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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.
Methods Mol Biol ; 2203: 187-204, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833213

RESUMO

Biotin-based proximity labeling circumvents major pitfalls of classical biochemical approaches to identify protein-protein interactions. It consists of enzyme-catalyzed biotin tags ubiquitously apposed on proteins located in close proximity of the labeling enzyme, followed by affinity purification and identification of biotinylated proteins by mass spectrometry. Here we outline the methods by which the molecular microenvironment of the coronavirus replicase/transcriptase complex (RTC), i.e., proteins located within a close perimeter of the RTC, can be determined by different proximity labeling approaches using BirAR118G (BioID), TurboID, and APEX2. These factors represent a molecular signature of coronavirus RTCs and likely contribute to the viral life cycle, thereby constituting attractive targets for the development of antiviral intervention strategies.


Assuntos
Coronavirus/patogenicidade , Enzimas/genética , Interações Hospedeiro-Patógeno/fisiologia , Proteômica/métodos , Proteínas Virais/metabolismo , Animais , Ascorbato Peroxidases/genética , Biotinilação , Carbono-Nitrogênio Ligases/genética , Linhagem Celular , Coronavirus/genética , Enzimas/metabolismo , Proteínas de Escherichia coli/genética , Imunofluorescência , Microrganismos Geneticamente Modificados , Proteínas Repressoras/genética , Proteínas Virais/química , Proteínas Virais/genética
4.
J Chromatogr A ; 1627: 461380, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823093

RESUMO

Microwave-ultrasonic assisted aqueous enzymatic extraction (MUAAEE) was applied to extract tiger nut oil (TNO). The conditions of MUAAEE were optimized by Plackett-Burman design followed Box-Behnken design. An oil recovery of 85.23% was achieved under optimum conditions of a 2% concentration of mixed enzyme including cellulase, pectinase and hemicellulase (1/1/1, w/w/w), particle size <600 µm, microwave power 300 W, ultrasonic power 460 W, radiation temperature 40 °C, time 30 min, enzymolysis temperature 45 °C, pH 4.9, liquid-to-solid ratio 10 mL/g and time 180 min. Oil by MUAAEE revealed the similar fatty acid compositions, triglyceride compositions, thermal behaviour and flavour compared with oil by Soxhlet extraction (SE), while the oil quality of MUAAEE is superior to that of SE. Scanning electron microscopy revealed that structural disruption of tiger nut caused by MUAAEE facilitated the oil extraction. Results suggest that MUAAEE could be an efficient and environment-friendly method for extraction of TNO.


Assuntos
Cyperus/química , Enzimas/metabolismo , Micro-Ondas , Nozes/química , Óleos Vegetais/química , Ultrassom , Varredura Diferencial de Calorimetria , Celulase/metabolismo , Análise Discriminante , Ácidos Graxos/química , Glicosídeo Hidrolases/metabolismo , Poligalacturonase/metabolismo , Análise de Componente Principal , Reprodutibilidade dos Testes , Temperatura , Triglicerídeos/análise , Água/química
5.
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
6.
BMC Bioinformatics ; 21(1): 327, 2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32703160

RESUMO

BACKGROUND: Managing and organizing biological knowledge remains a major challenge, due to the complexity of living systems. Recently, systemic representations have been promising in tackling such a challenge at the whole-cell scale. In such representations, the cell is considered as a system composed of interlocked subsystems. The need is now to define a relevant formalization of the systemic description of cellular processes. RESULTS: We introduce BiPOm (Biological interlocked Process Ontology for metabolism) an ontology to represent metabolic processes as interlocked subsystems using a limited number of classes and properties. We explicitly formalized the relations between the enzyme, its activity, the substrates and the products of the reaction, as well as the active state of all involved molecules. We further showed that the information of molecules such as molecular types or molecular properties can be deduced by automatic reasoning using logical rules. The information necessary to populate BiPOm can be extracted from existing databases or existing bio-ontologies. CONCLUSION: BiPOm provides a formal rule-based knowledge representation to relate all cellular components together by considering the cellular system as a whole. It relies on a paradigm shift where the anchorage of knowledge is rerouted from the molecule to the biological process. AVAILABILITY: BiPOm can be downloaded at https://github.com/SysBioInra/SysOnto.


