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1.
Chem Biol Interact ; 329: 109223, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32781033

RESUMO

Thromboembolism is a major cause of morbidity and mortality worldwide. Most therapeutic drugs for treating thrombosis can cause hemorrhage and have short half-lives within human blood circulation resulting in a need to discover and develop novel anticoagulants/antithrombotics. EuRP-61 has been isolated from a plant latex (Euphorbia resinifera) and characterized as a serine protease. In this study, EuRP-61 was able to hydrolyze all chains of human fibrin clots. The enzyme may have long term stability in blood circulation as its fibrinogenolytic activity was not affected by human blood circulating inhibitors such as α2-macroglobulin and antithrombin III. The enzyme may affect the extrinsic, intrinsic or common pathways of the human blood coagulation cascade as evidenced by its prolonged of both prothrombin (PT) and activated partial thromboplastin (APTT) time. Moreover, the enzyme inhibited platelet aggregation via the ADP-receptor pathway. EuRP-61 was not toxic to human red blood cells in the 4 common blood groups (A, B, O and AB) (all Rh+) or human peripheral blood mononuclear cells (hPBMCs). The enzyme may protect human peripheral blood cells from aggregation without destroying them. This study provides evidence that EuRP-61 may have potential as an agent for the treatment of thrombosis.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Euphorbia/enzimologia , Fibrinolíticos/farmacologia , Peptídeo Hidrolases/farmacologia , Proteínas de Plantas/farmacologia , Anticoagulantes/isolamento & purificação , Antitrombina III/antagonistas & inibidores , Antitrombina III/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Fibrinolíticos/isolamento & purificação , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Agregação Plaquetária/efeitos dos fármacos , alfa 2-Macroglobulinas Associadas à Gravidez/antagonistas & inibidores , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo
2.
Plant Mol Biol ; 104(3): 327-337, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761540

RESUMO

KEY MESSAGE: Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.


Assuntos
Apiaceae/enzimologia , Apiaceae/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/metabolismo , Proteínas de Plantas/metabolismo , Apiaceae/genética , China , Cromatografia Líquida de Alta Pressão/métodos , Coenzima A Ligases/genética , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Furocumarinas/química , Furocumarinas/genética , Regulação da Expressão Gênica de Plantas , Cinética , Medicina Tradicional Chinesa , Fenilalanina Amônia-Liase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Transcriptoma
3.
Food Chem ; 333: 127491, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659672

RESUMO

A cascade process for the sequential recovery of proteins and feruloylated arabinoxylan from wheat bran is proposed, involving a protein isolation step, enzymatic destarching and subcritical water extraction. The protein isolation step combining lactic acid fermentation and cold alkaline extraction reduced the recalcitrance of wheat bran, thus improving the total yields of the subsequent subcritical water extraction. The time evolution of subcritical water extraction of feruloylated arabinoxylan was compared at two temperatures (160 °C and 180 °C). Longer residence times enhanced the purity of target feruloylated arabinoxylans, whereas higher temperatures resulted in faster extraction at the expense of significant molar mass reduction. The radical scavenging activity of the extracted feruloylated arabinoxylans was preserved after the initial protein isolation step. This study opens new possibilities for the cascade valorization of wheat bran into enriched protein and non-starch polysaccharide fractions, which show potential to be used as functional food ingredients.


Assuntos
Fracionamento Químico/métodos , Ácidos Cumáricos/química , Fibras na Dieta/análise , Proteínas de Plantas/isolamento & purificação , Xilanos/química , Xilanos/isolamento & purificação , Temperatura Alta , Peso Molecular
4.
Food Chem ; 333: 127503, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32683258

RESUMO

Protein isolates were prepared from wet heat processed (APIp) and unprocessed alfalfa seeds (APIc) and characterized for composition and functionality at different pH. APIc and APIp exhibited high content of all the essential amino acids. Antinutrient content of APIp was lower in comparison to APIc and marked reduction in the trypsin inhibitor (85.97%) and lectin activity (100%) was observed. Processing did not cause much reduction of bioactive constituents and antioxidant activity of APIp. Alfalfa protein isolates exhibited complex polypeptide banding ranging from molecular weight of 11-75 kDa. APIp exhibited change in the conformation of protein discerned as alteration in interrelated nuances of ATR-FTIR spectra, XRD-pattern, morphology, charge on proteins and reduced solubility in comparison to APIc due to processing. APIp exhibited marked improvement in the functional properties in comparison to APIc discerned as improved hydration, surface active and gelation properties. Highest hydration and surface active properties were exhibited at pH 9.0, even though APIp at pH 7.0 showed fairly similar functional properties as APIc and APIp at pH 9.0. APIp exhibited reduced least gelation concentration in comparison to APIc at pH 7.0 and also engendered gelation at pH 4.0 and 9.0 contrary to APIc.


