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1.
Mar Drugs ; 18(2)2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32102373

RESUMO

A bifunctional alginate lyase (ALFA3) and mannuronate-specific alginate lyase (ALFA4) genes were found in the genome of polysaccharide-degrading marine bacterium Formosa algae KMM 3553T. They were classified to PL7 and PL6 polysaccharide lyases families and expressed in E. coli. The recombinant ALFA3 appeared to be active both on mannuronate- and guluronate-enriched alginates, as well as pure sodium mannuronate. For all substrates, optimum conditions were pH 6.0 and 35 °C; Km was 0.12 ± 0.01 mg/ml, and half-inactivation time was 30 min at 42 °C. Recombinant ALFA4 was active predominately on pure sodium mannuronate, with optimum pH 8.0 and temperature 30 °C, Km was 3.01 ± 0.05 mg/ml. It was stable up to 30 °C; half-inactivation time was 1h 40 min at 37 °C. 1H NMR analysis showed that ALFA3 degraded mannuronate and mannuronate-guluronate blocks, while ALFA4 degraded only mannuronate blocks, producing mainly disaccharides. Products of digestion of pure sodium mannuronate by ALFA3 at 200 µg/ml inhibited anchorage-independent colony formation of human melanoma cells SK-MEL-5, SK-MEL-28, and RPMI-7951 up to 17% stronger compared to native polymannuronate. This fact supports previous data and suggests that mannuronate oligosaccharides may be useful for synergic tumor therapy.


Assuntos
Flavobacteriaceae/enzimologia , Polissacarídeo-Liase/metabolismo , Clonagem Molecular , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Conformação Proteica
2.
Arch Microbiol ; 202(5): 1005-1013, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31932863

RESUMO

Pectinase is widely used in numerous industrial fields, including the food, wine, and paper industries. In this work, seven bacteria were isolated from orange peel and their pectinase production activity was assayed. One bacterium (OR-B2) identified as a Bacillus sp. showed the highest enzyme activity towards others. A gene encoding a pectate lyase designed as PelB-B2 in this work was amplified and heterogeneous expressed in E.coli. PelB-B2 was defined as a member of the PelB pectate lyase family after phylogenic tree analysis. 3D model of PelB-B2 was constructed by SWISS-MODEL and PelB-B2 showed conserved para-ß structure. After inducing culture and purified by Ni-affinity chromatography, the properties of the purified PelB-B2 were assayed. Optimal pH and temperature for PelB-B2 was pH 8.0 and 50 °C, respectively. PelB-B2 showed excellent pH stability and thermostability. It was stable within pH range 3.0-11.0 and retained more than 51% activity after incubation at 40 °C, 50 °C, or 60 °C for 1 h. Furthermore, we determined that PelB-B2 was a Ca2+-dependent pectinase and the pectin extracted from citrus was the benefit substrate for PelB-B2. The Km and Vmax of PelB-B2 were 1.64 g/L and 232.56 mol/(L min), respectively. The OR-B2 can be a new resource for pectinase production and the PelB-B2 has potential for industrial application. 7 bacteria were isolated from orange peel, namely OR-B1 to OR-B7 and their pectinase activities were assayed. One pectate lyase belongs to PelB family was cloned from OR-B2 and heterogeneous expressed in E. coli. Purified PelB-B2 was further studied with its properties. Effects of pH, temperature, chemicals, substrate on the enzyme activity were assayed and the enzyme kinetic was also measured.


