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Ryan Kenny, Fei Liu
Cooperativity is essential to proficient catalysis, and designing biomimetic, cooperative catalysis is a major avenue to finding new and efficient chemical reactions with practical applications. One challenge in designed cooperative catalysis is to access high catalytic proficiency (large enhancement in both rate and enantioselectivity) as seen in biocatalysis. Here described is an approach of developing and investigating trifunctional organocatalysts with three distinct catalytic functionalities, in order to understand how cooperativity could be organized for enantioselective activation that confers the observed proficiency in a tandem Michael-aldol-proton transfer elimination model reaction...
October 24, 2016: Chemical Record: An Official Publication of the Chemical Society of Japan ... [et Al.]
Chengning Xu, Wenying Dong, Junfen Wan, Xuejun Cao
Aqueous two-phase systems (ATPS) have the potential application in bioseparation and biocatalysis engineering. In this paper, a recyclable ATPS was developed by two thermo-responsive copolymers, PVBAm and PN. Copolymer PVBAm was copolymerized using N-vinylcaprolactam, Butyl methacrylate and Acrylamide as monomers, and PN was synthesized by N-isopropylacrylamide. The lower critical solution temperature (LCST) of PVBAm and PN were 45.0°C and 33.5°C, respectively. The recoveries of both polymers could achieve over 95...
October 11, 2016: Journal of Chromatography. A
Marco Muth, Stefanie Rothkötter, Steven Paprosch, Reiner P Schmid, Klaus Schnitzlein
Lipase-catalyzed hydrolysis of triglycerides yields glycerol and free fatty-acids, provided that the enzyme is non-regioselective. For an Sn-1,3 regioselective enzyme, such as lipase from Thermomyces lanuginosus, the final product is no longer glycerol but Sn-2 monoglyceride instead. However, surface active molecules generated by lipolysis may have a detrimental effect on the interfacial biocatalysis since it is known that low molecular weight surfactants can displace proteins from interfaces. By using drop profile analysis tensiometry, we evaluated the interfacial properties of the lipase-generated molecules and their competitive effect on the adsorption behavior of the lipase and on the proceeding lipolysis...
October 12, 2016: Colloids and Surfaces. B, Biointerfaces
Joel L Kaar
Widespread interest in the use of room-temperature ionic liquids (RTILs) as solvents in anhydrous biocatalytic reactions has largely been met with underwhelming results. Enzymes are frequently inactivated in RTILs as a result of the influence of solvent on the enzyme's microenvironment, be it through interacting with the enzyme or enzyme-bound water molecules. The purpose of this chapter is to present a rational approach to mediate RTIL-enzyme interactions, which is essential if we are to realize the advantages of RTILs over conventional solvents for biocatalysis in full...
2017: Methods in Molecular Biology
Xiaoxue Tong, Tania Triscari Barberi, Catherine H Botting, Sunil V Sharma, Mark J H Simmons, Tim W Overton, Rebecca J M Goss
BACKGROUND: Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis...
October 21, 2016: Microbial Cell Factories
Thomas J Lawton, Amy C Rosenzweig
Nature utilizes two groups of enzymes to catalyze methane conversions, methyl-coenzyme M reductases (MCRs) and methane monooxygenases (MMOs). These enzymes have been difficult to incorporate into industrial processes due to their complexity, poor stability, and lack of recombinant tractability. Despite these issues, new ways of preparing and stabilizing these enzymes have recently been discovered, and new mechanistic insight into how MCRs and MMOs break the C-H bond in nature's most inert hydrocarbon have been obtained...
October 18, 2016: Current Opinion in Chemical Biology
Carlos Molina-Santiago, Zulema Udaondo, María Gómez-Lozano, Soren Molin, Juan Luis Ramos
Pseudomonas putida strains are generally recognized as solvent tolerant, exhibiting varied sensitivity to organic solvents. Pan-genome analysis has revealed that 30% of genes belong to the core-genome of Pseudomonas. Accessory and unique genes confer high degree of adaptability and capabilities for the degradation and synthesis of a wide range of chemicals. For the use of these microbes in bioremediation and biocatalysis, it is critical to understand the mechanisms underlying these phenotypic differences. In this study, RNA-seq analysis compared the short- and long-term responses of the toluene-sensitive KT2440 strain and the highly-tolerant DOT-T1E strain...
October 21, 2016: Environmental Microbiology
Gaspar Morgado, Daniel Gerngross, Tania M Roberts, Sven Panke
Cell-free biosynthesis in the form of in vitro multi-enzyme reaction networks or enzyme cascade reactions emerges as a promising tool to carry out complex catalysis in one-step, one-vessel settings. It combines the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. Such cascades have been successfully applied to the synthesis of fine and bulk chemicals, monomers and complex polymers of chemical importance, and energy molecules from renewable resources as well as electricity...
October 19, 2016: Advances in Biochemical Engineering/biotechnology
Liao Peng, Anchao Feng, Senyang Liu, Meng Huo, Tommy Fang, Ke Wang, Yen Wei, Xiaosong Wang, Jinying Yuan
Potential-stimulated Pickering emulsions, using electrochemical responsive microgels as particle stabilizers, are prepared and used for biocatalysis. The microgels are constructed from the cyclodextrin functionalized 8-arm poly(ethylene glycol) (8A PEG-CD) and ferrocene modified counterparts (8A PEG-Fc) via a CD-Fc host-guest chemistry. Taking advantage of the redox reaction of Fc, the formation and deformation of the microgels and corresponding Pickering emulsions can be reversibly stimulated by external potential, and have been used for the hydrolysis of triacetin and kinetic resolution reaction of (R,S)-1-phenylethanol catalyzed by lipases...
October 14, 2016: ACS Applied Materials & Interfaces
Deqiang Zhu, Jianrong Wu, Xiaobei Zhan, Li Zhu, Zhiyong Zheng, Minjie Gao
OBJECTIVES: N-Acetyl-D-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1-9, 2016). RESULTS: PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated...
October 13, 2016: Biotechnology Letters
Philippe Jacques, Max Béchet, Muriel Bigan, Delphine Caly, Gabrielle Chataigné, François Coutte, Christophe Flahaut, Egon Heuson, Valérie Leclère, Didier Lecouturier, Vincent Phalip, Rozenn Ravallec, Pascal Dhulster, Rénato Froidevaux
Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes...
October 13, 2016: Bioprocess and Biosystems Engineering
Yingmiao Liu, Junzhi Wang, Chongmao Xu, Yong Chen, Junjie Yang, Dong Liu, Huanqing Niu, Yu Jiang, Sheng Yang, Hanjie Ying
Cytidine diphosphate choline (CDP-choline) has been applied for treating acute craniocerebral injury and allowing recovery of consciousness after brain surgery. In this study, an acetate kinase (ACK)/acetyl phosphate system was used to supply ATP and combined with Escherichia coli-overexpressed CMP kinase (CMK), NDP kinase (NDK), choline phosphate cytidylyltransferase (CCT), and choline kinase (CKI) to produce CDP-choline from CMP and choline chloride. Within 1 h, 49 mM CDP-choline was produced, for a molar yield of 89...
October 13, 2016: Applied Microbiology and Biotechnology
Sondes Mechri, Mouna Ben Elhoul Berrouina, Maroua Omrane Benmrad, Nadia Zaraî Jaouadi, Hatem Rekik, Emna Moujehed, Alif Chebbi, Sami Sayadi, Mohamed Chamkha, Samir Bejar, Bassem Jaouadi
The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40-60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa...
October 5, 2016: International Journal of Biological Macromolecules
Fabián Rueda, Brigitte Gasser, Alejandro Sánchez-Chardi, Mònica Roldán, Sandra Villegas, Verena Puxbaum, Neus Ferrer-Miralles, Ugutz Unzueta, Esther Vázquez, Elena Garcia-Fruitós, Diethard Mattanovich, Antonio Villaverde
BACKGROUND: Bacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity...
October 1, 2016: Microbial Cell Factories
Danijela Gregurec, Susana Velasco-Lozano, Sergio E Moya, Luis Vázquez, Fernando López-Gallego
Optimal immobilization of enzymes on porous microbeads enables the fabrication of highly active and stable heterogeneous biocatalysts to implement biocatalysis in synthetic and analytical chemistry. However, empirical procedures for enzyme immobilization still prevail over rational ones because there is an unmet need for more comprehensive characterization techniques that aid to understand and trace the immobilization process. Here, we present the use of atomic force spectroscopy (AFS) as an innovative solution to indirectly characterize immobilized proteins on porous materials and monitor the immobilization process in real time...
October 6, 2016: Soft Matter
Zewang Guo, Xihua Zhao, Yuanzhi He, Tianxing Yang, Huifang Gao, Ganxi Li, Feixue Chen, Meijing Sun, Jung-Kul Lee, Liaoyuan Zhang
Acetoin (AC) is a volatile platform compound with various potential industrial applications. AC contains two stereoisomeric forms: (3S)-AC and (3R)-AC. Optically pure AC is important potential intermediate and widely used as a precursor to synthesize novel optically materials. In this study, chiral (3R)-AC production from meso-2,3-butanediol (meso-2,3-BD) was obtained using recombinant Escherichia coli cells co-expressing meso-2,3-butanediol dehydrogenase (meso-2,3-BDH), NADH oxidase (NOX) and hemoglobin protein (VHB) from Serratia sp...
October 6, 2016: Journal of Microbiology and Biotechnology
Joshua G Gober, Eric M Brustad
Despite increasing interest in using enzymes as tools for synthesis, many reactions discovered through the creativity of synthetic chemists remain beyond the scope of biocatalysis. This vacancy in the field has compelled researchers to develop strategies to adapt protein scaffolds for new reactivity. Heme proteins have recently been shown to activate synthetic precursors to generate reactive metallocarbenoid and metallonitrenoid species that enable the biosynthetic construction of novel C-C, C-N, and other bonds using mechanisms not previously explored by Nature...
September 30, 2016: Current Opinion in Chemical Biology
Osama El-Sepelgy, Nurtalya Alandini, Magnus Rueping
A dual catalytic iron/lipase system has been developed and applied in the dynamic kinetic resolution of benzylic and aliphatic secondary alcohols. A detailed study of the Knölker-type iron complexes demonstrated the hydrogen autotransfer of alcohols to proceed under mild reaction conditions and allowed the combination with the enzymatic resolution. Different racemic alcohols were efficiently converted to chiral acetates in good yields and with excellent enantioselectivities.
October 17, 2016: Angewandte Chemie
Yeongseon Jang, Julie A Champion
Proteins are potent molecules that can be used as therapeutics, sensors, and biocatalysts with many advantages over small-molecule counterparts due to the specificity of their activity based on their amino acid sequence and folded three-dimensional structure. However, they also have significant limitations in their stability, localization, and recovery when used in soluble form. These opportunities and challenges have motivated the creation of materials from such functional proteins in order to protect and present them in a way that enhances their function...
October 18, 2016: Accounts of Chemical Research
Søren Heintz, Tim Börner, Rolf H Ringborg, Gustav Rehn, Carl Grey, Mathias Nordblad, Ulrich Krühne, Krist V Gernaey, Patrick Adlercreutz, John M Woodley
An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs)...
September 26, 2016: Biotechnology and Bioengineering
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