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1.
Food Chem ; 305: 125440, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494496

RESUMO

Tea (Camellia sinensis) contains two active glutamate decarboxylases (CsGADs), whose unclear properties were examined here. CsGAD1 was 4-fold higher than CsGAD2 in activity. Their Km values for L-glutamate were around 5 mM. CsGAD1 and CsGAD2 performed best at 55 and 40 °C, respectively, and were both stimulated by calcium/calmodulin (Ca2+/CaM). Over 40 °C, their calmodulin-binding domains degraded. CsGADs were most active at pH 5.6, and were stimulated by Ca2+/CaM at pH 5.6-6.6, but inactivated at pH 3.6. Ca2+/CaM restored the CsGAD1 activity suppressed by inhibitors. CsGADs and CsCaM were localized to the cytosol. CsGAD1 was more highly expressed in most tissues, while CsGAD2 expression was more induced under stresses. The characteristics we first elucidated here revealed that CsGAD1 is the predominant isoform in tea plant, with CsGAD2 exhibiting a supplementary role under certain conditions. The information will contribute to regulation of GABA tea quality.


Assuntos
Camellia sinensis/enzimologia , Glutamato Descarboxilase/metabolismo , Proteínas de Plantas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Secas , Estabilidade Enzimática , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Cinética , Temperatura Ambiente
2.
Adv Exp Med Biol ; 1148: 131-150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482498

RESUMO

Enzymes are biocatalysts that have found profound applications in the current biotherapeutic industry and play a crucial role in diagnosis, prevention, and biochemical analysis of major diseases. However, stability, protein degradation and immunogenicity in the body present unique challenges that are faced upon sustained use of such enzymes. The present chapter is an attempt to dissect the state-of-the-art in relation to the challenges of development of therapeutic enzymes and the recent advances to address them. At the very outset, diseases where enzymes have found effective applications and the various causes of enzyme instability have been discussed. In recent times, polymer or nano- conjugated resistant delivery methods, as well as mutagenesis have led to manifold increase in enzyme stability against thermal denaturation, acidic gut environment, proteolysis and immunogenicity. Further, methods of analytical characterization of proteins have been highlighted and explored to shape future research directions.


Assuntos
Estabilidade Enzimática , Enzimas/química , Enzimas/farmacologia , Proteólise
3.
Adv Exp Med Biol ; 1148: 151-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482499

RESUMO

Oral application of therapeutic enzymes is a promising and non-invasive administration that improves patient compliance. However, the gastrointestinal tract poses several challenges to the oral delivery of proteins, including harsh pH conditions and digestive proteases. A promising way to stabilise enzymes during their gastrointestinal route is by modification with polymers that can provide both steric shielding and selective interaction in different digestive compartments. We give an overview of modification technologies for oral enzymes ranging from functionalisation of native proteins, to site-specific mutation and protein-polymer engineering. We specifically focus on enzymes that are active directly in the gastrointestinal lumen and not systemically absorbed. In addition, we discuss examples of microparticle and nanoparticle encapsulated enzymes for improved oral delivery. The modification of orally administered enzymes offers a broad chemical variability and may be a promising tool for enhancing their gastrointestinal stability.


Assuntos
Sistemas de Liberação de Medicamentos , Enzimas/farmacologia , Trato Gastrointestinal , Nanopartículas , Engenharia de Proteínas , Administração Oral , Estabilidade Enzimática , Humanos , Peptídeo Hidrolases , Polímeros
4.
World J Microbiol Biotechnol ; 35(10): 152, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31552479

RESUMO

L-asparaginase is a critical part of the treatment of acute lymphoblastic leukaemia in children and adolescents, and has contributed to the improvement in patient outcomes over the last 40 years. The main products used in clinical treatment are L-asparaginase enzymes derived from Escherichia coli and Erwinia chrysanthemi. However, a very active area of research is the identification and characterisation of potential new L-asparaginase therapeutics, from existing or novel prokaryotic and eukaryotic sources, including mutations to improve function. In this review, we discuss the critical factors necessary to adequately characterise novel L-asparaginase therapeutic products, including enzyme kinetic parameters, glutaminase activity, and toxicity. One critical consideration is to ensure that the substrate affinity of novel enzymes, as measured by the Michaelis constant KM, is sufficiently low to enable efficient reaction rates in human clinical use. The activity of L-asparaginases towards glutamine as a substrate is discussed and reviewed in detail, as there is much debate in the scientific literature about the importance of this feature for therapeutic enzymes. The recent research in the area is reviewed, including identification of new sources of the enzyme, modulating glutaminase activity, and improving the thermal stability and immunogenic response. New research in the area may benefit from these considerations, to enable the next generation of therapeutic product design. Critical to future work in this area is a complete characterisation of novel enzymes with respect to performance for both L-asparagine and L-glutamine as substrates.