Assuntos
Bacillus/enzimologia , Pectinas/metabolismo , Poligalacturonase/metabolismo , Bacillus/genética , Bacillus/metabolismo , Citrus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Poligalacturonase/biossíntese , Polissacarídeo-Liase/metabolismo , Temperatura
3.
J Biosci Bioeng ; 129(1): 16-22, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31400994

RESUMO

The economical production of pectin oligosaccharides with a specific degree of polymerization and structure from agro-food waste is an industrially important process. This study identified a novel pectate lyase gene (plhy1) from the thermophilic cellulolytic fungus H. insolens Y1 and tested its ability to produce pectin oligosaccharides. The recombinant PLHY1 produced in Pichia pastoris was superior to other similar enzymes due to its high thermal and pH stability. PLHY1 demonstrated optimal enzymatic activity at 55°C and pH 10.0 in the presence of 0.4 mM Ca2+, and preferred methyl esterified substrates for digestion. High performance anion exchange chromatography-pulsed amperometric detector and ultra high performance liquid chromatography in combination with electrospray ionization tandem mass spectrometry analysis showed that galacturonic acid-oligosaccharides with a small degree of polymerization (4-6) were the major hydrolysates produced by the degradation of apple peel pectin by PLHY1. The properties of PLHY1 make it valuable for application in the agro-food industry for the production of pectin oligosaccharides.


Assuntos
Proteínas Fúngicas/química , Oligossacarídeos/metabolismo , Pectinas/química , Polissacarídeo-Liase/química , Sordariales/enzimologia , Biocatálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácidos Hexurônicos/química , Ácidos Hexurônicos/metabolismo , Pectinas/metabolismo , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sordariales/química , Sordariales/genética
4.
Food Chem ; 309: 125559, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31679850

RESUMO

Plant cell walls are complex structures that are modified throughout development. They are a major contributor to the properties of plant structure and act as barriers against pathogens. The primary cell walls of plants are composed of polysaccharides and proteins. The polysaccharide fraction is divided into components cellulose, hemicelluloses and pectin, are all modified during fruit ripening. Pectin plays an important role in intercellular adhesion and controlling the porosity of the wall. A large number of pectin degrading enzymes have been characterised from plants and they are involved in numerous aspects of plant development. The role of pectate lyases in plant development has received little attention, probably because they are normally associated with the action of plant pathogenic organisms. However their importance in plant development and ripening is now becoming well established and new information about the role of pectate lyases in plant development forms the focus of this review.


Assuntos
Frutas/enzimologia , Plantas/enzimologia , Polissacarídeo-Liase/metabolismo , Frutas/metabolismo , Frutas/fisiologia , Pectinas/metabolismo , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/metabolismo , Plantas/metabolismo
5.
Carbohydr Polym ; 229: 115497, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826447

RESUMO

Rhamnan-rich sulfated polysaccharides extracted from green algae (ulvan) constitute potentially useful natural materials for drug development. However, the characterization of their complex structures poses a challenge for their application. In this study, the structure of ulvan extracted from Ulva clathrata was analyzed with the assistance of an ulvan lyase belonging to the PL25 family. According to mass spectrometry and nuclear magnetic resonance analysis of the degraded oligosaccharides, the backbone of such a polysaccharide mainly consisted of →4)-ß-d-GlcA-(1→4)-α-l-Rha3S-(1→ and →4)-ß-d-Xyl-(1→4)-α-l-Rha3S-(1→ disaccharide repeating units, and the ratio is approximately 4:1. In addition, about 4% of the xylose moieties bear sulfate groups. Minor amounts of branches containing hexose and unsaturated glucuronic acid were found during the sequence analysis of hexa- to octasaccharides. These results indicated the presence of a long branch in the ulvan. The clarification of the detailed structure provides a foundation for ulvan modification and its structure-activity relationship studies.


Assuntos
Polissacarídeo-Liase/metabolismo , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Ulva/química , Fenômenos Químicos , Polissacarídeos/metabolismo
6.
Enzyme Microb Technol ; 132: 109397, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731972

RESUMO

Pectin lyase (from Rohapect 10 L) was immobilized on glutaraldehyde supports at low ionic strength at pH 5, 6.5 or 8 and later incubated at pH 8 for 48 h. The activity recovery of the biocatalysts versus pectin was quite low, under 10% for all of the immobilized biocatalyst at 20 °C. However, a high stabilization was found when the enzyme was immobilized at pH 5, (e.g., the immobilized enzyme kept 83% of the activity when the free enzyme was fully inactivated (pH 4.8 and 55 °C in 5 h)). This biocatalyst increased the activity versus pectin in an almost exponential way when temperature increased until reach the maximum temperature used in the study (90 °C), conditions where the free enzyme was almost inactive. The immobilized biocatalyst was also active even at pH 9, where the free enzyme was fully inactive. This biocatalyst could be reused for pectin hydrolysis 5 times for 72 h reaction cycles at 40 °C maintaining more than 90% of the initial activity.


