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1.
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
2.
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
3.
Mar Drugs ; 17(5)2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137680

RESUMO

Pseudomonas aeruginosa biofilms are typically associated with the chronic lung infection of cystic fibrosis (CF) patients and represent a major challenge for treatment. This opportunistic bacterial pathogen secretes alginate, a polysaccharide that is one of the main components of its biofilm. Targeting this major biofilm component has emerged as a tempting therapeutic strategy for tackling biofilm-associated bacterial infections. The enormous potential in genetic diversity of the marine microbial community make it a valuable resource for mining activities responsible for a broad range of metabolic processes, including the alginolytic activity responsible for degrading alginate. A collection of 36 bacterial isolates were purified from marine water based on their alginolytic activity. These isolates were identified based on their 16S rRNA gene sequences. Pseudoalteromonas sp. 1400 showed the highest alginolytic activity and was further confirmed to produce the enzyme alginate lyase. The purified alginate lyase (AlyP1400) produced by Pseudoalteromonas sp. 1400 showed a band of 23 KDa on a protein electrophoresis gel and exhibited a bifunctional lyase activity for both poly-mannuronic acid and poly-glucuronic acid degradation. A tryptic digestion of this gel band analyzed by liquid chromatography-tandem mass spectrometry confirmed high similarity to the alginate lyases in polysaccharide lyase family 18. The purified alginate lyase showed a maximum relative activity at 30 °C at a slightly acidic condition. It decreased the sodium alginate viscosity by over 90% and reduced the P. aeruginosa (strain PA14) biofilms by 69% after 24 h of incubation. The combined activity of AlyP1400 with carbenicillin or ciprofloxacin reduced the P. aeruginosa biofilm thickness, biovolume and surface area in a flow cell system. The present data revealed that AlyP1400 combined with conventional antibiotics helped to disrupt the biofilms produced by P. aeruginosa and can be used as a promising combinational therapeutic strategy.


Assuntos
Biofilmes/efeitos dos fármacos , Polissacarídeo-Liase/farmacologia , Pseudoalteromonas/enzimologia , Pseudomonas aeruginosa/efeitos dos fármacos , Alginatos/metabolismo , Antibacterianos/farmacologia , Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Carbenicilina/farmacologia , Ciprofloxacino/farmacologia , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Pseudoalteromonas/genética , Pseudomonas aeruginosa/fisiologia , RNA Ribossômico 16S/genética
4.
Mar Drugs ; 17(5)2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137685

RESUMO

Alginate oligosaccharides (AOS) show versatile bioactivities. Although various alginate lyases have been characterized, enzymes with special characteristics are still rare. In this study, a polysaccharide lyase family 7 (PL7) alginate lyase-encoding gene, aly08, was cloned from the marine bacterium Vibrio sp. SY01 and expressed in Escherichia coli. The purified alginate lyase Aly08, with a molecular weight of 35 kDa, showed a specific activity of 841 U/mg at its optimal pH (pH 8.35) and temperature (45 °C). Aly08 showed good pH-stability, as it remained more than 80% of its initial activity in a wide pH range (4.0-10.0). Aly08 was also a thermo-tolerant enzyme that recovered 70.8% of its initial activity following heat shock treatment for 5 min. This study also demonstrated that Aly08 is a polyG-preferred enzyme. Furthermore, Aly08 degraded alginates into disaccharides and trisaccharides in an endo-manner. Its thermo-tolerance and pH-stable properties make Aly08 a good candidate for further applications.


Assuntos
Organismos Aquáticos/enzimologia , Polissacarídeo-Liase/metabolismo , Temperatura Ambiente , Vibrio/enzimologia , Organismos Aquáticos/genética , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vibrio/genética
5.
Curr Microbiol ; 76(7): 879-887, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089795

