RESUMEN
Hyaluronidase, an enzyme that degrades hyaluronic acid (HA), is utilized in clinical settings to facilitate drug diffusion, manage extravasation, and address injection-related complications linked to HA-based fillers. In this study, a novel hyaluronate lyase EsHyl8 was cloned, expressed, and characterized from Escherichia sp. A99 of human intestinal origin. This lyase belongs to polysaccharide lyase (PL) family 8, and showed specific activity towards HA. EsHyl8 exhibited optimal degradation at 40 °C and pH 6.0. EsHyl8 exhibited a high activity of 376.32 U/mg among hyaluronidases of human gut microorganisms. EsHyl8 was stable at 37 °C and remained about 70 % of activity after incubation at 37 °C for 24 h, demonstrating excellent thermostability. The activity of EsHyl8 was inhibited by Zn2+, Cu2+, Fe3+, and SDS. EsHyl8 was an endo-type enzyme whose end-product was unsaturated disaccharide. This study enhances our understanding of hyaluronidases from human gut microorganisms.
Asunto(s)
Clonación Molecular , Polisacárido Liasas , Polisacárido Liasas/genética , Polisacárido Liasas/química , Polisacárido Liasas/aislamiento & purificación , Polisacárido Liasas/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Escherichia/genética , Escherichia/enzimología , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Estabilidad de Enzimas , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Concentración de Iones de Hidrógeno , Especificidad por SustratoRESUMEN
Hyaluronidases are a class of enzymes that can degrade hyaluronic acid and have a wide range of applications in the medical field. In this study, the marine bacterium Vibrio sp. ZG1, which can degrade HA, was isolated, leading to the discovery of two novel hyaluronan lyases, Vhylzx1 and Vhylzx2, through genome sequencing and bioinformatic analysis. These lyases belong to the polysaccharide lyase-8 family. Vhylzx1 and Vhylzx2 specifically degrade HA, with highest activity at 35 °C, pH 5.7 and 50 °C, pH 7.1. Vhylzx1 and Vhylzx2 are endo-type enzymes that can fully degrade HA into unsaturated disaccharides. Sequence homology assessment and site-directed mutagenesis revealed that the catalytic residues of Vhylzx1 are Asn231, His281, and Tyr290, and that the catalytic residues of Vhylzx2 are Asn227, His277, and Tyr286. Moreover, this study used consensus sequences to enhance the specific activity of Vhylzx2 mutants. Notably, the mutants V564I, N742D, L619F, and D658G increases the specific activity by 2.4, 2.2, 1.3, and 1.2-fold. These characteristics are useful for further basic research and applications, and have a promising application in the preparation of biologically active hyaluronic acid oligosaccharides.
Asunto(s)
Clonación Molecular , Ácido Hialurónico , Polisacárido Liasas , Vibrio , Vibrio/enzimología , Vibrio/genética , Polisacárido Liasas/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/química , Ácido Hialurónico/química , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/metabolismo , Secuencia de Aminoácidos , Especificidad por SustratoRESUMEN
Microbial pectinolytic enzymes are important in various industries, particularly food processing. This study focuses on uncovering insights into a novel pectin lyase, BvPelB, from Bacillus velezensis 16B, with the aim of enhancing fruit juice processing. The study examines the structural and functional characteristics of pectinolytic enzyme, underscoring the critical nature of substrate specificity and enzymatic reaction mechanisms. BvPelB was successfully expressed and purified, exhibiting robust activity under alkaline conditions and thermal stability. Structural analysis revealed similarities with other pectin lyases, despite limited sequence identity. Biochemical characterization showed BvPelB's preference for highly methylated pectins and its endo-acting mode of cleavage. Treatment with BvPelB significantly increased juice yield and clarity without generating excessive methanol, making it a promising candidate for fruit juice processing. Overall, this study provides valuable insights into the enzymatic properties of BvPelB and its potential industrial applications in improving fruit juice processing efficiency and quality.
