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1.
Enzyme Microb Technol ; 150: 109879, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489032

RESUMO

The impact of hydrophilic polymers in an organosilica matrix on the features and performance of immobilized methylotrophic yeast cells used as biocatalysts was investigated and described. Yeast cells were immobilized in a matrix made of tetraethoxysilane (TEOS) and methyltriethoxysilane (MTES) by one-step sol-gel route of synthesis in the presence of polyethylene glycol (PEG) or polyvinyl alcohol (PVA). Organosilica shells were spontaneously built around cells as a result of yeast immobilization at a TEOS to MTES ratio of 85/15 vol% and hydrophilic polymer (PEG or PVA). As a structure-directing agent, PVA produces organosilica films. Stable high-performance biocatalysts active for one year, if stored at -18 °C, have been obtained by entrapment of methylotrophic yeast cells. A trickling biofilter with and without active aeration was designed using entrapped yeast cells to treat methanol polluted wastewater. A biofilter model with active aeration could halve methanol input thus demonstrating better performance compared to treatment without active aeration.


Assuntos
Polímeros , Saccharomyces cerevisiae , Biocatálise , Interações Hidrofóbicas e Hidrofílicas , Álcool de Polivinil
2.
Enzyme Microb Technol ; 150: 109880, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489033

RESUMO

The ene reductases (ERs) from the old yellow enzymes (OYEs) family have the ability to reduce activated alkenes to generate up to two stereocenters, therefore they have been received extensive attention as powerful biocatalysts. In this study, through gene mining, four ERs were identified from the genomes of Ensifer adhaerens, Pseudomonas fluorescens, and Pseudomonas veronil. The biocatalytic properties of these four ERs were identified, and their applications in the synthesis process of dihydrocarvone and profen derivatives were further evaluated. Among them, three ERs (EaER2, PvER1, and PvER2) belonging to the classic OYEs showed the best catalytic activity at 30 °C and pH 7.0 (100 mM potassium phosphate buffer) and the PfER2, which belongs to the thermophilic-like OYEs exhibited the best catalytic at 40 °C and pH 7.0 (100 mM potassium phosphate buffer). When exploring the influence of organic solvents on the catalytic efficiency, it was found that the four ERs were more sensitive to toluene and had tolerance to several other selected organic solvents. In addition, EaER2, PfER2, PvER1 and PvER2 showed excellent catalytic activity toward carvone, and the stereoselectivity of PvER2 toward carvone could reach up to 88.7 % de. EaER2 and PfER2 can catalyze the synthesis of a variety of profen derivatives with a stereoselectivity over 99 % ee. Moreover, through homology modeling and molecular docking, we preliminarily explained the mechanism of catalytic activity and stereoselectivity of the four ERs, which provided a solid base on the rational design of their stereo-preference in the future. The discovery of EaER2, PfER2, PvER1, and PvER2 provides four new enzyme sources for the study of the OYEs family and enriches the biocatalytic toolbox of ERs. Our exploration of the enzymatic properties of these four ERs will provide the sufficient data basis for future research and industrialization progress.


Assuntos
Oxirredutases , Biocatálise , Monoterpenos Cicloexânicos , Simulação de Acoplamento Molecular , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Rhizobiaceae
3.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
4.
Nat Commun ; 12(1): 5004, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408154

RESUMO

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Biocatálise , Dimerização , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Processamento de Proteína Pós-Traducional
5.
Appl Microbiol Biotechnol ; 105(14-15): 5873-5882, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34342711

RESUMO

(S)-4-Chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol are key pharmaceutical intermediates for the synthesis of bepotastine and cloperastine, respectively. However, the biocatalytic approach to prepare these bulky diaryl ketones remains challenging because of the low activity of naturally occurring alcohol dehydrogenases (ADH). In the present study, ADH seq5, which has an adequate binding pocket volume and accepts bulky diaryl ketones, was further engineered with a binding pocket of increased hydrophobicity. Based on molecular simulation and binding free energy analyses, a small mutation library was constructed, and mutant seq5-D150I with a threefold increase in kcat and a low Km was obtained successfully. The comparison of kinetic parameters, binding free energy, docking conformation, and critical catalytic distances calculated by molecular dynamic simulations revealed the source of increased activity. To develop a practical approach with seq5-D150I, reaction conditions including pH, temperature, buffer, and metal ions were optimised and applied to synthesise (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol with high enantiomeric excess. The space-time yields for (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol increased dramatically to as high as 263.4 g∙L-1 day-1 and 150 g∙L-1 day-1, respectively, which, to our knowledge, is the highest reported yield to date. These results show that the biocatalytic approach with seq5-D150I may be practical for future industrial applications.Key points An alcohol dehydrogenase was engineered based on binding free energy analysis. The mutant seq5-D150I obtained a threefold increase in kcat and a low Km. Two important pharmaceutical intermediates were obtained with high space-time yield.