Assuntos
Ontologias Biológicas , Metabolismo , Bases de Dados Factuais , Enzimas/metabolismo , Bases de Conhecimento
7.
PLoS One ; 15(6): e0232549, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32484808

RESUMO

Prodigiosin is an important secondary metabolite produced by Serratia marcescens. It can help strains resist stresses from other microorganisms and environmental factors to achieve self-preservation. Prodigiosin is also a promising secondary metabolite due to its pharmacological characteristics. However, pigmentless S. marcescens mutants always emerge after prolonged starvation, which might be a way for the bacteria to adapt to starvation conditions, but it could be a major problem in the industrial application of S. marcescens. To identify the molecular mechanisms of loss of prodigiosin production, two mutants were isolated after 16 days of prolonged incubation of wild-type (WT) S. marcescens 1912768R; one mutant (named 1912768WR) exhibited reduced production of prodigiosin, and a second mutant (named 1912768W) was totally defective. Comparative genomic analysis revealed that the two mutants had either mutations or deletions in rpoS. Knockout of rpoS in S. marcescens 1912768R had pleiotropic effects. Complementation of rpoS in the ΔrpoS mutant further confirmed that RpoS was a positive regulator of prodigiosin production and that its regulatory role in prodigiosin biosynthesis was opposite that in Serratia sp. ATCC 39006, which had a different type of pig cluster; further, rpoS from Serratia sp. ATCC 39006 and other strains complemented the prodigiosin defect of the ΔrpoS mutant, suggesting that the pig promoters are more important than the genes in the regulation of prodigiosin production. Deletion of rpoS strongly impaired the resistance of S. marcescens to stresses but increased membrane permeability for nutritional competence; competition assays in rich and minimum media showed that the ΔrpoS mutant outcompeted its isogenic WT strain. All these data support the idea that RpoS is pleiotropic and that the loss of prodigiosin biosynthesis in S. marcescens 1912768R during prolonged incubation is due to a mutation in rpoS, which appears to be a self-preservation and nutritional competence (SPANC) trade-off.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Enzimas/metabolismo , Prodigiosina/biossíntese , Serratia marcescens/fisiologia , Sideróforos/biossíntese , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Permeabilidade da Membrana Celular/fisiologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Movimento/fisiologia , Regiões Promotoras Genéticas , Fator sigma/genética , Estresse Fisiológico
8.
Arch Microbiol ; 202(7): 1597-1615, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32451592

RESUMO

Extracellular enzymes produced from Streptomyces have the potential to replace toxic chemicals that are being used in various industries. The endorsement of this replacement has not received a better platform in developing countries. In this review, we have discussed the impact of chemicals and conventional practices on environmental health, and the role of extracellular enzymes to replace these practices. Burning of fossil fuels and agriculture residue is a global issue, but the production of biofuel using extracellular enzymes may be the single key to solve all these issues. We have discussed the replacement of hazardous chemicals with the use of xylanase, cellulase, and pectinase in food industries. In paper industries, delignification was done by the chemical treatment, but xylanase and laccase have the efficient potential to remove the lignin from pulp. In textile industries, the conventional method includes the chemicals which affect the nervous system and other organs. The use of xylanase, cellulase, and pectinase in different processes can give a safe and environment-friendly option to textile industries. Hazardous chemical pesticides can be replaced by the use of chitinase as an insecticide and fungicide in agricultural practices.


Assuntos
Proteínas de Bactérias/metabolismo , Enzimas/metabolismo , Microbiologia Industrial/tendências , Streptomyces/enzimologia , Agricultura , Biocombustíveis , Lignina/metabolismo
10.
Food Chem ; 328: 127135, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32473490

RESUMO

Watermelon seed, a watermelon processing industry by-product, is a good protein source for the preparation of antioxidant peptides due to its high protein content, low cost, special amino acid composition. Antioxidant hydrolysates obtained from watermelon seed protein (WSP) after slit divergent ultrasound (SDU) treatment were studied. The stepwise multiple linear regression model verified that the reducing power of watermelon seed protein hydrolysates (WSPHs) is positively related with -SH and ß-turn content of WSP (R2 = 0.931, p < 0.01). Using the degree of hydrolysis (DH) and reducing power as indicators, the WSPHs was prepared under the optimal conditions (ultrasound frequency: 20/28 kHz, time: 60 min, power density: 100 W/L) and divided into three components by ultrafiltration membrane (1 and 5 kDa). Compared with WSPHs and other fractions, WSPHs-I (Mw < 1 kDa) not only significantly protected HepG2 cells from H2O2-induced damage, but also greatly alleviated the liver injury caused by d-galactose in male SD rats.


