lab-on-PCB diagnostic platform. Copyright © 2016 Elsevier B.V. All rights reserved.

Seaweed Hydrocolloid Production: An Update on Enzyme Assisted Extraction and Modification Technologies

PubMed Central

Rhein-Knudsen, Nanna; Ale, Marcel Tutor; Meyer, Anne S.

2015-01-01

Agar, alginate, and carrageenans are high-value seaweed hydrocolloids, which are used as gelation and thickening agents in different food, pharmaceutical, and biotechnological applications. The annual global production of these hydrocolloids has recently reached 100,000 tons with a gross market value just above US$ 1.1 billion. The techno-functional properties of the seaweed polysaccharides depend strictly on their unique structural make-up, notably degree and position of sulfation and presence of anhydro-bridges. Classical extraction techniques include hot alkali treatments, but recent research has shown promising results with enzymes. Current methods mainly involve use of commercially available enzyme mixtures developed for terrestrial plant material processing. Application of seaweed polysaccharide targeted enzymes allows for selective extraction at mild conditions as well as tailor-made modifications of the hydrocolloids to obtain specific functionalities. This review provides an update of the detailed structural features of κ-, ι-, λ-carrageenans, agars, and alginate, and a thorough discussion of enzyme assisted extraction and processing techniques for these hydrocolloids. PMID:26023840

Seaweed hydrocolloid production: an update on enzyme assisted extraction and modification technologies.

PubMed

Rhein-Knudsen, Nanna; Ale, Marcel Tutor; Meyer, Anne S

2015-05-27

Agar, alginate, and carrageenans are high-value seaweed hydrocolloids, which are used as gelation and thickening agents in different food, pharmaceutical, and biotechnological applications. The annual global production of these hydrocolloids has recently reached 100,000 tons with a gross market value just above US$ 1.1 billion. The techno-functional properties of the seaweed polysaccharides depend strictly on their unique structural make-up, notably degree and position of sulfation and presence of anhydro-bridges. Classical extraction techniques include hot alkali treatments, but recent research has shown promising results with enzymes. Current methods mainly involve use of commercially available enzyme mixtures developed for terrestrial plant material processing. Application of seaweed polysaccharide targeted enzymes allows for selective extraction at mild conditions as well as tailor-made modifications of the hydrocolloids to obtain specific functionalities. This review provides an update of the detailed structural features of κ-, ι-, λ-carrageenans, agars, and alginate, and a thorough discussion of enzyme assisted extraction and processing techniques for these hydrocolloids.

Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase.

PubMed

Arjomand, Maryam Rezaei; Habibi-Rezaei, Mehran; Ahmadian, Gholamreza; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Asadifar, Mandana; Amanlou, Massoud

2016-11-01

Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment ( 74 YGSDVT 79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (V max , K m , k cat and, k cat /K m ) and activation energy (E a ) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance. Copyright © 2016. Published by Elsevier B.V.

Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase.

PubMed

Montgomery, H J; Romanov, V; Guillemette, J G

2000-02-18

Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum.

PubMed

Jortzik, Esther; Mailu, Boniface M; Preuss, Janina; Fischer, Marina; Bode, Lars; Rahlfs, Stefan; Becker, Katja

2011-06-15

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

Targeting the cell wall of Mycobacterium tuberculosis: a molecular modeling investigation of the interaction of imipenem and meropenem with L,D-transpeptidase 2.

PubMed

Silva, José Rogério A; Bishai, William R; Govender, Thavendran; Lamichhane, Gyanu; Maguire, Glenn E M; Kruger, Hendrik G; Lameira, Jeronimo; Alves, Cláudio N

2016-01-01

The single crystal X-ray structure of the extracellular portion of the L,D-transpeptidase (ex-LdtMt2 - residues 120-408) enzyme was recently reported. It was observed that imipenem and meropenem inhibit activity of this enzyme, responsible for generating L,D-transpeptide linkages in the peptidoglycan layer of Mycobacterium tuberculosis. Imipenem is more active and isothermal titration calorimetry experiments revealed that meropenem is subjected to an entropy penalty upon binding to the enzyme. Herein, we report a molecular modeling approach to obtain a molecular view of the inhibitor/enzyme interactions. The average binding free energies for nine commercially available inhibitors were calculated using MM/GBSA and Solvation Interaction Energy (SIE) approaches and the calculated energies corresponded well with the available experimentally observed results. The method reproduces the same order of binding energies as experimentally observed for imipenem and meropenem. We have also demonstrated that SIE is a reasonably accurate and cost-effective free energy method, which can be used to predict carbapenem affinities for this enzyme. A theoretical explanation was offered for the experimental entropy penalty observed for meropenem, creating optimism that this computational model can serve as a potential computational model for other researchers in the field.

An Internet Application To Relieve Constraints in the Flow of Technical Information--The Virtual Technology Market (VTM).

