Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 19.416
Filtrar
1.
Bioresour Technol ; 311: 123513, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32417661

RESUMO

An elastin-like polypeptide (ELP) sequence fused with Lactobacillus sp. B164 ß-galactosidase modified with 6x-Histidine (ß-Gal-LH) to produce recombinant ß-Gal-Linker-ELP-His (ß-Gal-LEH) was expressed in E. coli and purified via inverse thermal cycling (ITC) and nickel-nitrilotriacetic acid (Ni-NTA) resin. The ß-galactosidase integrated with ELP-system showed an improved purification at 1.75 M (NH4)2SO4 after 1 round ITC (95.66% recovery rate and 13.04 purification fold) with better enzyme activity parameters compared to Ni-NTA. The enzyme maintained an optimal temperature (40 °C) and pH (7.5) for both ß-Gal-LEH and ß-Gal-LH. The results further showed that the ELP-fusion system improved the enzyme's thermal and storage stability. Moreover, the enzyme secondary structure was not changed by ELP-tag. Enzyme activity was completely inactivated by Hg2+, Cd2+ and Cu2+, unaffected by Ca2+, EDTA and urea, but partially activated by Mn2+ at lower concentration. Compared to commercial ß-galactosidases, ß-Gal-LEH exhibited similar biocatalytic efficiency on lactose and could potentially catalyze transgalactosylation.


Assuntos
Elastina , Lactose , Escherichia coli , Hidrólise , Lactobacillus , Peptídeos , Proteínas Recombinantes de Fusão , beta-Galactosidase
2.
An Acad Bras Cienc ; 92(1): e20180609, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267306

RESUMO

The present study investigated the encapsulation of ß-galactosidase in carrageenan, pectin and its hybrid hydrogels by using the ionotropic gelation method. The material obtained was characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG/DTG) and scanning electron microscopy (SEM). The effects of pH, temperature and storage time were evaluated in terms of the catalytic activity of the free and encapsulated enzyme. Addition studies were conducted evaluating the performance of catalytic activity in vitro conditions. Carrageenan, pectin and hybrid hydrogels presented encapsulation efficiency of 58 ± 1%, 72 ± 1% and 77 ± 2%, respectively. The pectin hydrogel showed the higher ß-galactosidase activity in pH and temperature tests. However, the carrageenan hydrogel exhibited best stability after been stored for three months. Carrageenan and pectin hydrogels were 2.0 and 2.4 times more efficiently than commercial tablet in the releasing ß-galactosidase under in vitro conditions, respectively. The results suggest that pectin and carrageenan hydrogels may be useful for the development of new formulation of ß-galactosidase.


Assuntos
Carragenina/química , Portadores de Fármacos/química , Pectinas/química , beta-Galactosidase/química , Varredura Diferencial de Calorimetria , Química Farmacêutica , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Tecnologia Farmacêutica
3.
Food Chem ; 322: 126645, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32335492

RESUMO

ß-Galactosidase formulations can be added to infant milks prior to feeding to reduce the level of lactose and to avoid symptoms of lactose intolerance. The hydrolysis of lactose affects osmolality, which is an important property of infant milk. This paper introduces novel ultrasonic technology for precision, real-time, non-destructive monitoring of osmolality of infant milks, including breast milk, during enzymatic hydrolysis of lactose by supplemental ß-galactosidases. This technology can be utilised in the development and testing of ß-galactosidase formulations. Additionally, ultrasonic real-time measurements of the average degree of polymerisation and molar mass of milk saccharides throughout the hydrolysis are discussed. Comparison of the ultrasonic results with discontinuous data of osmometry and HPLC showed an excellent agreement between the different techniques. The results elucidate the osmolality dynamics involved in the enzymatic hydrolysis of lactose in milks.