Assuntos
Aminoácidos Essenciais/química , Medicago sativa/química , Proteínas de Plantas/química , Concentração de Íons de Hidrogênio , Medicago sativa/efeitos dos fármacos , Peso Molecular , Proteínas de Plantas/isolamento & purificação , Solubilidade , Inibidores da Tripsina/química
5.
Food Chem ; 330: 127313, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569932

RESUMO

Starch granule-surface proteins (SGSPs) and granule-channel proteins (SGCPs) are granule-associated proteins (SGAPs), which have been found to be distributed on the surface and channels of starch granules, respectively. To investigate the impacts of SGAPs on α-amylase hydrolysis of starch, SGCPs or SGAPs of waxy, low and high amylose rice starches were removed. Removal of SGAPs or SGCPs greatly increased hydrolysis rate of rice starches. Meanwhile, these granules incurred a greater number and size of pores on their surfaces during hydrolysis. Compared to low and high amylose starches, waxy starch before and after removing SGAPs exhibited a higher hydrolysis rate. Rice starch hydrolysis began with enlargement of cavity and channels both horizontally and vertically. XRD analysis revealed that removal of SGAPs decreased relative crystallinity (RC) of starch and advanced changes in RC during hydrolysis process. This study provides new information about the role of SGAPs in the mechanisms of α-amylase hydrolysis.


Assuntos
Oryza/química , Proteínas de Plantas/química , Amido/química , alfa-Amilases/metabolismo , Amilose/metabolismo , Hidrólise , Oryza/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Amido/metabolismo , Amido/farmacocinética , Difração de Raios X , alfa-Amilases/química
6.
Food Chem ; 330: 127217, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32521404

RESUMO

Low pH-shifting was firstly applied in the black turtle bean (Phaseolus vulgaris L.) protein isolate treatment by acidic (pH 1.0-3.0) buffer incubation for 8 h, then was adjusted to pH 7.2 and kept 3 h for protein stabilizing. Mild loss of secondary structure was confirmed in the protein isolate after low pH-shifting treatment by CD and FT-IR analyses. Intrinsic fluorescence, UV spectra, surface hydrophobicity, SH content and SDS-PAGE analyses indicated the protein conformation was unfolded with the exposure of much more buried hydrophobic residues, which would result in the enhancement of emulsifying properties, foaming properties and fat holding capacity, and lead to the reduction of solubility and water holding capacity. Furthermore, lower immunoreactivity was observed by the ELISA, and improved digestibility was found in in vitro digestion assay. Our results suggested the low pH-shifting treatments would broaden the application of bean protein isolate with better hydrophobic processing functions and safety.


Assuntos
Phaseolus/química , Proteínas de Plantas/química , Emulsões/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Imunoensaio , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Estrutura Secundária de Proteína , Solubilidade
7.
Food Chem ; 327: 126998, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32438264

RESUMO

Cold-pressed rapeseed meal with high protein content (38.76% protein dry weight basis) was used to prepare rapeseed protein isolates (RPIs) by alkaline extraction (pH 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0) and acid precipitation (pH 3.0, 3.5, 4.0, 4.5, 5.0 and 5.5). The protein with an intact structure and the highest yield (65.08%) was obtained at extraction pH 9.0 and precipitation pH 4.5, accompanied by the lowest D-amino acid content, the lightest colour and the lowest contents of glucosinolates (2.85 mmol/kg), phytic acid (1.05 mg/g) and sinapine (0.68 mg/g). Additionally, water/oil absorption, foaming and emulsifying capacities decreased with decreasing precipitation pH, while the solubility showed the reverse trend. During gastric simulation digestion, the α-polypeptide of cruciferin and napin in the RPIs showed digestive resistance. Overall, pH regulation might be an effective method to isolate high quality RPIs for use in the food processing industry.


