Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.746
Filtrar
1.
Biochim Biophys Acta Proteins Proteom ; 1868(5): 140379, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32032760

RESUMO

Exo-polygalacturonase (Exo-PG), an extracellular pectinolytic enzyme with great potential in the food industry, was produced from Penicillium janczewskii under solid-state fermentation using wheat bran as a lignocellulosic substrate. Optimization of various fermentation process variables revealed that a moisture level of 70%, d-mannitol (carbon source), yeast extract (nitrogen source) and ZnSO4/NiCl2 as metal salt inducers generated the maximum yield of Exo-PG. After ammonium sulfate precipitation (70% saturation level), the enzyme was characterized in terms of various kinetic (i.e., temperature, pH, inhibitors, Michaelis-Menten constants) and thermodynamic parameters (i.e., Ea, ΔH*, ΔS* and ΔG*). The temperature and pH optima were 45 °C and 6.0, respectively, and the Exo-PG presented stability at a wider range of temperature and pH values. Metal ions Mn+2 markedly improved the enzyme activity, while exposure to urea and ethylenediaminetetraacetic acid drastically inhibited the biocatalytic performance. The kinetic parameters i.e., km and Vmax were computed to be 10 mM and 41.67 U/mL, respectively. A profound increase in clarity, yield, and reduction in viscosity was achieved for different fruit juices (apple, mango, and peach) after treatment with Exo-PG. Total antioxidant and total phenolic contents were also ameliorated. In conclusion, the catalytic activity, thermal steadiness, and fruit juices clarification performance of Exo-PG manifests a great prospect for bio-industrial exploitation.


Assuntos
Aditivos Alimentares/química , Sucos de Frutas e Vegetais/normas , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Estabilidade Enzimática , Cinética , Penicillium/enzimologia
2.
J Agric Food Chem ; 68(10): 3195-3202, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32075368

RESUMO

d-Tagatose is a rare monosaccharide that is used in products in the food industry as a low-calorie sweetener. To facilitate biological conversion of d-tagatose, the agarolytic enzyme complexes based on the principle of the cellulosome structure were constructed through dockerin-cohesin interaction with the scaffoldin. The construction of agarolytic complexes composed of l-arabinose isomerase caused efficient isomerization activity on the agar-derived sugars. In a trienzymatic complex, the chimeric ß-agarase (cAgaB) and anhydro-galactosidase (cAhgA) from Zobellia galactanivorans could synergistically hydrolyze natural agar substrates and l-arabinose isomerase (LsAraA Doc) from Lactobacillus sakei 23K could convert d-galactose into d-tagatose. The trienzymatic complex increased the concentration of d-tagatose from the agar substrate to 4.2 g/L. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.4-fold increase in tagatose production, good thermostability, and reusability. A residual activity of 75% remained, and 52% of conversion was noted after five recycles. These results indicated that the dockerin-fused chimeric enzymes on the scaffoldin successfully isomerized d-galactose into d-tagatose with synergistic activity. Thus, the results demonstrated the possibility of advancing efficient strategies for utilizing red algae as a biomass source to produce d-tagatose in the industrial food field that uses marine biomass as the feedstock.


Assuntos
Aldose-Cetose Isomerases/química , Proteínas de Bactérias/química , Galactose/química , Galactosidases/química , Glicosídeo Hidrolases/química , Hexoses/química , Edulcorantes/química , Biocatálise , Flavobacteriaceae/enzimologia , Isomerismo , Lactobacillus sakei/enzimologia
3.
Nat Commun ; 11(1): 899, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060313

RESUMO

The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-ß-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man9GlcNAc2Asn substrate complex, and two EndoBT-3987-Man9GlcNAc and EndoBT-3987-Man5GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.


