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1.
BMC Cancer ; 22(1): 817, 2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35879690

RESUMO

BACKGROUND: Urothelial carcinoma (UC) is among the most prevalent malignancies. The muscle-invasive bladder cancer (MIBC) shows an invasive feature and has poor prognosis, while the non-muscle invasive bladder cancer (NMIBC) shows a better prognosis as compared with the MIBC. However, a significant proportion (10%-30%) of NMIBC cases progress to MIBC. Identification of efficient biomarkers for the prediction of the course of UC remains challenging nowadays. Recently, there is an emerging study showed that post-translational modifications (PTMs) by glycosylation is an important process correlated with tumor angiogenesis, invasion and metastasis. Herein, we reported a data-driven discovery and experimental validation of GANAB, a key regulator of glycosylation, as a novel prognostic marker in UC. METHODS: In the present study, we conducted immunohistochemistry (IHC) assay to evaluate the correlation between the expression levels of GANAB protein and the prognosis of UC in our cohort of 107 samples using whole slide image (WSI) analysis. In vitro experiments using RNAi were also conducted to investigate the biological functions of GANAB in UC cell lines. RESULTS: We observed that positive GANAB protein expression was significantly correlated with poor prognosis of UC in our cohort, with p-value of 0.0017 in Log-rank test. Notably, tumor cells at the invasive front of the tumor margin showed stronger GANAB expression than the tumor cells inside the tumor body in UCs. We further validated that the elevated expression levels of GANAB were significantly correlated with high grade tumors (p-values of 1.72 × 10-10), advanced stages (6.47 × 10-6), and elevated in luminal molecular subtypes. Moreover, knocking-down GANAB using RNAi in UM-UC-3 and T24 cells inhibited cell proliferation and migration in vitro. Knockdown of GANAB resulted in cell cycle arrest at G1 phase. We demonstrated that GANAB mediated HIF1A and ATF6 transcriptional activation in the ER stress signaling, and regulated the gene expression of cell cycle-related transcriptional factors E2F7 and FOXM1. CONCLUSIONS: The elevated expression of GANAB is a novel indicator of poorer prognosis of UC. Our data suggests that GANAB is not only a new and promising prognostic biomarker for UC, but also may provide important cues for the development of PTM-based therapeutics for UC treatment.


Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma de Células de Transição/patologia , Glucosidases , Glicoproteínas , Humanos , Invasividade Neoplásica , Prognóstico , Neoplasias da Bexiga Urinária/patologia , alfa-Glucosidases
2.
Food Res Int ; 156: 111344, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35651090

RESUMO

A new Aspergillus niger whole-cell catalyst was cultured for the cascade hydrolysis of hesperidin (HES) to produce high-value hesperetin-7-O-glucoside (HG) and hesperetin with high conversion (above 90%). Moreover, the inducers used were shown to be useful for cell growth and to induce cells to produce specific enzymes. Remarkably, the type of inducers determined whether the cells can hydrolyze HES. The product composition was also controllable by adjusting different inducers. Transcriptome analysis suggested that both naringin-vs-blank group and saccharose-vs-blank group had obviously difference in gene expression. The naringin-vs-blank group was mainly up-regulated differentially expressed genes (DEGs), while saccharose-vs-blank group was mainly down-regulated DEGs. The Gene Ontology (GO) analysis showed that whether naringin or saccharose was added as an inducer would greatly affect the catalytic activity of cells. Furthermore, 3 genes related to rhamnosidase, 14 genes related to glucosidase and 5 genes related to hydrolase activity were found. These genes were not only involved in rhamnosidase and glucosidase activities, but also spliceosome and the sucrose and starch metabolic pathways. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the results of transcriptome sequencing were reliable. This study gave a new approach to hydrolyze HES, and new perspectives to understand the mechanisms associated with the hydrolysis of whole-cell catalyst.


Assuntos
Citrus , Aspergillus , Aspergillus niger/genética , Flavonoides , Glucosidases , Hidrólise , Sacarose , Transcriptoma
3.
Mol Ecol ; 31(10): 2920-2934, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35344623

RESUMO

Soil microbiota increase their fitness to local habitats by adjusting their life history strategies. Yet, how such adjustments drive their ecological adaptations in xeric grasslands remains elusive. In this study, shifts in the traits that potentially represent microbial life history strategies were studied along two aridity gradients with different climates using metagenomic and trait-based approaches. The results indicated that resource acquisition (e.g., higher activities of ß-d-glucosidase and N-acetyl-ß-d-glucosidase, higher degradation rates of cellulose and chitin, as well as genes involved in cell motility, biodegradation, transportation and competition) and growth yield (e.g., higher biomass and respiration) strategies were depleted at higher aridity. However, maintenance of cellular and high growth potential (e.g., higher metabolic quotients and genes related to DNA replication, transcription, translation, central carbon metabolism and biosynthesis) and stress tolerance (e.g., genes involved in DNA damage repair, cation transportation, sporulation and osmolyte biosynthesis) strategies were enriched at higher aridity. This implied that microbiota have lower growth yields but are probably well primed for rapid responses to pulses of rainfall in more arid soils, whereas those in less arid soils may have stronger resource acquisition and growth yield abilities. By integrating a large amount of evidence from taxonomic, metagenomic, genomic and biochemical investigations, this study demonstrates that the ecological adaptations of soil microbiota to aridity made by adjusting and optimizing their life history strategies are universal in xeric grasslands and provides an underlying mechanistic understanding of soil microbial responses to climate changes.


Assuntos
Traços de História de Vida , Microbiota , Ecossistema , Glucosidases , Microbiota/genética , Solo , Microbiologia do Solo
4.
J Biol Chem ; 298(5): 101827, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35293315

RESUMO

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The kcat/Km values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.


Assuntos
Bactérias/enzimologia , Fungos/enzimologia , Glucosidases , Glicosídeo Hidrolases , Sequência de Aminoácidos , Bactérias/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Fungos/metabolismo , Glucosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Lactococcus lactis/enzimologia , Lactococcus lactis/metabolismo , Modelos Moleculares , Oligossacarídeos/metabolismo , Filogenia , Especificidade por Substrato
5.
J Biosci Bioeng ; 133(6): 560-566, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35314117

RESUMO

Kitchen waste (KW) is a vast potential source of fermentable substrates. To bio-convert the KW into high-value chemicals, we used KW as substrate for the production of fengycin by an artificial consortium containing Bacillus amyloliquefaciens HM618 producing fengycin and the engineering Pichia pastoris producing amylase, glucosidase, or lipases. The maximal amylase activity of the constructed amylase-producing engineering strain (recombinant P. pastoris GS115-amy98) reached 385.4 U‧mL-1. The engineering strain GS115-α-glu53 producing glucosidase reached an enzyme activity titer of 247.3 U‧mL-1, while the lipase activities of the engineering strains GS115-lip2, GS115-α-lip2, and GS115-lip7 were around 90.0 U‧mL-1, with no significant differences among them. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that the components of fengycin synthesized by B. amyloliquefaciens HM618 were complex, including C14-C18 fengycins A, C13-C14 fengycins B, C16-C18 fengycins B, C16 fengycin B2 and some fengycin homologues with unsaturated fatty acid chains. The levels of fengycin were 15.9 mg‧L-1 and 4.6 mg‧L-1 under the co-culture with strain HM618 and the recombinant strains producing amylase and lipase, respectively. The maximal titer of fengycin was 21.2 mg‧L-1 in the artificial consortia consisting of HM618 and the engineering strains producing glucosidase, amylase and lipase. Taken together, these results show that the co-culture of B. amyloliquefaciens HM618 and engineering strains producing amylase and lipase can promote the conversion of KW into fengycin. The work provides a new strategy for boosting the resource utilization of KW.


Assuntos
Bacillus amyloliquefaciens , Amilases , Bacillus amyloliquefaciens/genética , Técnicas de Cocultura , Glucosidases , Lipase/genética , Lipopeptídeos , Pichia/genética , Saccharomycetales
6.
Nanoscale ; 14(12): 4495-4510, 2022 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-35254362

RESUMO

PRKCSH, also known as glucosidase II beta, functions as a contributor to lung tumorigenesis by regulating the cell cycle in a p53-dependent manner under severe environmental stress. However, the prognostic value and molecular mechanisms by which the level of PRKCSH is significantly increased in cancer cells are not clearly understood. Here, we first generated a biological profile of PRKCSH expression changes in cancers by analysing bioinformatic data from cancer databases. We found that higher PRKCSH expression was correlated with a poorer prognosis and greater infiltration of most immune cell types in patients with lung cancer. In particular, PRKCSH expression showed significant negative correlations with the level of STAT6 (r = -0.31, p < 0.001) in lung cancer tissues. We further found that PRKCSH deficiency promoted G2/M arrest in response to zinc oxide nanoparticle (Nano ZnO) treatment in A549 cells. With regard to the mechanism, PRKCSH deficiency may induce STAT6 translocation to the nucleus to activate p53 expression through binding to the p53 promoter region from -365 bp to +126 bp. Eventually, activated p53 contributed to Nano-ZnO-induced G2/M arrest in lung cancer cells. Taken together, our data provide new insights into immunotherapy target choices and the prognostic value of PRKCSH. Since the G2/M cell cycle checkpoint is crucial for lung cancer prognosis, targeting PRKCSH expression to suppress the activation of the STAT6/p53 pathway is a potential therapeutic strategy for managing lung cancer.


Assuntos
Neoplasias Pulmonares , Óxido de Zinco , Apoptose , Proteínas de Ligação ao Cálcio/uso terapêutico , Linhagem Celular Tumoral , Biologia Computacional , Pontos de Checagem da Fase G2 do Ciclo Celular , Glucosidases/metabolismo , Glucosidases/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
7.
Food Funct ; 13(5): 2456-2464, 2022 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-35147627

RESUMO

Codonopsis pilosula (Franch.) Nannf. (CPN), mainly planted in the northwest region, is a traditional Chinese medicine/good health food for nourishing qi and promoting blood circulation. This study firstly evaluated the inhibitory effects of the CPN extraction (CPNE) on α-glucosidase in vitro and in vivo, and tentatively confirmed its chemical ingredients by employing UHPLC-Triple-TOF-MS/MS. The CPNE had strong inhibitory activities against mammalian α-glucosidase (sucrase and maltase) and yeast α-glycosidase with semi-inhibitory concentrations (IC50) of 0.241 mg mL-1, 0.326 mg mL-1 and 1.167 mg mL-1, respectively. In addition, the CPNE could significantly decrease the postprandial blood glucose (PBG) levels in the sucrose/maltose/starch tolerance assays of diabetic mice. Furthermore, a total of 29 compounds, including 3 alkaloids, 13 phenolic acids, 8 alcohol glycosides and 5 alkynosides, were assigned based on comparison with the standards and references, as well as the analysis of main fragments. These results demonstrated that CPN could be used as an adjuvant therapy or dietary supplements to effectively control the occurrence and development of diabetes.


Assuntos
Codonopsis , Diabetes Mellitus Experimental/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Hipoglicemiantes/farmacologia , Extratos Vegetais/farmacologia , Aloxano , Animais , Animais não Endogâmicos , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/química , Glucosidases/efeitos dos fármacos , Hipoglicemiantes/química , Concentração Inibidora 50 , Masculino , Camundongos , Fitoterapia , Extratos Vegetais/química , Raízes de Plantas
8.
Food Chem ; 384: 132497, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35219994

RESUMO

Current methods for vanilla bean curing are long and reduce the enzymatic activity necessary for flavor development. High hydrostatic pressure (HHP) at 50-600 MPa was used to improve phenolic compounds formation and ß-d-glucosidase activity in vanilla beans compared with scalded beans. Phenolics were analyzed by HPLC and ß-d-glucosidase activity by spectrophotometry. Vanillin was the main phenolic and it was formed by ß-d-glucovanillin hydrolysis and vanillyl alcohol oxidation. HHP improved vanillin content and influenced ß-d-glucosidase activity. At the beginning of the curing the highest increments of vanillin were produced at 400 MPa (up to 15%), while at the end, this was observed at 50 (138%) and 600 MPa (74%). Maximum increment of up to 400% in ß-d-glucosidase activity was observed from 100 to 300 MPa, which was attributed to tissue decompartmentalization, and conformational changes induced by pressure. HHP could be used during vanilla curing to improve vanillin content and ß-d-glucosidase activity.