Assuntos
Asparaginase/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Animais , Asparaginase/química , Asparaginase/genética , Asparaginase/metabolismo , Estabilidade Enzimática , Humanos , Cinética , Especificidade por Substrato
5.
J Med Microbiol ; 68(11): 1629-1640, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31553301

RESUMO

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.


Assuntos
Proteínas de Bactérias/imunologia , Esterases/imunologia , Hanseníase/microbiologia , Mycobacterium leprae/enzimologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocinas/genética , Citocinas/imunologia , Estabilidade Enzimática , Esterases/química , Esterases/genética , Feminino , Humanos , Concentração de Íons de Hidrogênio , Cinética , Hanseníase/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Mycobacterium leprae/química , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Óxido Nítrico/imunologia , Espécies Reativas de Oxigênio/imunologia , Alinhamento de Sequência
6.
J Agric Food Chem ; 67(42): 11553-11567, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31553885

RESUMO

Enzymes are macromolecular biocatalysts, widely used in food industry. In applications, enzymes are often immobilized on inert and insoluble carriers, which increase their efficiency due to multiple reusability. The properties of immobilized enzymes depend on the immobilization method and the carrier type. The choice of the carrier usually concerns the biocompatibility, chemical and thermal stability, insolubility under reaction conditions, capability of easy regeneration and reusability, as well as cost efficiency. In this review, we provide an overview of various carriers for enzyme immobilization, with the primary focus on food industry.


Assuntos
Enzimas Imobilizadas/química , Indústria Alimentícia , Estabilidade Enzimática , Enzimas Imobilizadas/economia , Indústria Alimentícia/economia
7.
Microb Cell Fact ; 18(1): 159, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542050

RESUMO

BACKGROUND: Xylanases randomly cleave the internal ß-1,4-glycosidic bonds in the xylan backbone and are grouped into different families in the carbohydrate-active enzyme (CAZy) database. Although multiple xylanases are detected in single strains of many filamentous fungi, no study has been reported on the composition, synergistic effect, and mode of action in a complete set of xylanases secreted by the same microorganism. RESULTS: All three xylanases secreted by Penicillium chrysogenum P33 were expressed and characterized. The enzymes Xyl1 and Xyl3 belong to the GH10 family and Xyl3 contains a CBM1 domain at its C-terminal, whereas Xyl2 belongs to the GH11 family. The optimal temperature/pH values were 35 °C/6.0, 50 °C/5.0 and 55 °C/6.0 for Xyl1, Xyl2, and Xyl3, respectively. The three xylanases exhibited synergistic effects, with the maximum synergy observed between Xyl3 and Xyl2, which are from different families. The synergy between xylanases could also improve the hydrolysis of cellulase (C), with the maximum amount of reducing sugars (5.68 mg/mL) observed using the combination of C + Xyl2 + Xyl3. Although the enzymatic activity of Xyl1 toward xylan was low, it was shown to be capable of hydrolyzing xylooligosaccharides into xylose. Xyl2 was shown to hydrolyze xylan to long-chain xylooligosaccharides, whereas Xyl3 hydrolyzed xylan to xylooligosaccharides with a lower degree of polymerization. CONCLUSIONS: Synergistic effect exists among different xylanases, and it was higher between xylanases from different families. The cooperation of hydrolysis modes comprised the primary mechanism for the observed synergy between different xylanases. This study demonstrated, for the first time, that the hydrolysates of GH11 xylanases can be further hydrolyzed by GH10 xylanases, but not vice versa.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/enzimologia , Polissacarídeos/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucuronatos/metabolismo , Temperatura Alta , Hidrólise , Família Multigênica , Oligossacarídeos/metabolismo , Penicillium chrysogenum/química , Penicillium chrysogenum/genética , Domínios Proteicos , Xilanos/metabolismo
8.
J Agric Food Chem ; 67(37): 10373-10379, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453692