ERIC Educational Resources Information Center

Beverly, James E.; Xue, Lan; Lee, Chung-Shing

1996-01-01

Reports on the use of the Internet and World Wide Web as a virtual technology market (VTM) for information and technology transfer. The project focuses on creating awareness of technology demand (problems) and linking it to technology supply (solutions) in the field of particle technology and multiphase processes in the chemical industry. Benefits…

SNAP Assay Technology.

PubMed

O'Connor, Thomas P

2015-12-01

The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System.

PubMed

Akama, Kenji; Shirai, Kentaro; Suzuki, Seigo

2016-07-19

Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.

Fluorescence Detection In Electrophoresis

NASA Astrophysics Data System (ADS)

Swarner, Susan

1988-04-01

Fluorescence detection is in common usage in forensic science laboratories for the visualization of three enzyme markers. The fluorogenic substrates, 4-methylumbelliferyl phosphate, 4-methylutbel-liveryl acetate, and fluorecein diacetate, are acted upon by the enzymes Erythrocyte Acid Phospha, tase, Esterase-D, and Carbonic Anhydrase-III, respectively, to produce compounds visible to the analyst when viewed with transmitted UV light at 365 nm. Additionally, the choice of fluorogenic corn, pounds may help detect a specific enzyme from a related enzyme. One of the responsibilities of a forensic science laboratory may be the analysis of blood for genetically controlled polymorphic enzymes and protein markers. The genetic markers are said to be polymorphic because each exhibits types which can be differentiated and allows for the inclusion or exclusion of possible-donors of the blood. Each genetic marker can be separated into these recognizable types by electrophoresis, a technique which separates compounds based on electrical charges. Electrophoresis is conducted by placing a portion or extract of each bloodstain into a support medium which will conduct electricity. This is known as a plate or membrane. By controlling the pH of the buffer and the potential that is applied to the plate, the analyst can achieve separation of the types within an enzyme marker. The types appear as differing patterns of bands. Once the bloodstain has been subjected to electrophoresis, the enzymes must be visualized. This is generally best accomplished by using the specific activity of the enzyme. For the enzymes described in the present work, the visualization is performed by over-layering the plate with a piece of filter paper that 'has been saturated with the appropriate non-fluorescent substrate and buffer. The bands of enzyme, which is now in discrete patterns, will act upon the non-fluorescent substrate to create a fluorescent compound. The plate is then viewed with transmitted UV light at 365 nm to locate the band patterns which will identify the phenotype of the blood source. The plate should be photographed to record the findings.

Synthetic fuels, and a sustainable set of civilizations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leonard, R.S.

Described in this paper is a concept that combines a set of technologies with a set of economic and social concepts that would allow people to create sustainable ecologies for their region or country. As such it describes a possible implementation path. The technologies are : solar electricity, power satellites, wireless power transmission, electrolytic hydrogen, and synthetic liquid fuels manufactured from air, water, and electricity. Economic initiatives and policies include creating sustainable economic development regions through the use of tax incentives and tax penalties. The technologies and economies are brought together by social concepts such as Technopolis and the theorymore » of self-organizing and self-energizing social systems, i.e. creating wealth where there was none through sweat equity. Existing organizational structures such as credit unions, kibbutz`s and agricultural and marketing cooperatives provide methods by which global marco-projects can be implemented on a local level. Some topics of this paper are : creating global markets by solving global problems or how to breakout of the chicken or egg paradox that has stymied the development of energy from space for so long ; and linking energy availability to self-help economic development programs that create sustainable cultures while benefiting both the local and global environment. 1 refs., 6 figs., 6 tabs.« less

Presidential Green Chemistry Challenge: 2004 Greener Reaction Conditions Award

EPA Pesticide Factsheets

Presidential Green Chemistry Challenge 2004 award winner, Buckman Laboratories International, developed Optimyze technology, which uses an esterase enzyme to remove sticky contaminants from paper products prior to recycling.

Technology Tips

ERIC Educational Resources Information Center

Santos-Trigo, Manuel

2004-01-01

A dynamic program for geometry called Cabri Geometry II is used to examine properties of figures like triangles and make connections with other mathematical ideas like ellipse. The technology tip includes directions for creating such a problem with technology and suggestions for exploring it.

TREND 2000

NASA Technical Reports Server (NTRS)

1996-01-01

The Educator Resource Center has created the Technology, Research, Education and Discovery (TREND) 2000 computer lab at NASA's John C. Stennis Space Center to facilitate the integration of technology into schools' curriculums by providing innovative and creative classroom strategies using state-of-the-art technology.

Alternate energy sources for catheter ablation.

