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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.
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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.
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In the process of sepsis, excessive occurrence of pyroptosis, a form of programmed cell death acting as a defense mechanism against pathogens, can disrupt immune responses, thus leading to tissue damage and organ dysfunction. Chitosan oligosaccharide (COS), derived from chitosan degradation, has demonstrated diverse beneficial effects. However, its impact on sepsis-induced pyroptosis remains unexplored. In the present study, ATP/LPS was utilized to induce canonical-pyroptosis in THP-1 cells, while bacterial outer membrane vesicles (OMV) were employed to trigger non-canonical pyroptosis in RAW264.7 cells. Our results revealed a dose-dependent effect of COS on both types of pyroptosis. This was evidenced by a reduction in the expression of pro-inflammatory cytokines, as well as crucial regulatory proteins involved in pyroptosis. In addition, COS inhibited the cleavage of caspase-1 and GSDMD, and reduced ASC oligomerization. The underlying mechanism revealed that COS acts an antioxidant, reducing the release of pyroptosis-induced ROS and malondialdehyde (MDA) by upregulation the expression and promoting the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2), which led to an elevation of glutathione peroxidase 4 (GPX4) and superoxide dismutase (SOD). Notably, the actions of COS were completely reversed by the Nrf2 inhibitor. Consequently, COS intervention increased the survival rate of sepsis.
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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.
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Alginatos , Estabilidade Enzimática , Polissacarídeo-Liases , Vibrio , Polissacarídeo-Liases/metabolismo , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/química , Vibrio/enzimologia , Vibrio/genética , Alginatos/metabolismo , Alginatos/química , Ligação Proteica , Domínio Catalítico , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Solubilidade , Sequência de Aminoácidos , Temperatura , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The application of chondroitinase requires consideration of the complex microenvironment of the target. Our previous research reported a marine-derived sodium dodecyl sulfate (SDS)-resistant chondroitinase VhChlABC. This study further investigated the mechanism of VhChlABC resistance to SDS. Focusing on the hydrophobic cluster on its strong hydrophilic surface, it was found that the reduction of hydrophobicity of surface residues Ala181, Met182, Met183, Ala184, Val185, and Ile305 significantly reduced the SDS resistance and stability. Molecular dynamics (MD) simulation and molecular docking analysis showed that I305G had more conformational flexibility around residue 305 than wild type (WT), which was more conducive to SDS insertion and binding. The affinity of A181G, M182A, M183A, V185A and I305G to SDS was significantly higher than that of WT. In conclusion, the surface hydrophobic microenvironment composed of six residues was the structural basis for SDS resistance. This feature could prevent the binding of SDS and the destruction of hydrophobic packaging by increasing the rigid conformation of protein and reducing the binding force of SDS-protein. The study provides a new idea for the rational design of SDS-resistant proteins and may further promote chondroitinase research in the targeted therapy of lung diseases under the pressure of pulmonary surfactant. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00201-1.
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AIMS AND OBJECTIVES: The aim of this study is to discuss the influence of endotoxin on insulin amyloid formation, to provide guidance for therapeutic insulin preparation and storage. MATERIALS AND METHODS: The ThT and ANS binding assays were applied to characterize the dynamics curve of insulin amyloid formation with the presence or absence of endotoxin. The morphological structures of intermediate and mature insulin fibrils were observed with SEM and TEM. Secondary structural changes of insulin during fibriliation were examined with CD, FTIR and Raman spectral analysis. The cytotoxic effects of oligomeric and amyloidogenic insulin aggregates were detected using a cck-8 cell viability assay kit. The influence of endotoxin on insulin efficacy was analyzed by monitoring the activation of insulin signal transduction. KEY FINDINGS: ThT analysis showed that endotoxin, regardless of species, accelerated insulin fibrils formation in a dose-dependent manner, as observed with a shorter lag phase. ANS binding assay demonstrated endotoxin provoked the exposure of insulin hydrophobic patches. The results of SEM and TEM data displayed that endotoxin drove insulin to cluster into dense and viscous form, with thicker and stronger filaments. Based on CD, FTIR and Raman spectra, endotoxin promoted the transition of α-helix to random coil and ß-strand secondary structures during insulin aggregation. Insulins in both oligomeric and amyloidogenic forms were cytotoxic to HepG2 cells, with the former being more severe. Finally, the efficacy of endotoxin treated insulin obviously decreased. SIGNIFICANCE: Our studies revealed that endotoxin disrupts the structural integrity of insulin and promotes its amyloidosis. These findings offered theoretical guidance for insulin storage and safe utilization, as well as pointing up a new direction for insulin resistance research.