Assuntos
Vanilla , Benzaldeídos/metabolismo , Cromatografia Líquida de Alta Pressão , Glucosidases/metabolismo , Pressão Hidrostática , Fenóis/metabolismo , Vanilla/metabolismo
9.
Fitoterapia ; 156: 105086, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34798164

RESUMO

Two new isoquinoline alkaloids, cryptowrayines A (1) and B (2), along with one known pavine alkaloid (-)-12-hydroxyeschscholtzidine (3), were isolated from the twigs of Cryptocarya wrayi. The structures of new compounds were elucidated by extensive spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Both compounds 1 and 2 exhibited moderate quinone reductase inducing activity in Hepa 1c1c7 cells.


Assuntos
Alcaloides/isolamento & purificação , Cryptocarya/química , Isoquinolinas/isolamento & purificação , Alcaloides/química , Alcaloides/metabolismo , Glucosidases/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Inibidores de Glicosídeo Hidrolases/metabolismo , Concentração Inibidora 50 , Isoquinolinas/química , Isoquinolinas/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/análise , Rotação Ocular
10.
FASEB J ; 35(12): e22014, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34751973

RESUMO

Spinal cord injury (SCI) is a complex pathological change that includes primary SCI and gradually evolves into secondary SCI. Accumulating evidence demonstrates that circular RNAs (circRNAs) are involved in the pathology of a variety of neurological diseases and injuries. However, the characteristics and function of circRNAs in SCI have yet to be elucidated. Although previous research demonstrated that circPrkcsh induces astrocytes to produce inflammatory factors and chemokines, the precise function and mechanism of circPrkcsh in microglia after SCI remains unknown. In this study, we constructed a mouse model of SCI by applying a SCI impactor. Quantitative Real-time PCR and Fluorescence in situ hybridization analysis revealed that circPrkcsh was upregulated in the microglia of SCI mice when compared to sham-operated mice. Gain- or loss-of-function experiments and in vivo assays further indicated that circPrkcsh promotes microglia M1 polarization both in vivo and in vitro. Furthermore, bioinformatics analysis, dual-luciferase assays, and RNA immunoprecipitation assays, confirmed that circPrkcsh serves as a competing endogenous RNA (ceRNA) to promote the expression of MEKK1 mRNA by sponging miR-488. Double knockout rescue experiments further showed that circPrkcsh regulates the MEKK1/JNK/p38 MAPK pathway via miR-488. Our research provides a better understanding of the mechanism of circPrkcsh in SCI and demonstrates that the circPrkcsh/miR-488/Mekk1 axis is a promising regulatory method for the treatment of SCI.


Assuntos
Glucosidases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , MicroRNAs/genética , RNA Circular/genética , Traumatismos da Medula Espinal/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose , Modelos Animais de Doenças , Regulação da Expressão Gênica , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Transdução de Sinais , Traumatismos da Medula Espinal/etiologia , Traumatismos da Medula Espinal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética
11.
Nat Commun ; 12(1): 5885, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620873

RESUMO

Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3ß-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3ß-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1's substrate specificity and enable the rational design of antifungal agents targeting Sgl1.


Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos , Domínio Catalítico , Criptococose , Cryptococcus neoformans/genética , Cristalografia por Raios X , Modelos Animais de Doenças , Descoberta de Drogas , Ergosterol , Feminino , Proteínas Fúngicas/genética , Glucosidases/química , Glucosidases/efeitos dos fármacos , Glucosidases/genética , Ensaios de Triagem em Larga Escala , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular
12.
Int J Biol Macromol ; 191: 996-1005, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34597698

RESUMO

In the present study, effects of maturity stage on structural characteristics and biosynthesis/hydrolysis-associated genes expression of glucans from Volvariella volvacea fruit body were well investigated. Elongation and pileus expansion stages decreased total soluble carbohydrate and protein contents to 17.09 mg/g and 8.33 mg/g, and significantly accumulated the total amino acids contents to 32.37 mg/g. Yields of crude polysaccharides significantly increased to 8.12% at egg stage and decreased to 3.72% at pileus expansion stage. Purified VVP I-a and VVP I-b were proved to be α-glucans. The maturity process affected the monosaccharide compositions, decreased the molecular weights of VVP I-a and VVP I-b with decreased transcription levels of glucan biosynthesis-associated enzyme genes vvugp and vvgls and increased glucan hydrolysis-associated glucanase gene vvexg2 expression with no significant effects on backbone structures including glycosidic linkages and configurations. The findings would benefit for understanding change patterns of V. volvacea glucan structures and their biosynthesis/hydrolysis-associated genes expression at maturity stages.


Assuntos
Agaricales/genética , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Glucosidases/metabolismo , Agaricales/enzimologia , Agaricales/crescimento & desenvolvimento , Carpóforos/genética , Carpóforos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Glucosidases/química , Glucosidases/genética
13.
J Chem Inf Model ; 61(9): 4554-4570, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34423980

RESUMO

Bacterial glycoside hydrolase 1 (GH1) enzymes with 6-phospho-ß-galactosidase and 6-phospho-ß-glucosidase activities have the important task of releasing phosphorylated and nonphosphorylated monosaccharides into the cytoplasm. Curiously, dual 6-phospho-ß-galactosidase/6-phospho-ß-glucosidase (dual-phospho) enzymes have broad specificity and are able to hydrolyze galacto- and gluco-derived substrates. This study investigates the structure and substrate specificity of a GH family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglC. The enzyme structure has been solved, and sequence analysis, molecular dynamics simulations, and binding free energy calculations offered evidence of dual-phospho activity. Both test ligands p-nitrophenyl-ß-d-galactoside-6-phosphate (PNP6Pgal) and p-nitrophenyl-ß-d-glucoside-6-phosphate (PNP6Pglc) demonstrated strong binding to BlBglC although the pose and interactions of the PNP6Pglc triplicates were slightly more consistent. Interestingly, known specificity-inducing residues, Gln23 and Trp433, bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-BlBglC complex and to the ligand O4 hydroxyl group in the PNP6Pglc-BlBglC complex. Additionally, the BlBglC-His124 residue is a major contributor of hydrogen bonds to the PNP6Pgal O3 hydroxyl group but does not form any hydrogen bonds with PNP6Pglc. On the other hand, BlBglC residues Tyr173, Tyr301, Gln302, and Thr321 form hydrogen bonds with PNP6Pglc but not PNP6Pgal. These findings provide important details of the broad specificity of dual-phospho activity GH1 enzymes.


Assuntos
Bacillus licheniformis , Glucosidases , Bacillus licheniformis/metabolismo , Galactosidases , Glucosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Especificidade por Substrato
14.
Biomolecules ; 11(8)2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34439895

RESUMO

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops-a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to "the proline rule". Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.


Assuntos
Aminoácidos/química , Oligo-1,6-Glucosidase/química , Engenharia de Proteínas/métodos , Dicroísmo Circular , Clonagem Molecular , Temperatura Baixa , Biologia Computacional , Estabilidade Enzimática , Exiguobacterium/enzimologia , Glucosidases/genética , Glucosidases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Pergelissolo , Prolina/química , Proteínas Recombinantes/química , Temperatura
15.
J Agric Food Chem ; 69(36): 10515-10526, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34463509

RESUMO

As a natural flavonolignan, silibinin is reported to possess multiple biological activities, while the inhibitory potential of silibinin on carbohydrate-hydrolyzing enzymes is still unclear. Therefore, in this study, the inhibitory effect and underlying mechanism of silibinin against α-amylase/α-glucosidase were investigated. The results indicated that silibinin showed a strong inhibitory efficiency against α-amylase/α-glucosidase in noncompetitive manners and exhibited synergistic inhibition against α-glucosidase with acarbose. However, interestingly, the inhibitory effect of silibinin was significantly hindered in various milk protein-rich environments, but this phenomenon disappeared after simulated gastrointestinal digestion of milk proteins in vitro. Furthermore, silibinin could combine with the inactive site of α-amylase/α-glucosidase and change the microenvironment and secondary structure of the enzymes, thereby influencing the catalytic efficiency of enzymes. This research suggested that silibinin could be used as a novel carbohydrate-hydrolyzing enzyme inhibitor, and milk beverages rich in silibinin had the potential for further application in antidiabetic dietary or medicine.


Assuntos
Acarbose , alfa-Glucosidases , Acarbose/farmacologia , Amilases , Glucosidases , Inibidores de Glicosídeo Hidrolases , Proteínas do Leite , Silibina , alfa-Amilases
16.
PLoS One ; 16(8): e0255669, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34407107

RESUMO

Soil enzymes mediate key processes and functions of the soils, such as organic matter decomposition and nutrient cycling in both natural and agricultural ecosystems. Here, we studied the activity of five extracellular soil enzymes involved in the C, N, and P-mineralizing process in both litter and surface soil layer of rainforest in the northwest region of the Colombian Amazon and the response of those soil enzymes to land use change. The experimental study design included six study sites for comparing long-term pasture systems to native forest and regeneration practices after pasture, within the main landscapes of the region, mountain and hill landscapes separately. Results showed considerable enzymatic activity in the litter layer of the forest, highlighting the vital role of this compartment in the nutrient cycling of low fertility soils from tropical regions. With the land use transition to pastures, changes in soil enzymatic activities were driven by the management of pastures, with SOC and N losses and reduced absolute activity of soil enzymes in long-term pastures under continuous grazing (25 years). However, the enzyme activities expressed per unit of SOC did not show changes in C and N-acquiring enzymes, suggesting a higher mineralization potential in pastures. Enzymatic stoichiometry analysis indicated a microbial P limitation that could lead to a high catabolic activity with a potential increase in the use of SOC by microbial communities in the search for P, thus affecting soil C sequestration, soil quality and the provision of soil-related ecosystem services.


Assuntos
Acetilglucosaminidase/análise , Fosfatase Ácida/análise , Agricultura/métodos , Celulose 1,4-beta-Celobiosidase/análise , Glucosidases/análise , Floresta Úmida , Solo/química , Xilosidases/análise , Carbono/análise , Colômbia , Conservação dos Recursos Naturais , Microbiota , Nitrogênio/análise , Fósforo/análise , Microbiologia do Solo , Clima Tropical
17.
Environ Health ; 20(1): 75, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193170

RESUMO

BACKGROUND: Cadmium (Cd) is a common heavy metal that mainly causes renal damage. There is a lack of research on the large-scale and systematic evaluation of the association between urinary Cd (U-Cd) and various effect biomarkers among Chinese residents. METHODS: Based on the establishment process of dietary Cd limit standards by European Food Safety Authority (EFSA), the dose-response relationships between U-Cd and four biomarkers, ß2-microglobulin (ß2-MG), N-acetyl-ß-glucosidase (NAG), microalbumin (mALB), and retinol binding Protein (RBP) were explored, respectively. Toxicokinetic model was used to derive the dietary Cd exposure limit for Chinese residents after critical U-Cd concentration was calculated. RESULTS: As the sensitive biomarkers of renal injury, ß2-MG and NAG were selected to estimate the 95% confidence interval lower limit of the U-Cd benchmark dose (BMDL5) to be 3.07 and 2.98 µg/g Cr, respectively. Dietary Cd exposure limit was calculated to be 0.28 µg/kg bw/day (16.8 µg/day, based on the body weight of 60 kg), which was lower than the average Chinese Cd exposure (30.6 µg/day) by the China National Nutrient and Health Survey. CONCLUSION: This study established an overall association between U-Cd and renal injury biomarkers, and explored the Chinese dietary Cd exposure limits, which helps improve Chinese Cd exposure risk assessment and provides a reference basis for formulating reasonable exposure standards.


Assuntos
Cádmio/urina , Exposição Dietética , Poluentes Ambientais/urina , Nefropatias/urina , Modelos Biológicos , Adulto , Albuminúria/epidemiologia , Monitoramento Biológico , Biomarcadores/urina , Cádmio/toxicidade , China/epidemiologia , Exposição Dietética/análise , Exposição Dietética/normas , Relação Dose-Resposta a Droga , Poluentes Ambientais/toxicidade , Feminino , Glucosidases/urina , Humanos , Nefropatias/epidemiologia , Masculino , Nível de Efeito Adverso não Observado , Proteínas de Ligação ao Retinol/urina , Microglobulina beta-2/urina
18.
Food Chem ; 364: 130412, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34174646

RESUMO

Nitraria tangutorum Bobr. (NTB), mainly distributed in the Qaidam Basin of Tibetan Plateau, have high economic, ecological and medicinal value. The chemical compositions in the NTB fruits were tentatively analyzed and characterized by applying UPLC-Q-TOF-MS/MS. Total 45 constituents, including 9 hydroxycinnamic acids derivatives, 12 flavonols, 4 flavonoids, 1 trolox derivative, 8 ß-carboline alkaloids, 4 tryptophan derivatives, and 7 other amino acid derivatives were identified by comparing with standard products, and analyzing their retention times, characteristic fragment ions and deprotonated molecule ions. The activity studies in vitro indicated that NTB-Z and NTB-C extracts had marked inhibitory effects against sucrase and maltase. Further sucrose/maltose/starch tolerance experiment demonstrated that both NTB-Z and NTB-C extracts at 400 mg/kg could markedly lower the postprandial blood glucose (PBG) level in diabetic animals. All these results indicated that the NTB fruits could be used as the functional health food or medicine for controlling postprandial blood glucose level.


Assuntos
Alcaloides , Frutas , Animais , Cromatografia Líquida de Alta Pressão , Glucosidases , Extratos Vegetais/farmacologia , Espectrometria de Massas em Tandem
19.
J Biotechnol ; 334: 51-57, 2021 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-33878390

RESUMO

Although coumaroyltyramine (CT) derivatives are one kind of phenolamides with remarkable biological activities, the low content in plants would inhibit their potential use in food and pharmaceutical industries. Therefore, it is necessary to screen an efficient method to produce CT derivatives. A green and efficient method by using lipase as catalyst to synthesize a series of CT derivatives, was thus proposed. To obtain optimum reaction conditions, the effects of various parameters on conversion rate were firstly evaluated. An in vitro α-glucosidase inhibitory assay of synthesized compounds was then carried out, and the structure-activity relationship of these compounds was conducted. Under the optimum conditions (MTBE, Nu/S: 2/1, E/S: 20/1, 50 °C and 24 h), the conversion rates of synthesized compounds were above 65 %. The bioassay results indicated that N-trans-caffeoyltyramine and N-trans-feruloyltyramine had potent activities against α-glucosidase with IC50 of 30.08 µM and 31.94 µM, respectively. The structure-activity relationship results showed that the presence of -OH or -OCH3 group at C-3 position could boost the activities of CT derivatives. Meanwhile, the presence of -OH group at C-4 position and double bound on caffeoyl moiety as well as the presence of -OH group at C-4' position was essential for the activities of CT derivatives.


Assuntos
Inibidores de Glicosídeo Hidrolases , alfa-Glucosidases , Glucosidases , Inibidores de Glicosídeo Hidrolases/farmacologia , Lipase , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , alfa-Glucosidases/metabolismo
20.
Molecules ; 26(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802808

RESUMO

Smoke taint has become a prominent issue for the global wine industry as climate change continues to impact the length and extremity of fire seasons around the world. Although the issue has prompted a surge in research on the subject in recent years, no singular solution has yet been identified that is capable of maintaining the quality of wine made from smoke-affected grapes. In this review, we summarize the main research on smoke taint, the key discoveries, as well as the prevailing uncertainties. We also examine methods for mitigating smoke taint in the vineyard, in the winery, and post production. We assess the effectiveness of remediation methods (proposed and actual) based on available research. Our findings are in agreement with previous studies, suggesting that the most viable remedies for smoke taint are still the commercially available activated carbon fining and reverse osmosis treatments, but that the quality of the final treated wines is fundamentally dependent on the initial severity of the taint. In this review, suggestions for future studies are introduced for improving our understanding of methods that have thus far only been preliminarily investigated. We select regions that have already been subjected to severe wildfires, and therefore subjected to smoke taint (particularly Australia and California) as a case study to inform other wine-producing countries that will likely be impacted in the future and suggest specific data collection and policy implementation actions that should be taken, even in countries that have not yet been impacted by smoke taint. Ultimately, we streamline the available information on the topic of smoke taint, apply it to a global perspective that considers the various stakeholders involved, and provide a launching point for further research on the topic.


Assuntos
Frutas/química , Fumaça/efeitos adversos , Vitis/química , Vinho/análise , Poluição do Ar/efeitos adversos , Austrália , California , Carvão Vegetal/química , Mudança Climática , Ciclodextrinas/química , Frutas/efeitos adversos , Glucosidases/química , Fenóis/análise , Vitis/efeitos adversos , Incêndios Florestais , Vinho/efeitos adversos , Vinho/normas
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