PubMed

Wang, P J; Homoud, M K; Link, M S; Estes III, N A

1999-07-01

Because of the limitations of conventional radiofrequency ablation in creating large or linear lesions, alternative energy sources have been used as possible methods of catheter ablation. Modified radiofrequency energy, cryoablation, and microwave, laser, and ultrasound technologies may be able to create longer, deeper, and more controlled lesions and may be particularly suited for the treatment of ventricular tachycardias and for linear atrial ablation. Future studies will establish the efficacy of these new and promising technologies.

The Challenges of Adopting a Culture of Mission Command in the US Army

DTIC Science & Technology

2015-05-23

NUMBER 6. AUTHOR(S) LTC(P) James W. Wright 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND...the development and implementation of high- end information technology creates a paradox for mission command. 15. SUBJECT TERMS Mission command...centralized control and less risk. Likewise, the development and implementation of high- end information technology creates a paradox for mission

Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5'-dAdo• "Free Radical" Is Never Free.

PubMed

Horitani, Masaki; Byer, Amanda S; Shisler, Krista A; Chandra, Tilak; Broderick, Joan B; Hoffman, Brian M

2015-06-10

Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S-C5' bond, which creates the highly reactive 5'-deoxyadenosyl radical (5'-dAdo•), the same radical generated by homolytic Co-C bond cleavage in B12 radical enzymes. The SAM surrogate S-3',4'-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of (13)C, (2)H, and (15)N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 "tames" the 5'-dAdo• radical, preventing it from carrying out harmful side reactions: this "free radical" in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S-C5' bond, thereby enabling the 5'-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ∼0.6 Å toward the target and ∼1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5' radical, with "van der Waals control" of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature.

Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5′-dAdo• “Free Radical” Is Never Free

PubMed Central

Horitani, Masaki; Byer, Amanda S.; Shisler, Krista A.; Chandra, Tilak; Broderick, Joan B.; Hoffman, Brian M.

2015-01-01

Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S–C5′ bond, which creates the highly reactive 5′-deoxyadenosyl radical (5′-dAdo•), the same radical generated by homolytic Co–C bond cleavage in B12 radical enzymes. The SAM surrogate S-3′,4′-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of 13C, 2H, and 15N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 “tames” the 5′-dAdo• radical, preventing it from carrying out harmful side reactions: this “free radical” in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S–C5′ bond, thereby enabling the 5′-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ~0.6 Å toward the target and ~1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5′ radical, with “van der Waals control” of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature. PMID:25923449

Technology-Supported Learning Environments in Science Classrooms in India

ERIC Educational Resources Information Center

Gupta, Adit; Fisher, Darrell

2012-01-01

The adoption of technology has created a major impact in the field of education at all levels. Technology-supported classroom learning environments, involving modern information and communication technologies, are also entering the Indian educational system in general and the schools in Jammu region (Jammu & Kashmir State, India) in…

State of Washington Strategic Information Technology Plan.

ERIC Educational Resources Information Center

Washington State Dept. of Information Services, Olympia. Policy and Regulation Div.

The Strategic Information Technology Plan of Washington is introduced and explained. The plan is mandated by state law to create a new framework for communication and collaboration to bring together agency technology planning with the achievement of statewide information technology goals and strategies. It provides a point of reference for the…

Navigating Schools Past the Technology On-Ramp.

ERIC Educational Resources Information Center

Wasser, Judith Davidson

1996-01-01

Discussion of how to support schools in the use of technology focuses on the Hanau Model Schools Partnership in Germany, supported by TERC and the National Science Foundation, that was developed to create broad technological access in schools and to support teachers in developing connections between the technology and the curriculum. (LRW)

Pre-School Children Creating and Communicating with Digital Technologies in the Home

ERIC Educational Resources Information Center

McPake, Joanna; Plowman, Lydia; Stephen, Christine

2013-01-01

There is a limited literature on pre-school children's experiences with "digital technologies" at home and little discussion of the ways in which children harness these technologies for their own purposes. This paper discusses findings drawn from three studies that investigated the role of "domestic technologies" and…

Computer Technology Standards of Learning for Virginia's Public Schools

ERIC Educational Resources Information Center

Virginia Department of Education, 2005

2005-01-01

The Computer/Technology Standards of Learning identify and define the progressive development of essential knowledge and skills necessary for students to access, evaluate, use, and create information using technology. They provide a framework for technology literacy and demonstrate a progression from physical manipulation skills for the use of…

Factors Affecting Use of Telepresence Technology in a Global Technology Company

ERIC Educational Resources Information Center

Agnor, Robert Joseph

2013-01-01

Telepresence uses the latest video conferencing technology, with high definition video, surround sound audio, and specially constructed studios, to create a near face-to-face meeting experience. A Fortune 500 company which markets information technology has organizations distributed around the globe, and has extensive collaboration needs among…

"Future Proofing" Faculty: The Struggle To Create Technical Lifelong Learners.

ERIC Educational Resources Information Center

Nay, Fred W.; Malm, Loren D.; Malone, Bobby G.; Oliver, Brad E.; Saunders, Nancy G.; Thompson, Jay C., Jr.