Assuntos
Bacteroides thetaiotaomicron/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides thetaiotaomicron/química , Bacteroides thetaiotaomicron/enzimologia , Bacteroides thetaiotaomicron/genética , Microbioma Gastrointestinal , Regulação Bacteriana da Expressão Gênica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Manose/química , Manose/metabolismo , Especificidade por Substrato
4.
Appl Microbiol Biotechnol ; 104(5): 2079-2096, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31980921

RESUMO

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the ß-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Enzimas Multifuncionais/metabolismo , Paenibacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Celulase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Hidrólise , Cinética , Manosidases/metabolismo , Simulação de Acoplamento Molecular , Enzimas Multifuncionais/química , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/isolamento & purificação , Mutação , Oligossacarídeos/biossíntese , Paenibacillus/genética , Paenibacillus/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xilosidases/metabolismo
5.
Carbohydr Polym ; 230: 115667, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887885

RESUMO

To gain desired viscoelastic properties of wheat flour and to expand the wheat flour current utility with more palatable foods, herein wheat starch, one of the important components of wheat flour, has been modified sequentially using ß-amylase (BA), transglucosidase (TG) and pullulanase (PUL). The results show that compared to native starch, percentage of shorter linear chains (DP 6-12) significantly increased after the BA→TG→PUL treatment, resulting in lower relative crystallinity but with enhanced ordered structure. Compared to the native starch paste viscosity of 1632.98 Cp, the final viscosity of BA/TG/PUL-modified starch pastes reached the maximum value of 3132.01 Cp when the TG treatment time was 20 h, which indicated the paste viscosity increased by 91.80 %. Furthermore, BA/TG/PUL-modified starch pastes exhibit higher shear resistance and gel strength.


Assuntos
Farinha/análise , Reologia/métodos , Amido/química , Triticum/metabolismo , Glicosídeo Hidrolases/química , Resistência ao Cisalhamento , Transglutaminases/química , Viscosidade , beta-Amilase/química
6.
Carbohydr Polym ; 231: 115738, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31888846

RESUMO

The chemical structure of pea pectin was delineated using pectin-degrading enzymes and biochemical methods. The molecular weight of the pea pectin preparation was 488,000, with 50 % arabinose content, and neutral sugar side chains attached to approximately 60 % of the rhamnose residues in rhamnogalacturonan-I (RG-I). Arabinan, an RG-I side chain, was highly branched, and the main chain was comprised of α-1,5-l-arabinan. Galactose and galactooligosaccharides were attached to approximately 35 % of the rhamnose residues in RG-I. Long chain ß-1,4-galactan was also present. The xylose substitution rate in xylogalacturonan (XGA) was 63 %. The molar ratio of RG-I/homogalacturonan (HG)/XGA in the backbone of the pea pectin was approximately 3:3:4. When considering neutral sugar side chain content (arabinose, galactose, and xylose), the molar ratio of RG-I/HG/XGA regions in the pea pectin was 7:1:2. These data will help understand the properties of pea pectin.


Assuntos
Estrutura Molecular , Ervilhas/química , Pectinas/química , Arabinose/química , Galactanos/química , Galactose/química , Glicosídeo Hidrolases/química , Ácidos Hexurônicos/química , Ervilhas/ultraestrutura , Pectinas/ultraestrutura , Polissacarídeos/química , Ramnose/química , Xilose/química
7.
Sci Rep ; 10(1): 1329, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992772

RESUMO

The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: ß-N-acetyl-glucosaminidases, ß-1,4-glucosidases/ß-xylosidases and macrolide ß-glucosidases. The RmNag3 with additional ß-lactamase domain clustered with the deepest rooted GH3-lineage of ß-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed ß-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide ß-glucosidases from Actinomycetes. The ß-xylosidases, RmXyl3A and RmXyl3B, and the ß-glucosidases RmBgl3A and RmBgl3C clustered within the major ß-glucosidases/ß-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed ß-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed ß-1,4-glucosidase/ß-xylosidase activity while RmBgl3C was active on pNP-ß-Glc and ß-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Família Multigênica , Rhodothermus/enzimologia , Rhodothermus/genética , Ativação Enzimática , Ordem dos Genes , Genes Bacterianos , Loci Gênicos , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/classificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Temperatura
8.
Chem Rec ; 20(1): 10-22, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30993894

RESUMO

Glycosidases are ubiquitous enzymes involved in a diversity of key biological processes such as energy uptake or cell wall degradation. The design of specific glycosidase inhibitors has been therefore the subject of intense research efforts in academia and pharmaceutical industry. However, until recently, the study of the impact of multivalency on glycosidase inhibition was almost completely neglected. The following account will review our ten year journey on the design of multivalent glycomimetics within our research group, from the discovery of the first strong multivalent effect in glycosidase inhibition to the high-resolution crystal structures of Jack bean α-mannosidase in complex with the multimeric inhibitor displaying the largest binding enhancements reported so far.


Assuntos
Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/química , Mimetismo Molecular
9.
Arch Biochem Biophys ; 679: 108189, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31726038

RESUMO

Flavonoid natural products are well known for their beneficial antimicrobial, antitumor, and anti-inflammatory properties, however, some of these natural products often are rhamnosylated, which severely limits their bioavailability. The lack of endogenous rhamnosidases in the human GI tract not only prevents many of these glycosylated compounds from being of value in functional foods but also limits the modification of natural product libraries being tested for drug discovery. RHA-P is a catalytically efficient, thermostable α-l-rhamnosidase from the marine bacterium Novosphingobium sp. PP1Y that selectively hydrolyzes α-1,6 and α-1,2 glycosidic linkages between a terminal rhamnose and a flavonoid moiety. This work reports the 2.2 Šresolution crystal structure of RHA-P, which is an essential step forward in the characterization of RHA-P as a potential catalyst to increase the bioavailability of rhamnosylated natural compounds. The structure shows highly conserved rhamnose- and calcium-binding residues in a shallow active site that is housed in the (ß/α)8 domain. In comparison to BT0986 (pdbID: 5MQN), the only known structure of an RHA-P homolog, the morphology, electrostatic potentials and amino acid composition of the substrate binding pocket are significantly different, offering insight into the substrate preference of RHA-P for glycosylated aryl compounds such as hesperidin, naringin, rutin, and quercitrin, over polysaccharides, which are preferred by BT0986. These preferences were further explored by using in silico docking, the results of which are consistent with the known kinetic data for RHA-P acting on different rhamnosylated flavonoids. Due to its promiscuity, relative thermostability compared to other known rhamnosidases, and catalytic efficiency even in significant concentrations of organic solvents, RHA-P continues to show potential for biocatalytic applications.


Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Sphingomonadaceae/enzimologia , Cristalografia por Raios X , Domínios Proteicos , Eletricidade Estática , Especificidade por Substrato
10.
J Sci Food Agric ; 100(3): 986-994, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31650545

RESUMO

BACKGROUND: Controlling the blood glucose level is an effective method to reduce type 2 diabetes and prevent diabetes-related complications. Ursolic acid is a plant extract that can reduce postprandial hyperglycemia effectively. This study aimed to explore the inhibitory effect and interaction mechanism of ursolic acid against α-amylase and α-glucosidase. RESULTS: In this study, the effect of ursolic acid on glycosidase was studied in vitro, in vivo, and in silico. The half-maximal inhibitory concentration (IC50 ) of ursolic acid on α-amylase and α-glucosidase was 0.482 ± 0.12 mg mL-1 and 0.213 ± 0.042 mg mL-1 , respectively. The results of enzymatic kinetics showed that ursolic acid inhibited α-amylase and α-glucosidase activity in a non-competitive manner. The fluorescence spectrum showed that the combination of ursolic acid and glycosidase caused the intrinsic fluorescence quenching of glycosidase. The observation of starch granules revealed that the activity of α-amylase was inhibited and the hydrolysis of starch granules was prevented in the presence of ursolic acid. Molecular docking results showed that ursolic acid bound to the inactive site of α-amylase and α-glucosidase through the formation of ursolic acid-glucosidase complex. Ursolic acid interacted with α-amylase and α-glucosidase mainly through hydrogen bonding. The postprandial hypoglycemic effect of ursolic acid in C57BL/6J mice showed that the high concentration of ursolic acid could quickly reduce postprandial blood glucose level. CONCLUSION: Ursolic acid can be considered as a natural ingredient in functional foods to control postprandial blood glucose levels and prevent diabetes by delaying the digestion of starch in foods. © 2019 Society of Chemical Industry.


Assuntos
Diabetes Mellitus Tipo 2/enzimologia , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/antagonistas & inibidores , Hipoglicemiantes/química , Triterpenos/química , Animais , Glicemia/metabolismo , Simulação por Computador , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Inibidores Enzimáticos/administração & dosagem , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Humanos , Hipoglicemiantes/administração & dosagem , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Período Pós-Prandial/efeitos dos fármacos , Triterpenos/administração & dosagem
11.
Mater Sci Eng C Mater Biol Appl ; 106: 110130, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753364

RESUMO

Chronic infection is a major cause of delayed wound-healing. It is recognized to be associated with infectious bacterial communities called biofilms. Currently used conventional antibiotics alone often reveal themselves ineffective, since they do not specifically target the wound biofilm. Here, we report a new conceptual tool aimed at overcoming this drawback: an antibiofilm drug delivery system targeting the bacterial biofilm as a whole, by inhibiting its formation and/or disrupting it once it is formed. The system consists of a micro/nanostructured poly(butylene-succinate-co-adipate) (PBSA)-based asymmetric membrane (AM) with controlled porosity. By the incorporation of hydrophilic porogen agents, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG), we were able to obtain AMs with high levels of porosity, exhibiting interconnections between pores. The PBSA-PEG membrane presented a dense upper layer with pores small enough to block bacteria penetration. Upon using such porogen agents, under dry and wet conditions, membrane's integrity and mechanical properties were maintained. Using bovine serum albumin (BSA) as a model protein, we demonstrated that protein loading and release from PBSA membranes were affected by the membrane structure (porosity) and the presence of residual porogen. Furthermore, the release curve profile consisted of a fast initial slope followed by a second slow phase approaching a plateau within 24 h. This can be highly beneficial for the promotion of wound healing. Cross-sectional confocal laser scanning microscopy (CLSM) images revealed a heterogeneous distribution of fluorescein isothiocyanate (FITC) labeled BSA throughout the entire membrane. PBSA membranes were loaded with dispersin B (DB), a specific antibiofilm matrix enzyme. Studies using a Staphylococcus epidermidis model, indicate significant efficiency in both inhibiting or dispersing preformed biofilm (up to 80 % eradication). The asymmetric PBSA membrane prepared with the PVP porogen (PBSA-PVP) displayed highest antibiofilm activity. Moreover, in vitro cytotoxicity assays using HaCaT and reconstructed human epidermis (RHE) models revealed that unloaded and DB-loaded PBSA-PVP membranes had excellent biocompatibility suitable for wound dressing applications.


Assuntos
Membranas Artificiais , Soroalbumina Bovina/química , Cicatrização , Adipatos/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bandagens , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Biofilmes/efeitos dos fármacos , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Humanos , Nanoestruturas/química , Polietilenoglicóis/química , Porosidade , Povidona/química , Staphylococcus epidermidis/fisiologia , Succinatos/química , Cicatrização/efeitos dos fármacos
12.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140294, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676454

RESUMO

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and ß-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Šfrom the active site, the single mutants had 15-40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for ß-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-63-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51-109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and ß-limit dextrin.


