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1.
Nucleic Acids Res ; 49(8): 4750-4767, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33856458

RESUMO

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , DNA/química , Histonas/química , Histonas/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/metabolismo , Dimerização , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Proteólise , Tripsina/química
2.
J Med Chem ; 64(3): 1611-1625, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33471524

RESUMO

In the S1 pocket, the serine proteases thrombin and trypsin commonly feature Asp189 and a Ala190Ser and Glu192Gln exchange. Nevertheless, thrombin cleaves peptide chains solely after Arg, and trypsin after Lys and Arg. Thrombin exhibits a Na+-binding site next to Asp189, which is missing in trypsin. The fragment benzylamine shows direct H-bonding to Asp189 in trypsin, while in thrombin, it forms an H-bond to Glu192. A series of fragments and expanded ligands were studied against both enzymes and mutated variants by crystallography and ITC. The selectivity-determining features of both S1 pockets are difficult to assign to one dominating factor. The Ala190Ser and Glu192Gln replacements may be regarded as highly conserved as no structural and affinity changes are observed between both proteases. With respect to charge distribution, Glu192, together with the thrombin-specific sodium ion, helps in creating an electrostatic gradient across the S1 pocket. This feature is definitely absent in trypsin but important for selectivity along with solvation-pattern differences in the S1 pocket.


Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Trombina/química , Tripsina/química , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Cinética , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Relação Estrutura-Atividade , Especificidade por Substrato , Trombina/genética , Tripsina/genética
3.
J Med Chem ; 64(2): 1001-1017, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33307695

RESUMO

Herein, we report our work exploring the essential requirements for fluorophore selection during the development of various fluorescence applications. We assembled a library of chromone-derived fluorophores with diverse structure-fluorescence properties, which allowed us to choose the fluorophore pairs with similar structures but differing fluorescence properties and compared the performance of the selected fluorophore pairs in three types of commonly used fluorescence applications. We found that the selection standard of a suitable fluorophore is variable depending on the application. (1) In fluorescence imaging, fluorophores with strong and constant fluorescence under various conditions, such as a large pH range, are preferred. Notably, (2) in the detection of bioactive species, fluorophores with relatively lower fluorescence quantum yield favor the detection sensitivity. Furthermore, (3) in enzymatic assays employing fluorescence, the key parameter is the binding affinity between the fluorophore and the enzyme.


Assuntos
Cromonas/química , Corantes Fluorescentes/química , Linhagem Celular Tumoral , Sobrevivência Celular , Enzimas/química , Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Imagem Óptica/métodos , Sensibilidade e Especificidade , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Tripsina/química
4.
Meat Sci ; 171: 108290, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32949821

RESUMO

A long ripening period is essential for developing dry-cured ham flavor, but the effect of ripening process on its in vitro digestion product has not been extensively studied. Here, we investigated the in vitro digestion profiles from Chinese dry-cured ham (Jinhua, Rugao and Xuanwei) with different ripening periods by particle size measurement, gel eletrophoresis analysis and nano liquid chromatography-tandem mass spectrometry. The results showed that the in vitro digestibility of ham was in a good agreement with the particle size of digestion products. Among the three types dry-cured ham, Xuanwei showed the highest digestibility (93.46%), followed by Jinhua (74.46%). In term of ripening period, the 2-year Xuanwei and Jinhua showed the diversity of peptides (especially for peptide with molecular weight < 2500 Da), besides their good digestibility. Moreover, the highest amount of peptides (404) was observed in 2-year Jinhua compared to other hams. Our finding gave a new insight into the digestion profiles and nutritional properties of Chinese dry-cured hams.