Assuntos
Brassica napus/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacocinética , Albuminas 2S de Plantas/farmacocinética , Aminoácidos/análise , Aminoácidos/química , Antígenos de Plantas , Precipitação Química , Cor , Digestão , Emulsificantes/química , Indústria de Processamento de Alimentos/métodos , Glucosinolatos/análise , Concentração de Íons de Hidrogênio , Ácido Fítico/análise , Proteínas de Plantas/química , Óleo de Brassica napus/química , Proteínas de Armazenamento de Sementes/farmacocinética , Solubilidade
8.
Food Chem ; 321: 126745, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32276148

RESUMO

The effect of high pressure (HP) and pulsed electric field (PEF) treatments combined or not with heat on denaturation and allergenicity of Pru p 3, the major allergenic protein of peach, was studied. Denaturation of Pru p 3, determined by ELISA using rabbit IgG, occurred when the protein was treated at 500 and 600 MPa at 20 °C and at 400 MPa at 50 °C. PEF treatment at 25 kV/cm at 50 °C denatures Pru p 3. Allergenicity of Pru p 3 was estimated in vitro by a competitive fluorescent immunoassay using three pools of sera from peach allergic patients. Any treatment applied did not show to influence the binding of Pru p 3 to IgE. When HP and PEF treated samples were tested by the prick test, the skin response was dependent on the particular sensitization of each patient, obtaining an increased reaction in more than 50% of individuals.


Assuntos
Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Hipersensibilidade Alimentar/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Prunus persica/química , Adulto , Animais , Antígenos de Plantas/isolamento & purificação , Estimulação Elétrica , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/metabolismo , Proteínas de Plantas/isolamento & purificação , Pressão , Desnaturação Proteica , Prunus persica/imunologia , Coelhos , Testes Cutâneos
9.
Food Chem ; 317: 126423, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32097824

RESUMO

The impact of pH (6-9) and NaCl concentration (0-0.5 mol.L-1) on sunflower protein extraction was studied through design of experiments. The considered criteria were protein extraction yield (total proteins, helianthinin and albumins), chlorogenic acids covalently bound to proteins, and free chlorogenic acid concentration in the aqueous extract. Statistical analysis showed that the obtained by design of experiments the polynomial models of each extraction criteria were reliable for predicting the responses. They were employed in an original multi-objective optimization methodology. The optimal conditions revealed to be pH 7.3/0.3 mol.L-1 NaCl yielded 46.83% and 59.16% of total protein and albumin extraction yield, 1.730 and 1.998 mg.g-1 of chlorogenic acids covalently bound to helianthinin and albumins in aqueous extract, respectively. The sunflower protein isolate obtained after extraction in this condition had good solubility (40-80% at pH 5-8), functional properties (foaming and emulsifying) and a satisfying color.


Assuntos
Helianthus/metabolismo , Extração Líquido-Líquido/métodos , Proteínas de Plantas/isolamento & purificação , Extração em Fase Sólida/métodos , Albuminas/análise , Albuminas/isolamento & purificação , Albuminas/metabolismo , Ácido Clorogênico/química , Ácido Clorogênico/metabolismo , Concentração de Íons de Hidrogênio , Isomerismo , Extração Líquido-Líquido/instrumentação , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Polifenóis/análise , Polifenóis/metabolismo , Ligação Proteica , Cloreto de Sódio/química , Extração em Fase Sólida/instrumentação
10.
Gene ; 743: 144484, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32081694

RESUMO

Glutathione S-transferases (GSTs) are a large complex family of enzymes (EC 2.5.1.18) that play vital roles in flavonoid metabolism and plant growth and development and are responsive to heavy metal stress. However, knowledge about GST genes in radish (a vegetable crop with an extraordinary capacity to adapt to heavy metal stresses) is limited. Therefore, it is critical to identify putative candidate GST genes responsible for heavy metal stress tolerance and anthocyanin biosynthesis. In this study, we first identified 82 R. sativus GST (RsGST) genes using various bioinformatic approaches, and their expression profiles were characterized from RNAseq data. These RsGST genes could be grouped into 7 major subclasses: tau (43 members), phi (21 members), tetrachlorohydroquinone dehalogenase (7 members), dehydroascorbat reductase (5 members), zeta (3 members), lambda (2 members) and theta (1 member). In addition, most of the RsGST genes showed organ-specific expression in our study. Moreover, the transcripts of RsGSTF12-1 and RsGSTF12-2, belonging to the phi class, might be candidates encoding anthocyanin transporters in carmine radish, whereas the tau class, consisting of RsGSTU13-1, RsGSTU19, RsGSTU24-1, and RsGSTU3, and theta class, consisting of RsGSTT1-1, might be defend radish against adverse heavy metal stresses. These results will aid in understanding the functions of the GST family related to heavy metal stress and anthocyanin biosynthesis, thereby potentially improving radish breeding programs for high-pigment-content material as well as HM-tolerant material.