Assuntos
Fórmulas Infantis/química , Lactose/química , Leite Humano/química , Análise Espectral/métodos , Ultrassom/métodos , Animais , Bovinos , Feminino , Humanos , Hidrólise , Lactente , Peso Molecular , Oligossacarídeos/química , Concentração Osmolar , beta-Galactosidase/química
4.
Gene ; 748: 144676, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32305635

RESUMO

OBJECTIVE: lacZ encodes for ß-galactosidase within the galactose operon of bacterial cells. When used as a reporter gene, bacterial "ß-galactosidase" expression is often insufficient for detection in mammalian cells. We intended to optimize the lacZ codon usage according to the most frequently used codons for the seven major proteins in cow's milk, in order to pave a way for the enhancement of transgenic genes expression in eukaryotes. RESULTS: We constructed modified lacZ (named olacZ) according to optional codons used for proteins expressed in cow's milk. The expression of lacZ and olacZ was then compared in HC11 (a murine mammary gland epithelial line), 293T, HeLa, Cos7, and NIH 3T3 cells. While there was no significant difference at the mRNA level between lacZ and olacZ (P > 0.05). The quantification of ß-galactosidase activity and in situ staining experiments showed a 1.2-fold to 3.3-fold expression improvement when comparing olacZ with lacZ. The levels of ß-galactosidase expression at the protein levels from olacZ were approximately 9.2-fold and 2.4-fold respectively for Cos7 and HC11 cells. Furthermore, a 1.9-fold tendency of enhanced expression of olacZ in mammary gland during lactation was observed in transgenic-olacZ mice. CONCLUSION: This study demonstrates an alternative choice for improving lacZ reporter expression in eukaryotes, especially in the mammary gland of cattle or goats.


Assuntos
Códon , Processamento Pós-Transcricional do RNA , beta-Galactosidase/genética , Animais , Bovinos , Linhagem Celular , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética
5.
Hum Genet ; 139(5): 657-673, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32219518

RESUMO

GM1-gangliosidosis, a lysosomal storage disorder, is associated with ~ 161 missense variants in the GLB1 gene. Affected patients present with ß-galactosidase (ß-Gal) deficiency in lysosomes. Loss of function in ER-retained misfolded enzymes with missense variants is often due to subcellular mislocalization. Deoxygalactonojirimycin (DGJ) and its derivatives are pharmaceutical chaperones that directly bind to mutated ß-Gal in the ER promoting its folding and trafficking to lysosomes and thus enhancing its activity. An Emirati child has been diagnosed with infantile GM1-gangliosidosis carrying the reported p.D151Y variant. We show that p.D151Y ß-Gal in patient's fibroblasts retained < 1% residual activity due to impaired processing and trafficking. The amino acid substitution significantly affected the enzyme conformation; however, p.D151Y ß-Gal was amenable for partial rescue in the presence of glycerol or at reduced temperature where activity was enhanced with ~ 2.3 and 7 folds, respectively. The butyl (NB-DGJ) and nonyl (NN-DGJ) derivatives of DGJ chaperoning function were evaluated by measuring their IC50s and ability to stabilize the wild-type ß-Gal against thermal degradation. Although NN-DGJ showed higher affinity to ß-Gal, it did not show a significant enhancement in p.D151Y ß-Gal activity. However, NB-DGJ promoted p.D151Y ß-Gal maturation and enhanced its activity up to ~ 4.5% of control activity within 24 h which was significantly increased to ~ 10% within 6 days. NB-DGJ enhancement effect was sustained over 3 days after washing it out from culture media. We therefore conclude that NB-DGJ might be a promising therapeutic chemical chaperone in infantile GM1 amenable variants and therefore warrants further analysis for its clinical applications.


Assuntos
1-Desoxinojirimicina/farmacologia , Fibroblastos/metabolismo , Gangliosidose GM1/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , beta-Galactosidase/metabolismo , 1-Desoxinojirimicina/química , Pré-Escolar , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Gangliosidose GM1/tratamento farmacológico , Gangliosidose GM1/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Chaperonas Moleculares/farmacologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Conformação Proteica , Transporte Proteico , beta-Galactosidase/química , beta-Galactosidase/genética
6.
BMC Med Genet ; 21(1): 59, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209057