Assuntos
Antioxidantes/farmacologia , Citrullus/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacologia , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Enzimas/metabolismo , Galactose/toxicidade , Células Hep G2 , Humanos , Peróxido de Hidrogênio/farmacologia , Hidrólise , Modelos Lineares , Masculino , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Ratos Sprague-Dawley , Sementes/química , Ultrassom/instrumentação , Ultrassom/métodos
11.
Nucleic Acids Res ; 48(W1): W85-W93, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32469073

RESUMO

Rapid progress in proteomics and large-scale profiling of biological systems at the protein level necessitates the continued development of efficient computational tools for the analysis and interpretation of proteomics data. Here, we present the piNET server that facilitates integrated annotation, analysis and visualization of quantitative proteomics data, with emphasis on PTM networks and integration with the LINCS library of chemical and genetic perturbation signatures in order to provide further mechanistic and functional insights. The primary input for the server consists of a set of peptides or proteins, optionally with PTM sites, and their corresponding abundance values. Several interconnected workflows can be used to generate: (i) interactive graphs and tables providing comprehensive annotation and mapping between peptides and proteins with PTM sites; (ii) high resolution and interactive visualization for enzyme-substrate networks, including kinases and their phospho-peptide targets; (iii) mapping and visualization of LINCS signature connectivity for chemical inhibitors or genetic knockdown of enzymes upstream of their target PTM sites. piNET has been built using a modular Spring-Boot JAVA platform as a fast, versatile and easy to use tool. The Apache Lucene indexing is used for fast mapping of peptides into UniProt entries for the human, mouse and other commonly used model organism proteomes. PTM-centric network analyses combine PhosphoSitePlus, iPTMnet and SIGNOR databases of validated enzyme-substrate relationships, for kinase networks augmented by DeepPhos predictions and sequence-based mapping of PhosphoSitePlus consensus motifs. Concordant LINCS signatures are mapped using iLINCS. For each workflow, a RESTful API counterpart can be used to generate the results programmatically in the json format. The server is available at http://pinet-server.org, and it is free and open to all users without login requirement.


Assuntos
Processamento de Proteína Pós-Traducional , Proteômica/métodos , Software , Animais , Gráficos por Computador , Enzimas/metabolismo , Humanos , Internet , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Fluxo de Trabalho
12.
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
13.
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
14.
BMC Bioinformatics ; 21(1): 186, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32410570

RESUMO

BACKGROUND: Continuous enzyme kinetic assays are often used in high-throughput applications, as they allow rapid acquisition of large amounts of kinetic data and increased confidence compared to discontinuous assays. However, data analysis is often rate-limiting in high-throughput enzyme assays, as manual inspection and selection of a linear range from individual kinetic traces is cumbersome and prone to user error and bias. Currently available software programs are specialized and designed for the analysis of complex enzymatic models. Despite the widespread use of initial rate determination for processing kinetic data sets, no simple and automated program existed for rapid analysis of initial rates from continuous enzyme kinetic traces. RESULTS: An Interactive Continuous Enzyme Kinetics Analysis Tool (ICEKAT) was developed for semi-automated calculation of initial rates from continuous enzyme kinetic traces with particular application to the evaluation of Michaelis-Menten and EC50/IC50 kinetic parameters, as well as the results of high-throughput screening assays. ICEKAT allows users to interactively fit kinetic traces using convenient browser-based selection tools, ameliorating tedious steps involved in defining ranges to fit in general purpose programs like Microsoft Excel and Graphpad Prism, while still maintaining simplicity in determining initial rates. As a test case, we quickly analyzed over 500 continuous enzyme kinetic traces resulting from experimental data on the response of the protein lysine deacetylase SIRT1 to small-molecule activators. CONCLUSIONS: ICEKAT allows simultaneous visualization of individual initial rate fits and the resulting Michaelis-Menten or EC50/IC50 kinetic model fits, as well as hits from high-throughput screening assays. In addition to serving as a convenient program for practicing enzymologists, ICEKAT is also a useful teaching aid to visually demonstrate in real-time how incorrect initial rate fits can affect calculated Michaelis-Menten or EC50/IC50 kinetic parameters. For the convenience of the research community, we have made ICEKAT freely available online at https://icekat.herokuapp.com/icekat.