Assuntos
Álcool Desidrogenase , Piperidinas , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Biocatálise , Interações Hidrofóbicas e Hidrofílicas , Cinética , Piridinas , Estereoisomerismo
6.
Nat Commun ; 12(1): 5065, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417452

RESUMO

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.


Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Reação de Cicloadição , Descarboxilação , Mononucleotídeo de Flavina/metabolismo , Cinética , Modelos Moleculares , Oxigênio/farmacologia , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Solventes , Relação Estrutura-Atividade , Especificidade por Substrato , Viscosidade
7.
Nat Commun ; 12(1): 4946, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400632

RESUMO

5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. Toward addressing the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, here we report coordinated reaction and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The biocatalyst and reaction conditions presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive blueprint for further development, giving hope for the creation of a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.


Assuntos
Furaldeído/análogos & derivados , Furaldeído/metabolismo , Galactose Oxidase/genética , Galactose Oxidase/metabolismo , Engenharia de Proteínas , Biocatálise , Catálise , Domínio Catalítico , Furanos , Galactose Oxidase/química , Mutagênese , Oxirredução
8.
Science ; 373(6553)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437092

RESUMO

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.


Assuntos
Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Biocatálise , Domínio Catalítico , Flavobacterium/enzimologia , Hidrólise , Cinética , Microfluídica , Modelos Moleculares , Mutação , Oxigênio/metabolismo , Fosfatos/metabolismo , Conformação Proteica , Dobramento de Proteína , Termodinâmica
9.
Molecules ; 26(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34443339

RESUMO

The treatment of environmental pollution by microorganisms and their enzymes is an innovative and socially acceptable alternative to traditional remediation approaches. Microbial biodegradation is often characterized with high efficiency as this process is catalyzed via degrading enzymes. Various naturally isolated microorganisms were demonstrated to have considerable ability to mitigate many environmental pollutants without external intervention. However, only a small fraction of these strains are studied in detail to reveal the mechanisms at the enzyme level, which strictly limited the enhancement of the degradation efficiency. Accordingly, this review will comprehensively summarize the function of various degrading enzymes with an emphasis on catalytic mechanisms. We also inspect the expanded applications of these pollutant-degrading enzymes in industrial processes. An in-depth understanding of the catalytic mechanism of enzymes will be beneficial for exploring and exploiting more degrading enzyme resources and thus ameliorate concerns associated with the ineffective biodegradation of recalcitrant and xenobiotic contaminants with the help of gene-editing technology and synthetic biology.


Assuntos
Biocatálise , Poluentes Ambientais/metabolismo , Enzimas/metabolismo , Poluentes Ambientais/isolamento & purificação
10.
Nat Commun ; 12(1): 4626, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330913

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development.Statement of significancePDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors.


Assuntos
Carcinoma Ductal Pancreático/genética , Dano ao DNA , Neoplasias Pancreáticas/genética , Proteína-Arginina N-Metiltransferases/genética , RNA/genética , Proteínas Repressoras/genética , Animais , Biocatálise/efeitos dos fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/prevenção & controle , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/prevenção & controle , Proteína-Arginina N-Metiltransferases/metabolismo , RNA/metabolismo , Interferência de RNA , Proteínas Repressoras/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
11.
Anal Chem ; 93(31): 11052-11059, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34324305

RESUMO

Functional DNA nanostructures have been widely used in various bioassay fields. Yet, the programmable assembly of functional DNA nanostructures in living cells still represents a challenging goal for guaranteeing the sensitive and specific biosensing utility. In this work, we report a self-catalytic DNA assembly (SDA) machine by using a feedback deoxyribozyme (DNAzyme)-amplified branched DNA assembly. This SDA system consists of catalytic self-assembly (CSA) and DNAzyme amplification modules for recognizing and amplifying the target analyte. The analyte initiates the CSA reaction, leading to the formation of Y-shaped DNA that carries two RNA-cleaving DNAzymes. One DNAzyme can then successively cleave the corresponding substrate and generate numerous additional inputs to activate new CSA reactions, thus realizing a self-catalytic amplification reaction. Simultaneously, the other DNAzyme is assembled as a versatile signal transducer for cleaving the fluorophore/quencher-modified substrate, leading to the generation of an amplified fluorescence readout. By incorporating a flexible auxiliary sensing module, the SDA system can be converted into a universal sensing platform for detecting cancerous biomarkers, e.g., a well-known oncogene microRNA-21 (miR-21). Moreover, the SDA system realized the precise intracellular miR-21 imaging in living cells, which is attributed to the reciprocal amplification property between CSA reactions and DNAzyme biocatalysis. This compact SDA amplifier machine provides a universal and facile toolbox for the highly efficient identification of cancerous biomarkers and thus holds great potential for early cancer diagnosis.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , MicroRNAs , Biocatálise , DNA , DNA Catalítico/metabolismo , Corantes Fluorescentes , MicroRNAs/metabolismo
12.
Chem Commun (Camb) ; 57(61): 7517-7520, 2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34235521

RESUMO

We demonstrate that a recombinase ribozyme achieves multiple functions in the same reaction network: self-reproduction, iterative elongation and circularization of other RNAs, leading to synthesis of diverse products predicted by a kinetic model. This shows that key mechanisms can be integrated and controlled toward Darwinian evolution in RNA reaction networks.


Assuntos
RNA Bacteriano/genética , RNA Catalítico/genética , RNA/genética , Azoarcus/enzimologia , Biocatálise , Fenômenos Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Sequências Repetidas Invertidas , Cinética , RNA/química , RNA Bacteriano/química , RNA Catalítico/química , Recombinases/química , Recombinases/genética
13.
Nat Commun ; 12(1): 4236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244493

RESUMO

The repertoire of peptides presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface is tailored by the ER-resident peptide loading complex (PLC), which contains the exchange catalyst tapasin. Tapasin stabilizes MHC-I molecules and promotes the formation of stable peptide-MHC-I (pMHC-I) complexes that serve as T cell antigens. Exchange of suboptimal by high-affinity ligands is catalyzed by tapasin, but the underlying mechanism is still elusive. Here we analyze the tapasin-induced changes in MHC-I dynamics, and find the catalyst to exploit two essential features of MHC-I. First, tapasin recognizes a conserved allosteric site underneath the α2-1-helix of MHC-I, 'loosening' the MHC-I F-pocket region that accomodates the C-terminus of the peptide. Second, the scoop loop11-20 of tapasin relies on residue L18 to target the MHC-I F-pocket, enabling peptide exchange. Meanwhile, tapasin residue K16 plays an accessory role in catalysis of MHC-I allotypes bearing an acidic F-pocket. Thus, our results provide an explanation for the observed allele-specificity of catalyzed peptide exchange.


Assuntos
Alelos , Apresentação do Antígeno/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Regulação Alostérica , Biocatálise , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Imunoglobulinas/metabolismo , Imunoglobulinas/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
14.
Nat Commun ; 12(1): 4417, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285212

RESUMO

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.


Assuntos
Aspergillus/enzimologia , Biocatálise , Proteínas Fúngicas/metabolismo , Oxigenases/metabolismo , Peróxidos/metabolismo , Aspergillus/genética , Domínio Catalítico , Cristalografia por Raios X , Compostos Ferrosos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Ácidos Cetoglutáricos/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Oxigênio/metabolismo , Oxigenases/genética , Oxigenases/isolamento & purificação , Oxigenases/ultraestrutura , Conformação Proteica em Folha beta , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Terpenos/metabolismo
15.
Sheng Wu Gong Cheng Xue Bao ; 37(7): 2256-2271, 2021 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-34327893

RESUMO

The development of biotechnology and the in-depth research on disease mechanisms have led to increased application of enzymes in the treatment of diseases. In addition, enzymes have shown great potential in drug manufacturing, particularly in production of non-natural organic compounds, due to the advantages of mild reaction conditions, high catalytic efficiency, high specificity, high selectivity and few side reactions. Moreover, the application of genetic engineering, chemical modification of enzymes and immobilization technologies have further improved the function of enzymes. This review summarized the advances of using enzymes as drugs for disease treatment or as catalysts for drug manufacturing, followed by discussing challenges, potential solutions and future perspectives on the application of enzymes in the medical and pharmaceutical field.


Assuntos
Biotecnologia , Enzimas , Biocatálise , Catálise , Composição de Medicamentos , Enzimas/metabolismo
16.
Molecules ; 26(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205848

RESUMO

In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates' molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles.


Assuntos
Butanonas/química , Lipase/metabolismo , Xilose/química , Biocatálise , Esterificação , Temperatura Alta , Ácido Oleico/química
17.
Molecules ; 26(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207073

RESUMO

Laccases are multicopper oxidases that have shown a great potential in various biotechnological and green chemistry processes mainly due to their high relative non-specific oxidation of phenols, arylamines and some inorganic metals, and their high redox potentials that can span from 500 to 800 mV vs. SHE. Other advantages of laccases include the use of readily available oxygen as a second substrate, the formation of water as a side-product and no requirement for cofactors. Importantly, addition of low-molecular-weight redox mediators that act as electron shuttles, promoting the oxidation of complex bulky substrates and/or of higher redox potential than the enzymes themselves, can further expand their substrate scope, in the so-called laccase-mediated systems (LMS). Laccase bioprocesses can be designed for efficiency at both acidic and basic conditions since it is known that fungal and bacterial laccases exhibit distinct optimal pH values for the similar phenolic and aromatic amines. This review covers studies on the synthesis of five- and six-membered ring heterocyclic cores, such as benzimidazoles, benzofurans, benzothiazoles, quinazoline and quinazolinone, phenazine, phenoxazine, phenoxazinone and phenothiazine derivatives. The enzymes used and the reaction protocols are briefly outlined, and the mechanistic pathways described.