RESUMO

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase, which is an important enzyme for efficient saccharification of agarose or preparation of bioactive oligosaccharides from agarose. Although many ß-agarases have been reported and characterized, there are only a few studies on α-agarases. Here, we cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately 200 kDa belonging to glycoside hydrolase family 96 from Catenovulum agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37 °C and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as agarobiose (A2), agarotetraose (A4), and agarohexaose (A6), in which A4 is the dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose (A3) and A6 into A2 and A4 but cannot act on A2, A3, or A4. This is the first report to characterize the α-agarase action on AOSs in detail. Therefore, CaLJ96 has potential for the manufacture of bioactive AOSs.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Alteromonadaceae/química , Alteromonadaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sefarose/química , Sefarose/metabolismo , Especificidade por Substrato
9.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
10.
J Agric Food Chem ; 67(34): 9611-9617, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31385500

RESUMO

Pullulanase is a commonly used debranching enzyme in the starch processing industry. Because the starch liquefaction process requires high temperature, a thermostable pullulanase is desired. Here, a novel hyperthermostable type II pullulanase gene (pulPY) was cloned from Pyrococcus yayanosii CH1, isolated from a deep-sea hydrothermal site. PulPY was optimally active at pH 6.6 and 95 °C, retaining more than 50% activity after incubation at 95 °C for 10 h. The thermostability was significantly higher than those of most pullulanases reported previously. To further improve its activity and thermostability, the N-terminal and C-terminal domains of PulPY were truncated. The optimum temperature of the combined truncation mutant Δ28N + Δ791C increased to 100 °C with a specific activity of 32.18 U/mg, which was six times higher than that of wild-type PulPY. PulPY and the truncation mutant enzyme could realize the combined use of pullulanase with α-amylase during the starch liquefaction process to improve hydrolysis efficiency.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Pyrococcus/enzimologia , Água do Mar/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Domínios Proteicos , Pyrococcus/química , Pyrococcus/genética , Pyrococcus/isolamento & purificação , Amido/metabolismo , Especificidade por Substrato , Temperatura Ambiente
11.
Food Chem ; 301: 125266, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387037

RESUMO

ß-Xylosidase, of the glycoside hydrolase family 43 from Bacillus sp. HJ14, was expressed in Escherichia coli. Recombinant ß-xylosidase (rHJ14GH43) exhibited maximum activity at 25 °C, approximately 15, 45, and 88% of maximum activity at 0, 10, and 20 °C, respectively, and poor stability at temperatures over 20 °C. rHJ14GH43 showed moderate or high activity, but poor stability, in NaCl, KCl, NaNO3, KNO3, Na2SO4, and (NH4)2SO4 at concentrations from 3.0 to 30.0% (w/v). The crystal structure of rHJ14GH43 was resolved and showed higher structural flexibility due to fewer salt bridges and hydrogen bonds compared to mesophilic and thermophilic ß-xylosidases. High structural flexibility is presumed to be a key factor for catalytic adaptations to low temperatures and high salt concentrations. Approximately one-third of the surface of rHJ14GH43 is positively charged, which may be the primary factor responsible for poor stability in high neutral salt environments.


Assuntos
Bacillus/enzimologia , Xilosidases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
12.
J Microbiol Biotechnol ; 29(8): 1310-1315, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370115

RESUMO

Hyaluronidases enhance therapeutic drug transport by breaking down the hyaluronan barrier to lymphatic and capillary vessels, facilitating their tissue absorption. Commercially available hyaluronidases are bovine in origin; however, they pose risks such as bovine spongiform encephalopathy. The present study aimed to develop a novel, highly active hyaluronidase and assess its function. Therefore, in order to find the most efficient active hyaluronidase, we produced several shortened hyaluronidases with partial removal of the N- or C-terminal regions. Moreover, we created an enzyme that connected six histidines onto the end of the hyaluronidase C-terminus. This simplified subsequent purification using Ni2+ affinity chromatography, making it feasible to industrialize this highly active recombinant hyaluronidase which exhibited catalytic activity equal to that of the commercial enzyme. Therefore, this simple and effective isolation method could increase the availability of recombinant hyaluronidase for research and clinical purposes.


Assuntos
Histidina/metabolismo , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Oligopeptídeos/metabolismo , Proteínas Recombinantes , Animais , Bovinos , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Células HEK293 , Humanos , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/isolamento & purificação , Concentração de Íons de Hidrogênio , Temperatura Ambiente
13.
J Agric Food Chem ; 67(35): 9868-9876, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31389242

RESUMO

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0-8.0 and 30-60 °C.


