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1.
Nat Commun ; 11(1): 4808, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968058

RESUMO

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.


Assuntos
Simulação por Computador , Evolução Molecular Direcionada/métodos , Enzimas/química , Evolução Química , Liases/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Enzimas/genética , Enzimas/metabolismo , Cinética , Liases/genética , Liases/metabolismo , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Engenharia de Proteínas
2.
Biomolecules ; 10(8)2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32752270

RESUMO

Posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubiquitin-like polypeptides regulate numerous cellular processes that are captured by viruses to promote infection, replication, and spreading. The importance of these protein modifications for the viral life cycle is underscored by the discovery that many viruses encode deconjugases that reverse their functions. The structural and functional characterization of these viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infections and the host's antiviral defense. Given the growing body of evidence demonstrating their key contribution to pathogenesis, the viral deconjugases are now recognized as attractive targets for the design of novel antiviral therapeutics.


Assuntos
Antivirais/farmacologia , Enzimas/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Viroses/metabolismo , Adenoviridae/enzimologia , Coronavirus/enzimologia , Enzimas/química , Herpesviridae/enzimologia , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Virais/química , Viroses/tratamento farmacológico
3.
Nat Commun ; 11(1): 4292, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855421

RESUMO

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.


Assuntos
Bioquímica/métodos , Butanóis/metabolismo , Enzimas/metabolismo , Acetolactato Sintase/química , Acetolactato Sintase/metabolismo , Trifosfato de Adenosina , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Bioquímica/instrumentação , Reatores Biológicos , Sistema Livre de Células , Evolução Molecular Direcionada , Enzimas/química , Enzimas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Temperatura , Termodinâmica
4.
Nat Commun ; 11(1): 2903, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518257

RESUMO

Direct transfer of protons and electrons between two tandem reactions is still a great challenge, because overall reaction kinetics is seriously affected by diffusion rate of the proton and electron carriers. We herein report a host-guest supramolecular strategy based on the incorporation of NADH mimics onto the surface of a metal-organic capsule to encapsulate flavin analogues for catalytic biomimetic monooxygenations in conjunction with enzymes. Coupling an artificial catalysis and a natural enzymatic catalysis in the pocket of an enzyme, this host-guest catalyst-enzyme system allows direct proton and electron transport between two catalytic processes via NADH mimics for the monooxygenation of both cyclobutanones and thioethers. This host-guest approach, which involves the direct coupling of abiotic and biotic catalysts via a NADH-containing host, is quite promising compared to normal catalyst-enzyme systems, as it offers the key advantages of supramolecular catalysis in integrated chemical and biological synthetic sequences.


Assuntos
Biomimética , Oxigênio/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Transporte de Elétrons , Enzimas/química , Ligação de Hidrogênio , Íons , Cinética , Ligantes , NAD/química , Solventes/química , Zinco/química
5.
Nucleic Acids Res ; 48(W1): W104-W109, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32392342

RESUMO

Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Despite genomic databases growing exponentially, classical biochemical characterization techniques are time-demanding, cost-ineffective and low-throughput. Therefore, computational methods are being developed to explore the unmapped sequence space efficiently. Selection of putative enzymes for biochemical characterization based on rational and robust analysis of all available sequences remains an unsolved problem. To address this challenge, we have developed EnzymeMiner-a web server for automated screening and annotation of diverse family members that enables selection of hits for wet-lab experiments. EnzymeMiner prioritizes sequences that are more likely to preserve the catalytic activity and are heterologously expressible in a soluble form in Escherichia coli. The solubility prediction employs the in-house SoluProt predictor developed using machine learning. EnzymeMiner reduces the time devoted to data gathering, multi-step analysis, sequence prioritization and selection from days to hours. The successful use case for the haloalkane dehalogenase family is described in a comprehensive tutorial available on the EnzymeMiner web page. EnzymeMiner is a universal tool applicable to any enzyme family that provides an interactive and easy-to-use web interface freely available at https://loschmidt.chemi.muni.cz/enzymeminer/.


Assuntos
Enzimas/química , Software , Biocatálise , Estabilidade Enzimática , Enzimas/metabolismo , Hidrolases/química , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Solubilidade
6.
Nucleic Acids Res ; 48(W1): W110-W115, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32406917

RESUMO

The CUPP platform includes a web server for functional annotation and sub-grouping of carbohydrate active enzymes (CAZymes) based on a novel peptide-based similarity assessment algorithm, i.e. protein grouping according to Conserved Unique Peptide Patterns (CUPP). This online platform is open to all users and there is no login requirement. The web server allows the user to perform genome-based annotation of carbohydrate active enzymes to CAZy families, CAZy subfamilies, CUPP groups and EC numbers (function) via assessment of peptide-motifs by CUPP. The web server is intended for functional annotation assessment of the CAZy inventory of prokaryotic and eukaryotic organisms from genomic DNA (up to 30MB compressed) or directly from amino acid sequences (up to 10MB compressed). The custom query sequences are assessed using the CUPP annotation algorithm, and the outcome is displayed in interactive summary result pages of CAZymes. The results displayed allow for inspection of members of the individual CUPP groups and include information about experimentally characterized members. The web server and the other resources on the CUPP platform can be accessed from https://cupp.info.


Assuntos
Metabolismo dos Carboidratos , Enzimas/química , Enzimas/genética , Anotação de Sequência Molecular , Software , Algoritmos , Enzimas/classificação , Enzimas/metabolismo , Internet , Peptídeos/química , Análise de Sequência de DNA , Análise de Sequência de Proteína
7.
J Vis Exp ; (159)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32420996

RESUMO

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics-even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.


Assuntos
Enzimas/metabolismo , Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Fragmentos de Peptídeos/metabolismo , Proteômica/métodos , Enzimas/química , Humanos , Fragmentos de Peptídeos/química , Conformação Proteica , Proteólise
8.
Food Chem ; 317: 126464, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32114275

RESUMO

Oat rice kernels were subjected to decortication (DOR), decortication and enzyme deactivation (DDOR), decortication and cooking (DCOR), as well as combined decortication, enzyme deactivation and cooking (DDCOR). The starch fractions were isolated and their structural features were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, solid-state 13C nuclear magnetic resonance, small angle X-ray scattering (SAXS), and scanning electron microscope. In the cooked oat rice samples (DCOR and DDCOR), in addition to losing a significant amount of the A-type crystalline structure, there was an enhancement in the proportion of V-type crystallinity. The cooking process completely destroyed the periodic lamellar structure of oat starch on the SAXS profile. The Mw values (1.195 × 107-1.459 × 107 g/mol) were in the following order: DOR > DDOR > DCOR > DDCOR. The data was in line with the results for crystallinity, double helix content, degree of order, melting enthalpy, and those obtained for textural parameters, resistant starch content, and bile acid binding capacity.


Assuntos
Avena/química , Culinária/métodos , Amido/química , Cristalização , Enzimas/química , Espectroscopia de Ressonância Magnética , Espalhamento a Baixo Ângulo , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
9.
Proc Natl Acad Sci U S A ; 117(11): 5977-5986, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123117

RESUMO

Understanding the molecular basis of adaptation to the environment is a central question in evolutionary biology, yet linking detected signatures of positive selection to molecular mechanisms remains challenging. Here we demonstrate that combining sequence-based phylogenetic methods with structural information assists in making such mechanistic interpretations on a genomic scale. Our integrative analysis shows that positively selected sites tend to colocalize on protein structures and that positively selected clusters are found in functionally important regions of proteins, indicating that positive selection can contravene the well-known principle of evolutionary conservation of functionally important regions. This unexpected finding, along with our discovery that positive selection acts on structural clusters, opens previously unexplored strategies for the development of better models of protein evolution. Remarkably, proteins where we detect the strongest evidence of clustering belong to just two functional groups: Components of immune response and metabolic enzymes. This gives a coherent picture of pathogens and xenobiotics as important drivers of adaptive evolution of mammals.


Assuntos
Adaptação Fisiológica , Evolução Molecular , Mamíferos/genética , Mamíferos/fisiologia , Seleção Genética , Animais , Meio Ambiente , Enzimas/química , Genômica , Imunidade , Mamíferos/imunologia , Modelos Moleculares , Filogenia , Conformação Proteica , Proteínas/química
10.
Food Chem ; 315: 126268, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32018083

RESUMO

Starch digestion in pulse cellular matrices is primarily determined by the hindrance of cell walls limiting enzyme diffusion as well as the retention of starch granular structure. However, the effect of hydrothermal treatment on structure and digestion properties of entrapped pulse starches is not fully elucidated. In present study, we reported the variations in structure and enzyme susceptibility of pulse cells isolated at 60 °C followed by heated at 70, 80, 90, 100 °C, which were higher than the starch gelatinization temperature. Based on the thermal and crystalline properties, entrapped starches in pulse cells were not fully gelatinized even treated at 100 °C. Whilst, the digestion of entrapped pulse starches increased with higher temperature, but still much lower than the isolated starch treated at the same temperature. In addition to physical barriers (intact cell wall) and starch structural features (partial ordered crystalline structure), the soluble/insoluble proteinaceous materials in cells also synergistically reduced the starch digestibility.


Assuntos
Cicer/química , Cotilédone/química , Phaseolus/química , Amido/química , Parede Celular/química , Cotilédone/citologia , Digestão , Enzimas/química , Enzimas/metabolismo , Gelatina/química , Temperatura Alta , Células Vegetais , Temperatura , Difração de Raios X
11.
Nat Commun ; 11(1): 838, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047166

RESUMO

Protein-protein interactions are spatially regulated in living cells to realize high reaction efficiency, as seen in naturally existing electron-transfer chains. Nevertheless, arrangement of chemical/biochemical components at the artificial device interfaces does not possess the same level of control. Here we report a tetrahedral DNA framework-enabled bulk enzyme heterojunction (BEH) strategy to program the multi-enzyme catalytic cascade at the interface of electrochemical biosensors. The construction of interpenetrating network of BEH at the millimeter-scale electrode interface brings enzyme pairs within the critical coupling length (CCL) of ~10 nm, which in turn greatly improve the overall catalytic cascade efficiency by ~10-fold. We demonstrate the BEH generality with a range of enzyme pairs for electrochemically detecting clinically relevant molecular targets. As a proof of concept, a BEH-based sarcosine sensor enables single-step detection of the metabolic biomarker of sarcosine with ultrasensitivity, which hold the potential for precision diagnosis of early-stage prostate cancer.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Técnicas Eletroquímicas/métodos , Eletrodos , Enzimas Imobilizadas , Técnicas Biossensoriais/instrumentação , Catálise , Técnicas de Química Analítica/métodos , Técnicas Eletroquímicas/instrumentação , Enzimas/química , Desenho de Equipamento , Humanos , Limite de Detecção , Nanopartículas Metálicas , Modelos Teóricos , Nanotecnologia/métodos , Sarcosina
12.
Proc Natl Acad Sci U S A ; 117(10): 5310-5318, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32079722

RESUMO

The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in αßα sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an α-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first αßα sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.


Assuntos
Enzimas/química , Enzimas/classificação , Evolução Molecular , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/classificação , Sequência de Aminoácidos , Sítios de Ligação , Bases de Dados de Proteínas , Ligantes , Ligação Proteica , Domínios Proteicos
13.
Ecotoxicol Environ Saf ; 191: 110215, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31978765

RESUMO

Information on the kinetic characteristics of soil enzymes under long-term arsenic (As) pollution in field soils is scarce. We investigated Michaelis-Menten kinetic properties of four soil enzymes including ß-glucosidase (BG), acid phosphatase (ACP), alkaline phosphatase (ALP), and dehydrogenase (DHA) in field soils contaminated by As resulting from long-term realgar mining activity. The kinetic parameters, namely the maximum reaction velocity (Vmax), enzyme-substrate affinity (Km) and catalytic efficiency (Vmax/Km) were calculated. Results revealed that the enzyme kinetic characteristics varied in soils and were significantly influenced by total nitrogen (N) and total As, which explained 31.8% and 30.7% of the variance in enzyme kinetics respectively. Enzyme pools (Vmax) and catalytic efficiency (Vmax/Km) of BG, ACP and DHA decreased with elevated As pollution, while the enzyme affinity for substrate (Km) was less affected. Redundancy analysis and stepwise regression suggested that the adverse influence of As on enzyme kinetics may offset or weakened by soil total N and soil organic matter (SOM). Concentration-response fitting revealed that the specific kinetic parameters expressed as the absolute enzyme kinetic parameters multiplied by normalized soil total N and SOM were more relevant than the absolute ones to soil total As. The arsenic ecological dose values that cause 10% decrease (ED10) in the specific enzyme kinetics were 20-49 mg kg-1, with a mean value of 35 mg kg-1, indicating a practical range of threshold for As contamination at field level. This study concluded that soil enzymes exhibited functional adaptation to long-term As stress mainly through the reduction of enzyme pools (Vmax) or maintenance of enzyme-substrate affinity (Km). Further, this study demonstrates that the specific enzyme kinetics are the better indicators of As ecotoxicity at field-scale compared with the absolute enzyme parameters.


Assuntos
Arsênico/toxicidade , Enzimas/química , Poluentes do Solo/toxicidade , Solo/química , Arsênico/análise , Arsenicais , Biomarcadores Ambientais/efeitos dos fármacos , Cinética , Mineração , Nitrogênio/análise , Compostos Orgânicos/análise , Poluentes do Solo/análise , Sulfetos/toxicidade
14.
PLoS Comput Biol ; 16(1): e1007600, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917825

RESUMO

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.


Assuntos
Enzimas , Método de Monte Carlo , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Especificidade por Substrato/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Azidas/química , Azidas/metabolismo , Sítios de Ligação/genética , Catálise , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Metionina tRNA Ligase/química , Metionina tRNA Ligase/genética , Metionina tRNA Ligase/metabolismo , Mutação/genética , Norleucina/análogos & derivados , Norleucina/química , Norleucina/metabolismo
15.
Chemosphere ; 243: 125399, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31995869

RESUMO

Nutrient removal efficiency in green sorption media such as biosorption activated media (BAM) for treating stormwater runoff can be heavily influenced either on a short- or long-term basis by varying field conditions of linear ditches due to the presence of copper in stormwater runoff. It is also noticeable that the linear ditch undergoes physical or mechanical impacts from the traffic compaction, chemical impact of carbon sources from the nearby farmland, and biological impact from potential animal activities (such as gopher tortoises, moles, and ants). In the nitrogen cycle, two denitrification pathways, the dissimilatory nitrate reduction to ammonia and common denitrification, are deemed critical for such assessment. A fixed-bed column study was set up to mimic different linear ditch field conditions for BAM applications and measure the effect of short-and long-term copper addition on microbial dynamics given the varying decomposition of dissolved organic nitrogen (DON). The findings confirm that, as the denitrifiers (in the second pathway) were the dominant species, their population continued to grow and maintain small-sized cells for extracellular sequestration under long-term copper impact. Furthermore, the study indicated that the ammonia oxidizer comammox was found in higher quantities than ammonia oxidizing bacteria or archaea. An enormous amount of DON was released during this process to bind the copper ion and reduce its toxicity as the enzymatic cascade effect appeared. In addition, the long-term copper exposure posed salient inhibitory effects on the microbial community regardless of varying field conditions in BAM. Short-term copper toxicity exerted an important but varying role in the enzymatic cascade effect over different linear ditch field conditions in BAM.


Assuntos
Cobre/química , Enzimas/metabolismo , Nitrogênio/isolamento & purificação , Purificação da Água/métodos , Amônia/metabolismo , Carbono , Cobre/metabolismo , Desnitrificação , Enzimas/química , Consórcios Microbianos/efeitos dos fármacos , Nitratos/metabolismo
16.
Biotechnol Lett ; 42(3): 357-373, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31950406

RESUMO

Recent past years have witnessed the development of several artificial enzymes, using different materials to mimic natural enzymes with respect to their structure and functions. The nanozymes are nanomaterials possessing similar characteristics to the natural enzymes and have emerged recently as an innovative class of artificial enzymes. The nanozymes have got remarkable attention from the researchers and notable developments have been achieved owing to their unique properties compared with natural enzymes and classic artificial enzymes. In this regard, several nanomaterials have been scrutinized so far to mimic different natural enzymes for wider applications ranging from imaging, sensing, water treatment, pollutant removal, and therapeutics. The applications of nanozymes in biomedicine research are fast-growing and various nanozymes have been implicated in diagnostic medicine, targeted cancer therapy. Such abilities make them an appropriate alternative for the development of affordable, sustainable and safe diagnostic as well as therapeutic agents.


Assuntos
Materiais Biomiméticos/química , Técnicas Biossensoriais , Enzimas/química , Nanoestruturas/química
17.
Crit Rev Biotechnol ; 40(2): 231-246, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31914816

RESUMO

It is generally accepted that oligomeric enzymes evolve from their monomeric ancestors, and the evolution process generates superior structural benefits for functional advantages. Furthermore, adjusting the transition between different oligomeric states is an important mechanism for natural enzymes to regulate their catalytic functions for adapting environmental fluctuations in nature, which inspires researchers to mimic such a strategy to develop artificially oligomerized enzymes through protein engineering for improved performance under specific conditions. On the other hand, transforming oligomeric enzymes into their monomers is needed in fundamental research for deciphering catalytic mechanisms as well as exploring their catalytic capacities for better industrial applications. In this article, strategies for developing artificially oligomerized and monomerized enzymes are reviewed and highlighted by their applications. Furthermore, advances in the computational prediction of oligomeric structures are introduced, which would accelerate the systematic design of oligomeric and monomeric enzymes. Finally, the current challenges and future directions in this field are discussed.


Assuntos
Enzimas/genética , Engenharia de Proteínas , Bioengenharia , Catálise , Enzimas/química , Enzimas/metabolismo , Estabilidade Proteica
18.
Artigo em Inglês | MEDLINE | ID: mdl-31922627

RESUMO

Hydrogels are formed using various triggers, including light irradiation, pH adjustment, heating, cooling, or chemical addition. Here, a new method for forming hydrogels is introduced: ultrasound-triggered enzymatic gelation. Specifically, ultrasound is used as a stimulus to liberate liposomal calcium ions, which then trigger the enzymatic activity of transglutaminase. The activated enzyme catalyzes the formation of fibrinogen hydrogels through covalent intermolecular crosslinking. The catalysis and gelation processes are monitored in real time and both the enzyme kinetics and final hydrogel properties are controlled by varying the initial ultrasound exposure time. This technology is extended to microbubble-liposome conjugates, which exhibit a stronger response to the applied acoustic field and are also used for ultrasound-triggered enzymatic hydrogelation. To the best of the knowledge, these results are the first instance in which ultrasound is used as a trigger for either enzyme catalysis or enzymatic hydrogelation. This approach is highly versatile and can be readily applied to different ion-dependent enzymes or gelation systems. Moreover, this work paves the way for the use of ultrasound as a remote trigger for in vivo hydrogelation.


Assuntos
Enzimas/química , Hidrogéis/química , Ondas Ultrassônicas , Cloreto de Cálcio/química , Catálise , Reagentes para Ligações Cruzadas/química , Fibrinogênio/química , Cinética , Lipossomos/química , Microbolhas , Fosfatidiletanolaminas/química , Fosforilcolina/química , Polietilenoglicóis/química
19.
Chem Soc Rev ; 49(1): 233-262, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31815263

RESUMO

Enzymes are versatile catalysts and their synthetic potential has been recognized for a long time. In order to exploit their full potential, enzymes often need to be re-engineered or optimized for a given application. (Semi-) rational design has emerged as a powerful means to engineer proteins, but requires detailed knowledge about structure function relationships. In turn, directed evolution methodologies, which consist of iterative rounds of diversity generation and screening, can improve an enzyme's properties with virtually no structural knowledge. Current diversity generation methods grant us access to a vast sequence space (libraries of >1012 enzyme variants) that may hide yet unexplored catalytic activities and selectivity. However, the time investment for conventional agar plate or microtiter plate-based screening assays represents a major bottleneck in directed evolution and limits the improvements that are obtainable in reasonable time. Ultrahigh-throughput screening (uHTS) methods dramatically increase the number of screening events per time, which is crucial to speed up biocatalyst design, and to widen our knowledge about sequence function relationships. In this review, we summarize recent advances in uHTS for directed enzyme evolution. We shed light on the importance of compartmentalization to preserve the essential link between genotype and phenotype and discuss how cells and biomimetic compartments can be applied to serve this function. Finally, we discuss how uHTS can inspire novel functional metagenomics approaches to identify natural biocatalysts for novel chemical transformations.


Assuntos
Evolução Molecular Direcionada , Enzimas/metabolismo , Ensaios de Triagem em Larga Escala , Biocatálise , Enzimas/química , Enzimas/genética , Engenharia de Proteínas
20.
Anal Bioanal Chem ; 412(2): 499-506, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31807805

RESUMO

Although a massive research has been devoted on the exploration of noble metal-based nanozyme, less progress has been made in the investigation of palladium (Pd) nanozyme and the interaction between ions and Pd nanozyme. In this study, a new type of Pd nanozyme was prepared by a facile one-pot approach by using carboxylated chitosan as the stabilizer. Owing to the synergistic effect of carboxylated chitosan stabilized Pd nanoparticles (CC-PdNPs) can effectively catalyze the H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine sulfate (TMB) accompanied by a blue color change (oxidized TMB), indicating the peroxidase-like activity of CC-PdNPs. Furthermore, the Michaelis-Menten constants and catalytic stability of CC-PdNPs render them suitable for environmental analysis and bio-detection. Here, we found that while introducing the iodine ions (I-) into the reaction medium, the peroxidase-like activity of CC-PdNPs has been rapidly and effectively inhibited through the formation of Pd-I bond; thus, the active sites of PdNPs can be blocked by I-. Based on this specific inhibition by I-, a facile colorimetric assay has been performed for the detection of I- with an extremely low limit of detection (0.19 nM). Furthermore, the practicality of the proposed sensor also has been demonstrated in tap water, and the satisfactory recoveries were obtained. Our study not only demonstrated a novel Pd-based nanozyme but also provided guidance for I- sensing for environmental analysis, food inspection, and bio-detection. Graphical abstract.


Assuntos
Ácidos Carboxílicos/química , Quitosana/química , Colorimetria/instrumentação , Enzimas/química , Iodo/análise , Nanoestruturas/química , Paládio/química , Ânions , Limite de Detecção , Abastecimento de Água
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