Asunto(s)
Bacillus , Proteínas Bacterianas , Manipulación de Alimentos , Jugos de Frutas y Vegetales , Polisacárido Liasas , Bacillus/enzimología , Bacillus/química , Jugos de Frutas y Vegetales/análisis , Polisacárido Liasas/metabolismo , Polisacárido Liasas/química , Polisacárido Liasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Especificidad por Sustrato , Estabilidad de Enzimas , Pectinas/metabolismo , Pectinas/química , Frutas/química , Frutas/enzimología , Frutas/microbiologíaRESUMEN
To overcome the trade-off challenge encountered in the engineering of alginate lyase AlyG2 from Seonamhaeicola algicola Gy8T and to expand its potential industrial applications, we devised a two-step strategy encompassing activity enhancement followed by thermal stability engineering. To enhance the specific activity of efficient AlyG2, we strategically substituted residues with bulky steric hindrance proximal to the active pocket with glycine or alanine. This led to the generation of three promising positive mutants, with particular emphasis on the T91S mutant, exhibiting a 1.91-fold specific activity compared to the wild type. To mitigate the poor thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were screened out. Notably, the S72Ya mutant not only displayed 17.96 % further increase in specific activity but also exhibited improved stability compared to T91S, manifesting as a remarkable 30.97 % increase in relative activity following a 1-hour incubation at 42 °C. Furthermore, enhanced kinetic stability was observed. To gain deeper insights into the mechanism underlying the enhanced thermostability of the S72Ya mutant, we conducted molecular dynamics simulations, principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. The results unveiled a reduction in the flexibility of the surface loop, a stronger correlation dynamic and a narrower motion subspace in S72Ya system, along with the formation of more stable hydrogen bonds. Collectively, our findings suggest amino acids substitutions resulting in smaller side chains proximate to the active site can positively impact enzyme activity, while reducing the flexibility of surface loops emerges as a pivotal factor in conferring thermal stability. These insights offer valuable guidance and a framework for the engineering of other enzyme types.
Asunto(s)
Estabilidad de Enzimas , Simulación de Dinámica Molecular , Polisacárido Liasas , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Cinética , Temperatura , Ingeniería de Proteínas/métodos , Mutación , Sustitución de Aminoácidos , Mutagénesis Sitio-DirigidaRESUMEN
Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a "Pro-Asp-Thr(PDT)" fragment and two PL-7 catalytic domains (CD I and CD II). The "PDT" fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the "PDT" fragment may serve as a fusion tag in the soluble expression of recombinant proteins.
Asunto(s)
Alginatos , Estabilidad de Enzimas , Polisacárido Liasas , Vibrio , Polisacárido Liasas/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/química , Vibrio/enzimología , Vibrio/genética , Alginatos/metabolismo , Alginatos/química , Unión Proteica , Dominio Catalítico , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Solubilidad , Secuencia de Aminoácidos , Temperatura , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Pseudoalteromonas fuliginea sp. PS47 is a recently identified marine bacterium that has extensive enzymatic machinery to metabolize polysaccharides, including a locus that targets pectin-like substrates. This locus contains a gene (locus tag EU509_03255) that encodes a pectin-degrading lyase, called PfPL1, that belongs to polysaccharide lyase family 1 (PL1). The 2.2â Å resolution X-ray crystal structure of PfPL1 reveals the compact parallel ß-helix fold of the PL1 family. The back side of the core parallel ß-helix opposite to the active site is a meandering set of five α-helices joined by lengthy loops. A comparison of the active site with those of other PL1 enzymes suggests a catalytic mechanism that is independent of metal ions, such as Ca2+, but that substrate recognition may require metal ions. Overall, this work provides the first structural insight into a pectinase of marine origin and the first structure of a PL1 enzyme in subfamily 2.
Asunto(s)
Dominio Catalítico , Modelos Moleculares , Polisacárido Liasas , Pseudoalteromonas , Pseudoalteromonas/enzimología , Pseudoalteromonas/genética , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Cristalografía por Rayos X , Secuencia de Aminoácidos , Pectinas/metabolismo , Pectinas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Especificidad por Sustrato , Conformación ProteicaRESUMEN
Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn2+, Co2+, Ca2+, Mg2+, Ni2+, and Ba2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na+, and upon treatment with Mn2+, its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed.
