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Rashida Akter, Damien W M Arrigan
A label-free electrochemical strategy for the detection of a cancer biomarker, prostate specific membrane antigen (PSMA), at picomolar concentrations without the use of antibodies, was investigated. The approach is based on the assisted ion transfer of protons, generated by a series of enzymatic reactions, at an array of microinterfaces between two immiscible electrolyte solutions (μ-ITIES). This nonredox electrochemical approach based on biocatalysis-coupled proton transfer at the μ-ITIES array opens a new way to detect the prostate cancer biomarker, with detection capability achieved at concentrations below those indicative of disease presence...
December 6, 2016: Analytical Chemistry
Qi Sun, Briana Aguila, Jason Perman, Nicholas Nguyen, Shengqian Ma
The combination of two or more reactive centers working in concert on a substrate to facilitate the reaction is now considered state of the art in catalysis, yet there still remains a tremendous challenge. Few heterogeneous systems of this sort have been exploited, as the active sites spatially separated within the rigid framework are usually difficult to cooperate. It is now shown that this roadblock can be surpassed. The underlying principle of the strategy presented here is the integration of catalytic components with excellent flexibility and porous heterogeneous catalysts, as demonstrated by the placement of linear ionic polymers in close proximity to surface Lewis acid active sites anchored on the walls of a covalent organic framework (COF)...
December 7, 2016: Journal of the American Chemical Society
O K L Rapheeha, M P Roux-van der Merwe, J Badenhorst, V Chhiba, M L Bode, K Mathiba, D Brady
AIMS: The aim of the current study was to explore the bacterial soil diversity for nitrile biocatalysts, in particular those for hydrolysis of β-substituted nitriles, to the corresponding carboxamides and acids that may be incorporated into peptidomimetics. To achieve this we needed to compare the efficiency of isolation methods and determine the influence of land use and geographical origin of the soil sample. METHODS AND RESULTS: Nitrile-utilizing bacteria were isolated from various soil environments across a thousand kilometre long transect of South Africa, including agricultural soil, a gold mine tailings dam, and uncultivated soil...
December 8, 2016: Journal of Applied Microbiology
Johannes Döbber, Martina Pohl
Easy, fast and gentle immobilization for the efficient reuse of important biocatalysts is highly demanded. We used the commercially available HaloTag™ technology (Promega), so far relatively unknown in the context of biocatalysis, to immobilize the benzaldehyde lyase from P. fluorescence (PfBAL). Immobilization mediated by this fusion tag proceeds rapidly within minutes from crude extracts yielding covalently attached enzymes in high purity, making expensive and laborious previous chromatographic purification steps obsolete, which strongly reduces the costs for biocatalyst immobilization...
December 3, 2016: Journal of Biotechnology
Souvik Basak, Sumanta Kumar Ghosh, Vinay Deep Punetha, Ashish N Aphale, Prabir K Patra, Nanda Gopal Sahoo
A carbonyl reductase (cr) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regenerating E. coli host to convert Ethyl-4-chloro-3-oxobutanoate (COBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363mM with highest turnover number (112.77±3.95s(-1)). Solitary recombineering of such catalytic cell reproduced CHBE 161.04g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp (crp*, F136I) into this heterologous E...
December 3, 2016: International Journal of Biological Macromolecules
A Pinsolle, F Charmantray, L Hecquet, F Sarrazin
We present a continuous-flow reactor at the millifluidic scale coupled with an online, non-intrusive spectroscopic monitoring method for determining the kinetic parameters of an enzyme, transketolase (TK) used in biocatalysis for the synthesis of polyols by carboligation. The millifluidic system used is based on droplet flow, a well-established method for kinetic chemical data acquisition. The TK assay is based on the direct quantitative measurement of bicarbonate ions released during the transketolase-catalysed reaction in the presence of hydroxypyruvic acid as the donor, thanks to an irreversible reaction: bicarbonate ions react with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase as the first auxiliary enzyme...
November 2016: Biomicrofluidics
Santosh Kumar Padhi
Hydroxynitrile lyases (HNLs) have grown in importance from laboratory to industry due to their potential to catalyze stereoselective C-C bond-formation reactions in the synthesis of several chiral intermediates, such as enantiopure α-cyanohydrins, β-nitro alcohols, and their derivatives with multiple functional groups. With these wide applications, the demand for finding new HNLs has increased, and this has led to exploration not only of new HNLs but also of new ways to discover them. An exclusive review article on HNLs by Asano et al...
November 29, 2016: Chembiochem: a European Journal of Chemical Biology
Marvin Kadisch, Mattijs K Julsing, Manfred Schrewe, Nico Jehmlich, Benjamin Scheer, Martin von Bergen, Andreas Schmid, Bruno Bühler
It is a common misconception in whole-cell biocatalysis to refer to an enzyme as the biocatalyst, thereby neglecting the structural and metabolic framework provided by the cell. Here, the low whole-cell biocatalyst stability, that is, the stability of specific biocatalyst activity, in a process for the terminal oxyfunctionalization of renewable fatty acid methyl esters was investigated. This reaction, which is difficult to achieve by chemical means, is catalyzed by Escherichia coli featuring the monooxygenase system AlkBGT and the uptake facilitator AlkL from Pseudomonas putida GPo1...
November 7, 2016: Biotechnology and Bioengineering
Hao Liu, Xing Wu, Jianliang Sun, Shicheng Chen
It has been well known that laccases can directly or indirectly catalyze oxidation of a broad species of phenols, amines and many other electron acceptors. However, laccases as biocatalyst in "green" ionic liquids (ILs) media instead of conventional solvents are less known and regarded as an innovative research direction. The presence of ILs can either inhibit or stimulate laccase activity, strongly depending on water-miscibility and kosmotropic natures of ILs. In addition, enzyme source, mediator, pH as well as water content are very important factors which influence laccase activity and stability...
