catalyst engineering
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Enzymes are the natural catalysts that execute biochemical reactions upholding life. Their natural effectiveness has been fine-tuned as a result of millions of years of natural evolution. Such catalytic effectiveness has prompted the use of biocatalysts from multiple sources on different applications, including the industrial production of goods (food and beverages, detergents, textile, and pharmaceutics), environmental protection, and biomedical applications. Natural enzymes often need to be improved by protein engineering to optimize their function in non-native environments. Recent technological advances have greatly facilitated this process by providing the experimental approaches of directed evolution or by enabling computer-assisted applications. Directed evolution mimics the natural selection process in a highly accelerated fashion at the expense of arduous laboratory work and economic resources. Theoretical methods provide predictions and represent an attractive complement to such experiments by waiving their inherent costs. Computational techniques can be used to engineer enzymatic reactivity, substrate specificity and ligand binding, access pathways and ligand transport, and global properties like protein stability, solubility, and flexibility. Theoretical approaches can also identify hotspots on the protein sequence for mutagenesis and predict suitable alternatives for selected positions with expected outcomes. This review covers the latest advances in computational methods for enzyme engineering and presents many successful case studies.
Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and α4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (ΔTm = -5 to 6 °C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear β-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties.
- Publikační typ
- časopisecké články MeSH
Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + I211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4'-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L + I211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.
- Publikační typ
- časopisecké články MeSH
Enzymes are efficient and specific catalysts for many essential reactions in biotechnological and pharmaceutical industries. Many times, the natural enzymes do not display the catalytic efficiency, stability or specificity required for these industrial processes. The current enzyme engineering methods offer solutions to this problem, but they mainly target the buried active site where the chemical reaction takes place. Despite being many times ignored, the tunnels and channels connecting the environment with the active site are equally important for the catalytic properties of enzymes. Changes in the enzymatic tunnels and channels affect enzyme activity, specificity, promiscuity, enantioselectivity and stability. This review provides an overview of the emerging field of enzyme access tunnel engineering with case studies describing design of all the aforementioned properties. The software tools for the analysis of geometry and function of the enzymatic tunnels and channels and for the rational design of tunnel modifications will also be discussed. The combination of new software tools and enzyme engineering strategies will provide enzymes with access tunnels and channels specifically tailored for individual industrial processes.
Fabrication of porous and biologically inspired biomaterials that mimic the formation of microstructural structures of nacre in the form of calcite (CaCO3) and evaluation of the biocompatibility of such organic-inorganic composite scaffold for bone tissue engineering, are focus of this paper. Nacre's self-assembly characteristics are concerned about the development of calcite filled biomineralized scaffold following the nature based biomineralization process and biomimetic applications. The PVP-CMC hydrogel film, comprised of PVP:0.2, CMC:0.8, PEG:1.0, Agar:2.0, Glycerene:1.0 and water:95.0 w/v%; acts as catalyst and template for the nucleation and growth of the inorganic CaCO3 within the scaffold. The PVP-CMC hydrogel (in the dry state) was immersed in ionic solutions (g/100 ml) of Na2CO3 and CaCl2·H2O in different concentrations sets i.e. Set-1: 10.50/14.70; Set-2: 5.25/7.35; Set-3: 4.20/5.88; Set-4: 2.10/2.94; Set-5: 1.05/1.47, Set-6: 0.55/0.55 for 90 min. As a result, "PVP-CMC-CaCO3" hydrogel scaffold was fabricated having bio-inspired structural and functional properties. Cell proliferation and cell viability were examined until 7 days in the presence of "PVP-CMC-CaCO3" scaffolds using permanent cell lines MG63 (human osteosarcoma), L929 (murine fibroblasts) as well as cultures from mouse bone explants (CC-MBE), confirmed that the said hydrogel scaffolds are biocompatible. But, from mechanical strength as well as biocompatibility point of view, scaffolds prepared in Set-1 to 3 ionic solutions were superior. In conclusion, these three calcite filled hydrogel scaffolds are recommended and can be used for osseointegration.
- MeSH
- biokompatibilní materiály chemie MeSH
- buněčné linie MeSH
- difrakce rentgenového záření MeSH
- hydrogely chemie MeSH
- mikroskopie elektronová rastrovací MeSH
- myši MeSH
- osteointegrace fyziologie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- viabilita buněk fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Zeolites are microporous tectosilicates of natural or synthetic origin, which have been extensively used in various technological applications, e.g. as catalysts and as molecular sieves, for separating and sorting various molecules, for water and air purification, including removal of radioactive contaminants, for harvesting waste heat and solar heat energy, for adsorption refrigeration, as detergents, etc. These applications of zeolites were typically related with their porous character, their high adsorption capacity, and their ion exchange properties. This review is focused on potential or already practically implemented applications of zeolites in biotechnology and medicine. Zeolites are promising for environment protection, detoxication of animal and human organisms, improvement of the nutrition status and immunity of farm animals, separation of various biomolecules and cells, construction of biosensors and detection of biomarkers of various diseases, controlled drug and gene delivery, radical scavenging, and particularly tissue engineering and biomaterial coating. As components of scaffolds for bone tissue engineering, zeolites can deliver oxygen to cells, can stimulate osteogenic cell differentiation, and can inhibit bone resorption. Zeolites can also act as oxygen reservoirs, and can improve cell performance in vascular and skin tissue engineering and wound healing. When deposited on metallic materials for bone implantation, zeolite films showed anticorrosion effects, and improved the osseointegration of these implants. In our studies, silicalite-1 films deposited on silicon or stainless steel substrates improved the adhesion, growth, viability and osteogenic differentiation of human osteoblast-like Saos-2 cells. Zeolites have been clinically used as components of haemostatics, e.g. in the Advanced Clotting Sponge, as gastroprotective drugs, e.g. Absorbatox® 2.4D, or as antioxidative agents (Klinobind®). Some zeolites are highly cytotoxic and carcinogenic, e.g. erionite. However, in other zeolites, the antiproliferative and pro-apoptotic effects can be used for tumor therapy.
- MeSH
- biotechnologie metody MeSH
- cytostatické látky chemie farmakologie terapeutické užití MeSH
- lidé MeSH
- protinádorové látky chemie farmakologie terapeutické užití MeSH
- zachování přírodních zdrojů metody MeSH
- zeolity chemie farmakologie terapeutické užití toxicita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
A significant enhancement in the catalytic performance due to enzymes immobilization is a great way to enhance the economics of biocatalytic processes. The soybean peroxidase (SP) immobilization under ferroxyte and the ferulic acid removal by the enzyme free and immobilized were investigated. The immobilization via silica-coated ferroxyte nanoparticles was effective, and immobilization yield of 39%. The scanning electron microscopy (SEM) images showed significant changes in the materials morphology. Substantial differences were observed in the particles' Fourier Transform Infrared (FTIR) spectra. The magnetic catalyst revealed a better performance than the free enzyme in the ferulic acid conversion, presenting a good V max/K m ratio when compared with the free enzyme. The reuse evaluated by ten cycles exhibited excellent recycling, remaining constant between the sixth and seventh cycles. The use of magnetic nanocatalyst becomes possible to eliminate the high operational costs, and complicated steps of the conventional enzymatic processes. Thus, a viable industrial route for the use of the enzyme as catalyst is possible.
The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.
- MeSH
- aminohydrolasy genetika metabolismus MeSH
- Bacteria enzymologie genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- biodegradace MeSH
- biokatalýza MeSH
- databáze proteinů MeSH
- fungální proteiny genetika metabolismus MeSH
- houby enzymologie genetika MeSH
- metagenomika MeSH
- nitrily metabolismus MeSH
- proteinové inženýrství metody MeSH
- rekombinantní proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Smart materials of biological origin are attracting a lot of attention nowadays, especially as catalysts, carriers or adsorbents. Among them, magnetically modified biomaterials are especially important due to their response to external magnetic field. This report demonstrates that naturally occurring micrometer sized, high aspect ratio material (native and autoclaved Leptothrix sp. sheaths) efficiently bind synthetically prepared magnetite and maghemite nanoparticles and their aggregates. Magnetic modification of Leptothrix sheaths enables to prepare a promising material for advanced biotechnology and environmental technology applications. The prepared magnetically responsive sheaths were tested as inexpensive adsorbent for crystal violet removal from aqueous solutions. The observed maximum adsorption capacity was 243.1mg of dye per 1g of adsorbent.
- MeSH
- genciánová violeť chemie MeSH
- Leptothrix chemie MeSH
- magnetické nanočástice chemie MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: In recent years, enzymes modifying N-acetylhexosamine substrates have emerged in numerous theoretical studies as well as practical applications from biology, biomedicine, and biotechnology. Advanced enzyme engineering techniques converted them into potent synthetic instruments affording a variety of valuable glycosides. SCOPE OF REVIEW: This review presents the diversity of engineered enzymes active with N-acetylhexosamine carbohydrates: from popular glycoside hydrolases and glycosyltransferases to less known oxidases, epimerases, kinases, sulfotransferases, and acetylases. Though hydrolases in natura, engineered chitinases, β-N-acetylhexosaminidases, and endo-β-N-acetylglucosaminidases were successfully employed in the synthesis of defined natural and derivatized chitooligomers and in the remodeling of N-glycosylation patterns of therapeutic antibodies. The genes of various N-acetylhexosaminyltransferases were cloned into metabolically engineered microorganisms for producing human milk oligosaccharides, Lewis X structures, and human-like glycoproteins. Moreover, mutant N-acetylhexosamine-active glycosyltransferases were applied, e.g., in the construction of glycomimetics and complex glycostructures, industrial production of low-lactose milk, and metabolic labeling of glycans. In the synthesis of biotechnologically important compounds, several innovative glycoengineered systems are presented for an efficient bioproduction of GlcNAc, UDP-GlcNAc, N-acetylneuraminic acid, and of defined glycosaminoglycans. MAJOR CONCLUSIONS: The above examples demonstrate that engineering of N-acetylhexosamine-active enzymes was able to solve complex issues such as synthesis of tailored human-like glycoproteins or industrial-scale production of desired oligosaccharides. Due to the specific catalytic mechanism, mutagenesis of these catalysts was often realized through rational solutions. GENERAL SIGNIFICANCE: Specific N-acetylhexosamine glycosylation is crucial in biological, biomedical and biotechnological applications and a good understanding of its details opens new possibilities in this fast developing area of glycoscience.
- MeSH
- glykoproteiny biosyntéza MeSH
- glykosidhydrolasy metabolismus MeSH
- glykosylace MeSH
- glykosyltransferasy metabolismus MeSH
- hexosaminy metabolismus MeSH
- katalýza MeSH
- oligosacharidy biosyntéza MeSH
- proteinové inženýrství * MeSH
- sulfotransferasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