Assuntos
Antocianinas/biossíntese , Glutationa Transferase/genética , Metais Pesados/efeitos adversos , Proteínas de Plantas/genética , Raphanus/enzimologia , Adaptação Fisiológica , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glutationa Transferase/isolamento & purificação , Glutationa Transferase/metabolismo , Filogenia , Melhoramento Vegetal , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Raízes de Plantas , RNA-Seq , Raphanus/genética , Raphanus/metabolismo , Estresse Fisiológico/efeitos dos fármacos
11.
BMC Res Notes ; 13(1): 60, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32028996

RESUMO

OBJECTIVE: Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. RESULTS: We compared three different protein extraction methods, i.e.,1.5 M Tris-HCl (pH 8.8), 50 mM Tris-HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)-acetone, in a liverwort Dumortiera hirsuta. Among these methods, 50 mM Tris-HCl (pH 7.5) extraction, followed by 20% TCA-acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum. Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Hepatófitas/metabolismo , Proteínas de Plantas/isolamento & purificação , Ecossistema , Células Germinativas Vegetais/metabolismo
12.
Food Chem ; 314: 126168, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31951889

RESUMO

In this work, a kinetic model for protein extraction from Camellia oleifera seed cake using high voltage electrical discharge extraction (HVED) was built with discharge energy inputs as primary variables. The results showed that both the equilibrium yields and the mass transfer coefficient of HVED were highly dependent on the HVED specific energy input per pulse (kJ/kg). After linear and nonlinear fitting with five different basic functions, the best model satisfied the power function relationship through optimizing the influence of specific energy input per pulse on the equilibrium yields and the mass transfer coefficients. Based on the observations, a predictive model that correlates the energy input, mass of raw material and kinetics of HVED extraction was proposed. The validity of the predictive model was verified, and the observed deviation was found to be less than 10%. This could provide a model basis for optimization of HVED at different processing capacities.


Assuntos
Camellia/química , Fracionamento Químico/métodos , Técnicas Eletroquímicas/métodos , Extratos Vegetais/isolamento & purificação , Fracionamento Químico/instrumentação , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Cinética , Modelos Teóricos , Extratos Vegetais/química , Proteínas de Plantas/isolamento & purificação , Reprodutibilidade dos Testes , Sementes/química
13.
Food Chem ; 313: 126154, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31931425

RESUMO

The aim of this study was to develop a scalable crossflow diafiltration/ultrafiltration procedure for quinoa 11S globulin purification starting at the bench scale using Ultra15 centrifugal filter devices. The electrophoretic profiles of centrifugal ultrafiltration fractions showed a high heterogeneity in the bands, while crossflow ultrafiltration reduced the phenomena of protein sticking to the membrane, avoiding aggregate formation. In the crossflow protein concentration, flux decline curves were studied according to Hermia's fouling mechanisms and the resistance in a series model. High reversible resistance was related to external mechanisms due to complete blockage of the membrane surface followed by cake formation. The crossflow ultrafiltration was the most efficient technique for obtaining 57 kDa chenopodin isolate with higher processing capacity, purity and protein yield. The diafiltration/ultrafiltration process proved to be adequate and easy to handle to scale up the production of the 11S quinoa globulin.


Assuntos
Proteínas de Plantas/isolamento & purificação , Ultrafiltração/métodos , Centrifugação/métodos , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ultrafiltração/instrumentação
14.
Sci Rep ; 10(1): 1381, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992824

RESUMO

Natural products isolation using protein based methods is an attractive for obtaining bioactive compounds. To discover neural stem cell (NSC) differentiation activators, we isolated eight inhibitors of Hes1 dimer formation from Psidium guajava using the Hes1-Hes1 interaction fluorescent plate assay and one inhibitor from Terminalia chebula using the Hes1-immobilized beads method. Of the isolated compounds, gallic acid (8) and 4-O-(4"-O-galloyl-α-L-rhamnopyranosyl)ellagic acid (11) showed potent Hes1 dimer formation inhibitory activity, with IC50 values of 10.3 and 2.53 µM, respectively. Compound 11 accelerated the differentiation activity of C17.2 NSC cells dose dependently, increasing the number of neurons with a 125% increase (5 µM) compared to the control.


Assuntos
Ácido Elágico/química , Ácido Gálico/química , Proteínas de Plantas , Multimerização Proteica , Psidium/química , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Terminalia/química
15.
J Biol Chem ; 295(6): 1598-1612, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31914404

RESUMO

Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) is an ancient aquatic plant of medicinal value because of antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline structural subclasses and displaying a predominant R-enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1-5 and encoding polypeptides sharing >40% amino acid sequence identity, were expressed in Escherichia coli Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R- and S-substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S-enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-benzylisoquinoline intermediates. Km values for NnOMT1 and NnOMT5 were 20 and 13 µm for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of alkaloid content, OMT activity in crude extracts, and OMT gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant N. nucifera.


Assuntos
Benzilisoquinolinas/metabolismo , Metiltransferases/metabolismo , Nelumbo/enzimologia , Proteínas de Plantas/metabolismo , Alcaloides/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Nelumbo/química , Nelumbo/genética , Nelumbo/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Alinhamento de Sequência
16.
Biosci Biotechnol Biochem ; 84(3): 563-574, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31790634

RESUMO

Two kinds of Kunitz-type protease inhibitors, AKPI1 and AKPI2, were purified from Apios americana tubers by four steps of column chromatographies and their cDNA cloning was performed. AKPI1 cDNA consist of 809 nucleotides, and the matured protein had 190 amino acids with 20,594 Da. AKPI2 cDNA consist of 794 nucleotides, and the matured protein had 177 amino acids with 19,336 Da. P1 site of AKPI2 was Leu88, suggested the target enzyme was chymotrypsin. On the other hand, Gly85-Ile86-Ser87 was positioned around P1 site of AKTI1. Sequence analysis suggested that two forms (single-chain and two-chain form) of AKPI2 protein were present in the tubers. Recombinant AKPI2 expressed by E.coli system showed inhibitory activity toward serine proteases and heat stability. The Ki values toward chymotrypsin and trypsin were 4 × 10-7 M and 6 × 10-6 M, respectively.Abbreviations: AAL: Apios americana lectin; AATI: Apios americana Bowman-Birk type trypsin inhibitor; ACE: angiotensin-converting enzyme; IPTG: isopropyl-ß-D-thio-galactopyranoside; Ki: inhibition constant; KPIs: Kunitz-type protease inhibitors; L-BAPA: Benzoyl-L-arginine p-nitroanilide monohydrochloride; L-BTPA: Benzoyl-L-tyrosine p-nitroanilide; PFLNA: Pyr-Phe-Leu-p-nitroanilide; RP-HPLC: reverse-phase high-performance liquid chromatography; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLIC: sequence and ligation independent cloning; STANA: N-Succinyl-Ala-Ala-Ala-p-nitroanilide; SHR: spontaneously hypertensive rats; TFA: trifluoroacetic acid; UTR: untranslated region.


Assuntos
Clonagem Molecular , DNA Complementar/genética , Fabaceae/metabolismo , Peptídeos/genética , Proteínas de Plantas/genética , Tubérculos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida/métodos , Escherichia coli/genética , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
17.
Prep Biochem Biotechnol ; 50(2): 133-140, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31702437

RESUMO

Watermelon seed is the potential source of value-added proteins, oils, and carbohydrates. The present study evaluates the extraction, and functional properties of watermelon seed protein (WMSP) obtained by ultrasound-assisted extraction (UAE) method from watermelon seed (WMS). The optimization of various operating parameters, such as pH (9), WMS powder to solvent ratio (1:50 w/v), temperature (30 ± 2 °C), ultrasound power (90 W), frequency (25 kHz), and duty cycle (75%) has been carried out. The extraction yield obtained was 87% and the extraction time was lowered down to 9 min from 120 min of conventional batch extraction. It contains all essential amino acids in an adequate amount required for adults as per FAO/WHO guidelines while for 2-5 years old children, the content of valine and isoleucine are above the required range. Methionine and lysine contents are adequate for both children and adults. Functional properties of ultrasonic extracted proteins were found superior to conventionally extracted proteins.highlightsThe UAE method is more efficient for watermelon seed protein extraction.Impact of extraction parameters on the extraction yield was studied.Protein isolate with enhanced functional properties was obtained.Essential amino acid content was determined.


Assuntos
Citrullus/embriologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/fisiologia , Sementes/metabolismo , Sonicação , Adulto , Aminoácidos/metabolismo , Carboidratos/análise , Pré-Escolar , Humanos , Temperatura
18.
J Sci Food Agric ; 100(3): 1344-1349, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31605384

RESUMO

BACKGROUND: Water-soluble proteins extracted from the heterotrophically cultivated microalga Chlorella protothecoides have been shown to have a good solubility over a broad pH range, which makes them a promising candidate for beverage formulations. This study investigated the sensory properties of dispersions of a protein-rich extract from C. protothecoides at neutral and pH 3. RESULTS: Sensory acceptance tests of the pure extract revealed an overall low acceptance at pH 7 without sucrose addition. Sensory acceptance was significantly (P ≤ 0.05) increased by lowering the pH to 3 with citric acid, and the addition of 50 g kg-1 sucrose. Here, overall positive sensory acceptance ratings were achieved up to a protein extract concentration of 40 g kg-1 . Basic taste evaluations showed only low bitterness scores and no significant (P > 0.05) increase in bitterness with decreasing pH. CONCLUSION: It is suggested that protein-rich extracts from C. protothecoides have promising sensory properties in beverage formulations. © 2019 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Assuntos
Chlorella/química , Extratos Vegetais/química , Proteínas de Plantas/química , Bebidas/análise , Chlorella/crescimento & desenvolvimento , Processos Heterotróficos , Humanos , Concentração de Íons de Hidrogênio , Microalgas/química , Microalgas/crescimento & desenvolvimento , Extratos Vegetais/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Solubilidade , Paladar
19.
Food Chem ; 305: 125457, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505414

RESUMO

Taking into account a growing market and small number of articles related to honeydew honey, a metabolomic approach associated with multivariate analysis and modelling was proposed to discriminate five varieties of honey. Advanced analytical techniques were used for determination of 20 elements, 14 carbohydrates and stable carbon isotope ratio. No chemical marker has been found within sugar compounds, but several elements (Ba, Ca, Mg, Sr, Mn, Al, Co, Ni, Se) were marked as characteristic of honey type and allow classification of three botanical origins (Abies alba, Quercus frainetto, Quercus ilex). Sugars turanose, trehalose, arabinose and raffinose, elements Ba, Sr, P, Cd and Se, and δ13C values of honey, have different concentrations in honeys of the same botanical origin but harvested in different season. In addition to a confirmation of authenticity in terms of production, the values of δ13C of protein could be a good indicator of botanical origin.


Assuntos
Mel/análise , Espectrometria de Massas/métodos , Quercus/metabolismo , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Análise Discriminante , Análise dos Mínimos Quadrados , Proteínas de Plantas/análise , Proteínas de Plantas/isolamento & purificação , Análise de Componente Principal , Álcoois Açúcares/análise , Açúcares/análise
20.
Food Chem ; 309: 125671, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31670129

RESUMO

A pure glycoprotein (BGP4-I) was obtained from tartary buckwheat seeds by aqueous extraction followed by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The average molecular weight of BGP4-I, as determined by high performance gel permeation chromatography, was 123.43 kDa. The structure of BGP4-I was characterized based on Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy, etc. Based on the nano-liquid chromatography-coupled electrospray ionization mass spectrometry analysis of the amino acid sequence of BGP4-I, belongs unequivocally to the glycosyl hydrolase family 1 in the Carbohydrate Active Enzymes database by alignment studies. The specific activity of BGP4-I was 18.44 µmol/min/mg on the substrate p-nitrophenyl-ß-d-glucopyranoside. Furthermore, BGP4-I is unique in its specificity for some substrates. These results suggest that the BGP4-I from tartary buckwheat seeds is a novel specific ß-glucosidase setting the foundation for potential applications in the food industry.


Assuntos
Fagopyrum/metabolismo , Glicoproteínas/química , Proteínas de Plantas/química , Sementes/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Peso Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Espectrometria de Massas em Tandem
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