1,721,094 research outputs found
A comprehensive practical laboratory course on protein engineering: Evolution of a glycine oxidase variant active on the herbicide glyphosate
No full textProtein engineering represents a modern approach to generate novel proteins for the different fields of biotechnology. Here, we report about an 8-day laboratory activity in which students generate enzyme variants to degrade the herbicide glyphosate. The students conduct a true research experiment in an important field (bioremediation by novel, engineered enzymes) and are introduced to widely used techniques in molecular biology and protein biochemistry laboratories. Based on a docking analysis of glycine (the original substrate) and of glyphosate into the active site of glycine oxidase, residues putatively involved in substrate selectivity are identified that will become the target of site-saturation mutagenesis. Each group of students focuses on the library generated at one position and selects the most active variant based on colorimetric screening. Following protein overexpression in Escherichia coli, the selected glycine oxidase variants are purified and their kinetic properties on glycine and glyphosate assessed. The best variant identified by the whole class is then used for detecting the herbicide in water. With the help of the professor, students can improve technical skills, ability to evaluate results, team work activity, and critical thinking. (c) 2019 International Union of Biochemistry and Molecular Biology, 2019
PEG-DAAO conjugate: A promising tool for cancer therapy optimized by protein engineering
No full textThe flavoenzyme D-amino acid oxidase (DAAO) represents a potentially good option for cancer enzyme prodrug therapy as it produces H2O2 using D-amino acids as substrates, compounds present at low concentration in vivo and that can be safely administered to regulate H2O2 production. We optimized the cytotoxicity of the treatment by: i) using an efficient enzyme variant active at low O2 and D-alanine concentrations (mDAAO); ii) improving the stability and half-life of mDAAO and the enhanced permeability and retention effect by PEGylation; and iii) inhibiting the antioxidant cellular system by a heme oxygenase-1 inhibitor (ZnPP). A very low amount of PEG-mDAAO (10 mU, 50 ng of enzyme) induces cytotoxicity on various tumor cell lines. Notably, PEG-mDAAO seems well suited for in vivo evaluation as it shows the same cytotoxicity at air saturation (21%) and 2.5% O2, a condition resembling the microenvironment found in the central part of tumors
AN ENZYMATIC TOOL-BOX FOR LIGNIN OXIDATION/DEGRADATION
No full textLignin is an amorphous polymer characterized by a wide range of molecular mass components, a disordered and branched three-dimensional structure, insoluble in water and in most common solvents. In order to perform lignin degradation, enzymatic treatment could represent an environmentally friendly alternative to chemical methods[1].
The main purpose of this work was to develop an “enzymatic tool-box” for an efficient oxidation and degradation of lignin into aromatic monomers. Biochemical properties of a number of commercial and recombinant ligninolytic oxidative enzymes (laccases, Mn peroxidases and lignin peroxidases) were evaluated under identical experimental conditions, with the final goal to identify interesting biocatalysts for lignin degradation[2]. The effect of pH, temperature, NaCl, DMSO and Tween-80 on the enzymatic activity has been investigated. The activity of novel enzymes, such as the membrane-bound polyphenol oxidase from the marine bacterium Marinomonas mediterranea[3] and a peroxidase produced by Nonomuraea gerenzanensis, was also evaluated[4].
A new high-throughput colorimetric screening to assay the oxidation/degradation of lignin by different enzymes was developed: this method facilitates the identification of optimal conditions for a lignin treatment based on the combined use of various laccases and peroxidases[5]. On this side, coupling the colorimetric assay with a size-exclusion chromatography analysis allows to identify changes in lignin molecular mass distribution due to enzymatic treatment. Notably, the enzymatic tool-box also comprises etherases, cathecol oxidase and demethylase activities. Finally, a chemo-enzymatic process to depolymerise lignin was carried out on lignin linkage model compounds and technical lignins.
Altogether, the combination of chemical and enzymatic approaches could represent an innovative and feasible way for valorisation of lignin under mild conditions.
This work was done as part of Biorefill (ID42611813) and ValorPlus (no FP7-KBBE-2013-7-613802) projects.
[1] Pollegioni, L.; Tonin, F.; Rosini, E. FEBS Journal 2015, 282(7), 1190-1213.
[2] Tonin, F.; Melis, R.; Cordes, A.; Sanchez-Amat, A.; Pollegioni, L.; Rosini, E. New Biotechnology 2016,
33(3), 387-398.
[3] Tonin, F.; Rosini, E.; Piubelli, L.; Sanchez-Amat, A.; Pollegioni, L. Protein Expression and Purification
2016, (123), 60-69.
[4] Casciello, C.; Tonin, F.; Berini, F.; Fasoli, E.; Marinelli, F.; Pollegioni, L.; Rosini, E. Biotechnology Re- ports 2017, (13), 49-57.
[5] Tonin, F.; Vignali, E.; Pollegioni, L.; D’Arrigo, P.; Rosini, E. Enzyme and Microbial Technology 2017,
(96), 143-150
Reactive oxygen species as a double-edged sword: The role of oxidative enzymes in antitumor therapy
No full textA number of approaches have been developed over the years to manage cancer, such as chemotherapy using low-molecular-mass molecules and radiotherapy. Here, enzymes can also find useful applications. Among them, oxidases have attracted attention because of their ability to produce reactive oxygen species (ROS, especially hydrogen peroxide) in tumors and potentially modulate the production of this cytotoxic compound when enzymes active on substrates present in low amounts are used, such as the d-amino acid oxidase and d-amino acid couple system. These treatments have been also developed for additional cancer treatment approaches, such as phototherapy, nutrient starvation, and metal-induced hydroxyl radical production. In addition, to improve tumor specificity and decrease undesired side effects, oxidases have been targeted by means of nanotechnologies and protein engineering (i.e., by designing chimeric proteins able to accumulate in the tumor). The most recent advances obtained by using six different oxidases (i.e., the FAD-containing enzymes glucose oxidase, d- and l-amino acid oxidases, cholesterol oxidase and xanthine oxidase, and the copper-containing amine oxidase) have been reported. Anticancer therapy based on oxidase-based ROS production has now reached maturity and can be applied in the clinic
Advances in enzymatic synthesis of D-amino acids
No full textIn nature, the D-enantiomers of amino acids (D-AAs) are not used for protein synthesis and during evolution acquired specific and relevant physiological functions in different organisms. This is the reason for the surge in interest and investigations on these “unnatural” molecules observed in recent years. D-AAs are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. In past years, a number of methods have been devised to produce D-AAs based on enantioselective enzymes. With the aim to increase the D-AA derivatives generated, to improve the intrinsic atomic economy and cost-effectiveness, and to generate processes at low environmental impact, recent studies focused on identification, engineering and application of enzymes in novel biocatalytic processes. The aim of this review is to report the advances in synthesis of D-AAs gathered in the past few years based on five main classes of enzymes. These enzymes have been combined and thus applied to multi-enzymatic processes representing in vitro pathways of alternative/exchangeable enzymes that allow the generation of an artificial metabolism for D-AAs synthetic purposes
Analytical methods for the investigation of enzyme-catalyzed degradation of polyethylene terephthalate
Full text linkThe polyester PET (poly(ethylene terephthalate)) plastic is chemically inert and remarkably persistent, posing relevant and global pollution concerns due to its accumulation in ecosystems across the globe. In past years, research focused on identifying bacteria active on PET and on the specific enzymes responsible for its degradation. Here, the enzymatic degradation of PET can be considered as an ‘erosion process’ that takes place on the surface of an insoluble material and results in an unusual, substrate-limited kinetic condition. In this review, we report on the most suitable models to evaluate the kinetics of PET-hydrolyzing enzymes, which takes into consideration the amount of enzyme adsorbed on the substrate, the enzyme-accessible ester bonds, and the product inhibition effects. Careful kinetic analysis is especially relevant to compare enzymes from different sources and evolved variants generated by protein engineering studies as well. Furthermore, the analytical methods most suitable to screen natural bacteria and recombinant variant libraries generated by protein engineering have been also reported. These methods rely on different detection systems and are performed both on model compounds and on different PET samples (e.g., nanoparticles, microparticles, and waste products). All this meaningful information represents an optimal starting point and boosts the process of identifying systems able to biologically recycle PET waste products
Whole-Cell Bioconversion of Renewable Biomasses-Related Aromatics to cis,cis-Muconic Acid
Full text linkLignin and wheat bran represent renewable feedstocks for generation of useful and value-added compounds such as vanillin (a popular flavoring agent) and cis,cis-muconic acid (ccMA, a building block for the synthesis of plastic materials). In the present work, we report on the setup of an efficient and green process for producing such valuable compounds based on (a) the optimization of the extraction procedures for vanillin from lignin and ferulic acid from wheat bran and (b) the genetic engineering of an Escherichia coli strain with up to three plasmids differing in copy numbers to modulate the expression of up to seven recombinant enzymes. In detail, we used two sequential reactions catalyzed by the decarboxylase Fdc and the dioxygenase Ado to convert wheat bran-derived ferulic acid into vanillin: nature-identical vanillin was produced in one pot with a >85% yield in 20 h. Next, the dehydrogenase LigV, the demethylase VanAB, the decarboxylase AroY, and the dioxygenase C12O converted lignin-derived vanillin into ccMA with a >95% conversion yield and a productivity of 4.2 mg of ccMA/g of Kraft lignin in 30 min. Finally, when the optimized E. coli strain expressing all the abovementioned enzymes was used, ccMA was produced with a >95% conversion yield starting from ferulic acid in 10 h following product isolation, corresponding to 0.73 g of ccMA/g of ferulic acid, 1.4 g of ccMA/L, and 2.2 g of ccMA/g of wheat bran biomass. The optimized whole-cell system represents a sustainable and cost-competitive process for producing high value-added products from renewable resources
Substitution of arginine 120 in human D-amino acid oxidase favors FAD-binding and nuclear mistargeting
Full text linkThe peroxisomal enzyme human D-amino acid oxidase (hDAAO) is attracting attention owing to its role in degrading D-serine, the main co-agonist of N-methyl D-aspartate receptors in brain, and its involvement in brain functions and diseases. Here, we focused on arginine 120, a residue located at the protein interface, 20 Å from the assumed second ligand-binding site, showing a different orientation of the side chain in the hDAAO-benzoate complex, and corresponding to Ser119 in rat DAAO, which is part of a putative nuclear translocation signal (NTS). By substituting Arg120 in hDAAO with a glutamate (to mimic the active NTS) or a leucine (to eliminate the positive charge) the protein conformation, thermal stability, and kinetic properties are slightly altered, while the dimeric structure and the ligand-binding properties are unchanged. The most relevant alteration in Arg120 variants is the strongest interaction with FAD. Nevertheless, the activity assayed at low D-serine and FAD concentrations (resembling physiological conditions) was quite similar for wild-type and Arg120 hDAAO variants. These results resemble the ones obtained substituting another residue located at the interface region (i.e., the W209R variant), indicating that substitutions at the monomer-monomer interface mainly affects the FAD binding in hDAAO. Indeed, U87 glioblastoma cells transiently transfected for hDAAO variants show that substitution of Arg120 favors mistargeting: the increase in cytosolic localization observed for the variants promotes nuclear targeting, especially for the R120E hDAAO, without affecting cell viability. Notably, mistargeting to the nucleus is an innate process as it is apparent for the wild-type hDAAO, too: whether such a process is related to specific pathologic processes is still unknown
Biosensors for D-Amino Acids: Detection Methods and Applications
No full textD-enantiomers of amino acids (D-AAs) are only present in low amounts in nature, frequently at trace levels, and for this reason, their biological function was undervalued for a long time. In the past 25 years, the improvements in analytical methods, such as gas chromatography, HPLC, and capillary electrophoresis, allowed to detect D-AAs in foodstuffs and biological samples and to attribute them specific biological functions in mammals. These methods are time-consuming, expensive, and not suitable for online application; however, life science investigations and industrial applications require rapid and selective determination of D-AAs, as only biosensors can offer. In the present review, we provide a status update concerning biosensors for detecting and quantifying D-AAs and their applications for safety and quality of foods, human health, and neurological research. The review reports the main challenges in the field, such as selectivity, in order to distinguish the different D-AAs present in a solution, the simultaneous assay of both L- and D-AAs, the production of implantable devices, and surface-scanning biosensors. These innovative tools will push future research aimed at investigating the neurological role of D-AAs, a vibrant field that is growing at an accelerating pace
Determination of D-amino acids using a D-amino acid oxidase biosensor with spectrophotometric and potentiometric detection
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