Assuntos
Enzimas Imobilizadas/metabolismo , Glutaral/química , Polissacarídeo-Liase/metabolismo , Estabilidade Enzimática , Enzimas , Concentração de Íons de Hidrogênio , Hidrólise , Temperatura
7.
Biochem Biophys Res Commun ; 523(2): 441-445, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31875842

RESUMO

Ulvan is a complex water-soluble sulfated polysaccharide in the cell wall of green algae belonging to genus Ulva. It is composed of l-rhamnose-3-sulfate (Rha3S), glucuronic acid (GluA), iduronic acid (IduA), and d-xylose (Xyl) distributed in three repetition moieties. The first step of a bacterial ulvan degradation is the cleavage of the ß-glycosidic bond between Rha3S and GluA/IduA through a ß-elimination mechanism by a ulvan lyase to produce oligo-ulvans with unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronate (Δ) at the non-reducing end. We have identified an ulvan associated polysaccharide utilization locus (PUL) residing between two ulvan lyase genes belonging to families of polysaccharide lyase 24 (PL24) and PL25 in the genome of a ulvan-utilizing bacterium Glaciecola KUL10 strain. The PUL contains many genes responsible for oligo-ulvan degradation. Among them, we demonstrated that both KUL10_26540 and KUL10_26770 had an unsaturated ß-glucuronyl hydrolase activity to produce Rha3S and oligosaccharides, such as Rha3S-GluA-Rha3S, Rha3S-IduA-Rha3S and, Rha3S-Xyl-Rha3S, by releasing 5-dehydro-4-deoxy-d-glucuronate. KUL10_26540 showed much higher activity than KUL10_26770 and was more active on disaccharide than tetrasaccharide. We also found a rhamnosidase activity on four KUL10 gene products, although they could not react on the sulfated rhamnose.


Assuntos
Alteromonadaceae/enzimologia , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/metabolismo , Ulva/química , Alteromonadaceae/genética , Alteromonadaceae/metabolismo , Glicosídeo Hidrolases/genética , Cinética , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Polissacarídeos/isolamento & purificação
8.
Proc Natl Acad Sci U S A ; 116(49): 24729-24737, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31740605

RESUMO

The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles-remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs.


Assuntos
Biodiversidade , Evolução Biológica , Besouros/genética , Transferência Genética Horizontal , Genoma de Inseto , Animais , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Celulases/genética , Celulases/metabolismo , Besouros/enzimologia , Besouros/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Fungos/genética , Herbivoria/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lignina/química , Lignina/metabolismo , Filogenia , Plantas/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo
9.
Life Sci ; 238: 116894, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31626789

RESUMO

AIMS: MicroRNAs (miRs) and their importance in development, normal physiology, and disease have become increasingly recognized. Our laboratory is interested in miR-29 and its effects on lung development. These studies set out to identify optimal conditions for the measurement of miR-29 in heparinized, biobanked samples and to compare isoform expression patterns. MATERIALS AND METHODS: The efficiency of three distinct heparinases were tested using reverse transcriptase polymerase chain reaction (RT-PCR): recombinant F. Heparinum heparinase I; recombinant P. heparinus heparinase II; recombinant P. heparinus heparinase III; and heparinase I (B. efferthii-derived). The effects of freeze/thaws, and the relative expression of different miR-29 isoforms were also assessed using RT-PCR. KEY FINDINGS: Our investigations determined that heparinase 1 (recombinant F. Heparinum) and 2 (recombinant P. heparinus) at 1 or 2 h incubation efficiently neutralized heparin activity and prevented interference with the PCR. Also, a single freeze/thaw did not affect the measurement of miR-29-3p but multiple freeze/thaw cycles decreased the measureable miR levels. Finally, the -3p strand was most abundantly expressed in all three isoforms in both human and mouse plasma. SIGNIFICANCE: Our findings illustrate that specific conditions need to be optimized for the particular miR and the type of sample being tested.