Asunto(s)
Alginatos , Kelp , Metagenómica , Polisacárido Liasas , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Polisacárido Liasas/química , Metagenómica/métodos , Kelp/genética , Alginatos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Especificidad por Sustrato , Chloroflexi/genética , Chloroflexi/enzimologíaRESUMEN
Proline metabolism plays a crucial role in both environmental stress responses and plant growth. However, the specific mechanism by which proline contributes to abiotic stress processes remains to be elucidated. In this study, we utilized atrzf1 (Arabidopsis thaliana ring zinc finger 1) as a parental line for T-DNA tagging mutagenesis and identified a suppressor mutant of atrzf1, designated proline content alterative 31 (pca31). The pca31 mutant suppressed the insensitivity of atrzf1 to dehydration stress during early seedling growth. Using Thermal Asymmetric Interlaced-PCR, we found that the T-DNA of pca31 was inserted into the promoter region of the At2g22620 gene, which encodes the cell wall enzyme rhamnogalacturonan lyase 1 (RGL1). Enzymatic assays indicated that RGL1 exhibited rhamnogalacturonan lyase activity, influencing cell wall pectin composition. The decrease in RGL1 gene expression suppressed the transcriptomic perturbation of the atrzf1 mutant. Silencing of the RGL1 gene in atrzf1 resulted in a sensitive phenotype similar to pca31 under osmotic stress conditions. Treatment with mannitol, salt, hydrogen peroxide, and abscisic acid induced RGL1 expression. Furthermore, we uncovered that RGL1 plays a role in modulating root growth and vascular tissue development. Molecular, physiological, and genetic experiments revealed that the positive modulation of RGL1 during abiotic stress was linked to the AtRZF1 pathway. Taken together, these findings establish that pca31 acts as a suppressor of atrzf1 in abiotic stress responses through proline and cell wall metabolisms.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Pectinas , Prolina , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Pared Celular/metabolismo , Deshidratación , Pectinas/metabolismo , Plantas Modificadas Genéticamente , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Prolina/metabolismo , Estrés Fisiológico , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2-4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation.
Asunto(s)
Alginatos , Estabilidad de Enzimas , Simulación del Acoplamiento Molecular , Oligosacáridos , Polisacárido Liasas , Oligosacáridos/química , Oligosacáridos/metabolismo , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Alginatos/química , Alginatos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , BiocatálisisRESUMEN
Enzymatic degradation of lignocellulosic biomass provides an eco-friendly approach to produce value-added macromolecules, e.g., bioactive polysaccharides. A novel acidophilic GH5 ß-1,4-endoglucanase (termed TaCel5) from Trichoderma asperellum ND-1 was efficiently expressed in Komagataella phaffii (â¼1.5-fold increase, 38.42 U/mL). TaCel5 displayed both endoglucanase (486.3 U/mg) and alginate lyase (359.5 U/mg) enzyme activities. It had optimal pH 3.0 and strong pH stability (exceed 86 % activity retained over pH range 3.0-5.0). 80 % activity (both endoglucanase and alginate lyase) was retained in the presence of 15 % ethanol or 3.42 M NaCl. Analysis of action mode revealed that hydrolytic activity of TaCel5 required at least three glucose (cellotriose) residues, yielding mainly cellobiose. Glu241 and Glu352 are essential catalytic residues, while Asp106, Asp277 and Asp317 play auxiliary roles in cellulose degradation. TaCel5 displayed high hydrolysis efficiency for glucan and alginate substrates. ESI-MS analysis indicated that the enzymatic hydrolysates of alginate mainly contained disaccharides and heptasaccharides. This is the first detailed report of a bifunctional GH5 endoglucanase/alginate lyase enzyme from T. asperellum. Thus TaCel5 has strong potential in food and feed industries as a catalyst for bioconversion of cellulose- and alginate-containing waste materials into value-added products oligosaccharides, which was of great benefit both for the economy and environment.
Asunto(s)
Alginatos , Celulasa , Celulosa , Oligosacáridos , Alginatos/metabolismo , Alginatos/química , Celulasa/metabolismo , Celulasa/química , Oligosacáridos/metabolismo , Oligosacáridos/química , Hidrólisis , Celulosa/metabolismo , Concentración de Iones de Hidrógeno , Hypocreales/enzimología , Especificidad por Sustrato , Polisacárido Liasas/metabolismo , Polisacárido Liasas/química , Polisacárido Liasas/genéticaRESUMEN
Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.