College faculty can avoid investing valuable time and resources in inappropriate technologies by staying in step with technological progress. A "future proof" approach to technology recognizes and welcomes small failures, considering them part of the ongoing process of absorbing technology into the learning process. Future proofing attempts to…

Using Information and Communication Technology (ICT) to the Maximum: Learning and Teaching Biology with Limited Digital Technologies

ERIC Educational Resources Information Center

Van Rooy, Wilhelmina S.

2012-01-01

Background: The ubiquity, availability and exponential growth of digital information and communication technology (ICT) creates unique opportunities for learning and teaching in the senior secondary school biology curriculum. Digital technologies make it possible for emerging disciplinary knowledge and understanding of biological processes…

Applying Sustainable Systems Development Approach to Educational Technology Systems

ERIC Educational Resources Information Center

Huang, Albert

2012-01-01

Information technology (IT) is an essential part of modern education. The roles and contributions of technology to education have been thoroughly documented in academic and professional literature. Despite the benefits, the use of educational technology systems (ETS) also creates a significant impact on the environment, primarily due to energy…

TTC Fellowship Program | NCI Technology Transfer Center | TTC

Cancer.gov

The TTC has fellowship opportunities available to qualified candidates in the field of technology transfer. This Fellowship starts with your science, legal, and/or business background to create a new competency in technology transfer, preparing you for technology transfer positions within academia, industry, or the federal government.

The Change Book: A Blueprint for Technology Transfer.

ERIC Educational Resources Information Center

Addiction Technology Transfer Centers.

This document was developed by the Addiction Technology Transfer Center (ATTC) National Network to improve understanding about how valuable effective technology transfer is to the fields of substance abuse treatment and prevention. Technology transfer involves creating a mechanism by which a desired change is accepted, incorporated, and reinforced…

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, LEAD IN DUST WIPE MEASUREMENT TECHNOLOGY, NITON LLC, X-RAY FLUORESCENCE SPECTRUM ANALYZER, XLT-700

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) design efficient processes for conducting has created the Environmental Technology perfofl1lance tests of innovative technologies. Verification Program (E TV) to facilitate the deployment of innovative or improved environmental techn...

The Time Is Now! Creating Technology Competencies for Teacher Educators

ERIC Educational Resources Information Center

Foulger, Teresa S.; Graziano, Kevin J.; Slykhuis, David; Schmidt-Crawford, Denise; Trust, Torrey

2016-01-01

The way preservice teachers learn to use technology within their practice varies widely depending on the learning opportunities available (e.g., technology-infused teacher preparation program vs. standalone education technology course), and the knowledge, skills, and attitudes of the teacher educators within their teacher preparation programs.…

Creating Technology-Enriched Classrooms: Implementational Challenges in Turkish Education

ERIC Educational Resources Information Center

Kurt, Serhat

2014-01-01

This paper provides an overview of the status of educational technology in Turkey. In the face of severe social and economic challenges, many developing nations, including Turkey, are looking to education as a potential remedy. Recognizing that in an increasingly technology-dependent world, information and communications technology skills and…

Advanced Launch Technology Life Cycle Analysis Using the Architectural Comparison Tool (ACT)

NASA Technical Reports Server (NTRS)

McCleskey, Carey M.

2015-01-01

Life cycle technology impact comparisons for nanolauncher technology concepts were performed using an Affordability Comparison Tool (ACT) prototype. Examined are cost drivers and whether technology investments can dramatically affect the life cycle characteristics. Primary among the selected applications was the prospect of improving nanolauncher systems. As a result, findings and conclusions are documented for ways of creating more productive and affordable nanolauncher systems; e.g., an Express Lane-Flex Lane concept is forwarded, and the beneficial effect of incorporating advanced integrated avionics is explored. Also, a Functional Systems Breakdown Structure (F-SBS) was developed to derive consistent definitions of the flight and ground systems for both system performance and life cycle analysis. Further, a comprehensive catalog of ground segment functions was created.

Enhancing Readiness Through Environmental Quality Technology

DTIC Science & Technology

1996-05-01

mercury . Up coming technologies for heavy metal soil contamination include phytoremediation and electrokinetics. Plants have also been shown to uptake... phytoremediation could be that process. Many plants have been found that have a nitroreductase enzyme. These plants can degrade explosive contaminants. This... phytoremediation in a wetland environment for explosive contaminated groundwater. But, this could be transferred directly to soils if proven successful

Europe Report, Science and Technology.

DTIC Science & Technology

1986-09-29

for example, research is being conducted on the synthesis of antibiotics from slime bacteria (myxobacteria), the formation of pigments with...nannocystis, and the formation of steroids. In cooperation with the enzyme technology division, work is also being done on cofactor regeneration in L-amino...also supporting work in product development at Asea Cerama in Robertfors, where they are working with silicon carbide and zirconium dioxide. 12507

Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases

PubMed Central

Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

2015-01-01

Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines. PMID:26184194

Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases.