Assuntos
Bancos de Espécimes Biológicos/normas , Heparina/sangue , MicroRNAs/sangue , Animais , Estudos de Coortes , Flavobacterium/enzimologia , Heparina Liase/metabolismo , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Polissacarídeo-Liase/metabolismo , Proteínas Recombinantes/metabolismo
10.
Mar Drugs ; 17(10)2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31597240

RESUMO

Ulvan lyases can degrade ulvan to oligosaccharides with potent biological activity. A new ulvan lyase gene, ALT3695, was identified in Alteromonas sp. A321. Soluble expression of ALT3695 was achieved in Escherichia coli BL21 (DE3). The 1314-bp gene encoded a protein with 437 amino acid residues. The amino acid sequence of ALT3695 exhibited low sequence identity with polysaccharide lyase family 25 (PL25) ulvan lyases from Pseudoalteromonas sp. PLSV (64.14% identity), Alteromonas sp. LOR (62.68% identity), and Nonlabens ulvanivorans PLR (57.37% identity). Recombinant ALT3695 was purified and the apparent molecular weight was about 53 kDa, which is different from that of other polysaccharide-degrading enzymes identified in Alteromonas sp. A321. ALT3695 exhibited maximal activity in 50 mM Tris-HCl buffer at pH 8.0 and 50 °C. ALT3695 was relatively thermostable, as 90% activity was observed after incubation at 40 °C for 3 h. The Km and Vmax values of ALT3695 towards ulvan were 0.43 mg·mL-1 and 0.11 µmol·min-1·mL-1, respectively. ESI-MS analysis showed that enzymatic products were mainly disaccharides and tetrasaccharides. This study reports a new PL25 family ulvan lyase, ALT3695, with properties that suggest its great potential for the preparation of ulvan oligosaccharides.


Assuntos
Alteromonas/metabolismo , Polissacarídeo-Liase/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Clonagem Molecular/métodos , Flavobacteriaceae/metabolismo , Concentração de Íons de Hidrogênio , Oligossacarídeos/metabolismo , Pseudoalteromonas/metabolismo
11.
Int J Mol Sci ; 20(18)2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31540110

RESUMO

Bacterial biofilm causes severe antibiotic resistance. An extracellular polymeric substance (EPS) is the main component in the bacterial biofilm. Alginate is a key EPS component in the biofilm of Pseudomonas aeruginosa and responsible for surface adhesion and stabilization of biofilm. Alginate lyase has emerged as an efficient therapeutic strategy targeting to degrade the alginate in the biofilm of P. aeruginosa. However, the application of this enzyme is limited by its poor stability. In this study, chitosan nanoparticles (CS-NPs) were synthesized using low molecular weight chitosan and alginate lyase Aly08 was immobilized on low molecular weight chitosan nanoparticles (AL-LMW-CS-NPs). As a result, the immobilization significantly enhanced the thermal stability and reusability of Aly08. In addition, compared with free Aly08, the immobilized AL-LMW-CS-NPs exhibited higher efficiency in inhibiting biofilm formation and interrupting the established mature biofilm of P. aeruginosa, which could reduce its biomass and thickness confirmed by confocal microscopy. Moreover, the biofilm disruption greatly increased the antibiotic sensitivity of P. aeruginosa. This research will contribute to the further development of alginate lyase as an anti-biofilm agent.


Assuntos
Alginatos/química , Biofilmes/efeitos dos fármacos , Quitosana/química , Nanopartículas/química , Polissacarídeo-Liase/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/farmacologia , Matriz Extracelular de Substâncias Poliméricas/química , Peso Molecular , Nanopartículas/ultraestrutura , Polissacarídeo-Liase/química , Polissacarídeo-Liase/metabolismo , Temperatura
12.
Biofabrication ; 11(4): 045020, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31387086

RESUMO

Bioink is of paramount importance in the process of three-dimensional extrusive bioprinting technology. Alginate is extensively used in cell-laden extrusive bioprinters with the advantage of biocompatibility, gelling and crosslinking features; however, the bioinert properties of alginate made it hard to degrade in vivo, and restrict cellular adhesion, extension and migration. In this study, we incorporated two concentrations of alginate lyase (0.5 mU ml-1 and 5 mU ml-1) into alginate/gelatin bioink to improve its degradation properties and effects on cellular behavior. The enzymatically degradable bioink demonstrated lower stiffness and higher porosity. Cellular proliferation, adhesion and extension were facilitated in the degradable bioink without sacrifice of cell viability. Additionally, the property of degradation still worked in vivo, with cellular infiltration and retention being observed in the grafted bioprinted constructs. The results suggest that alginate lyase could be incorporated into alginate/gelatin bioink. Degradation properties and cellular behavior could be promoted both in vitro and in vivo, providing a new avenue for the upgrade and modification of alginate-based bioink for further applications.


Assuntos
Alginatos/metabolismo , Fibroblastos/citologia , Gelatina/metabolismo , Tinta , Polissacarídeo-Liase/metabolismo , Animais , Bioimpressão , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Impressão Tridimensional , Ratos , Tecidos Suporte/química
13.
Biotechnol Lett ; 41(10): 1187-1200, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418101

RESUMO

OBJECTIVES: Bifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate. RESULTS: A new BP-2 strain that produces alginate lyase was screened and identified from rotted Sargassum. A new alginate lyase, Alg17B, belonging to the polysaccharide lyase family 17, was isolated and purified from BP-2 fermentation broth by freeze-drying, dialysis, and ion exchange chromatography. The enzymatic properties of the purified lyase were investigated. The molecular weight of Alg17B was approximately 77 kDa, its optimum reaction temperature was 40-45 °C, and its optimum reaction pH was 7.5-8.0. The enzyme was relatively stable at pH 7.0-8.0, with a temperature range of 25-35 °C, and the specific activity of the purified enzyme reached 4036 U/mg. A low Na+ concentration stimulated Alg17B enzyme activity, but Ca2+, Zn2+, and other metal ions inhibited it. Substrate specificity analysis, thin-layer chromatography, and mass spectrometry showed that Alg17B is an alginate lyase that catalyses the hydrolysis of sodium alginate, polymannuronic acid (polyM) and polyguluronic acid to produce monosaccharides and low molecular weight oligosaccharides. Alg17B is also bifunctional, exhibiting both endolytic and exolytic activities toward alginate, and has a wide substrate utilization range with a preference for polyM. CONCLUSIONS: Alg17B can be used to saccharify the main carbohydrate, alginate, in the ethanolic production of brown algae fuel as well as in preparing and researching oligosaccharides.


Assuntos
Organismos Aquáticos/enzimologia , Gammaproteobacteria/enzimologia , Polissacarídeo-Liase/isolamento & purificação , Polissacarídeo-Liase/metabolismo , Sargassum/microbiologia , Alginatos/metabolismo , Ácido Algínico/metabolismo , Organismos Aquáticos/classificação , Organismos Aquáticos/genética , Organismos Aquáticos/isolamento & purificação , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Monossacarídeos/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeos Bacterianos/metabolismo , Especificidade por Substrato , Temperatura
14.
Int J Biol Macromol ; 139: 879-885, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31381910

RESUMO

Enzymatic digestion of sodium alginate to produce specific oligosaccharides has attracted great attention. However, commercial enzymes that efficiently produce specific oligosaccharides are still unavailable. In the present study, a novel gene encoding an alginate lyase (designated alg7A) was cloned from the marine bacterium Vibrio sp. W13 and expressed in E. coli. The recombinant Alg7A shows high activities toward alginate, poly-α-l-guluronate (polyG), poly-ß-d-mannuronate (polyM) and polyMG, and more preferred to polyMG. Moreover, the enzyme contains a highly conserved domain of the Polysaccharide Lyase (PL) 7 family (R*E*R, Q*H and Y*KAG*Y*Q), which indicates that it belongs to PL7. Furthermore, the thin layer chromatography and ESI-MS analysis showed that Alg7A mainly releases trisaccharides from alginate. These results demonstrated that Alg7A has a great potential to be used to produce oligosaccharides from alginate.


Assuntos
Alginatos/metabolismo , Polissacarídeo-Liase/metabolismo , Trissacarídeos/metabolismo , Vibrio/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , Hidrólise , Modelos Moleculares , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Conformação Proteica , Especificidade por Substrato , Vibrio/genética
15.
Food Chem ; 299: 125142, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31325715

RESUMO

Alginate lyases can be used for alginate oligosaccharide production and for structural characterization or modification of alginates. For these applications it is important to obtain detailed information on mode of action and substrate specificities of alginate lyases. In this study, five alginate lyase genes were cloned from Cellulophaga algicola DSM 14237 genomic DNA, heterologously expressed, and characterized by using HPSEC-RI and HPAEC-PAD/MS. It was demonstrated that these analytical approaches can provide detailed information on preferred substrates, extent of hydrolysis, and the liberated products. The recombinant enzymes cleaved alginates endolytically (CaAly1, CaAly2, CaAly3) or exolytically (CaAly4, CaAly5). The three endolytic alginate lyases predominantly hydrolyzed guluronic acid-rich alginates, only CaAly1 also showed activity on mannuronic acid-rich alginates. The oligosaccharide profiles further demonstrated that the endolytic enzymes have rather narrow but slightly different substrate specificities and that the two exolytic alginate lyases mainly cleaved unsaturated guluronic acid oligosaccharides to monomers.


Assuntos
Alginatos/metabolismo , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Flavobacteriaceae/enzimologia , Polissacarídeo-Liase/metabolismo , Alginatos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Flavobacteriaceae/genética , Ácidos Hexurônicos/metabolismo , Hidrólise , Polissacarídeo-Liase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
16.
Int J Mol Sci ; 20(12)2019 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-31234557

RESUMO

Pectate lyases play an important role in pectin degradation, and therefore are highly useful in the food and textile industries. Here, we report on the cloning of an alkaline pectate lyase gene (pppel9a) from Paenibacillus polymyxa KF-1. The full-length gene (1350 bp) encodes for a 449-residue protein that belongs to the polysaccharide lyase family 9 (PL9). Recombinant PpPel9a produced in Escherichia coli was purified to electrophoretic homogeneity in a single step using Ni2+-NTA affinity chromatography. The enzyme activity of PpPel9a (apparent molecular weight of 45.3 kDa) was found to be optimal at pH 10.0 and 40 °C, with substrate preference for homogalacturonan type (HG) pectins vis-à-vis rhamnogalacturonan-I (RG-I) type pectins. Using HG-type pectins as substrate, PpPel9a showed greater activity with de-esterified HGs. In addition, PpPel9a was active against water-soluble pectins isolated from different plants. Using this lyase, we degraded citrus pectin, purified fractions using Diethylaminoethyl (DEAE)-sepharose column chromatography, and characterized the main fraction MCP-0.3. High-performance gel permeation chromatography (HPGPC) analysis showed that the molecular mass of citrus pectin (~230.2 kDa) was reduced to ~24 kDa upon degradation. Ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS) and monosaccharide composition analyses demonstrated that PpPel9a worked as an endo-pectate lyase, which acted primarily on the HG domain of citrus pectin. In vitro testing showed that the degradation product MCP-0.3 significantly promotes the growth of Lactobacillus plantarum and L. rhamnosus. In this regard, the enzyme has potential in the preparation of pharmacologically active pectin products.