Asunto(s)
Antibacterianos , Biopelículas , Paenibacillus , Polisacárido Liasas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Polisacárido Liasas/metabolismo , Polisacárido Liasas/genética , Antibacterianos/farmacología , Paenibacillus/genética , Paenibacillus/enzimología , Paenibacillus/efectos de los fármacos , Gentamicinas/farmacología , Amicacina/farmacología , Fermentación , Pruebas de Sensibilidad Microbiana , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Alginatos/metabolismoRESUMEN
Ulvan is a complex sulfated polysaccharide extracted from Ulva, and ulvan lyases can degrade ulvan through a ß-elimination mechanism to obtain oligosaccharides. In this study, a new ulvan lyase, EPL15085, which belongs to the polysaccharide lyase (PL) 28 family from Tamlana fucoidanivorans CW2-9, was characterized in detail. The optimal pH and salinity are 9.0 and 0.4 M NaCl, respectively. The Km and Vmax of recombinant EPL15085 toward ulvan are 0.80 mg·mL-1 and 11.22 µmol·min -1 mg-1·mL-1, respectively. Unexpectedly, it is very resistant to high temperatures. After treatment at 100 °C, EPL15085 maintained its ability to degrade ulvan. Molecular dynamics simulation analysis and site-directed mutagenesis analysis indicated that the strong rigidity of the disulfide bond between Cys74-Cys102 in the N-terminus is related to its thermostability. In addition, oligosaccharides with disaccharides and tetrasaccharides were the end products of EPL15085. Based on molecular docking and site-directed mutagenesis analysis, Tyr177 and Leu134 are considered to be the crucial residues for enzyme activity. In conclusion, our study identified a new PL28 family of ulvan lyases, EPL15085, with excellent heat resistance that can expand the database of ulvan lyases and provide the possibility to make full use of ulvan.
Asunto(s)
Estabilidad de Enzimas , Polisacárido Liasas , Polisacáridos , Polisacárido Liasas/genética , Polisacárido Liasas/química , Polisacárido Liasas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Cinética , Calor , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Especificidad por Sustrato , Simulación del Acoplamiento Molecular , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ulva/química , Ulva/enzimología , Ulva/genética , Simulación de Dinámica MolecularRESUMEN
Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.
Asunto(s)
Aspergillus niger , Jugos de Frutas y Vegetales , Proteínas Fúngicas , Polisacárido Liasas , Aspergillus niger/enzimología , Aspergillus niger/genética , Jugos de Frutas y Vegetales/análisis , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Polisacárido Liasas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Concentración de Iones de Hidrógeno , Manipulación de Alimentos , Ácidos/química , Ácidos/metabolismo , Ácidos/farmacología , Citrus sinensis/química , Pectinas/química , Pectinas/metabolismo , Estabilidad de EnzimasRESUMEN
Plant cell death is regulated in plant-pathogen interactions. While some aspartic proteases (APs) participate in regulating programmed cell death or defense responses, the defense functions of most APs remain largely unknown. Here, we report on a virulence factor, PlPeL8, which is a pectate lyase found in the hemibiotrophic pathogen Peronophythora litchii. Through in vivo and in vitro assays, we confirmed the interaction between PlPeL8 and LcAP1 from litchi, and identified LcAP1 as a positive regulator of plant immunity. PlPeL8 induced cell death associated with NbSOBIR1 and NbMEK2. The 11 conserved residues of PlPeL8 were essential for inducing cell death and enhancing plant susceptibility. Twenty-three LcAPs suppressed cell death induced by PlPeL8 in Nicotiana benthamiana depending on their interaction with PlPeL8. The N-terminus of LcAP1 was required for inhibiting PlPeL8-triggered cell death and susceptibility. Furthermore, PlPeL8 led to higher susceptibility in NbAPs-silenced N. benthamiana than the GUS-control. Our results indicate the crucial roles of LcAP1 and its homologs in enhancing plant resistance via suppression of cell death triggered by PlPeL8, and LcAP1 represents a promising target for engineering disease resistance. Our study provides new insights into the role of plant cell death in the arms race between plants and hemibiotrophic pathogens.
Asunto(s)
Ascomicetos , Proteasas de Ácido Aspártico , Resistencia a la Enfermedad , Litchi , Proteínas de Plantas , Polisacárido Liasas , Secuencia de Aminoácidos , Ascomicetos/genética , Ascomicetos/patogenicidad , Proteasas de Ácido Aspártico/metabolismo , Proteasas de Ácido Aspártico/genética , Muerte Celular , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las Plantas , Litchi/genética , Nicotiana/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Polisacárido Liasas/metabolismo , Polisacárido Liasas/genética , Unión ProteicaRESUMEN
Horizontal gene transfer (HGT) is an important evolutionary force in the formation of prokaryotic and eukaryotic genomes. In recent years, many HGT genes horizontally transferred from prokaryotes to eukaryotes have been reported, and most of them are present in arthropods. The Pacific white shrimp Litopenaeus vannamei, an important economic species of arthropod, has close relationships with bacteria, providing a platform for horizontal gene transfer (HGT). In this study, we analyzed bacteria-derived HGT based on a high-quality genome of L. vannamei via a homology search and phylogenetic analysis, and six HGT genes were identified. Among these six horizontally transferred genes, we found one gene (LOC113799989) that contains a bacterial chondroitinase AC structural domain and encodes an unknown glycosaminoglycan (GAG) lyase in L. vannamei. The real-time quantitative PCR results showed that the mRNA expression level of LOC113799989 was highest in the hepatopancreas and heart, and after stimulation by Vibrio parahaemolyticus, its mRNA expression level was rapidly up-regulated within 12 h. Furthermore, after injecting si-RNA and stimulation by V. parahaemolyticus, we found that the experimental group had a higher cumulative mortality rate in 48 h than the control group, indicating that the bacteria-derived GAG lyase can reduce the mortality of shrimp with respect to infection by V. parahaemolyticus and might be related to the resistance of shrimp to bacterial diseases. Our findings contribute to the study of the function of GAGs and provide new insights into GAG-related microbial pathogenesis and host defense mechanisms in arthropods.
Asunto(s)
Transferencia de Gen Horizontal , Penaeidae , Filogenia , Vibrio parahaemolyticus , Animales , Penaeidae/inmunología , Penaeidae/microbiología , Penaeidae/genética , Vibrio parahaemolyticus/fisiología , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Hepatopáncreas/microbiología , Hepatopáncreas/inmunología , Hepatopáncreas/metabolismo , Bacterias , Inmunidad Innata/genética , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Vibriosis/inmunologíaRESUMEN
Polysaccharide-degrading bacteria are key participants in the global carbon cycle and algal biomass recycling. Herein, a polysaccharide lyase-producing strain HB226069 was isolated from Sargassum sp. from Qingge Port, Hainan, China. Results of the phylogenetic of the 16S rRNA gene and genotypic analysis indicated that the isolate should be classified as Microbulbifer thermotolerans. The whole genome is a 4,021,337 bp circular chromosome with a G+C content of 56.5%. Analysis of the predicted genes indicated that strain HB226069 encoded 161 carbohydrate-active enzymes (CAZymes), and abundant putative enzymes involved in polysaccharide degradation were predicted, including alginate lyase, fucosidase, agarase, xylanase, cellulase, pectate lyase, amylase, and chitinase. Three of the putative polysaccharide lyases from PL7 and PL17 families were involved in alginate degradation. The alginate lyases of strain HB226069 showed the maximum activity of 117.4 U/mL at 50 °C, pH 7.0, and 0.05 M FeCl3, while exhibiting the best stability at 30 °C and pH 7.0. The Thin Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analyses indicated that the alginate oligosaccharides (AOSs) degraded by the partially purified alginate lyases contained oligosaccharides of DP2-DP5 and monosaccharide while reacting for 36 h. The complete genome of M. thermotolerans HB226069 enriches our understanding of the mechanism of polysaccharide lyase production and supports its potential application in polysaccharide degradation.
