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1.
Appl Microbiol Biotechnol ; 105(13): 5281-5298, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34223948

RESUMO

The enzyme ß-galactosidase has great potential for application in the food and pharmaceutical industries due to its ability to perform the hydrolysis of lactose, a disaccharide present in milk and in dairy by-products. It can be used in free form, in batch processes, or in immobilized form, which allows continuous operation and provides greater enzymatic stability. The choice of method and support for enzyme immobilization is essential, as the performance of the biocatalyst is strongly influenced by the properties of the material used and by the interaction mechanisms between support and enzyme. Therefore, this review showed the main enzyme immobilization techniques, and the most used supports for the constitution of biocatalysts. Also, materials with the potential for immobilization of ß-galactosidases and the importance of their biotechnological application are presented. KEY POINTS: • The main methods of immobilization are physical adsorption, covalent bonding, and crosslinking. • The structural conditions of the supports are determining factors in the performance of the biocatalysts. • Enzymatic hydrolysis plays an important role in the biotechnology industry.


Assuntos
Enzimas Imobilizadas , Lactose , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrólise , beta-Galactosidase/metabolismo
2.
Appl Microbiol Biotechnol ; 105(9): 3601-3610, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33937931

RESUMO

The food industry has developed a wide range of products with reduced lactose to allow people with intolerance to consume dairy products. Although ß-galactosidase has extensive applications in the food, pharma, and biotechnology industries, the enzymes are high-cost catalysts, and their use makes the process costly. Immobilization is a viable strategy for enzyme retention inside a reactor, allowing its reuse and application in continuous processes. Here, we studied the immobilization of ß-galactosidase from Bacillus licheniformis in ion exchange resin. A central composite rotational design (CCRD) was proposed to evaluate the immobilization process in relation to three immobilization solution variables: offered enzyme activity, ionic strength, and pH. The conditions that maximized the response were offered enzyme activity of 953 U, 40 mM ionic strength, and pH 4.0. Subsequently, experiments were performed to provide additional stabilization for biocatalyst, using a buffer solution pH 9.0 at 25 °C for 24 h, and crosslinking with different concentrations of glutaraldehyde. The stabilization step drastically impacted the activity of the immobilized enzyme, and the reticulation with different concentrations of glutaraldehyde showed significant influence on the activity of the immobilized enzyme. In spite of substantially affecting the initial activity of the immobilized enzyme, higher reagent concentrations (3.5 g L-1) were effective for maintaining stability related to the number of cycles of the enzyme immobilized. The ß-galactosidase from Bacillus licheniformis immobilized in Duolite A568 is a promising technique to produce reduced or lactose-free dairy products, as it allows reuse of the biocatalyst, decreasing operational costs.Key Points• Immobilization of ß-galactosidase from Bacillus licheniformis in batch reactor• Influence of buffer pH and ionic concentration and offered enzyme activity on immobilization• Influence of glutaraldehyde on operational stability.


Assuntos
Bacillus licheniformis , Bacillus licheniformis/metabolismo , Indústria de Laticínios , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lactose , Temperatura , beta-Galactosidase/metabolismo
3.
Food Chem ; 359: 129890, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-33934029

RESUMO

A new support for the immobilization of ß-d-galactosidase from Kluyveromyces lactis was developed, consisting of mesoporous silica/titania with a chitosan coating. This support presents a high available surface area and adequate pore size for optimizing the immobilization efficiency of the enzyme and, furthermore, maintaining its activity. The obtained supported biocatalyst was applied in enzyme hydrolytic activity tests with o-NPG, showing high activity 1223 Ug-1, excellent efficiency (74%), and activity recovery (54%). Tests of lactose hydrolysis in a continuous flow reactor showed that during 14 days operation, the biocatalyst maintained full enzymatic activity. In a batch system, after 15 cycles, it retained approximately 90% of its initial catalytic activity and attained full conversion of the lactose 100% (±12%). Additionally, with the use of the mesoporous silica/titania support, the biocatalyst presented no deformation and fragmentation, in both systems, demonstrating high operational stability and appropriate properties for applications in food manufacturing.