Assuntos
Produtos da Carne/análise , Peptídeos/química , Proteólise , Animais , Digestão , Manipulação de Alimentos/métodos , Tamanho da Partícula , Pepsina A/química , Suínos , Tripsina/química
5.
Nat Commun ; 11(1): 2918, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522984

RESUMO

Coarse-graining of fully atomistic molecular dynamics simulations is a long-standing goal in order to allow the description of processes occurring on biologically relevant timescales. For example, the prediction of pathways, rates and rate-limiting steps in protein-ligand unbinding is crucial for modern drug discovery. To achieve the enhanced sampling, we perform dissipation-corrected targeted molecular dynamics simulations, which yield free energy and friction profiles of molecular processes under consideration. Subsequently, we use these fields to perform temperature-boosted Langevin simulations which account for the desired kinetics occurring on multisecond timescales and beyond. Adopting the dissociation of solvated sodium chloride, trypsin-benzamidine and Hsp90-inhibitor protein-ligand complexes as test problems, we reproduce rates from molecular dynamics simulation and experiments within a factor of 2-20, and dissociation constants within a factor of 1-4. Analysis of friction profiles reveals that binding and unbinding dynamics are mediated by changes of the surrounding hydration shells in all investigated systems.


Assuntos
Modelos Teóricos , Benzamidinas/química , Sítios de Ligação , Simulação de Dinâmica Molecular , Ligação Proteica , Cloreto de Sódio/química , Termodinâmica , Tripsina/química , Água/química
6.
PLoS One ; 15(5): e0232253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365084

RESUMO

Proteases have been implicated in the tumorigenesis and aggressiveness of a variety of cancer types. In fact, proteases have proven to be very clinically useful as tumor biomarkers in the blood of patients. Proteases are typically involved in complex systems of substrates, activators, and inhibitors, thus making our ability to establish their exact function in cancer more difficult. Trypsin, perhaps the most famous of proteases, has been shown to play a role in cancer progression, but its functional role in ovarian cancer has not been much studied. PAR2, a transmembrane receptor that is known to be activated by trypsin, has been reported to be associated with ovarian cancer. Here, we found that stimulation of ovarian cancer cell lines with trypsin or PAR2 activating peptide markedly increased MAPK signaling and cell proliferation. Additionally, HE4, a WAP-family glycoprotein and ovarian cancer biomarker, was found to inhibit trypsin degradation, thereby retaining its activity. Patient data seemed to support this phenomenon, as the serum of ovarian cancer patients with high HE4 expression, revealed significantly elevated trypsin levels. These data support the hypothesis that trypsin plays a tumorigenic role in ovarian cancer, which can be mediated by its receptor PAR2, and potentiated by HE4.


Assuntos
Carcinoma Epitelial do Ovário/genética , Neoplasias Ovarianas/genética , Receptor PAR-2/metabolismo , Tripsina/genética , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/metabolismo , Carcinoma Epitelial do Ovário/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Ovarianas/metabolismo , Proteólise , Receptor PAR-2/genética , Tripsina/química , Tripsina/metabolismo , Regulação para Cima
7.
Artigo em Inglês | MEDLINE | ID: mdl-32361468

RESUMO

The present study aimed to evaluate the effect of the immobilization method of trypsin on biochar on the hydrolysis of casein from different sources, when compared to the process using trypsin in native form, to obtain bioactive peptides. The modification of the surface of biochar with glutaraldehyde was effective, as shown by the results of FTIR assay and the texture profile of the materials. Both activated and functionalized biochar showed high immobilization efficiency (greater than 87%) and high binding capacity (greater than 91 mg/g). During hydrolysis, the biocatalyst obtained by enzyme immobilization on the functionalized biochar presented a higher hydrolysis capacity for the different caseins when compared to the enzyme immobilized by adsorption, with values of 3.05 and 2.73 U/mg for goat casein, 2.36 and 1.85 U/mg for bovine casein, and 2.60 and 2.37 U/mg for buffalo, casein, respectively, with 60 min of reaction. The results of inhibitory activity in this study ranged from 93.5% and 25.5% for trypsin in its free form and immobilized on functionalized activated carbon, respectively, under the same reaction conditions. The immobilization methods were efficient, presenting high immobilization capacity. The proteolytic activity of trypsin immobilized via covalent binding was higher when compared the immobilization by adsorption. Thus, the functionalized biochar has proven to be potential support for enzyme immobilization, and the biocatalyst can be reused for more than 4 cycles. Despite lower ACE inhibition values of hydrolyzed obtained with the immobilized enzymes compared to free enzymes, biocatalysts present advantage due to the possibility of reuse.