Asunto(s)
Genoma Bacteriano , Filogenia , Polisacárido Liasas , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , ARN Ribosómico 16S/genética , China , Sargassum/microbiología , Sargassum/metabolismo , Alginatos/metabolismo , Polisacáridos/metabolismo , Composición de Base , Bacteroidetes/genética , Bacteroidetes/enzimología , Bacteroidetes/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguishable pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes and as a target gene for the molecular identification of these two pathotypes. KEY POINTS: ⢠Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed. ⢠The expression of Rs-pels is different in two pathotypes of Radopholus similis. ⢠A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target gene was established.
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Tylenchoidea , Animales , Tylenchoidea/genética , Raíces de Plantas , Polisacárido Liasas/genética , PlantonesRESUMEN
Glycosaminoglycan (GAG) lyases are important tools for investigating the structure of GAGs and preparing low-molecular-weight GAGs. The PL35 family, a recently established polysaccharide lyase family, should be further investigated. In this study, we discovered a new GAG lyase, CHa1, which belongs to the PL35 family. When expressed heterologously in Escherichia coli (BL21), CHa1 exhibited high expression levels and solubility. The optimal activity was observed in Tris-HCl buffer (pH 7.0) or sodium phosphate buffer (pH 8.0) at 30 °C. The specific activities towards HA, CSA, CSC, CSD, CSE, and HS were 3.81, 13.03, 36.47, 18.46, 6.46, and 0.50 U/mg protein, respectively. CHa1 digests substrate chains randomly that acting as an endolytic lyase and shows a significant preference for GlcA-containing structures, prefers larger oligosaccharides (≥UDP8) and can generate a series of oligosaccharides composed mainly of the A unit when digesting CSA. These oligosaccharides include ΔC-A, ΔC-A-A, ΔC-A-A-A, ΔC-A-A-A-A, and ΔC-A-A-A-A-A. The residues Tyr257 and His421 play crucial roles in the catalytic process, and Ser211, Asn212, Asn213, Trp214, Gln216, Lys360, Arg460 and Gln462 may participate in the binding process of CHa1. This study on CHa1 contributes to our understanding of the PL35 family and provides valuable tools for investigating the structure of GAGs.
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Polisacárido Liasas , Polisacárido Liasas/química , Polisacárido Liasas/metabolismo , Polisacárido Liasas/genética , Especificidad por Sustrato , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Escherichia coli/genética , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/química , Secuencia de Aminoácidos , Oligosacáridos/química , Oligosacáridos/metabolismoRESUMEN
A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg-1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like â4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 â 4)ΔUA/ΔUA2S[1 â and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.
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Heparina , Polisacárido Liasas , Heparina/análisis , Heparina/química , Heparina/metabolismo , Liasa de Heparina/genética , Liasa de Heparina/química , Liasa de Heparina/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Sitios de UniónRESUMEN
Alginate lyases with unique characteristics for degrading alginate into size-defined oligosaccharide fractions, were considered as the potential agents for disrupting Pseudomonas aeruginosa biofilms. In our study, a novel endolytic PL-7 alginate lyase, named AlyG2, was cloned and expressed through Escherichia coli. This enzyme exhibited excellent properties: it maintained more than 85% activity at low temperatures of 4 °C and high temperatures of 70 °C. After 1 h of incubation at 4 °C, it still retained over 95% activity, demonstrating the ability to withstand low temperature. The acid-base and salt tolerance properties shown it preserves more than 50% activity in the pH range of 5.0 to 11.0 and in a high salt environment at 3000 mM NacCl, indicating its high stability in several aspects. More importantly, AlyG2 in our research was revealed to be effective at removing mature biofilms and inhibiting biofilm formation produced by Pseudomonas aeruginosa, and the inhibition and disruption rates were 47.25 ± 4.52% and 26.5 ± 6.72%, respectively. Additionally, the enzyme AlyG2 promoted biofilm disruption in combination with antibiotics, particularly manifesting the synergistic effect with erythromycin (FIC=0.5). In all, these results offered that AlyG2 with unique characteristics may be an effective technique for the clearance or disruption of biofilm produced by P. aeruginosa.