Assuntos
Paenibacillus polymyxa/enzimologia , Pectinas/metabolismo , Polissacarídeo-Liase/metabolismo , Clonagem Molecular , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Especificidade por Substrato
17.
Mol Biotechnol ; 61(9): 681-693, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31218650

RESUMO

In this paper, we report cloning of a pectate lyase gene from Bacillus amyloliquefaciens S6 (pelS6), and biochemical characterization of the recombinant pectate lyase. PelS6 was found to be identical with B. subtilis 168 pel enzyme with 100% amino acid sequence homology. Although these two are genetically very close, they are distinctly different in physiology. pelS6 gene encodes a 421-aa protein with a molecular mass of 65,75 kDa. Enzyme activity increased from 12.8 ± 0.3 to 49.6 ± 0.4 units/mg after cloning. The relative enzyme activity of the recPel S6 ranged from 80% to 100% at pH between 4 and 14. It was quite stable at different temperature values ranging from 15 to 90 °C. The recPEL S6 showed a maximal activity at pH 10 and at 60 °C. 0.5 mM of CaCl2 is the most effective metal ion on the recPEL S6 as demonstrated by its increased relative activity with 473%. recPEL S6 remained stable at - 20 °C for 18 months. In addition recPEL S6 increased juice clarity. This study introduces a novel bacterial pectate lyase enzyme with its characteristic capability of being highly thermostable, thermotolerant, and active over a wide range of pH, meaning that it can work at both acidic and alkaline environments, which are the most preferred properties in the industry.


Assuntos
Bacillus amyloliquefaciens/enzimologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Pectinas/metabolismo , Polissacarídeo-Liase/metabolismo , Sequência de Aminoácidos , Bacillus amyloliquefaciens/química , Bacillus subtilis/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cátions Bivalentes , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Polissacarídeo-Liase/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura
18.
J Biol Chem ; 294(28): 10760-10772, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31167793

RESUMO

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h Our insight into PelAh's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.


Assuntos
Biofilmes/efeitos dos fármacos , Glicosídeo Hidrolases/metabolismo , Polissacarídeo-Liase/ultraestrutura , Anti-Infecciosos/metabolismo , Aspergillus fumigatus/metabolismo , Biofilmes/crescimento & desenvolvimento , Domínio Catalítico , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Glicosídeo Hidrolases/fisiologia , Hidrólise , Polissacarídeo-Liase/metabolismo , Polissacarídeos/metabolismo , Pseudomonas aeruginosa/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Especificidade por Substrato/fisiologia , Virulência
19.
Bioengineered ; 10(1): 240-249, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31181994

RESUMO

Gellan is a widely used microbial polysaccharide and one of the more effective ways to expand its application value would be to investigate the mechanism of gellan lyase and to produce gellan oligosaccharide. In this study, efficient gellan degrading bacteria were screened. One of the strains with high efficient gellan degradation capacity was labeled PE1. Through physiological and biochemical analysis of 16S rDNA, the species was identified as Pseudoalteromonas hodoensis. The optimum conditions for enzymatic activity and how it was affected by metal ions were determined, and the results showed that the lyase activities were much higher than those of previously reported (about 20 times). The gellan degradation products were determined by thin-layer chromatography and the oligosaccharides were determined by high-efficiency liquid chromatography to analyze the action site of lyase. This study laid a solid foundation which elucidates the production and application of gellan oligosaccharides. Research highlights ● High efficiency gellan lyase producing bacteria ● Optimization of reaction conditions for gellan degradation ● Oligosaccharides were detected by TLC and HPLC to speculate the lyase action sites.


Assuntos
Polissacarídeo-Liase/metabolismo , Pseudoalteromonas/enzimologia , DNA Ribossômico/metabolismo , Polissacarídeos Bacterianos/metabolismo
20.
Mar Drugs ; 17(6)2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31159265

RESUMO

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of -1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.


Assuntos
Alginatos/metabolismo , Organismos Aquáticos/enzimologia , Bacteroidetes/química , Bacteroidetes/enzimologia , Polissacarídeo-Liase/metabolismo , Cromatografia em Camada Delgada , Microbiologia Industrial , Polissacarídeo-Liase/química , Espectrometria de Massas por Ionização por Electrospray
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