RESUMO

The goal of this study was to elucidate the role of the outer membrane protein A (ompA) gene of Xanthomonas axonopodis pv. glycines in bacterial pustule pathogenesis of soybean. An ompA mutant of X. axonopodis pv. glycines KU-P-SW005 was shown to significantly decrease cellulase, pectate lyase, and polysaccharide production. The production of these proteins in the ompA mutant was approximately five times lower than that of the wildtype. The ompA mutant also exhibited modified biofilm development. More importantly, the mutant reduced disease severity to the soybean. Ten days after inoculation, the virulence rating of the susceptible soybean cv. SJ4 inoculated with the ompA mutant was 11.23%, compared with 87.98% for the complemented ompA mutant. Production of cellulase, pectate lyase, polysaccharide was restored, biofilm, and pustule numbers were restored in the complemented ompA mutant that did not differ from the wild type. Taken together, these data suggest that OmpA-mediated invasion plays an important role in protein secretion during pathogenesis to soybean.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Doenças das Plantas/microbiologia , Soja/microbiologia , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidade , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Celulase/metabolismo , Teste de Complementação Genética , Mutação , Folhas de Planta/microbiologia , Polissacarídeo-Liase/metabolismo , Polissacarídeos Bacterianos/metabolismo , Virulência/genética
6.
Appl Microbiol Biotechnol ; 103(13): 5231-5241, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31028436

RESUMO

Cold-active enzymes have become attractive biocatalysts in biotechnological applications for their ability to retain high catalytic activity below 30 °C, which allows energy reduction and cost saving. Here, a 1041 bp gene pel1 encoding a 34.7 KDa pectate lyase was cloned from a facultatively psychrophilic Antarctic bacterium Massilia eurypsychrophila and heterologously expressed in Escherichia coli. PEL1 presented the highest 66% identity to the reported mesophilic pectate lyase PLXc. The purified PEL1 exhibits the optimum temperature and pH of 30 °C and 10 toward polygalacturonic acid, respectively. PEL1 is a cold-active enzyme that can retain 60% and 25% relative activity at 10 °C and 0 °C, respectively, while it loses most of activity at 40 °C for 10 min. PEL1 has the highest specific activity (78.75 U mg-1) than all other reported cold-active pectinase, making it a better choice for use in industry. Based on the detailed sequence and structure comparison between PEL1 and PLXc and mutation analysis, more flexible structure and some loop regions may contribute to the cold activity and thermal instability of PEL1. Our investigations of the cold-active mechanism of PEL1 might guide the rational design of PEL1 and other related enzymes.


Assuntos
Temperatura Baixa , Oxalobacteraceae/enzimologia , Polissacarídeo-Liase/metabolismo , Regiões Antárticas , Biocatálise , Clonagem Molecular , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Oxalobacteraceae/genética , Polissacarídeo-Liase/genética , Especificidade por Substrato
7.
Mol Biotechnol ; 61(6): 451-460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997666

RESUMO

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Periplasma/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo
8.
J Agric Food Chem ; 67(19): 5486-5495, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31012315

RESUMO

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.


Assuntos
Cannabis/microbiologia , Proteínas Fúngicas/metabolismo , Pleurotus/enzimologia , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Galactosidases/química , Galactosidases/genética , Galactosidases/metabolismo , Glicosilação , Lacase/química , Lacase/genética , Lacase/metabolismo , Lignina/metabolismo , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Peroxidases/química , Peroxidases/genética , Peroxidases/metabolismo , Caules de Planta/microbiologia , Pleurotus/classificação , Pleurotus/genética , Pleurotus/crescimento & desenvolvimento , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Transporte Proteico
9.
Regul Toxicol Pharmacol ; 104: 157-162, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30904430

RESUMO

Korean red ginseng and its extract have been used as traditional medicines and functional foods in countries worldwide. Pectin lyase-modified red ginseng extract (GS-E3D) was newly developed as a dietary supplement for obesity, diabetes-related renal dysfunction, etc. In this study, the safety of GS-E3D on acute toxicity and genotoxicity was evaluated. For acute study, Sprague-Dawley rats were administrated by oral gavage at a dose of 5000 mg/kg GS-E3D. To evaluate genotoxicity of GS-E3D, we conducted three-battery tests, which are Ames test using Escherichia coli (WP2uvrA pKM101) and Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537), chromosomal aberration test -using Chinese hamster lung cells, and micronucleus test using ICR mice. In acute toxicity studies, there were no dead animals or abnormal necropsy findings in the control group and GS-E3D (5000 mg/kg) treated group. GS-E3D did not induce mutagenicity in the bacterial test, chromosomal aberrations in Chinese hamster lung cells and micronuclei in bone marrow cells of mice. Conclusively, the approximate lethal dose of GS-E3D was greater than 5000 mg/kg bw and GS-E3D has no genotoxic potential in the three-battery tests on genotoxicity.


Assuntos
Ginsenosídeos/metabolismo , Panax/química , Extratos Vegetais/metabolismo , Extratos Vegetais/toxicidade , Polissacarídeo-Liase/metabolismo , Animais , Peso Corporal , Linhagem Celular , Cricetinae , Cricetulus , Escherichia coli/efeitos dos fármacos , Feminino , Ginsenosídeos/química , Masculino , Camundongos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Salmonella typhimurium/efeitos dos fármacos
10.
J Agric Food Chem ; 67(10): 2936-2945, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30781951

RESUMO

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. We describe a thermostable alginate lyase belonging to Polysaccharide Lyase family 7 (PL7), which can be used to degrade brown seaweed, Saccharina latissima, at conditions also suitable for a commercial cellulase cocktail (Cellic CTec2). This enzyme, AMOR_PL7A, is a ß-d-mannuronate specific (EC 4.2.2.3) endoacting alginate lyase, which degrades alginate and poly mannuronate within a broad range of pH, temperature and salinity. At 65 °C and pH 6.0, its Km and kcat values for sodium alginate are 0.51 ± 0.09 mg/mL and 7.8 ± 0.3 s-1 respectively. Degradation of seaweed with blends of Cellic CTec2 and AMOR_PL7A at 55 °C in seawater showed that the lyase efficiently reduces viscosity and increases glucose solublization. Thus, AMOR_PL7A may be useful in development of efficient protocols for enzymatic seaweed processing.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Fontes Hidrotermais/microbiologia , Polissacarídeo-Liase/química , Regiões Árticas , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Fontes Hidrotermais/química , Cinética , Metagenômica , Feófitas/química , Filogenia , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Polissacarídeos/química , Alga Marinha/química , Especificidade por Substrato , Temperatura Ambiente
11.
Enzyme Microb Technol ; 121: 68-77, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30554647

RESUMO

Self-assembling amphipathic peptides (SAPs) have been used as stabilization tags to improve enzyme stability but do not function uniformly well with all target enzymes. Here, the key factors involved in SAPs stabilization were identified as the SAP length and linker length and flexibility, using S1 (AEAEAKAK)2 as an originated SAP and polygalacturonate lyase (PGL) as model protein. Biochemical analysis demonstrated that SAPs could induce loose protein oligomerization via intermolecular hydrophobic interactions. Based on this mechanism, a comprehensive protein stabilization strategy was proposed, in which a library of stabilizing tags through random combination of different SAPs and linker peptides was developed to design the fusion composition while the sodium chloride (NaCl) was used to enhance the intermolecular hydrophobic interactions. By using the strategy, the PGL, lipoxygenase (LOX) and L-asparaginase exhibited 33.25-, 17.55- and 15.6-fold increases, respectively, in the t1/2 value relative to that of the corresponding wild-type enzyme. The SAP library therefore shows great application potential in stability enhancement of enzymes/proteins.


Assuntos
Asparaginase/química , Bactérias/enzimologia , Lipoxigenase/química , Fragmentos de Peptídeos/química , Polissacarídeo-Liase/química , Proteínas Recombinantes de Fusão/química , Asparaginase/genética , Asparaginase/metabolismo , Estabilidade Enzimática , Interações Hidrofóbicas e Hidrofílicas , Lipoxigenase/genética , Lipoxigenase/metabolismo , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Temperatura Ambiente
12.
Food Chem ; 276: 503-510, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30409626

RESUMO

Pectinolytic enzymes are used in diverse industrial applications. We sought to isolate a pectate lyase from Aspergillus luchuensis var. saitoi, a filamentous fungus used in traditional food and beverage preparation in Japan. The identified enzyme, named AsPelA, is orthologous to PelA from A. luchuensis mut. kawachii (AkPelA); the enzymes exhibit 99% amino acid sequence identity, with Ile140 and Val197 of AsPelA being replaced by Val and Asp in AkPelA, respectively. AsPelA activity decreased to 71%, 61%, and 46% of maximal activity after 60-min incubation at 60 °C, 70 °C, and 80 °C, whereas AkPelA activity dropped to 16%, 10%, and 8.5%, respectively, indicating that AsPelA is more thermostable than AkPelA. Furthermore, AsPelA was stable within a neutral-to-alkaline pH range, as well as in the presence of organic solvents, detergents, and metal ions. Our findings suggest that AsPelA represents a candidate pectate lyase for applications in food, paper, and textile industries.


Assuntos
Aspergillus/enzimologia , Polissacarídeo-Liase/metabolismo , Temperatura Ambiente , Sequência de Aminoácidos , Detergentes/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Metais/farmacologia , Polissacarídeo-Liase/química , Alinhamento de Sequência , Solventes/farmacologia
13.
Protein Expr Purif ; 153: 97-104, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30201400

RESUMO

Alginate lyase digestion is an efficient way to degrade alginate into oligosaccharides, which are useful in various areas including chemistry, pharmacy and food industry. Here we determined the sequence of Vibrio sp. QY102 sourced alginate lyase, and set up its heterologous expression in E. coli. We improved its secretion efficiency by replacing the original secretive sequence by E. coli specific signal peptide ompA. We successfully purified the full-length protein in shake flask culture, however, degradation happened during fed batch cultivation. By domain and disorder examination, we found that the protein was completely functional by expressing the C terminal fragment alone. For the final strain we constructed (HMS-ompA-CF), the extracellular enzyme activity reached 375 U/ml in shake flask and 1789 U/ml in fed batch cultivation (5 L bioreactor). And the final protein yield reached 0.58 g/L in fed batch cultivation. We determined that the optimal pH and temperature for the shortened alginate lyase were 7.0 and 39 °C, respectively.


Assuntos
Alginatos/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Polissacarídeo-Liase/genética , Vibrio/enzimologia , Alginatos/metabolismo , Sequência de Aminoácidos , Organismos Aquáticos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Cromatografia de Afinidade , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeo-Liase/química , Polissacarídeo-Liase/isolamento & purificação , Polissacarídeo-Liase/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Temperatura Ambiente , Vibrio/genética
15.
Int J Mol Sci ; 19(8)2018 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104475

RESUMO

Next-generation sequencing (NGS) of the Flammulina elastica (wood-rotting basidiomycete) genome was performed to identify carbohydrate-active enzymes (CAZymes). The resulting assembly (31 kmer) revealed a total length of 35,045,521 bp (49.7% GC content). Using the AUGUSTUS tool, 12,536 total gene structures were predicted by ab initio gene prediction. An analysis of orthologs revealed that 6806 groups contained at least one F. elastica protein. Among the 12,536 predicted genes, F. elastica contained 24 species-specific genes, of which 17 genes were paralogous. CAZymes are divided into five classes: glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyltransferases (GTs), and auxiliary activities (AA). In the present study, annotation of the predicted amino acid sequences from F. elastica genes using the dbCAN CAZyme database revealed 508 CAZymes, including 82 AAs, 218 GHs, 89 GTs, 18 PLs, 59 CEs, and 42 carbohydrate binding modules in the F. elastica genome. Although the CAZyme repertoire of F. elastica was similar to those of other fungal species, the total number of GTs in F. elastica was larger than those of other basidiomycetes. This genome information elucidates newly identified wood-degrading machinery in F. elastica, offers opportunities to better understand this fungus, and presents possibilities for more detailed studies on lignocellulosic biomass degradation that may lead to future biotechnological and industrial applications.


Assuntos
Flammulina/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Bases de Dados Genéticas , Flammulina/enzimologia , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Filogenia , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
16.
Biochim Biophys Acta Gen Subj ; 1862(9): 1862-1869, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29864445

RESUMO

Noncatalytic carbohydrate binding modules (CBMs) have been demonstrated to play various roles with cognate catalytic domains. However, for polysaccharide lyases (PLs), the roles of CBMs remain mostly unknown. AlyB is a multidomain alginate lyase that contains CBM32 and a PL7 catalytic domain. The AlyB structure determined herein reveals a noncanonical alpha helix linker between CBM32 and the catalytic domain. More interestingly, CBM32 and the linker does not significantly enhance the catalytic activity but rather specifies that trisaccharides are predominant in the degradation products. Detailed mutagenesis, biochemical and cocrystallization analyses show "weak but important" CBM32 interactions with alginate oligosaccharides. In combination with molecular modeling, we propose that the CBM32 domain serves as a "pivot point" during the trisaccharide release process. Collectively, this work demonstrates a novel role of CBMs in the activity of the appended PL domain and provides a new avenue for the well-defined generation of alginate oligosaccharides by taking advantage of associated CBMs.


Assuntos
Alginatos/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeo-Liase/química , Polissacarídeo-Liase/metabolismo , Vibrio/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Modelos Moleculares , Polissacarídeo-Liase/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Especificidade por Substrato
17.
Appl Microbiol Biotechnol ; 102(16): 6987-6996, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29948117

RESUMO

Carbohydrates are the product of carbon dioxide fixation by algae in the ocean. Their polysaccharides are depolymerized by marine bacteria, with a vast array of carbohydrate-active enzymes. These enzymes are important tools to establish biotechnological processes based on algal biomass. Green tides, which cover coastal areas with huge amounts of algae from the genus Ulva, represent a globally rising problem, but also an opportunity because their biomass could be used in biorefinery processes. One major component of their cell walls is the anionic polysaccharide ulvan for which the enzymatic depolymerization remains largely unknown. Ulvan lyases catalyze the initial depolymerization step of this polysaccharide, but only a few of these enzymes have been described. Here, we report the cloning, overexpression, purification, and detailed biochemical characterization of the endolytic ulvan lyase from Formosa agariphila KMM 3901T which is a member of the polysaccharide lyase family PL28. The identified biochemical parameters of the ulvan lyase reflect adaptation to the temperate ocean where the bacterium was isolated from a macroalgal surface. The NaCl concentration has a high influence on the turnover number of the enzyme and the affinity to ulvan. Divalent cations were shown to be essential for enzyme activity with Ca2+ likely being the native cofactor of the ulvan lyase. This study contributes to the understanding of ulvan lyases, which will be useful for future biorefinery applications of the abundant marine polysaccharide ulvan.


Assuntos
Flavobacterium/enzimologia , Polissacarídeo-Liase/metabolismo , Polissacarídeos/metabolismo , Flavobacterium/isolamento & purificação , Taiwan
18.
Mar Drugs ; 16(5)2018 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-29751659

RESUMO

An alginate lyase encoding gene sagl from Flavobacterium sp. H63 was codon optimized and recombinantly expressed at high level in P.pastoris through high cell-density fermentation. The highest yield of recombinant enzyme of sagl (rSAGL) in yeast culture supernatant reached 226.4 µg/mL (915.5 U/mL). This was the highest yield record of recombinant expression of alginate lyase so far. The rSAGL was confirmed as a partially glycosylated protein through EndoH digestion. The optimal reaction temperature and pH of this enzyme were 45 °C and 7.5; 80 mM K⁺ ions could improve the catalytic activity of the enzyme by 244% at most. rSAGL was a thermal stable enzyme with T5015 of 57⁻58 °C and T5030 of 53⁻54 °C. Its thermal stability was better than any known alginate lyase. In 100 mM phosphate buffer of pH 6.0, rSAGL could retain 98.8% of the initial activity after incubation at 50 °C for 2 h. Furthermore, it could retain 61.6% of the initial activity after 48 h. The specific activity of the purified rSAGL produced by P. pastoris attained 4044 U/mg protein, which was the second highest record of alginate lyase so far. When the crude enzyme of the rSAGL was directly used in transformation of sodium alginate with 40 g/L, 97.2% of the substrate was transformed to di, tri, tetra brown alginate oligosaccharide after 32 h of incubation at 50 °C, and the final concentration of reducing sugar in mixture reached 9.51 g/L. This is the first report of high-level expression of thermally stable alginate lyase using P. pastoris system.


Assuntos
Alginatos/metabolismo , Proteínas de Bactérias/metabolismo , Oligossacarídeos/metabolismo , Pichia/metabolismo , Polissacarídeo-Liase/metabolismo , Fermentação/fisiologia , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
19.
Int J Biol Macromol ; 115: 1063-1070, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29698759

RESUMO

Marine polysaccharide-degrading enzymes play an important role in marine algae degradation and carbon cycling, especially the alginate lyases. Although many alginate lyases have been characterized, the enzymes with industrial potential are still rather rare. A gene, encoding a new alginate lyase AlgNJ04, has been cloned from the marine bacterium Vibrio sp. NJ04. The recombinant alginate lyase was characterized followed by purification on Ni-NTA Sepharose. It exhibited an optimum activity (2416 U/mg) at pH 7.0 and 40 °C. Notably, the AlgNJ04 retained more than 80% of its maximum activity at a broad pH range of pH 4.0 and 10.0, which exhibited excellent pH stability. Additionally, it possessed broader substrate specificity, showing activities towards both poly ß-D-mannuronate (polyM) and poly α-L-guluronate (polyG). Furthermore, the Km values of AlgNJ04 towards sodium alginate (0.49 mM) and polyG (0.24 mM) were lower than that towards polyM (0.86 mM). Notably, the activity of AlgNJ-04 could be activated by NaCl with certain concentrations, which was partly caused by the removal of bound water from sodium alginate molecules or by the effects of charges in forming the alginate-enzyme complex. The ESI-MS analysis suggested that it mainly released oligosaccharides with DP of 2-5 as end products in an endolytic manner. Therefore, it may be a potent tool to produce alginate oligosaccharides with lower DPs.


Assuntos
Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sais/farmacologia , Vibrio/enzimologia , Vibrio/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Polissacarídeo-Liase/química , Polissacarídeo-Liase/isolamento & purificação , Especificidade por Substrato
20.
Mar Drugs ; 16(4)2018 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-29642383

RESUMO

Alginate lyases are a group of enzymes that catalyze the depolymerization of alginates into oligosaccharides or monosaccharides. These enzymes have been widely used for a variety of purposes, such as producing bioactive oligosaccharides, controlling the rheological properties of polysaccharides, and performing structural analyses of polysaccharides. The algM4 gene of the marine bacterium Vibrio weizhoudaoensis M0101 encodes an alginate lyase that belongs to the polysaccharide lyase family 7 (PL7). In this study, the kinetic constants Vmax (maximum reaction rate) and Km (Michaelis constant) of AlgM4 activity were determined as 2.75 nmol/s and 2.72 mg/mL, respectively. The optimum temperature for AlgM4 activity was 30 °C, and at 70 °C, AlgM4 activity dropped to 11% of the maximum observed activity. The optimum pH for AlgM4 activity was 8.5, and AlgM4 was completely inactive at pH 11. The addition of 1 mol/L NaCl resulted in a more than sevenfold increase in the relative activity of AlgM4. The secondary structure of AlgM4 was altered in the presence of NaCl, which caused the α-helical content to decrease from 12.4 to 10.8% and the ß-sheet content to decrease by 1.7%. In addition, NaCl enhanced the thermal stability of AlgM4 and increased the midpoint of thermal denaturation (Tm) by 4.9 °C. AlgM4 exhibited an ability to degrade sodium alginate, poly-mannuronic acid (polyM), and poly-guluronic acid (polyG), resulting in the production of oligosaccharides with a degree of polymerization (DP) of 2-9. AlgM4 possessed broader substrate, indicating that it is a bifunctional alginate lyase. Thus, AlgM4 is a novel salt-activated and bifunctional alginate lyase of the PL7 family with endolytic activity.


Assuntos
Organismos Aquáticos/enzimologia , Proteínas de Bactérias/química , Polissacarídeo-Liase/química , Cloreto de Sódio/farmacologia , Vibrio/enzimologia , Alginatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Endopeptidases , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/isolamento & purificação , Polissacarídeo-Liase/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente
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