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1.
J Food Sci ; 86(5): 1778-1790, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33884619

RESUMO

Aspergillus fumigatus was found to produce thermostable exo-inulinase (EC 3.8.1.80; 38 U/ml) on inulin-rich infusions. Exo-inulinase (14.6 U/mg) was immobilized on glutaraldehyde activated Ca-alginate beads for continuous generation of fructose by hydrolyzing sucrose, chicory, and dandelion substrates. Immobilization of enzyme was confirmed by microscopic and spectroscopic techniques. The exo-inulinase was purified using ion-exchange (1.30-folds) and size-exclusion chromatography (2.71-folds). The purified exo-inulinase showed 64 kDa band on gel and was optimally active at 60 °C and pH 6.0. Kinetic constants, Km and Vmax of purified exo-inulinase, were 5.88 mM and 1.66 µM/min, respectively, and its relative activity was found to be enhanced (125.8%) in the presence of calcium ion. Immobilized preparation was utilized for continuous generation of fructose from chicory juice (26 to 70%) and dandelion root extracts (16 to 24%) by recycling upto five cycles, respectively. In comparison to other sweeteners, such as sucrose, fructose is considered as a healthy alternative. The present study demonstrated the use of immobilized exo-inulinase in continuous generation of fructose from some underutilized plant sources that can be used in food industry. PRACTICAL APPLICATION: Thermostable exo-inulinase produced by A. fumigatus was immobilized on calcium alginate matrix and was employed for continuous hydrolysis of chicory juice and dandelion root extract for generation of fructose syrup.


Assuntos
Aspergillus fumigatus/enzimologia , Enzimas Imobilizadas/metabolismo , Frutose/biossíntese , Glicosídeo Hidrolases/metabolismo , Chicória/química , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Hidrólise , Inulina/metabolismo , Raízes de Plantas/química , Taraxacum/química
2.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805072

RESUMO

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus: a GH1 ß-gluco-/ß-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of ß-gluco-/ß-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan.


Assuntos
Glucanos/química , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/química , Sulfolobus solfataricus/enzimologia , Xilanos/química , Glicosídeo Hidrolases/química , Hidrólise , Proteínas Recombinantes/química , Sementes/metabolismo , Tamarindus/metabolismo , Temperatura , Xilosidases/metabolismo
3.
Molecules ; 26(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806149

RESUMO

Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC-MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.


Assuntos
Antifúngicos , Bacillus subtilis/enzimologia , Proteínas de Bactérias , Fusarium/crescimento & desenvolvimento , Glicosídeo Hidrolases , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/farmacologia
4.
Molecules ; 26(6)2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808840

RESUMO

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other ß-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters Km of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the Kcat/Km were 181.5 s-1 mM-1 and 197.8 s-1 mM-1, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C20 position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.


Assuntos
Substituição de Aminoácidos , Bacillus subtilis , Proteínas de Bactérias , Ginsenosídeos/química , Glicosídeo Hidrolases , Mutagênese Sítio-Dirigida , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Mutação de Sentido Incorreto , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
5.
Molecules ; 26(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673063

RESUMO

This paper aims to investigate the effects of some salts (NaCl, (NH4)2SO4 and Na2SO4) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH4)2SO4 generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.


Assuntos
Estabilidade Enzimática/efeitos dos fármacos , Enzimas Imobilizadas/química , Sais/química , Catalase/química , Enzimas Imobilizadas/antagonistas & inibidores , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Cinética , Lacase/química , Lipase/química , Compostos Orgânicos/química , Penicilina Amidase/química , Peptídeo Hidrolases/química , Sais/farmacologia , Soluções/química , Soluções/farmacologia , Temperatura
6.
Carbohydr Polym ; 258: 117691, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33593564

RESUMO

The aim of the present study is to evaluate the potential of chitosan and chitosan nanoparticles (ChNPs) in enhancing the growth and yield of finger millet under greenhouse condition. Foliar application of ChNPs significantly enhanced the growth, yield and mineral content (Fe, Zn, Mn, P, Ca, Mg) when compared to the chitosan and untreated control. ChNPs also induced several defense related enzymes (chitinase, ß-1,3 glucanase, chitosanase, protease inhibitors, peroxidase, polyphenol oxidase) in leaves of finger millet plants their by enhancing the innate immune response. This quantitative difference in defense enzymes was also detected qualitatively on polyacrylamide gels. Our results suggest that ChNPs application can be used as an ecofriendly approach to enhance yield and mineral content in finger millet for sustainable production.


Assuntos
Quitosana/química , Eleusine/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Nanopartículas/química , Folhas de Planta/efeitos dos fármacos , Catecol Oxidase/química , Quitinases/química , Eleusine/imunologia , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Peroxidase/química , Doenças das Plantas/imunologia , Brotos de Planta/metabolismo , Inibidores de Proteases/química , Sementes/metabolismo
7.
Nat Commun ; 12(1): 367, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446650

RESUMO

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Cinética , Modelos Moleculares , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Xanthomonas/química , Xanthomonas/genética , Xilose/química , Xilose/metabolismo
8.
J Agric Food Chem ; 69(1): 78-87, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33393308

RESUMO

Chitooligosaccharides (COS) generated from either chitin (chitin oligosaccharides) or chitosan (chitosan oligosaccharides) have a wide range of applications in agriculture, medicine, and other fields. Here, we report the characterization of a chitosanase from Bacillus amyloliquefaciens (BamCsn) and the importance of a tryptophan (Trp), W204, for BamCsn activity. BamCsn hydrolyzed the chitosan polymer by an endo mode. It also hydrolyzed chitin oligosaccharides and interestingly exhibited transglycosylation activity on chitotetraose and chitopentaose. Mutation of W204, a nonconserved amino acid in chitosanases, to W204A abolished the hydrolytic activity of BamCsn, with a change in the structure that resulted in a decreased affinity for the substrate and impaired the catalytic ability. Phylogenetic analysis revealed that BamCsn could belong to a new class of chitosanases that showed unique properties like transglycosylation, cleavage of chitin oligosaccharides, and the presence of W204 residues, which is important for activity. Chitosanases belonging to the BamCsn class showed a high potential to generate COS from chitinous substrates.


Assuntos
Bacillus amyloliquefaciens/enzimologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/biossíntese , Bacillus amyloliquefaciens/química , Bacillus amyloliquefaciens/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Quitina/metabolismo , Quitosana/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Especificidade por Substrato
9.
Molecules ; 26(2)2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33467076

RESUMO

Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 ß-xylosidase from Geobacillus stearothermophilus with dual activity of ß-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original ß-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the K m value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.


Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Reagentes para Ligações Cruzadas/química , Geobacillus stearothermophilus/enzimologia , Glutaral/química , Glicosídeo Hidrolases/química , Agregados Proteicos , Proteínas de Bactérias/genética , Geobacillus stearothermophilus/genética , Glicosídeo Hidrolases/genética , Mutação de Sentido Incorreto
10.
Carbohydr Polym ; 251: 117056, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33142608

RESUMO

We describe a method for permitting efficient modification by transglucosidase (TGA), from glycoside hydrolase family 31 (GH31), sequentially after the pre-treatment by maltogenic α-amylases (MA) from GH13. TGA treatment without MA pre-treatment had negligible effects on native starch, while TGA treatment with MA pre-treatment resulted in porous granules and increased permeability to enzymes. MA→TGA treatments lead to decreased molecular size of amylopectin molecules, increased α-1,6 branching, and increased amounts of amylopectin chains with the degree of polymerization (DP)<10 and decreased amounts of DP 10-28 after debranching. Wide-angle X-ray scattering (WAXS) data showed a general decrease in crystallinity except for a long term (20 h) TGA post-treatment which increased the relative crystallinity back to normal. MA→TGA treatment significantly lowered the starch retrogradation of starch and retarded the increase of storage- and loss moduli during storage. This work demonstrates the potential of sequential addition of starch active enzymes to obtain granular starch with improved functionality.