Assuntos
Compostos Heterocíclicos/síntese química , Lacase/química , Lacase/metabolismo , Bactérias/metabolismo , Biocatálise/efeitos dos fármacos , Fungos/metabolismo , Oxirredução/efeitos dos fármacos
18.
Org Biomol Chem ; 19(30): 6650-6656, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34264250

RESUMO

The exquisite chemodiversity of terpenoids is the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue (Cys440 in Nicotiana tabacum 5-epi-aristolochene synthase) proximal to an ion-binding motif common to all TPSs and named the preNSE/DTE residue, which determines the product specificity of sesquiterpene synthases from different plant species. In sesquiterpene synthases catalyzing 1,10-cyclization (1,10-cyclases) of farnesyl diphosphate, mutation of the residue in both specific and promiscuous 1,10-cyclases from different lineages leads to the accumulation of monocyclic germacrene A-11-ol, which is "short-circuited" from complex cyclization cascades, suggesting a key role of this residue in generating the first common intermediate of 1,10-cyclization. Altering this residue in a specific 1,11-cyclase results in alternative 1,10-cyclization products. Moreover, the preNSE/DTE residue can be harnessed to engineer highly specific sesquiterpene synthases for an improved proportion of high-value terpenoids, such as patchoulol, a main constituent of several traditional Chinese medicines that could treat SARS-CoV-2.


Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Biocatálise , Alquil e Aril Transferases/genética , Domínio Catalítico , Ciclização , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Tabaco/enzimologia
19.
Inorg Chem ; 60(15): 11206-11213, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34289695

RESUMO

The catalytic cycle of a peroxidase-mimicking heme-DNAzyme involves an iron(IV)oxo porphyrin π-cation radical intermediate known as compound I formed through heterolytic O-O bond cleavage of an Fe3+-bound hydroperoxo ligand (Fe-OOH) in compound 0, like that of a heme enzyme such as horseradish peroxidase (HRP). Peroxidase assaying of complexes composed of chemically modified hemes possessing various electron densities of the heme iron atom (ρFe) and parallel-stranded tetrameric G-quadruplex DNAs of oligonucleotides d(TTAGGG), d(TTAGGGT), and d(TTAGGGA) was performed to elucidate the effects of the heme electronic structure and local heme environment on the catalytic activity of the heme-DNAzyme. The study revealed that the DNAzyme activity is enhanced through an increase in the ρFe and general base catalysis of the adenine base adjacent to the heme, which are reminiscent of the "push" and "pull" mechanisms in the catalytic cycle of HRP, respectively, and that the activity of the heme-DNAzyme can be independently controlled through the heme electronic structure and local heme environment. These findings allow a deeper understanding of the structure-function relationship of the peroxidase-mimicking heme-DNAzyme.


Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , DNA Catalítico/química , DNA Catalítico/metabolismo , Heme/química , Heme/metabolismo , Peroxidase/metabolismo , Biocatálise , Elétrons
20.
Chem Biol Interact ; 346: 109577, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34274336

RESUMO

The introduction of enzyme nanoreactors in medicine is relatively new. However, this technology has already been experimentally successful in cancer treatments, struggle against toxicity of reactive oxygen species in inflammatory processes, detoxification of drugs and xenobiotics, and correction of metabolic and genetic defects by using encapsulated enzymes, acting in single or cascade reactions. Biomolecules, e.g. enzymes, antibodies, reactive proteins capable of inactivating toxicants in the body are called bioscavengers. In this review, we focus on enzyme-containing nanoreactors for in vivo detoxification of organophosphorous compounds (OP) to be used for prophylaxis and post-exposure treatment of OP poisoning. A particular attention is devoted to bioscavenger-containing injectable nanoreactors operating in the bloodstream. The nanoreactor concept implements single or multiple enzymes and cofactors co-encapsulated in polymeric semi-permeable nanocontainers. Thus, the detoxification processes take place in a confined space containing highly concentrated bioscavengers. The article deals with historical and theoretical backgrounds about enzymatic detoxification of OPs in nanoreactors, nanoreactor polymeric enveloppes, realizations and advantages over other approaches using bioscavengers.


Assuntos
Nanoestruturas/química , Compostos Organofosforados/metabolismo , Xenobióticos/metabolismo , Biocatálise , Coenzimas/química , Coenzimas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Inativação Metabólica , Nanotecnologia , Compostos Organofosforados/química , Polímeros/química
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