RESUMO

BACKGROUND: Intellectual disability (ID) is both a clinically diverse and genetically heterogeneous group of disorder, with an onset of cognitive impairment before the age of 18 years. ID is characterized by significant limitations in intellectual functioning and adaptive behaviour. The identification of genetic variants causing ID and neurodevelopmental disorders using whole-exome sequencing (WES) has proven to be successful. So far more than 1222 primary and 1127 candidate genes are associated with ID. METHODS: To determine pathogenic variants causative of ID in three unrelated consanguineous Pakistani families, we used a combination of WES, homozygosity-by-descent mapping, de-deoxy sequencing and bioinformatics analysis. RESULTS: Rare pathogenic single nucleotide variants identified by WES which passed our filtering strategy were confirmed by traditional Sanger sequencing and segregation analysis. Novel and deleterious variants in VPS53, GLB1, and MLC1, genes previously associated with variable neurodevelopmental anomalies, were found to segregate with the disease in the three families. CONCLUSIONS: This study expands our knowledge on the molecular basis of ID as well as the clinical heterogeneity associated to different rare genetic causes of neurodevelopmental disorders. This genetic study could also provide additional knowledge to help genetic assessment as well as clinical and social management of ID in Pakistani families.


Assuntos
Consanguinidade , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Polimorfismo Genético , Proteínas de Transporte Vesicular/genética , beta-Galactosidase/genética , Criança , Pré-Escolar , Família , Feminino , Genes Recessivos/genética , Heterogeneidade Genética , Testes Genéticos , Homozigoto , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/patologia , Masculino , Transtornos do Neurodesenvolvimento/complicações , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Paquistão/epidemiologia , Linhagem , Sequenciamento Completo do Exoma
7.
Chem Commun (Camb) ; 56(18): 2731-2734, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32022000

RESUMO

We herein develop two ß-galactosidase (ß-Gal) activatable NIR fluorescent probes for visualizing ovarian cancers. Particularly, probe BOD-M-ßGal produced NIR-II emission light at 900-1300 nm upon ß-Gal activation. By using our activatable and target specific NIR-II probe for deep-tissue imaging of ß-Gal overexpressed ovarian cancer cells, rapid and accurate imaging of ovarian tumors in nude mice was achieved.


Assuntos
Corantes Fluorescentes/química , Imagem Óptica , Neoplasias Ovarianas/diagnóstico por imagem , beta-Galactosidase/química , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Corantes Fluorescentes/metabolismo , Células Endoteliais da Veia Umbilical Humana/química , Humanos , Raios Infravermelhos , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/metabolismo , beta-Galactosidase/metabolismo
8.
Nat Commun ; 11(1): 55, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896756

RESUMO

The introduction of local resolution has enormously helped the understanding of cryo-EM maps. Still, for any given pixel it is a global, aggregated value, that makes impossible the individual analysis of the contribution of the different projection directions. We introduce MonoDir, a fully automatic, parameter-free method that, starting only from the final cryo-EM map, decomposes local resolution into the different projection directions, providing a detailed level of analysis of the final map. Many applications of directional local resolution are possible, and we concentrate here on map quality and validation.


Assuntos
Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Anisotropia , Complexo de Endopeptidases do Proteassoma/química , Ribossomos/química , beta-Galactosidase/química
9.
Nucleic Acids Res ; 48(5): e28, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980824

RESUMO

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive ß-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased ß-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.


Assuntos
Bioensaio , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Transcrição Genética , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Sistemas CRISPR-Cas , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Repressores Lac/deficiência , Repressores Lac/genética , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
10.
Biotechnol Adv ; 39: 107465, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31689470

RESUMO

ß-Galactosidases, an important class of glycosidases, naturally catalyze the hydrolysis of ß-galactosidic bonds in oligosaccharides and polysaccharides. Traditionally, these enzymes have been used to degrade lactose in dairy products, which are beneficial for lactose-intolerant people. Attractively, ß-galactosidases exhibit glycosyl transfer activity under certain conditions in vitro. They are capable of synthesizing carbohydrates from cheap starting substrates in a facile, efficient, and environment-friendly way. The condensation of lactose into the well-known prebiotic galacto-oligosaccharides by ß-galactosidases has become a key aspect of the industrial interest in the synthetic activity in recent years. At present, the transglycosylation activity of these enzymes has been greatly extended. It can be used not only in building glycan blocks of crucial glycoconjugates to elucidate their biological functions, but also in glycosylation of vital molecules, which have been applied in food, medicine and cosmetic industries to improve solubility, stability and bioactivity. Further molecular engineering of ß-galactosidases has significantly improved their synthetic activity, expanded the substrate spectrum and made them more powerful in carbohydrate synthesis. This review covers the classification, structure and mechanism of ß-galactosidases, galactosylation reactions catalyzed by these enzymes, and various strategies of enzyme engineering, with an emphasis on recent advances.