Assuntos
Proteínas de Bactérias/química , Glucosiltransferases/química , Thermus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Conformação Proteica , Alinhamento de Sequência , Thermus/química , Thermus/genética
14.
J Agric Food Chem ; 67(37): 10505-10512, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31462045

RESUMO

An aspartic protease gene (Bsapa) was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris. The recombinant BsAPA showed maximal activity at pH 3.0 and 75 °C and remained stable at 70 °C and below, indicating the thermostable nature of BsAPA. However, heat inactivation still limits the application of BsAPA. To further improve its thermostability, an autocatalysis site (L205-F206) in BsAPA was identified and three mutants (F193W, K204P, and A371V) were generated based on the analysis of the structure neighboring the autocatalysis site. These mutants have improved thermostability, and their half-life at 75 °C increased by 0.5-, 0.2-, and 0.3-fold, respectively. A triple-site mutant (F193W/K204P/A371V) was generated, with 1.5-fold increased half-life at 80 and a 10.7 °C increased Tm, compared with those of the wild-type. These results indicate that autocatalysis of aspartic protease reduces enzyme thermostability. Furthermore, site-directed mutagenesis at regions near the autocatalysis site is an efficient approach to improve aspartic protease thermostability.


Assuntos
Ascomicetos/enzimologia , Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Ascomicetos/química , Ascomicetos/genética , Ácido Aspártico Proteases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Temperatura Alta , Cinética , Mutagênese Sítio-Dirigida , Mutação , Pichia/genética , Pichia/metabolismo
15.
J Microbiol ; 57(10): 900-909, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31463786

RESUMO

In the present study, a laccase gene (BaLc) from a lignin degrading bacterium, Bacillus atrophaeus, has been cloned and expressed in Escherichia coli. The optimal catalytic activity of the protein was achieved at 5.5 pH and 35°C temperature, measured by oxidation of ABTS. The Km and Vmax values were determined as 1.42 mM and 4.16 µmole/min, respectively. To achieve the enzyme recovery, the biocatalyst (BaLc) was covalently attached onto the functionalized iron magnetic-nanoparticles. The nanoparticles were characterized by zeta-potential and FTIR analyses. The immobilized BaLc enzyme was physico-kinetically characterized, exhibiting retention of 60% of the residual activity after ten reaction cycles of ABTS oxidation. The immobilized biocatalyst system was tested for its biotechnological exploitability in plant juice processing, achieving 41-58% of phenol reduction, 41-58% decolorization, 50-59% turbidity reduction in the extracts of banana pseudo-stem and sweet sorghum stalk, and apple fruit juice. This is the first study to demonstrate the use of nanoparticle-laccase conjugate in juice clarification. The findings suggest that B. atrophaus laccase is a potential catalytic tool for plant juice bioprocessing activities.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Lacase/química , Bacillus/química , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Manipulação de Alimentos , Sucos de Frutas e Vegetais/análise , Concentração de Íons de Hidrogênio , Cinética , Nanopartículas
16.
J Enzyme Inhib Med Chem ; 34(1): 1474-1480, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31414611

RESUMO

The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive. High-throughput screening combined with novel computational tools can reduce these factors. Thus, we developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent to the in-silico library screening. While the in-silico screening yields in the identification of potential allosteric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lipase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay conditions were adjusted to CRL's properties including pH, temperature and substrate specificity for two different substrates. The optimised assay conditions were validated and were used to characterise Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reproducible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated high-throughput screening workflow.


Assuntos
Lipase/metabolismo , Miniaturização , Regulação Alostérica , Candida/enzimologia , Cristalografia por Raios X , Estabilidade Enzimática , Ensaios de Triagem em Larga Escala , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Limite de Detecção , Lipase/química , Reprodutibilidade dos Testes , Especificidade por Substrato , Temperatura Ambiente
17.
Aquat Toxicol ; 214: 105254, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31357109

RESUMO

Intracellular copper/zinc superoxide dismutase (icCuZnSOD) is a member of superoxide dismutase family that is capable of catalyzing the superoxide radicals into either hydrogen peroxide (H2O2) or ordinary molecular oxygen (O2). Unlike mammals, the study of icCuZnSOD in aquatic animals is still in the infancy stage. Here, we identified the cDNA of na-iccuznsod from yellow drum (Nibea albiflora, Richardson 1846) and obtained its fusion protein for the first time. The mRNA expressions of na-iccuznsod were investigated in different tissues, and the dominant distribution was found in head-kidney, followed by brain, liver, heart, and gill. The effects of ammonia-N/nitrite-N on the mRNA expressions of na-iccuznsod were investigated. Na-iccuznsod transcription levels showed a general tendency of an initial up-regulation followed by a down-regulation in liver, gill, and head-kidney when yellow drum were exposed to ammonia-N/nitrite-N at the lethal concentration 50 at 96 h post-treatment, suggesting the important role of Na-icCuZnSOD in eliminating reactive oxygen species (ROS) induced by ammonia-N/nitrite-N. In addition, the characteristics of Na-icCuZnSOD protein and its comparative analysis with Na-ecCuZnSOD were investigated. Na-icCuZnSOD protein showed high enzyme stabilities over a wide range of temperature (10 to 60 °C) and pH (4.9 to 11.0), indicating its broad in vitro applications in many industries. Furthermore, the comparative analysis of Na-icCuZnSOD and Na-ecCuZnSOD gives a new perspective for the study of their structure-function relationship. Collectively, the present study will advance our understanding of the toxicity of ammonia-N/nitrite-N on yellow drum through testing the mRNA expression of iccuznsod gene, and broaden our knowledge of the protein characteristics of icCuZnSOD from fish.


Assuntos
Amônia/toxicidade , Cobre/metabolismo , Regulação Enzimológica da Expressão Gênica , Nitritos/toxicidade , Perciformes/genética , Estresse Fisiológico/genética , Superóxido Dismutase/genética , Zinco/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Estabilidade Enzimática/efeitos dos fármacos , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Temperatura Ambiente , Fatores de Tempo , Poluentes Químicos da Água/toxicidade
18.
J Agric Food Chem ; 67(33): 9307-9313, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352784

RESUMO

Porphyra is one of the most consumed types of red algae. Porphyran is the major polysaccharide extracted from Porphyra, and it is composed of alternating 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) residues. ß-Porphyranases are promising tools for degrading porphyran; however, few enzymes have been reported, and the biochemical properties of porphyranases are still unclear. Here, a novel GH16 ß-porphyranase, designated as Por16A_Wf, was cloned from Wenyingzhuangia fucanilytica and expressed in Escherichia coli. Its biochemical properties and hydrolysis pattern were characterized. Por16A_Wf exhibited stable activity on a wide pH scale from 3.5 to 11.0. Glycomics analysis using LC-MS revealed that Por16A_Wf specifically hydrolyzed the glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-l-galactopyranose and methyl-d-galactose in -2 and +2 subsites, respectively. Por16A_Wf could be applied as a biotechnological tool for tailoring porphyran, which would serve in directional preparation of its disaccharide, producing products with various molecular weights and facilitating investigation of the structural heterogeneity of Porphyra polysaccharides.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sefarose/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Filogenia , Porphyra/química , Porphyra/metabolismo , Sefarose/química , Sefarose/metabolismo , Alinhamento de Sequência
19.
Chem Biol Interact ; 310: 108756, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325422

RESUMO

Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37°C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.


Assuntos
Butirilcolinesterase/metabolismo , Engenharia de Proteínas/métodos , Butirilcolinesterase/genética , Substâncias para a Guerra Química/metabolismo , Dimerização , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Inseticidas/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Compostos Organofosforados/metabolismo
20.
Chemistry ; 25(54): 12576-12582, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31314132

RESUMO

Nature has evolved enzymes with exquisite active sites that catalyze biotransformations with high efficiency. However, the exploitation of natural enzymes is often hampered by poor stability, and natural enzyme production and purification are costly. Supramolecular self-assembly allows the construction of biomimetic active sites, although it is challenging to produce such artificial enzymes with catalytic activity and stability that rival those of natural enzymes. We report herein a strategy to produce a horseradish peroxidase (HRP) mimic based on the assembly of chitosan with a G-quadruplex DNA (G-DNA)/hemin complex. A network-like morphology of the assembled nanomaterial was observed together with a remarkable enhancement of peroxidase activity induced by the chitosan and G-DNA components. The turnover frequency and catalytic efficiency of the enzyme-mimicking material reached or even surpassed those of HRP. Moreover, the catalytic complex exhibited higher tolerance than HRP to harsh environments, such as extremely low pH or high temperatures. In accord with the experimental and simulated results, it is concluded that the spatial distribution of the G-DNA and chitosan components and the exposure of the catalytic center may facilitate the coordination of substrates by the hemin iron, leading to the superior activity of the material. Our work provides a simple and affordable avenue to produce highly active and robust enzyme-mimicking catalytic nanomaterials.


Assuntos
Materiais Biomiméticos/química , Quitosana/química , Quadruplex G , Hemina/química , Peroxidase do Rábano Silvestre/química , Nanoestruturas/química , Catálise , Domínio Catalítico , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Dinâmica Molecular , Oxirredução , Conformação Proteica , Temperatura Ambiente , Termodinâmica
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