Assuntos
Glicosídeo Hidrolases/química , Hordeum/enzimologia , Proteínas de Plantas/química , Amilopectina/química , Sítios de Ligação , Catálise , Dextrinas/química , Glucanos/química , Glicosídeo Hidrolases/genética , Modelos Moleculares , Pichia/genética , Proteínas de Plantas/genética , Domínios Proteicos , Proteínas Recombinantes/química , Especificidade por Substrato
13.
Food Chem ; 302: 125332, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31404871

RESUMO

Wheat bran (WB) was treated using xylanase and arabinofuranosidase from Thermotoga maritima and added to steamed breads on 15% flour weight basis. The antioxidant capacity and water and oil retention capacity of brans were increased while their soluble xylooligosaccharides and phenolic acids content were increased. Two enzymes treatment was found to be more effective in decreasing the resistance to extension, softening degree, water absorption and development time, and in increasing the extensibility, stability time, porosity and sensorial characteristics of the steamed breads. Two enzymes treatment had significantly (P < 0.05) greater specific volume, springiness and cohesiveness and lower crumb firmness, gumminess, chewiness than single enzyme treatment. All results highlighted that combination of xylanase and arabinofuranosidase can improve the degrees hydrolysis of WB and its soluble AX xylooligosaccharides produced, having a synergetic effect on the dough rheology and nutritional and quality characteristics of steamed bread.


Assuntos
Pão , Fibras na Dieta , Endo-1,4-beta-Xilanases/química , Glicosídeo Hidrolases/química , Antioxidantes/análise , Antioxidantes/química , Fibras na Dieta/análise , Farinha , Qualidade dos Alimentos , Humanos , Hidrólise , Valor Nutritivo , Reologia , Vapor , Paladar , Triticum
14.
Protein Expr Purif ; 166: 105519, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31629955

RESUMO

Chitosanase (EC 3.2.1.132) is an important chitosan-degrading enzyme involved in industrial applications. In this study, a chitosanase gene (BbCSN-1) from Beauveria bassiana, an insect fungal pathogen, was cloned and expressed in Pichia pastoris. The amount of BbCSN-1 in the fermentation broth of P. pastoris gradually increased after induction with methanol from one to 6 d, reaching 398 µg/ml on the 6th day. The molecular characteristics of BbCSN-1 were measured with colloidal chitosan as a substrate. The purified BbCSN-1 exhibited optimum activity at pH 5 and 30 °C and was stable at pH 2-8 and below 40 °C. The Km value of BbCSN-1 was approximately 0.8 mg/ml at 30 °C (pH 6.0). The activity of BbCSN-1 was significantly enhanced by Mn2+ but inhibited by Co2+ and Cu2+. These results indicated that BbCSN-1 from B. bassiana could be easily expressed in P. pastoris, which provided a basis for further study on its application.


Assuntos
Beauveria/genética , Glicosídeo Hidrolases/genética , Pichia/genética , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Cátions Bivalentes/química , Clonagem Molecular , Cobalto/química , Cobre/química , Expressão Gênica , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Manganês/química , Pichia/enzimologia , Ligação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Temperatura , Termodinâmica
15.
Commun Biol ; 2: 474, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31886414

RESUMO

Pseudoalteromonas is a globally distributed marine-associated genus that can be found in a broad range of aquatic environments, including in association with macroalgal surfaces where they may take advantage of these rich sources of polysaccharides. The metabolic systems that confer the ability to metabolize this abundant form of photosynthetically fixed carbon, however, are not yet fully understood. Through genomics, transcriptomics, microbiology, and specific structure-function studies of pathway components we address the capacity of newly isolated marine pseudoalteromonads to metabolize the red algal galactan carrageenan. The results reveal that the κ/ι-carrageenan specific polysaccharide utilization locus (CarPUL) enables isolates possessing this locus the ability to grow on this substrate. Biochemical and structural analysis of the enzymatic components of the CarPUL promoted the development of a detailed model of the κ/ι-carrageenan metabolic pathway deployed by pseudoalteromonads, thus furthering our understanding of how these microbes have adapted to a unique environmental niche.


Assuntos
Organismos Aquáticos/metabolismo , Carragenina/metabolismo , Redes e Vias Metabólicas , Pseudoalteromonas/metabolismo , Sítios de Ligação , Carragenina/química , Ordem dos Genes , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Fases de Leitura Aberta , Ligação Proteica , Pseudoalteromonas/genética , Relação Estrutura-Atividade
16.
Parasit Vectors ; 12(1): 508, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666116

RESUMO

BACKGROUND: New candidate protective antigens for tick vaccine development may be identified by selecting and testing antigen candidates that play key biological functions. After blood-feeding, tick midgut overexpresses proteins that play essential functions in tick survival and disease transmission. Herein, Ornithodoros erraticus midgut transcriptomic and proteomic data were examined in order to select functionally significant antigens upregulated after feeding to be tested as vaccine candidate antigens. METHODS: Transcripts annotated as chitinases, tetraspanins, ribosomal protein P0 and secreted proteins/peptides were mined from the recently published O. erraticus midgut transcriptome and filtered in a second selection step using criteria based on upregulation after feeding, predicted antigenicity and expression in the midgut proteome. Five theoretical candidate antigens were selected, obtained as recombinant proteins and used to immunise rabbits: one chitinase (CHI), two tetraspanins (TSPs), the ribosomal protein P0 (RPP0) and one secreted protein PK-4 (PK4). RESULTS: Rabbit vaccination with individual recombinant candidates induced strong humoral responses that mainly reduced nymph moulting and female reproduction, providing 30.2% (CHI), 56% (TSPs), 57.5% (RPP0) and 57.8% (PK4) protection to O. erraticus infestations and 19.6% (CHI), 11.1% (TSPs), 0% (RPP0) and 8.1% (PK4) cross-protection to infestations by the African tick Ornithodoros moubata. The joint vaccine efficacy of the candidates was assessed in a second vaccine trial reaching 66.3% protection to O. erraticus and 25.6% cross-protection to O. moubata. CONCLUSIONS: These results (i) indicate that argasid chitinases and RPP0 are promising protective antigens, as has already been demonstrated for ixodid chitinases and RPP0, and could be included in vaccines targeting multiple tick species; (ii) reveal novel protective antigens tetraspanins and secreted protein PK-4, never tested before as protective antigens in ticks; and (iii) demonstrate that multi-antigenic vaccines increased vaccine efficacy compared with individual antigens. Lastly, our data emphasize the value of the tick midgut as a source of protective candidate antigens in argasids for tick control.


Assuntos
Proteínas de Artrópodes/imunologia , Ornithodoros/química , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Quitinases/química , Epitopos/química , Feminino , Glicosídeo Hidrolases/química , Ornithodoros/classificação , Ornithodoros/imunologia , Filogenia , Sinais Direcionadores de Proteínas , Coelhos , Proteínas Recombinantes/imunologia , Proteínas Ribossômicas/imunologia , Alinhamento de Sequência , Tetraspaninas/química , Tetraspaninas/imunologia , Tetraspaninas/isolamento & purificação
17.
Molecules ; 24(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671673

RESUMO

Chitosanase plays an important role in the production of chitooligosaccharides (CHOS), which possess various biological activities. Herein, a glycoside hydrolase (GH) family 46 chitosanase-encoding gene, csnB, was cloned from marine bacterium Bacillus sp. BY01 and heterologously expressed in Escherichia coli. The recombinant chitosanase, CsnB, was optimally active at 35 °C and pH 5.0. It was also revealed to be a cold-adapted enzyme, maintaining 39.5% and 40.4% of its maximum activity at 0 and 10 °C, respectively. Meanwhile, CsnB showed wide pH-stability within the range of pH 3.0 to 7.0. Then, an improved reaction condition was built to enhance its thermostability with a final glycerol volume concentration of 20%. Moreover, CsnB was determined to be an endo-type chitosanase, yielding chitosan disaccharides and trisaccharides as the main products. Overall, CsnB provides a new choice for enzymatic CHOS production.