Assuntos
Quitosana , Enzimas Imobilizadas/metabolismo , Kluyveromyces/enzimologia , Dióxido de Silício , Titânio , beta-Galactosidase/metabolismo , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Hidrólise , Lactose/metabolismo
4.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923711

RESUMO

There has been a recent increase in the exploration of cold-active ß-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active ß-galactosidases. A screening revealed 81 out of 304 strains with ß-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41-62% of its ß-galactosidase activity in the cold (4 °C-15 °C). Three ß-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active ß-galactosidase producer.


Assuntos
Temperatura Baixa , Rahnella/enzimologia , beta-Galactosidase/metabolismo , Aclimatação , Estabilidade Enzimática , Rahnella/genética , beta-Galactosidase/química , beta-Galactosidase/genética
5.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803589

RESUMO

During their life span, cells have two possible states: a non-cycling, quiescent state (G0) and a cycling, activated state. Cells may enter a reversible G0 state of quiescence or, alternatively, they may undergo an irreversible G0 state. The latter may be a physiological differentiation or, following a stress event, a senescent status. Discrimination among the several G0 states represents a significant investigation, since quiescence, differentiation, and senescence are progressive phenomena with intermediate transitional stages. We used the expression of Ki67, RPS6, and beta-galactosidase to identify healthy cells that progressively enter and leave quiescence through G0-entry, G0 and G0-alert states. We then evaluated how cells may enter senescence following a genotoxic stressful event. We identified an initial stress stage with the expression of beta-galactosidase and Ki67 proliferation marker. Cells may recover from stress events or become senescent passing through early and late senescence states. Discrimination between quiescence and senescence was based on the expression of RPS6, a marker of active protein synthesis that is present in senescent cells but absent in quiescent cells. Even taking into account that fixed G0 states do not exist, our molecular algorithm may represent a method for identifying turning points of G0 transitional states that continuously change.


Assuntos
Ciclo Celular , Senescência Celular , Antígeno Ki-67/metabolismo , Proteína S6 Ribossômica/metabolismo , Estresse Fisiológico , beta-Galactosidase/metabolismo , Humanos , Modelos Biológicos , Fenótipo
6.
J Food Biochem ; 45(4): e13699, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33694174

RESUMO

UV-cured epoxy-based polymeric film was prepared from glycidyl methacrylate, trimethylolpropane triacrylate, and poly(ethylene glycol) methylether acrylate. 2-hydroxy-2- methylpropiophenone was used as photo initiator. Covalent binding through epoxy groups was employed to immobilize ß-galactosidase from Escherichia coli onto this film, and immobilization conditions were optimized by the response surface methodology. ATR-Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) analysis was carried out to characterize the epoxy-based polymeric film. Immobilization yield of ß-galactosidase on the material was calculated as 3.57 mg/g and the highest enzyme activity for the immobilized enzyme recorded at pH 6.5°C and 60°C. The immobilized enzyme preserved 51% of its activity at the end of 12 runs. Free and immobilized enzyme hydrolyzed 163.8 and 172.3 µM lactose from 1% lactose, respectively. Kinetic parameters of both free and immobilized ß-galactosidase were also investigated, and Km values were determined to be 0.647 and 0.7263 mM, respectively. PRACTICAL APPLICATIONS: In our study we prepared a UV-cured epoxy-based polymeric film and optimized the immobilization conditions of ß-galactosidase from Escherichia coli onto this polymeric film by using response surface methodology (RSM). For this purpose, three-level and three-factor Box-Behnken design, which is an independent, rotatable or nearly rotatable, quadratic design, was applied. Optimal levels of three variables, namely, the amount of enzyme, immobilization time, and pH were determined using Box-Behnken experimental design. Lactose hydrolysis studies were performed from milk and lactose samples using free and immobilized enzyme. In addition, kinetic parameters, storage stability, and re-usability of immobilized ß-galactosidase were examined.


Assuntos
Enzimas Imobilizadas , Lactose , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Cinética , beta-Galactosidase/metabolismo
7.
Mol Med Rep ; 23(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33786633

RESUMO

Targeting microRNAs (miRs) using small chemical molecules has become a promising strategy for disease treatment. miR­216a has been reported to be a potential therapeutic target in endothelial senescence and atherosclerosis via the Smad3/NF­κB signaling pathway. Ginsenoside Rb2 (Rb2) is the main bioactive component extracted from the plant Panax ginseng, and is a widely used traditional Chinese medicine. In the present study, Rb2 was identified to have a high score for miR­216a via bioinformatics analysis based on its sequence and structural features. The microscale thermophoresis experiment further demonstrated that Rb2 had a specific binding affinity for miR­216a and the dissociation constant was 17.6 µM. In both young and senescent human umbilical vein endothelial cells (HUVECs), as well as human aortic endothelial cells, Rb2 decreased the expression of endogenous miR­216a. Next, a replicative endothelial senescence model of HUVECs was established by infection with pre­miR­216a recombinant lentiviruses (Lv­miR­216a) and the number of population­doubling level (PDL) was calculated. Stable overexpression of miR­216a induced a premature senescent­like phenotype, whereas the senescent features and increased activity of senescence­associated ß­galactosidase (SA­ß­gal) were reversed after Rb2 treatment. The percentage of SA­ß­gal­positive cells in senescent PDL25 cells transfected with Lv­miR­216a was decreased 76% by Rb2 treatment compared with the Lv­miR­216a group without Rb2 treatment (P=0.01). Mechanistically, miR­216a inhibited Smad3 protein expression, promoted IκBα degradation and activated NF­κB­responsive genes, such as vascular cell adhesion molecule 1 (VCAM1), which promoted the adhesiveness of endothelial cells to monocytes. These pro­inflammatory effects of miR­216a were significantly suppressed by Rb2 treatment. When Smad3 was suppressed by small interfering RNA, the elevated expression levels of intercellular adhesion molecule 1 and VCAM1 induced by miR­216a were significantly reversed. Collectively, to the best of our knowledge, the present study demonstrated for the first time that Rb2 exerted an anti­inflammation effect on the process of endothelial cell senescence and could be a potential therapeutic drug by targeting miR­216a.


Assuntos
Anti-Inflamatórios/farmacologia , Senescência Celular , Ginsenosídeos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , MicroRNAs/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , MicroRNAs/genética , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
8.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668968

RESUMO

The unique chemical, optical, and electrical characteristics of nanoparticles make their utilization highly successful in every field of biological sciences as compared to their bulk counterpart. These properties arise as a result of their miniature size, which provides them an excellent surface area-to-volume ratio, inner structure, and shape, and hence increases their surface characteristics. Therefore, this study was undertaken to engineer gold nanoparticles (AuNPs) for improving their catalytic activity and stability in biotechnological processes. The characterization of AuNPs was performed by XRD, UV spectra, and TEM. The synthesized AuNPs were surface-modified by polyvinyl alcohol (PVA) for binding the enzyme in excellent yield. The developed immobilized enzyme system (PVA-AuNPs-ß-galactosidase) displayed pH optima at pH 7.0 and temperature optima at 40 °C. Moreover, the stability of PVA-AuNPs-ß-galactosidase was significantly enhanced at wider pH and temperature ranges and at higher galactose concentrations, in contrast to the free enzyme. ß-galactosidase bound to PVA-modified AuNPs exhibited greater operational activity, even after its sixth reuse. The developed nanosystem may prove useful in producing lactose-free dairy products for lactose-intolerant patients.


Assuntos
Laticínios , Ouro/química , Lactose/química , Nanopartículas Metálicas/química , beta-Galactosidase/química , Laticínios/análise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ouro/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Kluyveromyces/enzimologia , Lactose/metabolismo , Intolerância à Lactose/metabolismo , Teste de Tolerância a Lactose , Tamanho da Partícula , Propriedades de Superfície , Temperatura , beta-Galactosidase/metabolismo
9.
Food Chem ; 349: 129050, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33556730

RESUMO

The objective of this research was to evaluate the immobilization of the enzyme ß-galactosidase in a genipin-activated chitosan support. The influence of the number of spheres and substrate concentration on immobilization yield (IY) and enzyme activity (EA) was analyzed using experimental design. Thermal, operational and storage stabilities were assessed, and the enzymatic derivatives were characterized by thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The TGA showed that the enzymatic derivatives kept their thermal behavior, and the SEM images revealed smooth surfaces in all the spheres. The optimized conditions for the immobilization process were 4.57 mg·mL-1 of spheres and a substrate concentration of 10 mM (IY = 84.13%; EA = 24.97 U·g-1). Thermal stability was enhanced at 10 and 37 °C, enabling four successive cycles of lactose hydrolysis in diluted UHT milk. Therefore, the immobilized enzyme in genipin-activated chitosan has potential for lactose hydrolysis and applications in the food industry.


Assuntos
Quitosana/química , Enzimas Imobilizadas/química , Iridoides/química , Kluyveromyces/enzimologia , Leite/química , beta-Galactosidase/química , Animais , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrólise , Lactose/química , beta-Galactosidase/metabolismo
10.
Mar Drugs ; 19(1)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477853

RESUMO

ß-galactosidases (EC 3.2.1.23) catalyze the hydrolysis of ß-galactosidic bonds in oligosaccharides and, under certain conditions, transfer a sugar moiety from a glycosyl donor to an acceptor. Cold-active ß-galactosidases are identified in microorganisms endemic to permanently low-temperature environments. While mesophilic ß-galactosidases are broadly studied and employed for biotechnological purposes, the cold-active enzymes are still scarcely explored, although they may prove very useful in biotechnological processes at low temperature. This review covers several issues related to cold-active ß-galactosidases, including their classification, structure and molecular mechanisms of cold adaptation. Moreover, their applications are discussed, focusing on the production of lactose-free dairy products as well as on the valorization of cheese whey and the synthesis of glycosyl building blocks for the food, cosmetic and pharmaceutical industries.


Assuntos
Biotecnologia , Temperatura Baixa , beta-Galactosidase/metabolismo , Adaptação Fisiológica , Hidrólise , Oligossacarídeos/metabolismo , beta-Galactosidase/química
11.
Nat Commun ; 12(1): 257, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431893

RESUMO

Advances in deep learning technology have enabled complex task solutions. The accuracy of image classification tasks has improved owing to the establishment of convolutional neural networks (CNN). Cellular senescence is a hallmark of ageing and is important for the pathogenesis of ageing-related diseases. Furthermore, it is a potential therapeutic target. Specific molecular markers are used to identify senescent cells. Moreover senescent cells show unique morphology, which can be identified. We develop a successful morphology-based CNN system to identify senescent cells and a quantitative scoring system to evaluate the state of endothelial cells by senescence probability output from pre-trained CNN optimised for the classification of cellular senescence, Deep Learning-Based Senescence Scoring System by Morphology (Deep-SeSMo). Deep-SeSMo correctly evaluates the effects of well-known anti-senescent reagents. We screen for drugs that control cellular senescence using a kinase inhibitor library by Deep-SeSMo-based drug screening and identify four anti-senescent drugs. RNA sequence analysis reveals that these compounds commonly suppress senescent phenotypes through inhibition of the inflammatory response pathway. Thus, morphology-based CNN system can be a powerful tool for anti-senescent drug screening.


Assuntos
Forma Celular , Senescência Celular , Aprendizado Profundo , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Redes Neurais de Computação , beta-Galactosidase/metabolismo
12.
J Dairy Sci ; 104(3): 2735-2747, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33455743

RESUMO

The activities of ß-galactosidases from bacteria and molds are affected by temperature, pH, and other factors in the processing of dairy products, limiting their application, so it is necessary to find alternative lactases. In this study, the ß-galactosidase gene from Bacillus coagulans T242 was cloned, co-expressed with a molecular chaperone in Escherichia coli BL21, and subjected to bioinformatic and kinetic analyses and lactase characterization. The results show that the enzyme is a novel thermostable neutral lactase with optimum hydrolytic activity at pH 6.8 and 50°C. The thermal stability and increased lactose hydrolysis activity of ß-galactosidase in the presence of Ca2+ indicated its potential application in the dairy industry.


Assuntos
Bacillus coagulans , Galactosidases , Animais , Clonagem Molecular , Biologia Computacional , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Lactose , Temperatura , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
13.
Protein Pept Lett ; 28(2): 221-228, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32798366

RESUMO

BACKGROUND: ß-galactosidases are enzymes that are utilized to hydrolyze lactose into galactose and glucose, and are is widely used in the food industry. OBJECTIVE: We describe the recombinant expression of an unstudied, heterodimeric ß-galactosidase originating from Lactobacillus brevis ATCC 367 in Escherichia coli. Furthermore, six different constructs, in which the two protein subunits were fused with different peptide linkers, were also investigated. METHODS: The heterodimeric subunits of the ß-galactosidase were cloned in expressed in various expression constructs, by using either two vectors for the independent expression of each subunit, or using a single Duet vector for the co-expression of the two subunits. RESULTS: The co-expression in two independent expression vectors only resulted in low ß-galactosidase activities, whereas the co-expression in a single Duet vector of the independent and fused subunits increased the ß-galactosidase activity significantly. The recombinant ß-galactosidase showed comparable hydrolyzing properties towards lactose, N-acetyllactosamine, and pNP-ß-D-galactoside. CONCLUSION: The usability of the recombinant L. brevis ß-galactosidase was further demonstrated by the hydrolysis of human, bovine, and goat milk samples. The herein presented fused ß-galactosidase constructs may be of interest for analytical research as well as in food- and biotechnological applications.


Assuntos
Escherichia coli/enzimologia , Lactobacillus brevis/enzimologia , Lactose/metabolismo , Leite/metabolismo , Fragmentos de Peptídeos/metabolismo , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Animais , Bovinos , Galactose/metabolismo , Glucose/metabolismo , Cabras , Humanos , Hidrólise , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidase/genética
14.
Int J Biol Macromol ; 166: 1106-1110, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33157142

RESUMO

Western blotting was attempted to analyze proteins separated by agarose native gel electrophoresis that was previously developed on His/Mes buffer system. This report shows a simple protocol for blotting agarose native gel to a PVDF membrane by soaking the gel in sodium dodecylsulfate-containing transfer buffer and 3 examples of such analysis. First example showed expression of a recombinant antibody in HEK293 cells by direct staining of the agarose native gels for both proteins and nucleic acids and staining of the blots for proteins and host cell proteins. These analyses demonstrated usefulness of agarose native gel electrophoresis, confirming that the recombinant antibody migrates toward the cathode while nucleic acids and a majority of host cell proteins migrate toward the anode. Second example demonstrated the phosphorylation state of MAP kinase in human lymphocyte cell line. Namely, agarose native gel can separate kinase, whose phosphorylation can be analyzed by Western blotting. Third example showed correlation of Escherichia coli ß-galactosidase expression between the oligomerization and enzyme activity using antibody and substrate staining.


Assuntos
Western Blotting , Eletroforese em Gel de Ágar , Proteínas/análise , Proteínas/isolamento & purificação , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/isolamento & purificação , Células HEK293 , Humanos , Fosforilação , Proteínas Quinases/metabolismo , Multimerização Proteica , beta-Galactosidase/metabolismo
15.
Angew Chem Int Ed Engl ; 60(8): 3999-4003, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33119955

RESUMO

Chemiluminescence imaging is imperative for diagnostics and imaging due to its intrinsically high sensitivity. To improve in vivo detection of biomarkers, chemiluminophores that simultaneously possess near-infrared (NIR) emission and modular structures amenable to construction of activatable probes are highly desired; however, these are rare. Herein, we report two chemiluminophores with record long NIR emission (>750 nm) via integration of dicyanomethylene-4H-benzothiopyran or dicyanomethylene-4H-benzoselenopyran with dioxetane unit. Caging of the chemiluminophores with different cleavable moieties produces NIR chemiluminescence probes (NCPs) that only produce signals upon reaction with reactive oxygen species or enzymes, for example, ß-galactosidase, with a tissue-penetration depth of up to 2 cm. Thus, this study provides NIR chemiluminescence molecular scaffolds applicable for in vivo turn-on imaging of versatile biomarkers in deep tissues.


Assuntos
Corantes Fluorescentes/química , Imagem Óptica/métodos , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/metabolismo , Humanos , Raios Infravermelhos , Limite de Detecção , Medições Luminescentes , Camundongos , Camundongos Nus , Neoplasias/diagnóstico por imagem , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Transplante Heterólogo , beta-Galactosidase/metabolismo
16.
FEBS J ; 288(2): 546-565, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32363751

RESUMO

To survive in cold environments, psychrophilic organisms produce enzymes endowed with high specific activity at low temperature. The structure of these enzymes is usually flexible and mostly thermolabile. In this work, we investigate the structural basis of cold adaptation of a GH42 ß-galactosidase from the psychrophilic Marinomonas ef1. This enzyme couples cold activity with astonishing robustness for a psychrophilic protein, for it retains 23% of its highest activity at 5 °C and it is stable for several days at 37 °C and even 50 °C. Phylogenetic analyses indicate a close relationship with thermophilic ß-galactosidases, suggesting that the present-day enzyme evolved from a thermostable scaffold modeled by environmental selective pressure. The crystallographic structure reveals the overall similarity with GH42 enzymes, along with a hexameric arrangement (dimer of trimers) not found in psychrophilic, mesophilic, and thermophilic homologues. In the quaternary structure, protomers form a large central cavity, whose accessibility to the substrate is promoted by the dynamic behavior of surface loops, even at low temperature. A peculiar cooperative behavior of the enzyme is likely related to the increase of the internal cavity permeability triggered by heating. Overall, our results highlight a novel strategy of enzyme cold adaptation, based on the oligomerization state of the enzyme, which effectively challenges the paradigm of cold activity coupled with intrinsic thermolability. DATABASE: Structural data are available in the Protein Data Bank database under the accession number 6Y2K.


Assuntos
Proteínas de Bactérias/química , Galactose/química , Marinomonas/química , beta-Galactosidase/química , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Galactose/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Marinomonas/enzimologia , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
17.
J Appl Microbiol ; 130(3): 865-877, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32741059

RESUMO

AIMS: Optimization of ß-galactosidase production by Trichoderma sp. under solid-state fermentation using wheat bran as solid substrate through an experimental design and its application targeting the recovery of galactooligosaccharides (GOS) from whey cheese. METHODS AND RESULTS: The ß-galactosidase production by Trichoderma sp. increased 2·3-fold (2·67 U g-1 of substrate) culturing the fungus at 30°C for 187 h, at an inoculum of 105 spores per ml, and a 1 : 1·65 (w/v) ratio of wheat bran to tap water. The best enzyme activity was obtained at 55°C and pH 4·5. The catalytic activity was maintained for up to 180 min incubating at 35-45°C, and above 50% at acidic or alkaline pH for up to 24 h. It also presented resistance to chemical compounds. ß-galactosidase catalysed the hydrolysis of the lactose and the transgalactosylation reaction leading to the production of GOS. CONCLUSION: Trichoderma sp. produced ß-galactosidase with transgalactosylation activity that may be used to recover GOS, products with high added value, from whey cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: ß-galactosidases are used in different industrial sectors. Therefore, the Trichoderma ß-galactosidase is a promising alternative for the production of GOS as prebiotic from the dairy effluents, contributing to the reduction in the environmental impact.


Assuntos
Galactose/metabolismo , Oligossacarídeos/metabolismo , Trichoderma/metabolismo , Soro do Leite/metabolismo , beta-Galactosidase/metabolismo , Queijo , Fibras na Dieta/metabolismo , Fermentação , Glicosilação , Hidrólise , Lactose/metabolismo , Prebióticos , Soro do Leite/química
18.
Food Chem ; 344: 128686, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33246685

RESUMO

To solve the potential problem of hindered ß-galactosidase activity by procyanidins, carboxymethylated Pachyman (CMP), a negatively-charged carboxymethylated (1 â†’ 3)-ß-d-glucan, was applied to mitigate inhibition by procyanidins. The mechanisms underlying this effect were explored through enzyme kinetic analysis, fluorescence quenching assays, circular dichroism, and molecular docking studies. The results indicated that the introduction of CMP could decrease the inhibition rate of high-concentration lotus seedpod oligomeric procyanidins (LSOPC) from 98.7 to 46.5%, and enabled low-concentration LSOPC to activate ß-galactosidase in vitro and in vivo. The competitive/noncompetitive inhibition constants, fluorescence quenching constants, and molecular docking results indicated that the mechanism of this effect might be CMP competing with ß-galactosidase to bind procyanidins, resulting in restoration of the catalytic centre and key active site of procyanidin-bound lactase. Additionally, it was affected by procyanidin-CMP noncovalent interactions. This study illustrates a promising strategy for mitigating the anti-nutritional properties of procyanidins and activating ß-galactosidase to promote intestinal health.


Assuntos
Biflavonoides/metabolismo , Catequina/metabolismo , Proantocianidinas/metabolismo , beta-Galactosidase/metabolismo , beta-Glucanas/química , beta-Glucanas/farmacologia , Cinética , Lotus/química , Metilação , Ligação Proteica/efeitos dos fármacos
19.
Mol Cell Biol ; 41(2)2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33077499

RESUMO

Senescence is a state of long-term cell cycle arrest that arises in cells that have incurred sublethal damage. While senescent cells no longer replicate, they remain metabolically active and further develop unique and stable phenotypes that are not present in proliferating cells. On one hand, senescent cells increase in size, maintain an active mTORC1 complex, and produce and secrete a substantial amount of inflammatory proteins as part of the senescence-associated secretory phenotype (SASP). On the other hand, these progrowth phenotypes contrast with the p53-mediated growth arrest typical of senescent cells that is associated with nucleolar stress and an inhibition of rRNA processing and ribosome biogenesis. In sum, translation in senescent cells paradoxically comprises both a global repression of translation triggered by DNA damage and a select increase in the translation of specific proteins, including SASP factors.


Assuntos
Senescência Celular/genética , Quimiocinas/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Biossíntese de Proteínas , Proteína Supressora de Tumor p53/genética , Biomarcadores/metabolismo , Tamanho Celular , Células Cultivadas , Quimiocinas/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fenótipo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
20.
J Mater Chem B ; 9(1): 170-175, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33230516

RESUMO

The development of non-invasive and sensitive optical probes for in vivo bioimaging of cancer-related enzymes is desirable for early diagnosis and effective cancer therapy. ß-galactosidase (ß-gal) is regarded as a key ovarian cancer biomarker, owing to its overexpression in primary ovarian cancer. Herein, we designed a sensitive near-infrared (NIR) probe (DCMCA-ßgal) for the detection and real-time imaging of ß-gal activity in ovarian tumors, thereby achieving the visualization of ovarian tumors by ß-gal activity detection. DCMCA-ß-gal could be triggered by ß-gal, resulting in the release of a NIR chromophore, DCM-NH2; the linear range of fluorescent response to ß-gal concentration was 0-1.2 U with a low detection limit of 1.26 × 10-3 U mL-1. We used DCMCA-ß-gal to detect and visualize ß-gal activity in SKOV3 human ovarian cancer cells, as well as for real-time imaging of ß-gal activity in ovarian cancer mouse models. DCMCA-ß-gal possessed high sensitivity, "turn-on" NIR emission, a large spectral shift, and high photostability in a dynamic living system and thus could serve as a highly sensitive sensor for real-time tracking of ß-gal activity in vivo and ovarian tumor imaging.


Assuntos
Corantes Fluorescentes/metabolismo , Imagem Óptica/métodos , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/metabolismo , beta-Galactosidase/metabolismo , Animais , Linhagem Celular Tumoral , Ativação Enzimática/fisiologia , Feminino , Corantes Fluorescentes/análise , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Espectroscopia de Luz Próxima ao Infravermelho/métodos , beta-Galactosidase/análise
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