November 22, 2016: Current Protein & Peptide Science
Christiaan C Barron, Brandon J D Sponagle, Pugazhendhi Arivalagan, Godwin B D'Cunha
Phenylalanine ammonia lyase (E.C., PAL) activity of Rhodotorula glutinis yeast has been demonstrated in four commonly used ionic liquids. PAL forward reaction was carried out in 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO4]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium lactate ([BMIM][lactate]). Our experiments have revealed that PAL is catalytically active in ionic liquids and the enzyme activity in ([BMIM][PF6]) is comparable to that obtained in aqueous buffer medium...
January 2017: Enzyme and Microbial Technology
Junwei Liu, Rimao Hua, Pei Lv, Jun Tang, Yi Wang, Haiqun Cao, Xiangwei Wu, Qing X Li
s-Triazine herbicides have been widely used in recent decades and caused serious concern over contamination of groundwater, surface water and soil. A novel bacterial strain JW-1 was isolated from activated sludge and identified as Leucobacter sp. based on comparative morphology, physiological characteristics and comparison of the 16S rDNA gene sequence. JW-1 was capable of using methylthio-s-triazine prometryn as a sole source of carbon and energy in pure culture. Favorable conditions for prometryn degradation were found at pH7...
November 17, 2016: Science of the Total Environment
Abid H Tanzil, Sujala T Sultana, Steven R Saunders, Liang Shi, Enrico Marsili, Haluk Beyenal
The biological synthesis of nanoparticles (NPs) by bacteria and biofilms via extracellular redox reactions has received attention because of the minimization of harmful chemicals, low cost, and ease of culturing and downstream processing. Bioreduction mechanisms vary across bacteria and growth conditions, which leads to various sizes and shapes of biosynthesized NPs. NP synthesis in biofilms offers additional advantages, such as higher biomass concentrations and larger surface areas, which can lead to more efficient and scalable biosynthesis...
December 2016: Enzyme and Microbial Technology
P Dydio, H M Key, A Nazarenko, J Y-E Rha, V Seyedkazemi, D S Clark, J F Hartwig
Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C-H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours(-1) turnover frequency...
October 7, 2016: Science
Thomas Heine, Kathryn Tucker, Nonye Okonkwo, Berhanegebriel Assefa, Catleen Conrad, Anika Scholtissek, Michael Schlömann, George Gassner, Dirk Tischler
The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation...
November 9, 2016: Applied Biochemistry and Biotechnology
Joyeeta Mukherjee, Munishwar Nath Gupta
Protein aggregation is implicated in diverse biochemical phenomena which include formation of inclusion bodies and amyloids. In recent years, inclusion bodies of many enzymes have been found to be catalytically active. Enzyme precipitates and their crystalline aggregates have found extensive applications in Biocatalysis in low water media. Protein aggregates also play a useful role in processed food. Enzymes are incorporated in detergents in the form of granulates. This review also looks at the various techniques which are used for characterizing protein aggregates...
November 5, 2016: International Journal of Biological Macromolecules
Paul Quehl, Jan Schüürmann, Joel Hollender, Joachim Jose
Anchorage of recombinant proteins onto the outer membrane of gram-negative bacteria is an attractive solution for protein library screening and whole cell biocatalysis if a membrane environment is required or mass transfer into the cell is limiting. Autotransporters have been successfully applied for surface display of various heterologous proteins. Still, many underlying parameters for achieving active enzymes are not known. Here, we systematically tested different linkers between passenger and the membrane embedded β-barrel of the autotransporter...
November 1, 2016: Biochimica et Biophysica Acta
Adrian Romero-Rivera, Marc Garcia-Borràs, Sílvia Osuna
Biocatalysis is based on the application of natural catalysts for new purposes, for which enzymes were not designed. Although the first examples of biocatalysis were reported more than a century ago, biocatalysis was revolutionized after the discovery of an in vitro version of Darwinian evolution called Directed Evolution (DE). Despite the recent advances in the field, major challenges remain to be addressed. Currently, the best experimental approach consists of creating multiple mutations simultaneously while limiting the choices using statistical methods...
November 4, 2016: Chemical Communications: Chem Comm
Hui Chen, Zhiguang Zhu, Rui Huang, Yi-Heng Percival Zhang
Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP(+) to NAD(+). Through amino acid-sequence alignment of NADP(+)- and NAD(+)-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP(+) were identified...
November 2, 2016: Scientific Reports
J Porter Hunt, Seung Ook Yang, Kristen M Wilding, Bradley C Bundy
Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis...
October 28, 2016: Bioengineered
Haidong Peng, Erman Wei, Jiali Wang, Yanan Zhang, Lin Cheng, Hongmin Ma, Zixin Deng, Xudong Qu
Piperidine and indolizidine are two basic units of alkaloids that are frequently observed in natural and synthetic compounds. Their biosynthesis in natural products is highly conserved and mostly derived from the incorporation of lysine cyclization products. Through in vitro reconstitution, we herein identified a novel pathway involving a group of polyketide-derived indolizidines, which comprises the processes of tandem two-electron thioester reduction, transamination, and imine reduction to convert acyl carrier protein (ACP)-tethered polyketide chains into the piperidine moieties of their indolizidine scaffolds...
November 3, 2016: ACS Chemical Biology
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