Assuntos
Glucosidases/química , Glicosídeo Hidrolases/química , Amido/química , Zea mays/química , Amilopectina/química , Glucosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Hidrólise , Porosidade , Difração de Raios X/métodos
11.
Food Chem ; 339: 128027, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32949915

RESUMO

κ-Carrageenase cleaves the ß-(1-4) linkages of κ-carrageenan into κ-carrageenan oligosaccharides (κ-COS), which exhibit various biological activities. In this study, a glycoside hydrolase (GH) family 16 κ-carrageenase gene, cgkA, was cloned from the marine bacterium Vibrio sp. SY01 and secretory expressed in a food-grade host, Yarrowia lipolytica. The specific activity of the purified CgkA was 12.5 U/mg. Determination of biochemical properties showed that CgkA was a thermo-tolerant enzyme, and 59.9% of the initial enzyme activity was recovered by immediately placing the sample at 20 °C for 30 min after enzymatic inactivation by boiling for 5 min. The recombinant CgkA was an endo-type enzyme, the main enzymatic product was κ-carradiaose (accounting for 87.6% of total products), and κ-carratetraose was the minimum substrate. Additionally, in vitro and in vivo analyses indicated that enzymatic κ-carradiaose possesses anti-oxidant activity. These features make CgkA as a promising candidate for biotechnological applications in the production of anti-oxidant κ-COS.


Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células CACO-2 , Carragenina/química , Carragenina/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Humanos , Hidrólise , Simulação de Acoplamento Molecular , Oligossacarídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vibrio/genética , Yarrowia/genética
12.
Carbohydr Polym ; 252: 117139, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33183598

RESUMO

Fucose-containing oligosaccharides (FCOs) have important applications in the food, medicine, and cosmetics industries owing to their unique biological activities. The degradation of microbial fucose-containing exopolysaccharide (FcEPS) is a promising strategy for obtaining FCOs, and bacteriophage-borne glycanase is a useful tool for degrading FcEPS. Here, we aimed to obtain FCOs using bacteriophage-borne glycanase to depolymerize FcEPS from Enterobacter sakazakii. The FcEPS was mainly composed of l-fucose (42.72 %), d-galactose (20.59 %), and d-glucose (21.81 %). Based on the results of nuclear magnetic resonance and mass spectrometry, the obtained FCOs were disaccharide fragments with backbones of ß-d-Glcp-(1→4)-ß-l-Fucp and α-d-Galp-(1→3)-ß-l-Fucp, respectively. So far, few studies of disaccharides prepared from FcEPS have been reported. This study demonstrated that the FcEPS of E. sakazakii was a reliable fucose-containing disaccharide source and that bacteriophage-borne glycanase was an effective degradation tool for obtaining FCOs fragments from FcEPS.


Assuntos
Cronobacter sakazakii/química , Dissacarídeos/química , Fucose/química , Bacteriófagos/enzimologia , Glicosídeo Hidrolases/química , Estrutura Molecular
13.
Biochim Biophys Acta Proteins Proteom ; 1869(1): 140523, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853774

RESUMO

Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free energy. Though the proteins display maximum stabilities at similar temperatures, a higher melting temperature in the wild-type is achieved by a combination of higher enthalpy and lower heat capacity of unfolding. Comparisons of the structural changes, and the activity and thermodynamic profiles allow us to infer that specific non-covalent interactions, and the existence of residual structure in the unfolded state, are crucial determinants of its thermostability. These features permit the enzyme to balance the preservation of structure at a higher temperature with the thermodynamic stability required for optimum catalysis.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Thermotoga maritima/química , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica , Thermotoga maritima/enzimologia
14.
Carbohydr Polym ; 253: 117276, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33278947

RESUMO

The cbm6e gene from Saccharophagus degradans 2-40 T was cloned and expressed in Escherichia coli. CBM6E contains a glycoside hydrolase family 128 (GH128) catalytic module and a C-terminal carbohydrate-binding module (CBM) grouped into CBM family 6. The purified recombinant CBM6E displayed high substrate specificity toward curdlan as an endo-ß-1,3-glucanase and had maximal activity at pH 6.0 and 35 ℃. The hydrolytic products against curdlan were predominantly laminaritriose (L3) and laminaritetraose (L4) along with a lower amount of laminaripentaose (L5) and laminarihexaose (L6). The CBM6 module selectively enhanced the enzyme activity against curdlan and displayed strict binding specificity to curdlan, no matter in its powder or high-set gel forms. This study laid a foundation for enzymatic degradation of curdlan to produce high-value ß-1,3-glucooligosaccharides at moderate temperatures and provided a novel CBM tag for enzyme immobilization on curdlan.


Assuntos
Celulase/química , Gammaproteobacteria/enzimologia , Glicosídeo Hidrolases/química , Polissacarídeos Bacterianos/química , beta-Glucanas/química , Biocatálise , Clonagem Molecular/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Gammaproteobacteria/genética , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/biossíntese , Especificidade por Substrato , Temperatura
15.
J Sci Food Agric ; 101(2): 693-702, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32700446

RESUMO

BACKGROUND: This study developed a feasible catalytic method for d-allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. RESULTS: d-Allulose 3-epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo-inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L-1 Mn2+ , the CSCA-linkerE-DAE fusion enzyme obtained through high cell-density cultivation displayed a maximal exo-inulinase activity of 21.8 U mg-1 and resulted in a yield of 6.3 g L-1 d-allulose and 39.2 g L-1 d-fructose using 60 g L-1 inulin as the raw material. Catechol-modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA-immobilized CSCA-linkerE-DAE (8 U g-1 ) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L-1 d-allulose and 32.7 g L-1 D-fructose from 60 g L-1 inulin. CONCLUSION: This study shed light on a feasible and cost-effective approach for the production of syrup containing d-allulose and D-fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry.


Assuntos
Frutose/química , Glicosídeo Hidrolases/química , Inulina/química , Racemases e Epimerases/química , Proteínas Recombinantes de Fusão/química , Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Tecnologia de Alimentos/economia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Inulina/genética , Inulina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
16.
Biochemistry ; 59(51): 4845-4855, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33326210

RESUMO

The P22 tailspike endorhamnosidase confers the high specificity of bacteriophage P22 for some serogroups of Salmonella differing only slightly in their O-antigen polysaccharide. We used several biophysical methods to study the binding and hydrolysis of O-antigen fragments of different lengths by P22 tailspike protein. O-Antigen saccharides of defined length labeled with fluorophors could be purified with higher resolution than previously possible. Small amounts of naturally occurring variations of O-antigen fragments missing the nonreducing terminal galactose could be used to determine the contribution of this part to the free energy of binding to be ∼7 kJ/mol. We were able to show via several independent lines of evidence that an unproductive binding mode is highly favored in binding over all other possible binding modes leading to hydrolysis. This is true even under circumstances under which the O-antigen fragment is long enough to be cleaved efficiently by the enzyme. The high-affinity unproductive binding mode results in a strong self-competitive inhibition in addition to product inhibition observed for this system. Self-competitive inhibition is observed for all substrates that have a free reducing end rhamnose. Naturally occurring O-antigen, while still attached to the bacterial outer membrane, does not have a free reducing end and therefore does not perform self-competitive inhibition.


Assuntos
Bacteriófago P22/enzimologia , Glicosídeo Hidrolases/metabolismo , Antígenos O/metabolismo , Oligossacarídeos/metabolismo , Proteínas da Cauda Viral/metabolismo , Domínio Catalítico , Corantes Fluorescentes/química , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/química , Hidrólise , Antígenos O/química , Oligossacarídeos/química , Ligação Proteica , Salmonella enterica/química , Proteínas da Cauda Viral/antagonistas & inibidores , Proteínas da Cauda Viral/química
17.
BMC Bioinformatics ; 21(1): 512, 2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33167861

RESUMO

BACKGROUND: An enzyme activity is influenced by the external environment. It is important to have an enzyme remain high activity in a specific condition. A usual way is to first determine the optimal condition of an enzyme by either the gradient test or by tertiary structure, and then to use protein engineering to mutate a wild type enzyme for a higher activity in an expected condition. RESULTS: In this paper, we investigate the optimal condition of an enzyme by directly analyzing the sequence. We propose an embedding method to represent the amino acids and the structural information as vectors in the latent space. These vectors contain information about the correlations between amino acids and sites in the aligned amino acid sequences, as well as the correlation with the optimal condition. We crawled and processed the amino acid sequences in the glycoside hydrolase GH11 family, and got 125 amino acid sequences with optimal pH condition. We used probabilistic approximation method to implement the embedding learning method on these samples. Based on these embedding vectors, we design a computational score to determine which one has a better optimal condition for two given amino acid sequences and achieves the accuracy 80% on the test proteins in the same family. We also give the mutation suggestion such that it has a higher activity in an expected environment, which is consistent with the previously professional wet experiments and analysis. CONCLUSION: A new computational method is proposed for the sequence based on the enzyme optimal condition analysis. Compared with the traditional process that involves a lot of wet experiments and requires multiple mutations, this method can give recommendations on the direction and location of amino acid substitution with reference significance for an expected condition in an efficient and effective way.


Assuntos
Biologia Computacional/métodos , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Redes Neurais de Computação
18.
Yakugaku Zasshi ; 140(9): 1165-1173, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32879248

RESUMO

Relationship between water molecules and parent and hydroxypropylated cyclodextrins (denoted as CD and HP-CD) was assessed in this paper. The trends in ad-desorption isotherms of CD for water molecule were quite different compared to those of HP-CD. Ad-desorption isotherms of CD showed the hysteresis under our experimental conditions. The molar ratio of hydration (R-value) using α-CD, ß-CD, γ-CD, HP-α-CD, HP-ß-CD, and HP-γ-CD was 7.1, 11.4, 13.5, 12.5, 14.0, and 16.7, respectively. These results indicated that the adsorption capability of water molecule of HP-CD was greater than that of CD. Additionally, the changes in characteristics of CD and HP-CD at different water activity conditions were demonstrated. X-ray diffraction patterns were significantly different between CD and HP-CD. The crystal structure of HP-α-CD, HP-ß-CD, and HP-γ-CD showed amorphous at different water activity conditions. Finally, sorption entropy and heat of sorption of water molecules were calculated in this experiment. In summary, these results provide useful information for understanding the relationship between water molecules and parent and hydroxypropylated cyclodextrins.


Assuntos
Glicosídeo Hidrolases/química , Água/química , 2-Hidroxipropil-beta-Ciclodextrina/química , Adsorção , Cristalização , Relação Estrutura-Atividade
19.
PLoS One ; 15(6): e0231513, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479540

RESUMO

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of ß-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of ß-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular ß-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing ß-1,2-linked arabinofuranose disaccharide (ß-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) ß-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the ß-Ara2 disaccharide. Three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of structural homologs in other GH families: GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 ß-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. ß-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the ß-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.


Assuntos
Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Bifidobacterium longum/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Alinhamento de Sequência
20.
Nat Chem Biol ; 16(8): 920-929, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32451508

RESUMO

The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.


Assuntos
Glucana 1,3-beta-Glucosidase/química , Glicosídeo Hidrolases/química , beta-Glucanas/química , Sequência de Aminoácidos/genética , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Glucana 1,3-beta-Glucosidase/metabolismo , Glucanos/química , Glicosídeos/química , Modelos Moleculares , Especificidade por Substrato/fisiologia
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