Assuntos
Adaptação Biológica , Bacillus/enzimologia , Temperatura Baixa , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sequência de Aminoácidos , Ácido Edético/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Glicerol/farmacologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Metais/farmacologia , Filogenia , Dodecilsulfato de Sódio/farmacologia
18.
Glycobiology ; 30(1): 49-57, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31701135

RESUMO

The opportunistic pathogen Clostridium perfringens possesses the ability to colonize the protective mucin layer in the gastrointestinal tract. To assist this, the C. perfringens genome contains a battery of genes encoding glycoside hydrolases (GHs) that are likely active on mucin glycans, including four genes encoding family 84 GHs: CpGH84A (NagH), CpGH84B (NagI), CpGH84C (NagJ) and CpGH84D (NagK). To probe the potential advantage gained by the expansion of GH84 enzymes in C. perfringens, we undertook the structural and functional characterization of the CpGH84 catalytic modules. Here, we show that these four CpGH84 catalytic modules act as ß-N-acetyl-D-glucosaminidases able to hydrolyze N- and O-glycan motifs. CpGH84A and CpGH84D displayed a substrate specificity restricted to terminal ß-1,2- and ß-1,6-linked N-acetyl-D-glucosamine (GlcNAc). CpGH84B and CpGH84C appear more promiscuous with activity on terminal ß-1,2-, ß-1,3- and ß-1,6-linked GlcNAc; both possess some activity toward ß-1,4-linked GlcNAc, but this is dependent upon which monosaccharide it is linked to. Furthermore, all the CpGH84s have different optimum pHs ranging from 5.2 to 7.0. Consistent with their ß-N-acetyl-D-glucosaminidase activities, the structures of the four catalytic modules revealed similar folds with a catalytic site including a conserved -1 subsite that binds GlcNAc. However, nonconserved residues in the vicinity of the +1 subsite suggest different accommodation of the sugar preceding the terminal GlcNAc, resulting in subtly different substrate specificities. This structure-function comparison of the four GH84 catalytic modules from C. perfringens reveals their different biochemical properties, which may relate to how they are deployed in the bacterium's niche in the host.


Assuntos
Clostridium perfringens/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Biocatálise , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Humanos , Modelos Moleculares , Conformação Proteica
19.
Microb Cell Fact ; 18(1): 183, 2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31655584

RESUMO

BACKGROUND: α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included. RESULTS: In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases. CONCLUSIONS: The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.


Assuntos
Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Lactobacillus plantarum/metabolismo , alfa-Amilases/química , Clonagem Molecular , Escherichia coli/genética , Polissacarídeos/metabolismo , Amido/metabolismo , Especificidade por Substrato
20.
Enzyme Microb Technol ; 131: 109389, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615669

RESUMO

Cross-linked enzyme aggregate (CLEA) is a technology to overcome the limitation of enzymes for its application in chemical industries. The inability of repeated use of enzymes, stability and ease of separation from reaction mixture limits its applications. Here, magnetic combi-CLEA has been synthesised by adding amino-functionalized magnetic nanoparticles into pectinase ultra-clear (containing pectinases, xylanases and cellulases). Enzymes were precipitated on the surface of amino-functionalized magnetic nanoparticles with ethanol and cross-linked using glutaraldehyde. The structural characterization of magnetic combi-CLEA was studied by Scanning Electron Microscopy. Thermal stability was performed at 70 °C for pectinase and 80 °C for xylanase and cellulase respectively. Half-life (t1/2) of the xylanase, cellulase and pectinase in free form remarkably enhance from 84.51, 29.36, and 25.29 min respectively to 533.07, 187.29 and 147.44 min in magnetic-combi CLEA respectively. Magnetic combi-CLEA can be efficiently reused till 12th cycle after which pectinase, xylanase and cellulase retain 86.45%, 90.3% and 88.62% activity respectively. Using this CLEA preparation bioethanol concentration increases to 1.82-fold as compared to free enzyme, when simultaneous saccharification and fermentation was performed using wheat straw as the substrate. Magnetic combi-CLEA can be used for a variety of industrial applications like food processing, textile industry and bioethanol production.


Assuntos
Celulase/isolamento & purificação , Celulase/metabolismo , Enzimas Imobilizadas/metabolismo , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Magnetismo , Nanopartículas Metálicas , Biotransformação , Celulase/química , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Temperatura Alta , Microscopia Eletrônica de Varredura , Temperatura , Triticum/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA