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1.
PLoS Genet ; 16(3): e1008617, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130226

RESUMO

The oligosaccharyl transferase (OST) protein complex mediates the N-linked glycosylation of substrate proteins in the endoplasmic reticulum (ER), which regulates stability, activity, and localization of its substrates. Although many OST substrate proteins have been identified, the physiological role of the OST complex remains incompletely understood. Here we show that the OST complex in C. elegans is crucial for ER protein homeostasis and defense against infection with pathogenic bacteria Pseudomonas aeruginosa (PA14), via immune-regulatory PMK-1/p38 MAP kinase. We found that genetic inhibition of the OST complex impaired protein processing in the ER, which in turn up-regulated ER unfolded protein response (UPRER). We identified vitellogenin VIT-6 as an OST-dependent glycosylated protein, critical for maintaining survival on PA14. We also showed that the OST complex was required for up-regulation of PMK-1 signaling upon infection with PA14. Our study demonstrates that an evolutionarily conserved OST complex, crucial for ER homeostasis, regulates host defense mechanisms against pathogenic bacteria.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Proteostase/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Hexosiltransferases/metabolismo , Imunidade Inata/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/metabolismo , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Regulação para Cima/fisiologia , Vitelogeninas/metabolismo
2.
Nat Chem Biol ; 16(4): 450-457, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152541

RESUMO

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-ß-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.


Assuntos
Glicosiltransferases/metabolismo , Antígenos O/metabolismo , Antígenos O/ultraestrutura , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Glicosiltransferases/fisiologia , Hexosiltransferases , Klebsiella pneumoniae/metabolismo , Lipopolissacarídeos/química , Polissacarídeos Bacterianos/química
3.
Int J Mol Sci ; 20(23)2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810196

RESUMO

Oligosaccharyltransferase (OST) is a multi-span membrane protein complex that catalyzes the addition of glycans to selected Asn residues within nascent polypeptides in the lumen of the endoplasmic reticulum. This process, termed N-glycosylation, is a fundamental post-translational protein modification that is involved in the quality control, trafficking of proteins, signal transduction, and cell-to-cell communication. Given these crucial roles, N-glycosylation is essential for homeostasis at the systemic and cellular levels, and a deficiency in genes that encode for OST subunits often results in the development of complex genetic disorders. A growing body of evidence has also demonstrated that the expression of OST subunits is cell context-dependent and is frequently altered in malignant cells, thus contributing to tumor cell survival and proliferation. Importantly, a recently developed inhibitor of OST has revealed this enzyme as a potential target for the treatment of incurable drug-resistant tumors. This review summarizes our current knowledge regarding the functions of OST in the light of health and tumor progression, and discusses perspectives on the clinical relevance of inhibiting OST as a tumor treatment.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Hexosiltransferases/genética , Proteínas de Membrana/genética , Neoplasias/genética , Processamento de Proteína Pós-Traducional/genética , Sequência de Aminoácidos/genética , Asparagina/genética , Progressão da Doença , Retículo Endoplasmático/genética , Glicosilação , Hexosiltransferases/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Neoplasias/tratamento farmacológico , Polissacarídeos/genética
4.
Science ; 366(6471): 1372-1375, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31831667

RESUMO

Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum. Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner. Here, we present high-resolution cryo-electron microscopy structures of human OST-A and OST-B. Although they have similar overall architectures, structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B. In OST-A, interactions with TMEM258 and STT3A allow ribophorin-I to form a four-helix bundle that can bind to a translating ribosome, whereas the equivalent region is disordered in OST-B. We observed an acceptor peptide and dolichylphosphate bound to STT3B, but only dolichylphosphate in STT3A, suggesting distinct affinities of the two OST complexes for protein substrates.


Assuntos
Hexosiltransferases/química , Proteínas de Membrana/química , Microscopia Crioeletrônica , Humanos , Conformação Proteica , Subunidades Proteicas/química
5.
World J Microbiol Biotechnol ; 36(1): 9, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31858269

RESUMO

Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. PglB, a glycosyltransferase, plays a crucial role of mediating glycosylation of numerous periplasmic proteins. It catalyzes N-glycosylation at the sequon D/E-X1-N-X2-S/T in its substrate proteins. Here we report that the PglB itself is a glycoprotein which self-glycosylates at N534 site in its DYNQS sequon by its own catalytic WWDYG motif. Site-directed mutagenesis, lectin Immunoblot, and mobility shift assays confirmed that the DYNQS is an N-glycosylation motif. PglB's N-glycosylation motif is structurally and functionally similar to its widely studied glycosylation substrate, the OMPH1. Its DYNQS motif forms a solvent-exposed crest. This motif is close to a cluster of polar and hydrophilic residues, which form a loop flanked by two α helices. This arrangement extremely apposite for auto-glycosylation at N534. This self-glycosylation ability of PglB could mediate C. jejuni's ability to colonize the intestinal epithelium. Further this capability may also bear significance for the development of novel conjugated vaccines and diagnostic tests.


Assuntos
Campylobacter jejuni/enzimologia , Glicoproteínas/química , Hexosiltransferases/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Glicosilação , Hexosiltransferases/genética , Hexosiltransferases/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína , Vacinas
6.
Int J Mol Sci ; 21(1)2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31877648

RESUMO

Given its potential role in the synthesis of novel prebiotics and applications in the pharmaceutical industry, a strong interest has developed in the enzyme levansucrase (LSC, EC 2.4.1.10). LSC catalyzes both the hydrolysis of sucrose (or sucroselike substrates) and the transfructosylation of a wide range of acceptors. LSC from the Gram-negative bacterium Erwinia tasmaniensis (EtLSC) is an interesting biocatalyst due to its high-yield production of fructooligosaccharides (FOSs). In order to learn more about the process of chain elongation, we obtained the crystal structure of EtLSC in complex with levanbiose (LBS). LBS is an FOS intermediate formed during the synthesis of longer-chain FOSs and levan. Analysis of the LBS binding pocket revealed that its structure was conserved in several related species. The binding pocket discovered in this crystal structure is an ideal target for future mutagenesis studies in order to understand its biological relevance and to engineer LSCs into tailored products.


Assuntos
Proteínas de Bactérias/metabolismo , Dissacarídeos/metabolismo , Erwinia/metabolismo , Frutanos/metabolismo , Hexosiltransferases/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Erwinia/química , Hexosiltransferases/química , Modelos Moleculares , Oligossacarídeos/metabolismo , Conformação Proteica , Sacarose/química
7.
J Agric Food Chem ; 67(37): 10392-10400, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31461615

RESUMO

The specificity of fructooligosaccharides as prebiotics depends on their size and structure, which in turn depend on their origin or the synthesis procedure. In this work we describe the application of an inulosucrase (IslA) from Leuconostoc citreum CW28 to produce high molecular weight inulin from sucrose alongside a commercial endoinulinase (Novozym 960) produced by Aspergillus niger for a simultaneous or sequential reaction to synthesize fructooligosaccharides (FOS). The simultaneous reaction resulted in a higher substrate conversion and a wide diversity of FOS when compared to the sequential reaction. A shotgun MS analysis of the commercial endoinulinase preparation surprisingly revealed an additional enzymatic activity: a fructosyltransferase, responsible for the synthesis of FOS from sucrose. Consequentially, the range of FOS obtained in reactions combining inulosucrase from Ln. citreum with the fructosyltransferase and endoinulinase from A. niger with sucrose as substrate may be extended and regulated.


Assuntos
Proteínas de Bactérias/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Hexosiltransferases/química , Inulina/química , Leuconostoc/enzimologia , Oligossacarídeos/química , Aspergillus niger/enzimologia , Biocatálise , Sacarose/química
8.
Arch Virol ; 164(11): 2789-2792, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31414286

RESUMO

Replication of the dengue virus (DENV) genome occurs in a vesicle in the endoplasmic reticulum by a complex of host and viral proteins. Two host proteins, STT3A and STT3B, as members of the oligosaccharyl transferase complex, have been implicated in playing structural roles in the vesicle in mammalian cells, and the absence of these proteins has been shown to decrease DENV replication. Aedes aegypti is the main vector of the virus and has been used previously as a model organism to study mosquito-virus interactions. In this study, we found that genes of the oligosaccharyl transferase complex have no effect on replication of DENV in mosquito cells.


Assuntos
Aedes/virologia , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/genética , Hexosiltransferases/genética , Proteínas de Membrana/genética , Replicação Viral/genética , Animais , Benzamidas/farmacologia , Linhagem Celular , Chlorocebus aethiops , Dengue/virologia , Retículo Endoplasmático/virologia , Genoma Viral/genética , Glicosilação , Hexosiltransferases/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Proteínas de Membrana/antagonistas & inibidores , RNA Viral/genética , Sulfonamidas/farmacologia , Células Vero
9.
Carbohydr Polym ; 223: 115044, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31427007

RESUMO

Inulin nanoparticles (INNPs) are a biocompatible material which has a potential application for enhancing solubility and preventing degradation of compounds. In this work, we demonstrated that INNPs could be synthesized from sucrose using inulosucrase from Lactobacillus reuteri 121. Noticeably, dynamic light scattering (DLS) analysis showed that the derived INNPs exhibited uniformity in size, which was easily controlled by the reaction temperature. The effect of enzyme and sucrose concentration, as well as reaction time, was explored. Moreover, the solubility of INNPs in various organic solvents was also investigated, and we found that the INNPs were freely regenerated in water even though they had precipitated by organic solvents. Essentially, we demonstrated that the derived INNPs could be applied for flavonoid encapsulation. The solubility and stability of quercetin and fisetin in the INNPs complexes was higher than those of free compounds. These results make the INNPs very promising for many applications.


Assuntos
Flavonoides/química , Hexosiltransferases/metabolismo , Inulina/biossíntese , Lactobacillus reuteri/enzimologia , Nanopartículas/química , Quercetina/química , Concentração de Íons de Hidrogênio , Inulina/química , Tamanho da Partícula , Solubilidade , Temperatura
10.
Enzyme Microb Technol ; 130: 109364, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421727

RESUMO

A high yielding and straightforward production system of fructooligosaccharide (FOS) was developed for industrial production of prebiotics. To increase conversion yield of FOS from sucrose, recombinant yeast secreting inulosucrase from Lactobacillus reuteri (LrInu) were constructed. Efficient secretion of LrInu was achieved by truncation of both amino- and carboxy-termini (LrInuΔNC) and by introducing an optimal secretion signal. The recombinant yeast produced 220 U/mL of recombinant LrInuΔNC into culture medium during fed-batch fermentation. By direct fermentation of recombinant yeast in medium containing sucrose, 128.4 g/L of FOS was produced with 85.6% conversion yield from 300 g/L sucrose, and the highest titer was 152.6 g/L from 400 g/L sucrose. The degree of polymerization of generated FOS was 2-20 indicating medium chain (mcFOS) range. This is the first report of industrially applicable production of mcFOS by recombinant yeast secreting bacterial inulosucrase.


Assuntos
Fermentação , Hexosiltransferases/metabolismo , Lactobacillus reuteri/enzimologia , Oligossacarídeos/metabolismo , Saccharomyces cerevisiae/genética , Microbiologia Industrial , Lactobacillus reuteri/genética , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Sacarose/metabolismo
11.
Environ Microbiol ; 21(11): 4151-4165, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31374141

RESUMO

Acetic acid bacteria (AAB) are associated with plants and insects. Determinants for the targeting and occupation of these widely different environments are unknown. However, most of these natural habitats share plant-derived sucrose, which can be metabolized by some AAB via polyfructose building levansucrases (LS) known to be involved in biofilm formation. Here, we propose two LS types (T) encoded by AAB as determinants for habitat selection, which emerged from vertical (T1) and horizontal (T2) lines of evolution and differ in their genetic organization, structural features and secretion mechanism, as well as their occurrence in proteobacteria. T1-LS are secreted by plant-pathogenic α- and γ-proteobacteria, while T2-LS genes are common in diazotrophic, plant-growth-promoting α-, ß- and γ-proteobacteria. This knowledge may be exploited for a better understanding of microbial ecology, plant health and biofilm formation by sucrase-secreting proteobacteria in eukaryotic hosts.


Assuntos
Ecossistema , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Proteobactérias/enzimologia , Animais , Hexosiltransferases/classificação , Insetos/microbiologia , Plantas/microbiologia , Proteobactérias/genética
12.
Molecules ; 24(17)2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31443364

RESUMO

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 °C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 °C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 µM, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 °C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.


Assuntos
Carboidratos Epimerases/química , Técnicas de Química Sintética , Flavonóis/síntese química , Glucosiltransferases/química , Hexosiltransferases/química , Açúcares de Uridina Difosfato/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Catálise , Linhagem Celular Tumoral , Flavonóis/farmacologia , Humanos
13.
Int J Biol Macromol ; 140: 1239-1248, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31437510

RESUMO

Levansucrase (LS) from Gram-positive bacteria generally produces a large quantity of levan polymer, a polyfructose with glucose at the end (GFn) but a small quantity of levan-type fructooligosaccharides (LFOs). The properties of levan and LFOs depend on their chain lengths, thereby determining their potential applications in food and pharmaceutical industries such as prebiotics and anti-tumor agents. Therefore, an ability to redesign and engineer the active site of levansucrase for synthesis of products with desired degree of polymerization (DP) is very beneficial. We employed computational protein design, docking and molecular dynamics to redesign and engineer the active site of Bacillus licheniformis RN-01 levansucrase for production of LFOs with DP up to five (GF4), using two approaches: 1) blocking oligosaccharide binding track of GF3-LS complex with large aromatic residues and 2) eliminating hydrogen bond interactions between terminal glucose of GF4 and side chains of binding residues of GF4-LS complex. The designed enzymes and their product patterns from these two approaches were experimentally characterized. The experimental results show that the first approach was successful in creating N251W and N251W/K372Y mutants that synthesized LFOs with DP up to five. This work illustrates how computer-aided approaches can offer novel opportunities to engineer enzymes for desired products.


Assuntos
Bacillus licheniformis/enzimologia , Frutanos/química , Hexosiltransferases/química , Simulação de Dinâmica Molecular , Oligossacarídeos/química , Domínio Catalítico , Hidrólise , Cinética
14.
Appl Microbiol Biotechnol ; 103(19): 7953-7969, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31407037

RESUMO

Two sustainable and cost-effective cascade enzymatic systems were developed to regenerate uridine diphosphate (UDP)-α-D-glucose and UDP-ß-L-rhamnose from sucrose. The systems were coupled with the UDP generating glycosylation reactions of UDP sugar-dependent glycosyltransferase (UGT) enzymes mediated reactions. As a result, the UDP generated as a by-product of the GT-mediated reactions was recycled. In the first system, YjiC, a UGT from Bacillus licheniformis DSM 13, was used for transferring glucose from UDP-α-D-glucose to naringenin, in which AtSUS1 from Arabidopsis thaliana was used to synthesize UDP-α-D-glucose and fructose as a by-product from sucrose. In the second system, flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from A. thaliana was used to transfer rhamnose from UDP-ß-L-rhamnose to quercetin, in which AtSUS1 along with UDP-ß-L-rhamnose synthase (AtRHM1), also from A. thaliana, were used to produce UDP-ß-L-rhamnose from the same starter sucrose. The established UDP recycling system for the production of naringenin glucosides was engineered and optimized for several reaction parameters that included temperature, metal ions, NDPs, pH, substrate ratio, and enzymes ratio, to develop a highly feasible system for large-scale production of different derivatives of naringenin and other natural products glucosides, using inexpensive starting materials. The developed system showed the conversion of about 37 mM of naringenin into three different glucosides, namely naringenin, 7-O-ß-D-glucoside, naringenin, 4'-O-ß-D-glucoside, and naringenin, 4',7-O-ß-D-diglucoside. The UDP recycling (RCmax) was 20.10 for naringenin glucosides. Similarly, the conversion of quercetin to quercetin 7-O-α-L-rhamnoside reached a RCmax value of 10.0.


Assuntos
Flavanonas/metabolismo , Glucosídeos/metabolismo , Glucuronosiltransferase/metabolismo , Hexosiltransferases/metabolismo , Quercetina/metabolismo , Sacarose/metabolismo , Arabidopsis/enzimologia , Bacillus licheniformis/enzimologia , Biocatálise , Glucuronosiltransferase/isolamento & purificação , Hexosiltransferases/isolamento & purificação
15.
J Basic Microbiol ; 59(10): 1004-1015, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31407369

RESUMO

Screening of 18 bacterial honey isolates revealed that all the isolates were levansucrase producers. The most potent isolate that achieved the highest activity (45.66 U/ml) was identified as Bacillus subtilis NRC based on morphological examination and 16S rRNA. The results recorded the necessity of starch (5 g/L), baker's yeast (12.5 g/L), and AlCl3 (5 mM) in improvement of the enzyme productivity. The Bacillus subtilis levansucrase was eluted as a single protein in one purification step. The enzyme molecular weight was (14 kDa). It showed its optimum activity at 45°C and could retain 60% of its activity after incubation at 50°C for 2 h. Its optimum activity was obtained at pH 8.2 and the enzyme showed great pH stability in both acidic and alkaline ranges. Unlike, most levansucrases all tested metals had an adverse effect in enzyme activity. The enzyme had antioxidant activities and were characterized as spherical micro- and nanoparticles by transmission electron microscopy. The effect of growth conditions and medium composition in levan structure and its fibrinolytic activity was evaluated.


Assuntos
Bacillus subtilis/metabolismo , Frutanos/metabolismo , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Aminoácidos , Antioxidantes/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Carboidratos , Meios de Cultura , Estabilidade Enzimática , Fibrinolíticos/metabolismo , Hexosiltransferases/isolamento & purificação , Hexosiltransferases/ultraestrutura , Mel/microbiologia , Concentração de Íons de Hidrogênio , Peso Molecular , RNA Ribossômico 16S/genética , Sais/metabolismo , Temperatura
16.
Appl Microbiol Biotechnol ; 103(19): 7891-7902, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401753

RESUMO

Functional carbohydrates are ideal substitutes for table sugar and make up a large share of the worldwide functional food market because of their numerous physiological benefits. Growing attention has been focused on levan, a ß-(2,6) fructan that possesses more favorable physicochemical properties, such as lower intrinsic viscosity and greater colloidal stability, than ß-(2,1) inulin. Levan can be used not only as a functional carbohydrate but also as feedstock for the production of levan-type fructooligosaccharides (L-FOSs). Three types of levan-degrading enzymes (LDEs), including levanase (EC 3.2.1.65), ß-(2,6)-fructan 6-levanbiohydrolase (LF2ase, EC 3.2.1.64), and levan fructotransferase (LFTase, EC 4.2.2.16), play significant roles in the biological production of L-FOSs. These three enzymes convert levan into different L-FOSs, levanbiose, and difructose anhydride IV (DFA IV), respectively. The prebiotic properties of both L-FOSs and DFA IV have been confirmed in recent years. Although levanase, LF2ase, and LFTase belong to the same O-glycoside hydrolase 32 family (GH32), their catalytic properties and product spectra differ significantly. In this paper, recent studies on these LDEs are reviewed, including those investigating microbial source and catalytic properties. Additionally, comparisons of LDEs, including those of their differing cleavage behavior and applications for different L-FOSs, are presented in detail.


Assuntos
Bactérias/enzimologia , Frutanos/metabolismo , Fungos/enzimologia , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Oligossacarídeos/metabolismo , Biotransformação
17.
J Sci Food Agric ; 99(14): 6315-6323, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31260112

RESUMO

BACKGROUND: To increase the low residual activity of levansucrase during long-time processing, an enhancement of its weak thermostability is needed. Here, the effect of metal ions and polyethylene glycol (PEG) on the thermostability of levansucrase from Brenneria sp. EniD312 were studied and evaluated. The residual activity was determined and the protein structure was evaluated by circular dichroism spectrum, fluorescence intensity (FI), and surface hydrophobicity (S0 ). RESULTS: As a result of incubation with 10% (w/v) PEG 4000, the enzyme activity was increased by 1.24-fold. After incubation with 5% PEG 4000 for 6 h, the residual activity at 35 and 45 °C was decreased to 55% and 60% of the initial activity, with an increase of 1.2- and 3.3-fold than the wild-type enzyme. Furthermore, the random coil content of enzyme was decreased from 53% of the wild-type enzyme to 33.9% of the PEG pre-incubated enzyme. Additionally, the FI was maximally increased and the S0 was decreased from 117 to 69. CONCLUSION: All of these results suggested that after incubation with PEG 4000, the secondary and tertiary structure of wild-type enzyme could be greatly maintained and then its thermostability could be increased. This study was the first report on the enhancement of levansucrase thermostability by PEG incubation and might be a good guideline to other researches on levansucrase. © 2019 Society of Chemical Industry.


Assuntos
Proteínas de Bactérias/química , Enterobacteriaceae/enzimologia , Hexosiltransferases/química , Polietilenoglicóis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Enterobacteriaceae/química , Enterobacteriaceae/genética , Estabilidade Enzimática , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Polietilenoglicóis/metabolismo
18.
Microb Pathog ; 135: 103626, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325573

RESUMO

Plaque-related diseases are amongst the most common ailments of the oral cavity. Streptococcus mutans is the causal agent of dental caries in animals and humans and is responsible for the formation and accumulation of plaques. This study aimed to identify and evaluate the role of the dental plaque isolates and its surrounding environment in plaque formation or inhibition. The study started with the identification of human dental plaque isolates from high caries index patients based on 16S rRNA and Mitis salivarius bacitracin agar (MSB) was used for S. mutans growing. Unexpectedly, the Streptococcus mutans was completely absent. The disc diffusion assay recorded that all the isolates had antimicrobial activity against the S. mutans growth. Enzymes assay revealed that the isolates produced dextransucrase, levansucrase and levanase activity with wide variation degrees. Also, the lactic acid production assay was done based in pH shift assessment. The highest pH shift and dextran yield were detected by the isolates Bacillus subtilis_AG1 and Bacillus mojavensis_AG3. The adherence test revealed that Lysinibacillus cresolivorans_W2 (MK411028) recorded the highest adhesion property (60%). Oligo- and polysaccharides were synthesized by the action of dextransucrase enzyme and their cytotoxicity tests were negative. Dextran with a molecular weight (117521 Da) recorded the highest antimicrobial efficacy against Bacillus subtilis_AG1 and Bacillusmojavensis_AG3 (65%, 63.5%) respectively. The results concluded that the dextran was the most important factor causing the dental plaque pathogenicity. Also, oral oligo- and polysaccharides might play a role in dental plaque control.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Placa Dentária/microbiologia , Streptococcus mutans/isolamento & purificação , Animais , Antibacterianos/farmacologia , Bacillaceae/isolamento & purificação , Bacillus/isolamento & purificação , Bacillus subtilis/isolamento & purificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Aderência Bacteriana , Linhagem Celular , Cárie Dentária/microbiologia , Dextranos/metabolismo , Glucosiltransferases , Hexosiltransferases , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Testes de Sensibilidade Microbiana , Boca/microbiologia , Filogenia , RNA Ribossômico 16S/genética , Streptococcus mutans/efeitos dos fármacos
19.
J Cell Biol ; 218(8): 2782-2796, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31296534

RESUMO

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.


Assuntos
Glicoproteínas/metabolismo , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Proteômica , Glicosilação , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos
20.
Food Chem ; 299: 125128, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31299517

RESUMO

A novel strategy was used to produce inulin fructotransferase from Arthrobacter aurescens (Aa-IFTase) embedded in curdlan-based mesoporous silica microspheres (CMSiM-Aa-IFTase). The CMSiM-Aa-IFTase was constructed by co-entrapping cross-linked Aa-IFTase aggregates and curdlan into biomemitic silica, and the curdlan was subsequently removed by digestion with endo-ß-1,3-glucanase. During this process, the curdlan served as an agent to introduce pores in silica microspheres. The resulting CMSiM-Aa-IFTase showed higher stability and activity than free Aa-IFTase and mCLEAs-Aa-IFTase (modified cross-linked enzyme aggregates with Aa-IFTase). Furthermore, the CMSiM-Aa-IFTase displayed good reusability and excellent storage stability. The excellent catalytic performances were due to the combinational structure from the cross-linked enzyme aggregates and hard shell of mesoporous silica microspheres, which might decrease the negative interaction between support and enzyme, and improve the mechanical properties. The CMSiM-Aa-IFTase was applicable for efficient production of Difructose Anhydride III (DFA III), and this approach should be highly valuable for preparing various mesoporous composites for catalysis.


Assuntos
Arthrobacter/enzimologia , Dissacarídeos/metabolismo , Hexosiltransferases/química , Hexosiltransferases/metabolismo , beta-Glucanas/química , Catálise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Hexosiltransferases/genética , Microesferas , Dióxido de Silício
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