Assuntos
Caseínas/química , Carvão Vegetal/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Tripsina/química , Tripsina/metabolismo , Adsorção , Animais , Biocatálise , Bovinos , Estabilidade Enzimática , Glutaral/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Ácidos Fosfóricos/química , Proteólise , Propriedades de Superfície , Temperatura
8.
BMC Bioinformatics ; 21(1): 143, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293241

RESUMO

BACKGROUND: Protein-protein interactions (PPIs) are fundamental in many biological processes and understanding these interactions is key for a myriad of applications including drug development, peptide design and identification of drug targets. The biological data deluge demands efficient and scalable methods to characterize and understand protein-protein interfaces. In this paper, we present ppiGReMLIN, a graph based strategy to infer interaction patterns in a set of protein-protein complexes. Our method combines an unsupervised learning strategy with frequent subgraph mining in order to detect conserved structural arrangements (patterns) based on the physicochemical properties of atoms on protein interfaces. To assess the ability of ppiGReMLIN to point out relevant conserved substructures on protein-protein interfaces, we compared our results to experimentally determined patterns that are key for protein-protein interactions in 2 datasets of complexes, Serine-protease and BCL-2. RESULTS: ppiGReMLIN was able to detect, in an automatic fashion, conserved structural arrangements that represent highly conserved interactions at the specificity binding pocket of trypsin and trypsin-like proteins from Serine-protease dataset. Also, for the BCL-2 dataset, our method pointed out conserved arrangements that include critical residue interactions within the conserved motif LXXXXD, pivotal to the binding specificity of BH3 domains of pro-apoptotic BCL-2 proteins towards apoptotic suppressors. Quantitatively, ppiGReMLIN was able to find all of the most relevant residues described in literature for our datasets, showing precision of at least 69% up to 100% and recall of 100%. CONCLUSIONS: ppiGReMLIN was able to find highly conserved structures on the interfaces of protein-protein complexes, with minimum support value of 60%, in datasets of similar proteins. We showed that the patterns automatically detected on protein interfaces by our method are in agreement with interaction patterns described in the literature.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Animais , Gráficos por Computador , Mineração de Dados , Complexos Multiproteicos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tripsina/química , Tripsina/metabolismo
9.
Nat Commun ; 11(1): 1318, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165630

RESUMO

Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies.


Assuntos
Replicação do DNA , Receptores Virais/metabolismo , Tripsina/química , Camptotecina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores Virais/química , Receptores Virais/genética
10.
J Fluoresc ; 30(2): 229-233, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32052243

RESUMO

The interaction of (2R, 3S)-hydroxyleucine (trypsin inhibitor) and ß-hydroxyvaline with trypsin has been studied by the steady-state fluorescence spectroscopy. The analysis of fluorescence spectra has revealed the mechanism of binding of these nonprotein amino acids to trypsin. According to the docking (2R, 3S)-hydroxyleucine form hydrogen bonds with trypsin having little effect on tryptophan and tyrosine residues in enzyme molecule. The results obtained in this study indicate that fluorescence of trypsin is quenched at high concentrations of amino acids. Thus fluorescence spectra analysis confirms data obtained by molecular docking.


Assuntos
Aminoácidos/química , Tripsina/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Leucina/análogos & derivados , Leucina/química , Leucina/farmacologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Espectrometria de Fluorescência , Tripsina/metabolismo
11.
Anal Chim Acta ; 1102: 1-10, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32043988

RESUMO

A microfluidic platform based on the integration of denaturation and online immobilized enzyme reactor (IMER) digestion for protein pretreatment was first developed on a glass chip. The design of three inlet channels and two groups of snake channel in glass chip can allow the protein solution, the reducing reagent and the alkylating agent to be simultaneously injected into the chip channel and ensured the reaction solution on-line efficient mixing and sufficient reacting. By thiol-ene click chemistry, the capillary-based and glass chip-based trypsin IMER on the surface of poly(trimethylolpropane trimethacrylate) monolith were fabricated. The wide range of flow rate tolerance (0.8-5.0 µL/min), and the acceptable reproducibility (RSD% = 3.1%, n = 5) and stability (13.8% decrease of enzyme activity in 2 months) indicated the feasibility of using IMER for online digestion of proteins. Compared with the solution denaturation-offline IMER digestion, the integrated microfluidic platform of chip denaturation-chip IMER and chip denaturation-online IMER have comparable protein identification ability for mouse liver protein with a similar number of protein (798 or 826 vs. 843) and unique peptides (3923 or 4593 vs. 3916). More importantly, the easy and fast digestion of protein samples and possible combination with MS revealed that this microfluidic platform can be a potential method for rapid proteomics analysis.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Proteoma/análise , Proteômica/métodos , Animais , Bovinos , Ditiotreitol/química , Enzimas Imobilizadas/química , Iodoacetamida/química , Fígado/química , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Desnaturação Proteica , Proteólise , Proteoma/química , Soroalbumina Bovina/química , Tripsina/química
12.
J Med Chem ; 63(6): 3274-3289, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32011145

RESUMO

Trypsin and thrombin, structurally similar serine proteases, recognize different substrates; thrombin cleaves after Arg, whereas trypsin cleaves after Lys/Arg. Both recognize basic substrate headgroups via Asp189 at the bottom of the S1 pocket. By crystallography and isothermal titration calorimetry (ITC), we studied a series of d-Phe/d-DiPhe-Pro-(amino)pyridines. Identical ligand pairs show the same binding poses. Surprisingly, one ligand binds to trypsin in protonated state and to thrombin in unprotonated state at P1 along with differences in the residual solvation pattern. While trypsin binding is mediated by an ordered water molecule, in thrombin, water is scattered over three hydration sites. Although having highly similar S1 pockets, our results suggest different electrostatic properties of Asp189 possibly contributing to the selectivity determinant. Thrombin binds a specific Na+ ion next to Asp189, which is absent in trypsin. The electrostatic properties across the S1 pocket are further attenuated by charged Glu192 at the rim of S1 in thrombin, which is replaced by uncharged Gln192 in trypsin.


Assuntos
Antitrombinas/metabolismo , Dipeptídeos/metabolismo , Piridinas/metabolismo , Trombina/metabolismo , Inibidores da Tripsina/metabolismo , Tripsina/metabolismo , Animais , Antitrombinas/síntese química , Sítios de Ligação , Calorimetria , Bovinos , Cristalografia por Raios X , Dipeptídeos/síntese química , Humanos , Ligantes , Ligação Proteica , Prótons , Piridinas/síntese química , Termodinâmica , Trombina/química , Tripsina/química , Inibidores da Tripsina/síntese química , Água/metabolismo
13.
Mikrochim Acta ; 187(2): 144, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31970520

RESUMO

Iron(III-immobilized magnetic nano-composites (MNCs) were first fabricated using one-step aqueous self-assembly of oligopeptides (Glu-Pro-Ala-Lys-Ala-Lys-Ala-Lys; EPAK-VI) for the highly selective capture of phosphopeptides from complex biological samples. Under physiological conditions, EPAK-VI can readily self-organize into a robust and complete coating layer mainly composed of ß-sheets and ß-turns on the surface of Fe3O4@GO and Fe3O4@C MNCs. Tailored by the cyclic structure of proline, the Glu-Pro motifs of EPAK-VI are vertically erected on the surface and thus serve as an effective linker to chelate Fe3+ through carboxyl (COO-) group in the glutamic acid (E) residues. The ionic hydrogen bonds between the ε-amino groups and the surface negative charges coupled with intermolecular hydrogen bonds render the EPAK-VI coating on the MNCs insusceptible to repeated extreme washing conditions. The Fe3+-EPAK-VI coated MNCs exhibit high enrichment efficiency for ß-casein tryptic digest (0.05 fmol µL-1), excellent selectivity from mixed digests (ß-casein/bovine serum albumin, mass ratio 1:500), and high recovery rate (over 80%). Graphical abstractSchematic representation of the fabrication of Fe3+-immobilized MNCs for phosphopeptide enrichment.


Assuntos
Nanopartículas de Magnetita/química , Nanocompostos/química , Oligopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Animais , Caseínas/sangue , Caseínas/química , Caseínas/isolamento & purificação , Bovinos , Grafite/química , Humanos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/isolamento & purificação , Fosfopeptídeos/sangue , Proteólise , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/química
14.
Arch Biochem Biophys ; 682: 108280, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-31996302

RESUMO

Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F-Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.


Assuntos
Citoesqueleto de Actina/química , Actinas/química , Cofilina 1/química , Tropomiosina/química , Animais , Citoesqueleto/química , Humanos , Camundongos , Modelos Moleculares , Mutação , Polimerização , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas , Coelhos , Saccharomyces cerevisiae , Tripsina/química
15.
Spectrochim Acta A Mol Biomol Spectrosc ; 230: 118036, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-31931358

RESUMO

In this work, the effects of I2 on the activities and conformational structures of digestive enzymes, trypsin and pepsin were studied. The results indicated that the enzyme activities were decreased to some extent in the presence of I2, especially trypsin. Upon gradual addition of I2, the intrinsic fluorescence quenching of trypsin and pepsin were observed by mainly static collision and hydrophobic forces. I2 is more likely to cause the fluorescence quenching of trypsin than that of pepsin. Compared with pepsin, trypsin has a greater ability to bind with I2. The synchronous fluorescence spectral results indicated that I2 induced the quaternary structure changes of trypsin/pepsin and changed the hydrophobicity of Tyr and Trp residues. In addition, molecular docking was used to obtain the binding mode and the various amino acid residues of trypsin and pepsin with I2. These investigations may constitute a solid work to further explain the process of migration and transformation of I2 in digestive system.


Assuntos
Iodo/farmacologia , Pepsina A/metabolismo , Inibidores de Proteases/farmacologia , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Animais , Simulação de Acoplamento Molecular , Pepsina A/antagonistas & inibidores , Pepsina A/química , Ligação Proteica , Estrutura Quaternária de Proteína/efeitos dos fármacos , Suínos , Tripsina/química
16.
Bioengineered ; 11(1): 1-10, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31795804

RESUMO

This study presents new methods for hydrolyzing bacterial cell in cyclic utilization of waste bacterial cell for L-tryptophan production by fermentation. Using enzymatic hydrolysis of the pre-treated bacterial cells which were collected from an L-tryptophan fermentation broth, trypsin was selected as the optimal protease for hydrolyzing the bacterial cell. The optimum conditions for hydrolysis were determined by the orthogonal test. Hydrolyzate was then dealt with a compound protease to further increase its content of free amino acids. With the optimum conditions of pH = 8, temperature of 37°C, treatment time of 6 h, and E/S of 4%, the final content of free amino acids in the hydrolyzate was 500.61 mg/g. The hydrolyzate and the yeast extract were added to the medium at the proportion of 1:1, which served as an organic nitrogen source for L-tryptophan production by fermentation. The production of L-tryptophan was 53.87 g/L, and the highest biomass was 53.45 g/L. As an organic nitrogen source, this hydrolyzate satisfies the requirements for L-tryptophan production by fermentation.


Assuntos
Escherichia coli/química , Escherichia coli/metabolismo , Nitrogênio/metabolismo , Tripsina/química , Triptofano/biossíntese , Biocatálise , Biomassa , Meios de Cultura/química , Meios de Cultura/metabolismo , Escherichia coli/crescimento & desenvolvimento , Fermentação , Hidrólise
17.
Food Chem ; 310: 125823, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31757489

RESUMO

Phenolic acids, which are important aromatic secondary metabolites, are widely distributed in plant foods. In this study, a simple, economical and fast on-line immobilized trypsin microreactor was developed for evaluating the inhibitory activity of phenolic acids by capillary electrophoresis. The Michaelis-Menten constant (Km) of immobilized trypsin was determined as 0.99 mM, and the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) of benzamidine were measured as 3.39 and 1.68 mM, respectively. Then, the developed strategy was applied to investigate the inhibitory activity of six phenolic acids on trypsin. The results showed that gallic acid, caffeic acid and ferulic acid had high inhibitory activity at concentration of 150 µM. Molecular docking results illustrated that gallic acid, caffeic acid and ferulic acid can interact indirectly with the catalytic and substrate-binding sites of trypsin. The developed strategy is an effective tool for evaluating inhibitory activity of phenolic acids on trypsin.


Assuntos
Eletroforese Capilar/instrumentação , Enzimas Imobilizadas/metabolismo , Hidroxibenzoatos/metabolismo , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Sítios de Ligação , Reatores Biológicos , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Ácidos Cafeicos/farmacologia , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Ácidos Cumáricos/farmacologia , Eletroforese Capilar/métodos , Enzimas Imobilizadas/química , Ácido Gálico/química , Ácido Gálico/metabolismo , Ácido Gálico/farmacologia , Hidroxibenzoatos/farmacologia , Simulação de Acoplamento Molecular , Tripsina/química , Inibidores da Tripsina/metabolismo
18.
Int J Biol Macromol ; 143: 462-471, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31759008

RESUMO

Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5'-4,4'-dimethyltryptamine-thymidine-succinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL-4B were 40 mg/mL trypsin solution, pH 7 at 4 °C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) by the immobilized trypsin were 55 °C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 µM of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.


Assuntos
Enzimas Imobilizadas/química , Proteínas de Peixes/química , Baço/enzimologia , Tripsina/química , Atum , Animais , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio
19.
Biosens Bioelectron ; 149: 111828, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31726275

RESUMO

The determination of cytochrome c in the human serum sample is a regular medical investigation performed to assess cancer diseases. Herein, we used interferometric reflectance spectroscopy (IRS) based biosensor for the determination of cytochrome c. For this purpose first, the nanoporous anodic alumina (NAA) was fabricated. Then, the NAA pore walls were functionalized with 3-aminopropyl trimethoxy silane (NAA-NH2). Subsequently, the trypsin enzyme was immobilized on the NAA pore walls. The sensing principle of proposed IRS sensor to cytochrome c is based on a change in the intensity of the reflected light to a charge-coupled device (CCD) detector after digesting of cytochrome c by immobilized trypsin enzymes on NAA-NH2 into the heme-peptide fragment. The heme-peptide fragment then oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to green color ABTS·- anion radical in the presence of hydrogen peroxide. The generated green color ABTS·- anion radical solution adsorbed the white light and therefore the intensity of the reflected light from NAA to the CCD decreased. The decrease in the intensity of the white light had a logarithmic relationship with the concentration of the cytochrome c in the range of 1-100 nM. The limit of detections (LOD) for cytochrome c was 0.5 nM. The proposed biosensor exhibited high selectivity, sensitivity, and good stability.


Assuntos
Técnicas Biossensoriais , Citocromos c/isolamento & purificação , Neoplasias/sangue , Tripsina/química , Óxido de Alumínio/química , Benzotiazóis/química , Citocromos c/sangue , Humanos , Peróxido de Hidrogênio/química , Interferometria , Nanoporos , Neoplasias/diagnóstico , Análise Espectral , Ácidos Sulfônicos/química
20.
J Pharm Biomed Anal ; 179: 112995, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31767225

RESUMO

An efficient deglycosylation process is a key requirement for the identification and characterization of glycosylation during the production and purification of therapeutic antibodies. PNGase F is widely used for the deglycosylation of N-linked glycans. The commonly-used in-solution deglycosylation method is relatively time-consuming and requires several hours up to overnight for complete removal of all N-linked glycans. In order to develop a simple and efficient method for the rapid release of N-linked glycans from glycoproteins, we fabricated trypsin- and PNGase F-impregnated polyacrylamide gels in a commercial 200 µL volume pipette tip. Our enzyme reactor is based on simple photochemical copolymerization of monomers using the following procedure: (1) a pipette tip was filled with a gel solution comprising acrylamide, N,N'-methylene-bis-acrylamide containing PNGase F or trypsin with 2,2-azobis(2-methyl-N-(2-hydroxyethyl) propionamide) as a photocatalytic initiator; and (2) in situ polymerization of gel solution approximately 30 mm from the tip was performed by irradiation with a 365 nm blue LED beam from a distance 10 mm. The fixed enzymes maintained their activities in the polyacrylamide gel and the reaction was completed by 40 iterations of suction and discharge with a pipette (hereafter referred to as manual pipetting times) for 8 min with each enzyme digestion. Capillary electrophoresis (CE) of released glycans labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) demonstrated quantitative recovery of glycans from selected glycoproteins.


Assuntos
Resinas Acrílicas/química , Glicoproteínas/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Polissacarídeos/química , Eletroforese Capilar/métodos , Glicosilação , Técnicas de Síntese em Fase Sólida , Tripsina/química
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