Assuntos
Enzimas/metabolismo , Sistemas On-Line , Software , Algoritmos , Concentração Inibidora 50 , Cinética , Sirtuína 1/genética
15.
Nat Chem Biol ; 16(6): 620-629, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32444835

RESUMO

In eukaryotes, chromatin remodeling and post-translational modifications (PTMs) shape the local chromatin landscape to establish permissive and repressive regions within the genome, orchestrating transcription, replication, and DNA repair in concert with other epigenetic mechanisms. Though cellular nutrient signaling encompasses a huge number of pathways, recent attention has turned to the hypothesis that the metabolic state of the cell is communicated to the genome through the type and concentration of metabolites in the nucleus that are cofactors for chromatin-modifying enzymes. Importantly, both epigenetic and metabolic dysregulation are hallmarks of a range of diseases, and this metabolism-chromatin axis may yield a well of new therapeutic targets. In this Perspective, we highlight emerging themes in the inter-regulation of the genome and metabolism via chromatin, including nonenzymatic histone modifications arising from chemically reactive metabolites, the expansion of PTM diversity from cofactor-promiscuous chromatin-modifying enzymes, and evidence for the existence and importance of subnucleocytoplasmic metabolite pools.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/genética , Cromatina/metabolismo , Eucariotos/metabolismo , Redes e Vias Metabólicas , Processamento de Proteína Pós-Traducional/fisiologia , Dano ao DNA , Reparo do DNA , Enzimas/metabolismo , Epigênese Genética , Histonas/metabolismo , Humanos
16.
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
17.
Arch Razi Inst ; 75(1): 131-136, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32292011

RESUMO

This review paper aimed to provides precious information about the function and use of different enzymes in dairy food applications. An enzyme is called a protein and catalyzes a specific reaction. Every enzyme is intended to initiate a particular reaction with a specific outcome. Moreover, numerous enzymes are present in the human body. Dairy food applications include the use of different enzymes, such as protease, to lessen the allergic properties of bovine milk products and lipase to improve the flavor of the cheese. Caseins, which are acid-soluble, are free from a flavor and can be suitable for addition to beverages and acidy foods by the limitation of proteolysis. The hydrolysates of casein are better to use in foods based on milk proteins for newborn children with allergy to bovine milk. Lipolysis makes a significant role in the flavor of Swiss cheese. The peppery flavor of Blue cheese is produced by short-chain unsaturated fats and methyl ketones. Many minor enzymes with limited application in dairy processes are sulphydryl oxidase, lactoperoxidase, glucose oxidase, catalase, lysozyme, and superoxide dismutase. Both catalase and glucose oxidase are utilized in food preservation processes. The scope minor enzymes in milk products needed for better production of dairy products and for the future of dairy technology. The worldwide market for the production of microbial enzymes used in dairy products processing is impressively increasing; however, there are a limited number of enzyme-producing industries in the market. The production of proteinase, lactase, lipase, and microbial rennet is increasing in the laboratory and small scales. In near future, the need for these enzymes will be undoubtedly significantly increasing essentially due to the requirement of significant nutritional valuable dairy products in the country to overcome malnutrition and obesity and shift toward low-fat and healthy foods.


Assuntos
Indústria de Laticínios , Enzimas/metabolismo , Tecnologia de Alimentos
18.
Int J Food Microbiol ; 323: 108595, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32224347

RESUMO

One of the main objectives of the food industry is to guarantee food safety by providing innocuous food products. Therefore, this sector must consider all the possible biotic or abiotic contamination routes from the entry of raw materials to the release of the final product. Currently, one important problem in this regard is the presence of biofilms on food contact surfaces which can transmit pathogens such as L. monocytogenes. In industrial conditions biofilms are found in a mature state, so it is essential that when carrying out removal effectiveness studies in vitro the tests are realized with models that produce these structures in a similarly mature state. The main objective of this study was to evaluate the effectiveness of an alternative treatment (i.e. enzymatic detergent that include natural antimicrobial agents) and a conventional treatment (i.e. chlorinated alkaline) for the elimination of mature L. monocytogenes biofilms. The results showed a cell detachment from the formed mature biofilms with an effectivity of between 74.75%-97.73% and 53.94%-94.02% for the enzymatic treatment and the chlorinated alkaline detergent, respectively. On a qualitative level, it was observed that the dispersion in the structure was much higher for the enzymatic treatment than for the chlorinated alkaline, which continued to show obvious structure integrity. All this leads to the conclusion that treatments with an enzymatic detergent have a significantly greater impact on the removal of mature L. monocytogenes biofilms, although a further disinfection process would be needed, enhancing even more the treatment effectivity. This may imply that the industrial approach to addressing this problem should be modified to include new perspectives that are more effective than traditional ones.