PubMed

Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

2015-07-08

Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a '3D well' was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

Titania-polymeric powder coatings with nano-topography support enhanced human mesenchymal cell responses.

PubMed

Mozumder, Mohammad Sayem; Zhu, Jesse; Perinpanayagam, Hiran

2012-10-01

Titanium implant osseointegration is dependent on the cellular response to surface modifications and coatings. Titania-enriched nanocomposite polymeric resin coatings were prepared through the application of advanced ultrafine powder coating technology. Their surfaces were readily modified to create nano-rough (<100 nm) surface nano-topographies that supported human embryonic palatal mesenchymal cell responses. Energy dispersive x-ray spectroscopy confirmed continuous and homogenous coatings with a similar composition and even distribution of titanium. Scanning electron microscopy (SEM) showed complex micro-topographies, and atomic force microscopy revealed intricate nanofeatures and surface roughness. Cell counts, mitochondrial enzyme activity reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to dark purple, SEM, and inverted fluorescence microscopy showed a marked increase in cell attachment, spreading, proliferation, and metabolic activity on the nanostructured surfaces. Reverse Transcription- Polymerase Chain Reaction (RT-PCR) analysis showed that type I collagen and Runx2 expression were induced, and Alizarin red staining showed that mineral deposits were abundant in the cell cultures grown on nanosurfaces. This enhancement in human mesenchymal cell attachment, growth, and osteogenesis were attributed to the nanosized surface topographies, roughness, and moderate wetting characteristics of the coatings. Their dimensional similarity to naturally occurring matrix proteins and crystals, coupled with their increased surface area for protein adsorption, may have facilitated the response. Therefore, this application of ultrafine powder coating technology affords highly biocompatible surfaces that can be readily modified to accentuate the cellular response. Copyright © 2012 Wiley Periodicals, Inc.

Nailing Digital Jelly to a Virtual Tree: Tracking Emerging Technologies for Learning

ERIC Educational Resources Information Center

Serim, Ferdi; Schrock, Kathy

2008-01-01

Reliable information on emerging technologies for learning is as vital as it is difficult to come by. To meet this need, the International Society for Technology in Education organized the Emerging Technologies Task Force. Its goal is to create a database of contributions from educators highlighting their use of emerging technologies to support…

Value Creation, Appropriation, and Product Design Strategies in Technology Ecosystems: Three Essays on the Role of Complementary Technologies

ERIC Educational Resources Information Center

Miller, Cameron Dee

2017-01-01

Firms are often embedded in a technology ecosystem comprised of complementary technologies that span multiple product markets. In this dissertation, I examine how complementarity between the firm's technologies influences its strategies to create and appropriate value in the ecosystem. I investigate this overarching question in two contexts:…

An impoverished machine: challenges to human learning and instructional technology.

PubMed

Taraban, Roman

2008-08-01

Many of the limitations to human learning and processing identified by cognitive psychologists over the last 50 years still hold true, including computational constraints, low learning rates, and unreliable processing. Instructional technology can be used in classrooms and in other learning contexts to address these limitations to learning. However, creating technological innovations is not enough. As part of psychological science, the development and assessment of instructional systems should be guided by theories and practices within the discipline. The technology we develop should become an object of research like other phenomena that are studied. In the present article, I present an informal account of my own work in assessing instructional technology for engineering thermodynamics to show not only the benefits, but also the limitations, in studying the technology we create. I conclude by considering several ways of advancing the development of instructional technology within the SCiP community, including interdisciplinary research and envisioning learning contexts that differ radically from traditional learning focused on lectures and testing.

Drying characteristic, enzyme inactivation and browning pigmentation kinetics of controlled humidity-convective drying of banana slices

NASA Astrophysics Data System (ADS)

Sarpong, Frederick; Yu, Xiaojie; Zhou, Cunshan; Oteng-Darko, Patricia; Amenorfe, Leticia Peace; Wu, Bengang; Bai, Junwen; Ma, Haile

2018-04-01

Investigating the kinetics of enzyme activities and browning indexes in food are very essential in understanding the enzyme inactivation and browning pigmentation reaction during drying processing. In order to understand and predict accurately the enzyme inactivation and browning pigmentation of banana slices using Relative Humidity (RH)-convective hot air dryer aided by ultrasound (US) pretreatment, this study was conducted. Drying was carried out with 20 kHz frequency of US-pretreatment using three durations (10 20 and 30 min) and RH (10 20 and 30%) conditions at 70 °C and 2.0 m/s air velocity. The kinetic study of both enzyme inactivation and browning pigmentation results were compared to their relevance of fit in terms of coefficient of correlation (R2), the root mean square error (RMSE) and the reduced chi-square (χ 2). First order and second-order polynomial kinetic model fitted well for enzyme inactivation and browning indexes respectively. Both enzymes inactivation kinetics and enzymatic browning index (EBI) declined significantly (p < 0.05) with increasing drying time in all drying conditions and rate of decrease intensified in longer US-pretreatment duration and lower RH conditions. However, shorter US-pretreatment duration and higher RH conditions reduced the non- enzymatic browning index (NBI) significantly. Again, longer US-pretreatment duration and lower RH shortened the drying time but adversely created more microspores from the micrograph study. Longer US pretreatment and lower RH decrease significantly (p < 0.05) the L* and b* values whereas the a* values was increased.

Engineering self-assembled bioreactors from protein microcompartments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Savage, David

2016-10-12

The goals of this research are to understand how organisms such as bacteria segregate certain metabolic processes inside of specific structures, or “microcompartments,” in the cell and apply this knowledge to develop novel engineered microcompartments for use in nanotechnology and metabolic engineering. For example, in some bacteria, self-assembling protein microcompartments called carboxysomes encapsulate the enzymes involved in carbon fixation, enabling the cell to utilize carbon dioxide more effectively than if the enzymes were free in the cell. The proposed research will determine how structures such as carboxysomes assemble and function in bacteria and develop a means for creating novel, syntheticmore » microcompartments for optimizing production of specific energy-rich compounds.« less

Enzymatic trends of fructooligosaccharides production by microorganisms.

PubMed

Ganaie, Mohd Anis; Lateef, Agbaje; Gupta, Uma Shanker

2014-02-01

Fructooligosaccharides are influential prebiotics that affect various physiological functions in such a way that they promote positive impact to health. They occur naturally in many fruits and vegetables in trace amounts. However, they are mainly produced commercially by the reaction of microbial enzymes with di- or polysaccharides, such as sucrose or inulin as a substrate. For maximum production of fructooligosaccharides on an industrial level, development of more enzymes with high activity and stability is required. This has attracted the attention of biotechnologists and microbiologists worldwide. This study aims to discuss the new trends in the production of fructooligosaccharide and its effect on numerous health qualities through which it creates great demand in the sugar market.

Technology Innovation

EPA Pesticide Factsheets

EPA produces innovative technologies and facilitates their creation in line with the Agency mission to create products such as the stormwater calculator, remote sensing, innovation clusters, and low-cost air sensors.

A comparison of the effect of macerating enzymes and pulsed electric fields technology on phenolic content and color of red wine.

PubMed

Puértolas, E; Saldaña, G; Condón, S; Alvarez, I; Raso, J

2009-01-01

The effect of the addition of 2 enzymatic preparations and the application of a pulsed electric fields treatment (PEF) on the phenolic content and color of Cabernet Sauvignon wine has been compared. The evolution of color intensity (CI), anthocyanic content (AC), and total polyphenol index (TPI) from crushing to 3 mo of aging in bottle was studied. The results demonstrated that both treatments promoted greater extraction of phenolic compounds, compared to the untreated wine. However, PEF technology was more effective. After 3 mo of storage, CI, AC, and TPI were 28%, 26%, and 11%, respectively, higher in PEF-wine than in control wine. By contrast, while both enzymatic preparations increased the CI of the wine around 5%, only one of them increased the AC and TPI by 11% and 3%, respectively, in comparison with the control. After 3 mo of aging in bottle, the phenolic composition was also analyzed by high-performance liquid chromatography (HPLC). The content of nonanthocyanic families was higher in PEF-wine than in the rest of the wines. In wines treated by enzymes, only an increase in phenolic acids and flavonols with respect to the control was detected. Practical Application: Pulsed electric fields is a novel food processing technology that poses a very promising future to the enological field, due to its capacity to improve the mass transfer phenomenon. The continuous development of this technology allows nowadays the application of treatments at the semi-industrial scale. In this article, it has been demonstrated that the application of a PEF treatment to the grape pomace before maceration/fermentation is more effective, in terms of color intensity and phenolic content, than the addition of macerating enzymes.

Learning by Creating and Exchanging Objects: The SCY Experience

ERIC Educational Resources Information Center

De Jong, Ton; Van Joolingen, Wouter R.; Giemza, Adam; Girault, Isabelle; Hoppe, Ulrich; Kindermann, Jorg; Kluge, Anders; Lazonder, Ard W.; Vold, Vibeke; Weinberger, Armin; Weinbrenner, Stefan; Wichmann, Astrid; Anjewierden, Anjo; Bodin, Marjolaine; Bollen, Lars; D'Ham, Cedric; Dolonen, Jan; Engler, Jan; Geraedts, Caspar; Grosskreutz, Henrik; Hovardas, Tasos; Julien, Rachel; Lechner, Judith; Ludvigsen, Sten; Matteman, Yuri; Meistadt, Oyvind; Naess, Bjorge; Ney, Muriel; Pedaste, Margus; Perritano, Anthony; Rinket, Marieke; Von Schlanbusch, Henrik; Sarapuu, Tago; Schulz, Florian; Sikken, Jakob; Slotta, Jim; Toussaint, Jeremy; Verkade, Alex; Wajeman, Claire; Wasson, Barbara; Zacharia, Zacharias C.; Van Der Zanden, Martine

2010-01-01

Science Created by You (SCY) is a project on learning in science and technology domains. SCY uses a pedagogical approach that centres around products, called "emerging learning objects" (ELOs) that are created by students. Students work individually and collaboratively in SCY-Lab (the general SCY learning environment) on "missions" that are guided…

Captivate MenuBuilder: Creating an Online Tutorial for Teaching Software

ERIC Educational Resources Information Center

Yelinek, Kathryn; Tarnowski, Lynn; Hannon, Patricia; Oliver, Susan

2008-01-01

In this article, the authors, students in an instructional technology graduate course, describe a process to create an online tutorial for teaching software. They created the tutorial for a cyber school's use. Five tutorial modules were linked together through one menu screen using the MenuBuilder feature in the Adobe Captivate program. The…

Environmental Technology Verification (ETV) Quality Program (Poster)

EPA Science Inventory

This is a poster created for the ETV Quality Program. The EPA Environmental Technology Verification Program (ETV) develops test protocols and verifies the performance of innovative technologies that have the potential to improve protection of human health and the environment. The...

Development of a Web-Based Periscope Simulator for Submarine Officer Training

DTIC Science & Technology

2014-09-01

31 2. The Evolution of Web-Based technology .........................................32...DEVELOPMENT ............................................................................65 A. TECHNOLOGY ...the possibility to deliver 3D simulations using the web browsers and web technology . The objective is to create an effective and efficient WBLE that

TECHcitement: Advances in Technological Education, 2007

ERIC Educational Resources Information Center

Patton, Madeline

2007-01-01

This publication presents the following nine articles: (1) ATE [Advanced Technological Education] Readies Technicians for International Competition; (2) Technicians in Demand Worldwide; (3) Accreditation Board for Engineering and Technology Endorses International Protocols for Technicians; (4) Entrepreneurial Educator Creates InnovaBio to Meet…

32 CFR 2400.25 - Access.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Other Regulations Relating to National Defense OFFICE OF SCIENCE AND TECHNOLOGY POLICY REGULATIONS TO IMPLEMENT E.O. 12356; OFFICE OF SCIENCE AND TECHNOLOGY POLICY INFORMATION SECURITY PROGRAM Safeguarding.... (c) The Director, Office of Science and Technology Policy may create special access programs to...

32 CFR 2400.25 - Access.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Other Regulations Relating to National Defense OFFICE OF SCIENCE AND TECHNOLOGY POLICY REGULATIONS TO IMPLEMENT E.O. 12356; OFFICE OF SCIENCE AND TECHNOLOGY POLICY INFORMATION SECURITY PROGRAM Safeguarding.... (c) The Director, Office of Science and Technology Policy may create special access programs to...

32 CFR 2400.25 - Access.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Other Regulations Relating to National Defense OFFICE OF SCIENCE AND TECHNOLOGY POLICY REGULATIONS TO IMPLEMENT E.O. 12356; OFFICE OF SCIENCE AND TECHNOLOGY POLICY INFORMATION SECURITY PROGRAM Safeguarding.... (c) The Director, Office of Science and Technology Policy may create special access programs to...

32 CFR 2400.25 - Access.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Other Regulations Relating to National Defense OFFICE OF SCIENCE AND TECHNOLOGY POLICY REGULATIONS TO IMPLEMENT E.O. 12356; OFFICE OF SCIENCE AND TECHNOLOGY POLICY INFORMATION SECURITY PROGRAM Safeguarding.... (c) The Director, Office of Science and Technology Policy may create special access programs to...

32 CFR 2400.25 - Access.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Other Regulations Relating to National Defense OFFICE OF SCIENCE AND TECHNOLOGY POLICY REGULATIONS TO IMPLEMENT E.O. 12356; OFFICE OF SCIENCE AND TECHNOLOGY POLICY INFORMATION SECURITY PROGRAM Safeguarding.... (c) The Director, Office of Science and Technology Policy may create special access programs to...

Integrating Behavioral Technology into Public Schools.

ERIC Educational Resources Information Center

Axelrod, Saul

1993-01-01

Suggests seven measures that behavioral educators can take to make effective educational procedures available in public schools: make dissemination of effective technology first priority; develop comprehensive educational systems; simplify existing effective procedures; create market for effective educational technology; obtain new measures of…

PERFORMANCE VERIFICATION OF STORMWATER TREATMENT DEVICES UNDER EPA�S ENVIRONMENTAL TECHNOLOGY VERIFICATION PROGRAM

EPA Science Inventory

The Environmental Technology Verification (ETV) Program was created to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The program�s goal is to further environmental protection by a...

USEPA SITE PROGRAM APPROACH TO TECHNOLOGY TRANSFER AND REGULATORY ACCEPTANCE

EPA Science Inventory

The SITE Program was created to meet the increased demand for innovative technologies for hazardous waste treatment. To accomplish this mission, the program seeks to advance the development, implementation and commercialization of innovative technologies for hazardous waste chara...

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, ENVIRONMENTAL DECISION SUPPORT SOFTWARE, DECISION FX, INC., GROUNDWATER FX

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has created the Environmental Technology Verification Program (ETV) to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the...

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, ENVIRONMENTAL DECISION SUPPORT SOFTWARE, DECISION FX, INC. SAMPLING FX

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has created the Environmental Technology Verification Program (ETV) to facilitate the deployment of innovative or improved environmental technologies through performance verification and dissemination of information. The goal of the...

Collaboration spotting for dental science.

PubMed

Leonardi, E; Agocs, A; Fragkiskos, S; Kasfikis, N; Le Goff, J M; Cristalli, M P; Luzzi, V; Polimeni, A

2014-10-06

The goal of the Collaboration Spotting project is to create an automatic system to collect information about publications and patents related to a given technology, to identify the key players involved, and to highlight collaborations and related technologies. The collected information can be visualized in a web browser as interactive graphical maps showing in an intuitive way the players and their collaborations (Sociogram) and the relations among the technologies (Technogram). We propose to use the system to study technologies related to Dental Science. In order to create a Sociogram, we create a logical filter based on a set of keywords related to the technology under study. This filter is used to extract a list of publications from the Web of Science™ database. The list is validated by an expert in the technology and sent to CERN where it is inserted in the Collaboration Spotting database. Here, an automatic software system uses the data to generate the final maps. We studied a set of recent technologies related to bone regeneration procedures of oro--maxillo--facial critical size defects, namely the use of Porous HydroxyApatite (HA) as a bone substitute alone (bone graft) or as a tridimensional support (scaffold) for insemination and differentiation ex--vivo of Mesenchymal Stem Cells. We produced the Sociograms for these technologies and the resulting maps are now accessible on--line. The Collaboration Spotting system allows the automatic creation of interactive maps to show the current and historical state of research on a specific technology. These maps are an ideal tool both for researchers who want to assess the state--of--the--art in a given technology, and for research organizations who want to evaluate their contribution to the technological development in a given field. We demonstrated that the system can be used for Dental Science and produced the maps for an initial set of technologies in this field. We now plan to enlarge the set of mapped technologies in order to make the Collaboration Spotting system a useful reference tool for Dental Science research.

Collaboration Spotting for oral medicine.

PubMed

Leonardi, E; Agocs, A; Fragkiskos, S; Kasfikis, N; Le Goff, J M; Cristalli, M P; Luzzi, V; Polimeni, A

2014-09-01

The goal of the Collaboration Spotting project is to create an automatic system to collect information about publications and patents related to a given technology, to identify the key players involved, and to highlight collaborations and related technologies. The collected information can be visualized in a web browser as interactive graphical maps showing in an intuitive way the players and their collaborations (Sociogram) and the relations among the technologies (Technogram). We propose to use the system to study technologies related to oral medicine. In order to create a sociogram, we create a logical filter based on a set of keywords related to the technology under study. This filter is used to extract a list of publications from the Web of Science™ database. The list is validated by an expert in the technology and sent to CERN where it is inserted in the Collaboration Spotting database. Here, an automatic software system uses the data to generate the final maps. We studied a set of recent technologies related to bone regeneration procedures of oro-maxillo-facial critical size defects, namely the use of porous hydroxyapatite (HA) as a bone substitute alone (bone graft) or as a tridimensional support (scaffold) for insemination and differentiation ex vivo of mesenchymal stem cells. We produced the sociograms for these technologies and the resulting maps are now accessible on-line. The Collaboration Spotting system allows the automatic creation of interactive maps to show the current and historical state of research on a specific technology. These maps are an ideal tool both for researchers who want to assess the state-of-the-art in a given technology, and for research organizations who want to evaluate their contribution to the technological development in a given field. We demonstrated that the system can be used in oral medicine as is produced the maps for an initial set of technologies in this field. We now plan to enlarge the set of mapped technologies in order to make the Collaboration Spotting system a useful reference tool for oral medicine research.

A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition

PubMed Central

Stieglitz, Kimberly A.; Pastra-Landis, Styliani C.; Xia, Jiarong; Tsuruta, Hiro; Kantrowitz, Evan R.

2005-01-01

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7Å resolution. Time-resolved small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low activity low affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase. PMID:15890205