Assuntos
beta-Galactosidase/metabolismo , Galactose , Lactose , Oligossacarídeos , Prebióticos
11.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140271, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494342

RESUMO

ß-Galactosidase is one of the most important industrial enzymes, that has been used for many decades in the dairy industry. The main application of ß-galactosidase is related to the production of low-lactose and lactose-free milk and dairy products, which are now common consumer goods in supermarket shelves. This is a well-established market that is expected to keep on growing as these products become more accessible to mid-income people worldwide. However, a fresh air has come into the ß-galactosidase business as non-conventional applications arose in recent decades based on its transgalactosylation activity. This capacity is certainly a major asset for a commodity enzyme that can be used now as a catalyst for the upgrading of readily available and cheap lactose into high added-value glycosides in processes of organic synthesis in tune with green chemistry principles within the framework of sustainability. This is a reflection of a paradigm shift, where enzymes are now being considered as apt catalysts for the synthesis of valuable organic compounds. This article reviews the main applications of ß-galactosidase, going from its conventional use related to its hydrolytic activity to the ongoing non-conventional applications in the synthesis of high added-value oligosaccharides based on its transgalactosylation activity.


Assuntos
beta-Galactosidase/química , Catálise , Lactose/química
12.
Carbohydr Polym ; 229: 115549, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826450

RESUMO

An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methyl-esterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10-100 kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. The data revealed the presence of GalA sequences, having different patterns of methyl-esterification, RG-I composed of unbranched segments and segments heavily substituted with ß-(1→4)-linked galactan chains of varying length. The rheological study showed the shear-thinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did form stable foams at high concentration.


Assuntos
Galactose/metabolismo , Cebolas/metabolismo , Polissacarídeos/química , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Esterificação , Ácidos Hexurônicos/metabolismo , Peso Molecular , Polissacarídeos/metabolismo , Reologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Galactosidase/metabolismo
13.
Int J Food Microbiol ; 316: 108476, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-31874325

RESUMO

This work aimed to investigate the ability of two human-derived bifidobacterial strains, i.e. Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809, to utilize various oligosaccharides (i.e., 4-galactosyl-kojibiose, lactulosucrose, lactosyl-oligofructosides, raffinosyl-oligofructosides and lactulose-derived galacto-oligosaccharides) synthesized by means of microbial glycoside hydrolases. With the exception of raffinosyl-oligofructosides, these biosynthetic oligosaccharides were shown to support growth acting as a sole carbon and energy source of at least one of the two studied strains. Production of short-chain fatty acids (SCFAs) as detected by HPLC analysis corroborated the suitability of most of the studied novel oligosaccharides as fermentable growth substrates for the two bifidobacterial strains, showing that acetic acid is the main metabolic end product followed by lactic and formic acids. Transcriptomic and functional genomic approaches carried out for B. breve UCC2003 allowed the identification of key genes encoding glycoside hydrolases and carbohydrate transport systems involved in the metabolism of 4-galactosyl-kojibiose and lactulosucrose. In particular, the role of ß-galactosidases in the hydrolysis of these particular trisaccharides was demonstrated, highlighting their importance in oligosaccharide metabolism by human bifidobacterial strains.


Assuntos
Bifidobacterium breve/metabolismo , Bifidobacterium longum/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Bactérias/genética , Bifidobacterium breve/crescimento & desenvolvimento , Bifidobacterium breve/isolamento & purificação , Bifidobacterium longum/crescimento & desenvolvimento , Bifidobacterium longum/isolamento & purificação , Metabolismo dos Carboidratos/genética , Ácidos Graxos Voláteis/biossíntese , Ácidos Graxos Voláteis/química , Fermentação , Glicosídeo Hidrolases/genética , Humanos , Oligossacarídeos/química , Transcriptoma , beta-Galactosidase/genética
14.
Food Chem ; 303: 125388, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31454757

RESUMO

Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis ß-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin-protein interaction. Circular dichroism shows that saponin changes the ß-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of ß-galactosidase with the artificial substrate o-nitrophenyl-ß-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.


Assuntos
Kluyveromyces/enzimologia , Saponinas/farmacologia , beta-Galactosidase/efeitos dos fármacos , Calorimetria , Dicroísmo Circular , Cinética , Nitrofenilgalactosídeos/metabolismo , Casca de Planta/química , Estrutura Secundária de Proteína , Quillaja/química , Espectrometria de Fluorescência , beta-Galactosidase/metabolismo
15.
Food Chem ; 305: 125481, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525592

RESUMO

Prebiotics are rising in interest in commercial scale productions due to increasing health awareness of consumers. Under bio-economic aspects, sweet and acid whey provide a suitable feed medium for the enzymatic generation of prebiotic lactulose. Since whey has a broad variation in composition, the influence of the feed composition on the concentration of generated lactulose was investigated. The influence of lactose and fructose concentration as well as enzymatic activity of two commercially available ß-galactosidases were investigated. The results were evaluated via response surface analysis with a quadratic model containing pairwise interaction terms. The optimal feed composition yielding a theoretical maximal amount of lactulose was determined as 1.28 or 0.74 mol/kg fructose and 0.17 or 0.19 mol/kg lactose with an enzymatic activity of 2.0 or 2.8 µkat/kg for acid (pH 4.4) or sweet (pH 6.6) whey. Furthermore, the major reaction product was isolated and subsequently, the structural identity was elucidated and verified via extensive NMR analysis.


Assuntos
Lactulose/metabolismo , Soro do Leite/metabolismo , beta-Galactosidase/metabolismo , Frutose/metabolismo , Concentração de Íons de Hidrogênio , Isomerismo , Lactose/metabolismo , Lactulose/isolamento & purificação , Espectroscopia de Ressonância Magnética , Soro do Leite/química
16.
J Enzyme Inhib Med Chem ; 35(1): 42-49, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31656110

RESUMO

Matricaria chamomilla L. contains antioxidant flavonoids that can have their bioactivity enhanced by enzymatic hydrolysis of specific glycosyl groups. This study implements an untargeted metabolomics approach based on ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry technique operating in MSE mode (UPLC-QTOF-MSE) and spectrophotometric analysis of chamomile aqueous infusions, before and after hydrolysis by hesperidinase and ß-galactosidase. Several phenolic compounds were altered in the enzymatically treated infusion, with the majority being flavonoid derivatives of apigenin, esculetin, and quercetin. Although enzymatically modifying the infusion only led to a small increase in antioxidant activity (DPPH• method), its inhibitory effect on pancreatic lipase was of particular interest. The enzymatically treated infusion exhibited a greater inhibitory effect (EC50 of 35.6 µM) than unmodified infusion and kinetic analysis suggested mixed inhibition of pancreatic lipase. These results are of great relevance due to the potential of enzymatically treated functional foods in human health.


Assuntos
Antioxidantes/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Lipase/antagonistas & inibidores , Matricaria/química , Antioxidantes/química , Antioxidantes/metabolismo , Compostos de Bifenilo/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Hidrólise , Lipase/metabolismo , Matricaria/metabolismo , Metabolômica , Estrutura Molecular , Picratos/antagonistas & inibidores , Relação Estrutura-Atividade , beta-Galactosidase/metabolismo
17.
J Dairy Sci ; 103(1): 166-171, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31704010

RESUMO

The ability to use lactose is critical for the application of Streptococcus thermophilus in fermented dairy products. Most studies have evaluated the use of lactose of S. thermophilus by measuring lactose utilization, but its correlation with ß-galactosidase and urease has rarely been investigated. In this study, 10 strains of S. thermophilus isolated from fermented yak milk exhibited a diversity of ß-galactosidase and urease activities, growth, and acid production in de Man, Rogosa, and Sharpe-lactose. Among the strains, 15G5 possessed the highest ß-galactosidase activity and showed the highest cell growth, lactic acid production, and titratable acidity during fermentation. In contrast, 7G10, with the weakest ß-galactosidase activity, produced the lowest lactic acid content and change in titratable acidity. Further investigation indicated that ß-galactosidase activity of S. thermophilus showed significant positive correlations with the growth of cell densities, the production of lactic acid, and titratable acidity, and urease activity of S. thermophilus showed a significant correlation with the use of lactose and the production of lactic acid and acetaldehyde. These findings suggest that the differences of ß-galactosidase and urease activities are essential for the performance in the lactose metabolism, growth, and acid production of S. thermophilus, providing new insights into strain selection and application.


Assuntos
Ácido Láctico/metabolismo , Lactose/metabolismo , Leite/enzimologia , Streptococcus thermophilus/enzimologia , Urease/metabolismo , beta-Galactosidase/metabolismo , Animais , Metabolismo dos Carboidratos , Fermentação
18.
Eur J Pharm Sci ; 141: 105089, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31626967

RESUMO

The goals of this work were to evaluate if high-speed electrospinning can be used as a gentle and continuous drying technology to produce protein-containing cyclodextrin-based fibers from an aqueous solution and to convert the produced protein-cyclodextrin fibers into a directly compressible powder. A 400 mL/h feeding rate was used during the electrospinning experiments, corresponding to a ~270 g/h production rate of the dried material. The produced fibers were collected in a cyclone. The fibers were found grindable without secondary drying, and the ground powder was mixed with tableting excipients and was successfully tableted by direct compression. The model protein-type drug (ß-galactosidase) remained stable during each of the processing steps (electrospinning, grinding, tableting) and after 6 months of storage at room temperature in the tablets. The obtained results demonstrate that high speed electrospinning can be a gentle alternative to traditional drying methods used for protein-type drugs, and that tablet formulation is achievable from the electrospun material prepared this way.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Tecnologia Farmacêutica/métodos , beta-Galactosidase/química , Dessecação , Estabilidade Enzimática , Pós , Comprimidos
19.
Carbohydr Polym ; 227: 115353, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31590885

RESUMO

Potato cell walls (PCW) are a low value by-product from the potato starch industry. Valorisation of PCW is hindered by its high water-binding capacity (WBC). The composition of polysaccharides and interactions between these entities, play important roles in regulating the WBC in the cell wall matrix. Here, we show that in vivo exo-truncation of RG-I ß-(1→4)-D-galactan side-chains decreased the WBC by 6-9%. In contrast, exo-truncation of these side-chains increased the WBC by 13% in vitro. We propose that degradation of RG-I galactan side-chains altered the WBC of PCW, due to cell wall remodelling and loosening that affected the porosity. Our findings reinforce the view that RG-I galactan side-chains play a role in modulating WBC, presumably by affecting polysaccharide architecture (spacing) and interactions in the matrix. Better understanding of structure-function relationships of pectin macromolecules is needed before cell wall by-products may be tailored to render added-value in food and biobased products.


Assuntos
Parede Celular/química , Galactanos/química , Solanum tuberosum , Água/química , Parede Celular/ultraestrutura , Microscopia Eletrônica , beta-Galactosidase/química
20.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801274

RESUMO

Acid sphingomyelinase (aSMase) is involved in the generation of metabolites that function as part of the sphingolipid signaling pathway. It catalyzes the breakdown of sphingomyelin into ceramide, a bioactive lipid that, among other roles, is involved in regulation of apoptosis. Dry drop blood test (DBS) and colorimetric 2-step enzymatic assay were used to assess the activity of human blood aSMase, beta-galactosidase, and beta-glucosidase, these enzymes are lysosomal hydrolases that catalyze the degradation of related sphingolipids, of sphingolipid signaling molecules. Blood was collected from a group of healthy volunteers and patients that were diagnosed with multiple myeloma (MM) in various stages of the disease. Additionally, activity of those enzymes in patients diagnosed with other hematological cancers was also assessed. We found that aSMase activity in the blood of patients with MM (at the time of diagnosis) was 305.43 pmol/spot*20 h, and this value was significantly lower (p < 0.030) compared to the healthy group 441.88 pmol/spot*20 h. Our collected data suggest a possible role of aSMase in pathogenesis of MM development.


Assuntos
Mieloma Múltiplo/sangue , Esfingolipídeos/sangue , Esfingomielina Fosfodiesterase/sangue , beta-Galactosidase/sangue , beta-Glucosidase/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Leucemia de Células Pilosas/sangue , Leucemia de Células Pilosas/diagnóstico , Leucemia de Células Pilosas/patologia , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Metabolismo dos Lipídeos , Linfoma de Zona Marginal Tipo Células B/sangue , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/patologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Estadiamento de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Mielofibrose Primária/sangue , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA