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enzyme engineering

Mahejibin Khan, Amit Kumar
The understanding of the 3-dimensional enzyme structure is important for the point of protein engineering and applications. Computer-based molecular modelling is a vital tool for theoretical predication of enzyme activities and finding their substrates and inhibitors. SMlipA lipase was cloned from forest soil metagenome and characterized as broad spectrum enzyme with high stability in various organic solvents. In the present study, to understand the mechanism of SMlipA lipase and to identify the key residues involved in enzyme-substrate interaction, three dimensional-computational model of SMlipA has been generated and validated for stereo-chemical and amino-acid environment quality using appropriate programs, and further validation of the active-site architecture was achieved by performing docking studies with different ligand...
October 14, 2016: Journal of Molecular Graphics & Modelling
Zhaoyue Wang, Xuejing Bai, Xuena Guo, Xiuping He
2-Phenylethanol (2-PE) is widely used in food, perfume and pharmaceutical industry, but lower production in microbes and less known regulatory mechanisms of 2-PE make further study necessary. In this study, crucial genes like ARO8 and ARO10 of Ehrlich pathway for 2-PE synthesis and key transcription factor ARO80 in Saccharomyces cerevisiae were re-regulated using constitutive promoter; in the meantime, the effect of nitrogen source in synthetic complete (SC) medium with L-phenylalanine (L-Phe) on Aro8/Aro9 and Aro10 was investigated...
October 21, 2016: Journal of Industrial Microbiology & Biotechnology
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
Durga Mahor, Anu Priyanka, Gandham S Prasad, Krishan Gopal Thakur
Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway...
2016: PloS One
Ioannis V Pavlidis, Martin S Weiß, Maika Genz, Paul Spurr, Steven P Hanlon, Beat Wirz, Hans Iding, Uwe T Bornscheuer
The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99...
November 2016: Nature Chemistry
Whitney D Hollinshead, Sarah Rodriguez, Hector Garcia Martin, George Wang, Edward E K Baidoo, Kenneth L Sale, Jay D Keasling, Aindrila Mukhopadhyay, Yinjie J Tang
BACKGROUND: Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism. RESULTS: Overexpression of edd and eda in E...
2016: Biotechnology for Biofuels
Alessandro Borri, Pasquale Palumbo, Abhyudai Singh
Synthetic biology combines different branches of biology and engineering aimed at designing synthetic biological circuits able to replicate emergent properties useful for the biotechnology industry, human health and environment. The role of negative feedback in noise propagation for a basic enzymatic reaction scheme is investigated. Two feedback control schemes on enzyme expression are considered: one from the final product of the pathway activity, the other from the enzyme accumulation. Both schemes are designed to provide the same steady-state average values of the involved players, in order to evaluate the feedback performances according to the same working mode...
October 2016: IET Systems Biology
Kwang Soo Shin, Sangwoo Kim, Sung Kuk Lee
BACKGROUND: Microbial production of oleochemicals has been actively studied in the last decade. Free fatty acids (FFAs) could be converted into a variety of molecules such as industrial products, consumer products, and fuels. FFAs have been produced in metabolically engineered Escherichia coli cells expressing a signal sequence-deficient acyl-CoA thioesterase I ('TesA). Nonetheless, increasing the expression level of 'TesA seems not to be an appropriate approach to scale up FFA production because a certain ratio of each component including fatty acid synthase and 'TesA is required for optimal production of FFAs...
2016: Biotechnology for Biofuels
Chaudhari Archana Somabhai, Ruma Raghuvanshi, G Nareshkumar
AIMS: To assess protective efficacy of genetically modified Escherichia coli Nissle 1917 (EcN) on metabolic effects induced by chronic consumption of dietary fructose. MATERIALS AND METHODS: EcN was genetically modified with fructose dehydrogenase (fdh) gene for conversion of fructose to 5-keto-D-fructose and mannitol-2-dehydrogenase (mtlK) gene for conversion to mannitol, a prebiotic. Charles foster rats weighing 150-200 g were fed with 20% fructose in drinking water for two months...
2016: PloS One
Grace Yim, Wenliang Wang, Maulik N Thaker, Stephanie Tan, Gerard D Wright
Modification of natural product backbones is a proven strategy for the development of clinically useful antibiotics. Such modifications have traditionally been achieved through medicinal chemistry strategies or via in vitro enzymatic activities. In an orthogonal approach, engineering of biosynthetic pathways using synthetic biology techniques can generate chemical diversity. Here we report the use of a minimal teicoplanin class glycopeptide antibiotic (GPA) scaffold expressed in a production-optimized Streptomyces coelicolor strain to expand GPA chemical diversity...
September 9, 2016: ACS Infectious Diseases
Heidi Stoll, Heidrun Steinle, Katharina Stang, Silju Kunnakattu, Lutz Scheideler, Bernd Neumann, Julia Kurz, Ilka Degenkolbe, Nadja Perle, Christian Schlensak, Hans Peter Wendel, Meltem Avci-Adali
Hemocompatibility and cytocompatibility of biomaterials codetermine the success of tissue engineering applications. DNA, the natural component of our cells, is an auspicious biomaterial for the generation of designable scaffolds with tailorable characteristics. In this study, a combination of rolling circle amplification and multiprimed chain amplification is used to generate hydrogels at centimeter scale consisting solely of DNA. Using an in vitro rotation model and fresh human blood, the reaction of the hemostatic system on DNA hydrogels is analyzed...
October 19, 2016: Macromolecular Bioscience
Dar-Chone Chow, Kacie Rice, Wanzhi Huang, Robert L Atmar, Timothy G Palzkill
The β-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A β-lactamases including the TEM-1 β-lactamase (Ki= 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 β-lactamase (Ki= 1.2 nM), which hydrolyzes virtually all clinically useful β-lactam antibiotics. The extent to which the specificity of a protein that binds a broad range of targets can be modified to display narrow specificity was explored in this study by engineering BLIP to bind selectively to KPC-2 β-lactamase...
October 18, 2016: ACS Infectious Diseases
Junli Zhang, Huanjiao Weng, Wenwen Ding, Zhen Kang
Five-Aminolevulinic acid (ALA), the universal precursor of all tetrapyrroles, has various applications in medicine and agriculture industries. Glutamyl-tRNA reductase (GluTR) as the first key enzyme of C5 pathway is feedback regulated by heme, and its N-terminus plays a critical role on its stability control. Here, the GluTR N-terminus was engineered by inserting different numbers of positively charged lysine and arginine residues. The results confirmed that insertion of lysine or arginine residues (especially one arginine residue) behind Thr2 significantly increased the stability of GluTR...
October 18, 2016: Bioengineered
Fabian B H Rehm, Shuxiong Chen, Bernd H A Rehm
Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts...
October 14, 2016: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Thavasyappan Thambi, V H Giang Phan, Doo Sung Lee
Stimuli-sensitive injectable polymeric hydrogels are one of the promising delivery vehicles for the controlled release of bioactive agents. In aqueous solutions, these polymers are able to switch sol-to-gel transitions in response to various stimuli including pH, temperature, light, enzyme and magnetic field. Therapeutic agents, including chemotherapeutic agents, protein drugs or cells, are easily mixed with the low-viscous polymer solution at room temperature. Therapeutic-agents-containing solutions are readily injected into target sites through syringe or catheter, which could form hydrogel depot and serve as bioactive molecules release carriers...
October 18, 2016: Macromolecular Rapid Communications
Shohei Hayashi, Yasuharu Satoh, Tetsuro Ujihara, Yusuke Takata, Tohru Dairi
In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 × ACPs...
October 18, 2016: Scientific Reports
Aymeric Goyer
Thiamin is essential for human health. While plants are the ultimate source of thiamin in most human diets, staple foods like white rice have low thiamin content. Therefore, populations whose diets are mainly based on low-thiamin staple crops suffer from thiamin deficiency. Biofortification of rice grain by engineering the thiamin biosynthesis pathway has recently been attempted, with up to 5-fold increase in thiamin content in unpolished seeds. However, polished seeds that retain only the starchy endosperm had similar thiamin content than that of non-engineered plants...
October 14, 2016: Current Opinion in Biotechnology
Khushboo Gualti, Krishna Mohan Poluri
BACKGROUND: Unraveling the comprehensive networks of molecular signaling in various cellular processes and redesign/rewire them as per human wish is the ultimate dream of the biomedical researchers. Recent advances in the experimental and computational biophysics have provided us with enormous amount of protein sequences and a wide variety of structural information. Protein engineering is a fledging field and a creative process to design the target proteins or signaling networks with desirable structure and functions...
October 13, 2016: Recent Patents on Biotechnology
Hao Meng, Yuan Liu, Bruce P Lee
: Mussel adhesive moiety, catechol, has been utilized to design a wide variety of biomaterials. However, the biocompatibility and biological responses associated with the byproducts generated during the curing process of catechol has never been characterized. An in situ curable polymer model system, 4-armed polyethylene glycol polymer end-capped with dopamine (PEG-D4), was used to characterize the production of hydrogen peroxide (H2O2) during the oxidative crosslinking of catechol. Although PEG-D4 cured rapidly (under 30s), catechol continues to polymerize over several hours to form a more densely crosslinked network over time...
October 12, 2016: Acta Biomaterialia
Benjamin W Arentson, Erin L Hayes, Weidong Zhu, Harkewal Singh, John J Tanner, Donald F Becker
Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase domains that catalyzes the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon-helix-helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability...
October 14, 2016: Bioscience Reports
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