Assuntos
Biofilmes , Detergentes/química , Desinfecção/métodos , Enzimas/metabolismo , Microbiologia de Alimentos/métodos , Indústria de Processamento de Alimentos/métodos , Listeria monocytogenes/fisiologia , Animais , Detergentes/normas , Suínos
19.
Phys Chem Chem Phys ; 22(16): 8699-8712, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32270839

RESUMO

The selectivity of halogenation versus hydroxylation in α-KG de-pendent halogenases is vital to their function and has been widely studied, particularly using the halogenase SyrB2 as a model. WelO5, a new member of α-KG dependent halogenases, catalyzes the chlorination of 12-epi-fischerindole U in the welwitindolinone biosynthetic pathway. Herein, we give a detailed insight into the selectivity of WelO5 through combined quantum mechanical/molecular mechanical (QM/MM) calculations for the whole catalytic cycle. O2 activation leads to a Fe(iv)[double bond, length as m-dash]O moiety which adopts an equatorial conformation (in the plane consisting of His164, chloride and Fe atom), in contrast to axial conformation (perpendicular to the plane). Key to the conformational selectivity is a serine residue (Ser189) in the equatorial plane, that brings the precursor of the Fe(iv)[double bond, length as m-dash]O intermediate (a Fe(ii)-peracid complex) to the equatorial conformation through hydrogen bonding. Hydrogen abstraction of the substrate by the equatorial Fe(iv)[double bond, length as m-dash]O leads to a five-coordinated HO-Fe(iii)-Cl complex, where the hydroxyl ligand is still equatorial and thus relatively far from the substrate radical in the axial direction compared to the chloride ligand. This smoothly explains the extremely high selectivity of chlorination in WelO5 and provides a microscopic explanation for the experimental finding that S189A WelO5 ceases to display any chlorination selectivity versus hydroxylation. Notably, although Ser189 is vital for the selectivity of the enzyme, it is not part of the substrate binding pocket. Therefore, WelO5 serves as an excellent example how chemoselectivity can be achieved in directed evolution without the tedious redesign of the substrate binding pocket.


Assuntos
Enzimas/metabolismo , Ferroproteínas não Heme/metabolismo , Halogenação , Hidroxilação , Ferroproteínas não Heme/química , Especificidade por Substrato
20.
Chemosphere ; 251: 126377, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32143081

RESUMO

Eight constructed wetland microcosm (CWM) units have been designed using three macrophytes for domestic wastewater treatment. The main aim of this study is to evaluate enzyme activities with respect to time and soil depth and their correlation with removal efficiency of pollutants within different CWM units. The findings of this study show that the activity of enzymes and pollutants removal efficiency vary to a great extent on the soil depth, time of the sampling and type of pollutants. The correlation between removal of soluble reactive phosphorus and total phosphorus was significant with phosphatase activity in most of the CWM units. Activity of urease and NH4+-N removal was positively correlated with significant positive correlation in CWM units planted with Phragmites karka, and Pistia stratiotes (Ph + Pi) and Typha latifolia, Phragmites karka and Pistia stratiotes (T + Ph + Pi). Urease activity was found to be both positively and negatively correlated with respect to removal of NO3--N and NO2--N in different CWM units. Dehydrogenase activity showed negative correlation with respect to biological oxygen demand (BOD) removal except in CWM units with Ph + Pi and T + Ph + Pi. Similarly, a moderate positive and negative correlation exists between fluorescein diacetate hydrolysis and BOD removal. Removal of BOD and microbial biomass carbon (MBC) was negatively correlated with each other in most of the CWM units. With respect to vertical variation, the top layer of CWM units expressed significantly higher activity of extracellular enzymes and were significantly different from the deeper layer. CWM units exhibited significant variations in enzyme activity with respect to time.


Assuntos
Enzimas/metabolismo , Eliminação de Resíduos Líquidos/métodos , Áreas Alagadas , Análise da Demanda Biológica de Oxigênio , Biomassa , Carbono , Nitrogênio/análise , Fósforo/análise , Poaceae , Solo , Typhaceae , Águas Residuárias
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