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Amiloidose , Insulina , Humanos , Amiloide/química , Amiloidose/metabolismo , Insulina/metabolismo , Estrutura Secundária de Proteína , Transdução de Sinais , EndotoxinasRESUMO
Angiogenesis refers to the process of growing new blood vessels from pre-existing capillaries or post-capillary veins. This process plays a critical role in promoting tumorigenesis and metastasis. As a result, developing antiangiogenic agents has become an attractive strategy for tumor treatment. Sirtuin6 (SIRT6), a member of nicotinamide adenine (NAD+)-dependent histone deacetylases, regulates various biological processes, including metabolism, oxidative stress, angiogenesis, and DNA damage and repair. Some SIRT6 inhibitors have been identified, but the effects of SIRT6 inhibitors on anti-angiogenesis have not been reported. We have identified a pyrrole-pyridinimidazole derivative 8a as a highly effective inhibitor of SIRT6 and clarified its anti-pancreatic-cancer roles. This study investigated the antiangiogenic roles of 8a. We found that 8a was able to inhibit the migration and tube formation of HUVECs and downregulate the expression of angiogenesis-related proteins, including VEGF, HIF-1α, p-VEGFR2, and N-cadherin, and suppress the activation of AKT and ERK pathways. Additionally, 8a significantly blocked angiogenesis in intersegmental vessels in zebrafish embryos. Notably, in a pancreatic cancer xenograft mouse model, 8a down-regulated the expression of CD31, a marker protein of angiogenesis. These findings suggest that 8a could be a promising antiangiogenic and cancer therapeutic agent.
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Neoplasias , Sirtuínas , Humanos , Camundongos , Animais , Transdução de Sinais , Neovascularização Patológica/metabolismo , Peixe-Zebra/metabolismo , Neoplasias/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Sirtuínas/metabolismo , Células Endoteliais da Veia Umbilical HumanaRESUMO
Helicobacter pylori (H. pylori) is recognized as a pathogen associated with several gastrointestinal diseases. The current treatments exhibit numerous drawbacks, including antibiotic resistance. H. pylori can adhere to and colonize the gastric mucosa through H. pylori adhesin A (HpaA), and antibodies against HpaA may be an effective therapeutic approach. The variable domain of immunoglobulin new antigen receptor (VNAR) is a novel type of single-domain antibody with a small size, good stability, and easy manufacturability. This study isolated VNARs against HpaA from an immune shark VNAR phage display library. The VNARs can bind both recombinant and native HpaA proteins. The VNARs, 2A2 and 3D6, showed high binding affinities to HpaA with different epitopes. Furthermore, homodimeric bivalent VNARs, biNb-2A2 and biNb-3D6, were constructed to enhance the binding affinity. The biNb-2A2 and biNb-3D6 had excellent stability at gastrointestinal pH conditions. Finally, a sandwich ELISA assay was developed to quantify the HpaA protein using BiNb-2A2 as the capture antibody and BiNb-3D6 as the detection antibody. This study provides a potential foundation for novel alternative approaches to treatment or diagnostics applications of H. pylori infection.
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Helicobacter pylori , Tubarões , Animais , Anticorpos , Ensaio de Imunoadsorção Enzimática , EpitoposRESUMO
Pancreatic cancer is a highly aggressive cancer, and is primarily treated with gemcitabine, with increasing resistance. SIRT6 as a member of sirtuin family plays important roles in lifespan and diverse diseases, such as cancer, diabetes, inflammation and neurodegenerative diseases. Considering the role of SIRT6 in the cytoprotective effect, it might be a potential anticancer drug target, and is associated with resistance to anticancer therapy. However, very few SIRT6 inhibitors have been reported. Here, we reported the discovery of a pyrrole-pyridinimidazole derivative, 8a, as a new non-competitive SIRT6 inhibitor, and studied its roles and mechanisms in the antitumor activity and sensitization of pancreatic cancer to gemcitabine. Firstly, we found a potent SIRT6 inhibitor compound 8a by virtual screening and identified by molecular and cellular SIRT6 activity assays. 8a could effectively inhibit SIRT6 deacetylation activity with IC50 values of 7.46 ± 0.79 µM in FLUOR DE LYS assay, and 8a significantly increased the acetylation levels of H3 in cells. Then, we found that 8a could inhibit the cell proliferation and induce cell apoptosis in pancreatic cancer cells. We further demonstrate that 8a sensitize pancreatic cancer cells to gemcitabine via reversing the activation of PI3K/AKT/mTOR and ERK signaling pathways induced by gemcitabine and blocking the DNA damage repair pathway. Moreover, combination of 8a and gemcitabine induces cooperative antitumor activity in pancreatic cancer xenograft model in vivo. Overall, we demonstrate that 8a, a novel SIRT6 inhibitor, could be a promising potential drug candidate for pancreatic cancer treatment.
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Neoplasias Pancreáticas , Sirtuínas , Humanos , Apoptose , Linhagem Celular Tumoral , Gencitabina , Neoplasias Pancreáticas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Pirróis/farmacologia , Pirróis/uso terapêutico , Sirtuínas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that infects patients by regulating virulence factors and biofilms through a quorum sensing (QS) system to protect itself from antibiotics and environmental stress. Therefore, the development of quorum sensing inhibitors (QSIs) is expected to become a new strategy for studying drug resistance to P. aeruginosa infections. Marine fungi are valuable resources for screening QSIs. A marine fungus, Penicillium sp. JH1, with anti-QS activity was isolated from the offshore waters of Qingdao (China), and citrinin, a novel QSI, was purified from secondary metabolites of this fungus. Citrinin could significantly inhibit the production of violacein in Chromobacterium violaceum CV12472 and the production of three virulence factors (elastase, rhamnolipid and pyocyanin) in P. aeruginosa PAO1. It could also inhibit the biofilm formation and motility of PAO1. In addition, citrinin downregulated the transcript levels of nine genes (lasI, rhlI, pqsA, lasR, rhlR, pqsR, lasB, rhlA and phzH) associated with QS. Molecular docking results showed that citrinin bound to PqsR and LasR with better affinity than the natural ligands. This study laid a foundation for the further study of the structure optimization and structure-activity relationship of citrinin.
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Citrinina , Percepção de Quorum , Humanos , Pseudomonas aeruginosa/fisiologia , Citrinina/farmacologia , Simulação de Acoplamento Molecular , Biofilmes , Fatores de Virulência/metabolismo , Antibacterianos/química , Proteínas de Bactérias/metabolismoRESUMO
Introduction: O-GlcNAcylation is a type of reversible post-translational modification on Ser/Thr residues of intracellular proteins in eukaryotic cells, which is generated by the sole O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Thousands of proteins, that are involved in various physiological and pathological processes, have been found to be O-GlcNAcylated. However, due to the lack of favorable tools, studies of the O-GlcNAcylation and OGT were impeded. Immunoglobulin new antigen receptor (IgNAR) derived from shark is attractive to research tools, diagnosis and therapeutics. The variable domain of IgNARs (VNARs) have several advantages, such as small size, good stability, low-cost manufacture, and peculiar paratope structure. Methods: We obtained shark single domain antibodies targeting OGT by shark immunization, phage display library construction and panning. ELISA and BIACORE were used to assess the affinity of the antibodies to the antigen and three shark single-domain antibodies with high affinity were successfully screened. The three antibodies were assessed for intracellular function by flow cytometry and immunofluorescence co-localization. Results: In this study, three anti-OGT VNARs (2D9, 3F7 and 4G2) were obtained by phage display panning. The affinity values were measured using surface plasmon resonance (SPR) that 2D9, 3F7 and 4G2 bound to OGT with KD values of 35.5 nM, 53.4 nM and 89.7 nM, respectively. Then, the VNARs were biotinylated and used for the detection and localization of OGT by ELISA, flow cytometry and immunofluorescence. 2D9, 3F7 and 4G2 were exhibited the EC50 values of 102.1 nM, 40.75 nM and 120.7 nM respectively. VNAR 3F7 was predicted to bind the amino acid residues of Ser375, Phe377, Cys379 and Tyr 380 on OGT. Discussion: Our results show that shark single-domain antibodies targeting OGT can be used for in vitro detection and intracellular co-localization of OGT, providing a powerful tool for the study of OGT and O-GlcNAcylation.
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Anticorpos de Domínio Único , Anticorpos , Acetilglucosaminidase , AminoácidosRESUMO
Alginate lyase has received extensive attention as an important tool for oligosaccharide preparation, pharmaceutical production, and energy biotransformation. Noncatalytic module carbohydrate-binding modules (CBM) have a major impact on the function of alginate lyases. Although the effects of two different families of CBMs on enzyme characteristics have been reported, the effect of two combined CBM32s on enzyme function has not been elucidated. Herein, we cloned and expressed a new multimodular alginate lyase, VxAly7C, from Vibrioxiamenensis QY104, consisting of two CBM32s at N-terminus and a polysaccharide lyase family 7 (PL7) at C-terminus. To explore the function of CBM32s in VxAly7C, full-length (VxAly7C-FL) and two truncated mutants, VxAly7C-TM1 (with the first CBM32 deleted) and VxAly7C-TM2 (with both CBM32s deleted), were characterized. The catalytic efficiency of recombinant VxAly7C-TM2 was 1.82 and 4.25 times higher than that of VxAly7C-TM1 and VxAly7C-FL, respectively, indicating that CBM32s had an antagonistic effect. However, CBM32s improved the temperature stability, the adaptability in an alkaline environment, and the preference for polyG. Moreover, CBM32s contributed to the production of tri- and tetrasaccharides, significantly affecting the end-product distribution. This study advances the understanding of module function and provides a reference for broader enzymatic applications and further enzymatic improvement and assembly.
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Alginatos , Polissacarídeo-Liases , Alginatos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Oligossacarídeos/metabolismo , Polissacarídeo-Liases/metabolismo , Especificidade por Substrato , VibrioRESUMO
A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) was cloned from the genome of Yersinia sp. 298 and subjected to recombinant expression. The specific activity of the recombinase YsHyl8A was 11.19 U/mg, with an optimal reaction temperature of 40 °C and 50% of its specific activity remaining after thermal incubation at 30 °C for 1 h. In addition, its optimal reaction pH was 7.5, and while it was most stable at pH 6.0 in Na2HPO4-citric acid buffer, it remained highly stable at pH 6.0-11.0. Further, its enzymatic activity was increased five-fold with 0.1 M NaCl. YsHyl8A, as an endo-lyase, can degrade both HA and CS, producing disaccharide end-products. These properties suggested that YsHyl8A possessed both significant alkalophilic and cold-adapted features while being dependent on NaCl, likely resulting from its marine source. Yersinia is a typical fish pathogen, with glycosaminoglycan lyase (GAG lyase) as a potential pathogenic factor, exhibiting strong hyaluronidase and chondroitinase activity. Further research on the pathogenic mechanism of GAG lyase may benefit the prevention and treatment of related diseases.
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Glicosaminoglicanos , Liases , Animais , Sulfatos de Condroitina , Ácido Hialurônico/química , Concentração de Íons de Hidrogênio , Polissacarídeo-Liases/química , Cloreto de Sódio , Yersinia/genética , Yersinia/metabolismoRESUMO
Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the ß-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The Km and kcat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s-1, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.
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Proteínas de Bactérias , Gammaproteobacteria/enzimologia , Polissacarídeo-Liases , Polissacarídeos/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Gammaproteobacteria/genética , Conformação Molecular , Filogenia , Polissacarídeo-Liases/química , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/isolamento & purificação , Proteínas Recombinantes/química , Cloreto de Sódio/químicaRESUMO
Glycosaminoglycan lyase is an effective tool for the functional studies of glycosaminoglycans and for the preparation of oligosaccharides. In this study, a new glycosaminoglycan lyase HCLaseV with a molecular weight of 90 kDa was cloned, expressed, and characterized from Vibrio sp. H240. The lyase belonged to the polysaccharide lyase (PL)- 8 family. HCLaseV showed enzyme activities toward chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, and hyaluronic acid. HCLaseV exhibited the highest activity against HA at pH 7.0 and 40 °C. HCLaseV was an endo-type enzyme whose degradation end-product was unsaturated disaccharides. Ca2+ inhibited the activity of HCLaseV to a certain extent, which was different from most of the enzymes in the PL-8 family. Mutagenesis studies showed that the Ca2+ inhibition might be related to the Asn244 residue. The sequence homology was evaluated by mutagenesis studies, and the catalytic residues in HCLaseV were presumed to be His278, Trp485, and Tyr287. These characteristics are helpful for further basic research and application.
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Liases , Vibrio , Clonagem Molecular , Glicosaminoglicanos , Polissacarídeo-Liases/genética , Vibrio/genéticaRESUMO
A new α-agarase AgaE belonging to glycoside hydrolase (GH) family 96 was identified and cloned from marine bacterium Thalassomonas sp. LD5. AgaE consists of 926 amino acids with a theoretical molecular mass of 97 kDa. The optimum temperature and pH for recombinant AgaE were 35 °C and 7.0, respectively. In contrast to known α-agarases, the activity of AgaE does not depend on Ca2+, but on Na+. Thin-layer chromatography and 13C NMR analysis revealed that AgaE endohydrolytic of agarose to produce agarotetraose and agarohexaose as the final main products. Extensive site-directed mutagenesis studies on the conserved carboxylic amino acids of GH96 revealed two essential amino acids for AgaE, D779 and D781. Replacing D779 with G779 leads to complete inactivation of the enzyme, while D781G results in 70% loss of activity. Later studies showed that site D781 involved in the binding of Na+, and its mutation raised the optimal concentration of Na+ 4 times higher than that of the wild type. However, attempts to rescue the mutant's activities with sodium azide were failed. Kinetic parameters comparison of AgaE, AgaD, another α-agarase from LD5, and their mutants revealed that the former aspartic acid plays critical role in the catalysis.
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Aminoácidos Essenciais , Gammaproteobacteria/enzimologia , Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Aminoácidos , Catálise , Gammaproteobacteria/genética , Glicosídeo Hidrolases/genética , Hidrólise , Proteínas Recombinantes , Análise EspectralRESUMO
Alginate oligosaccharides (AOS) and their derivatives become popular due to their favorable biological activity, and the key to producing functional AOS is to find efficient alginate lyases. This study showed one alginate lyase TsAly7A found in Thalassomonas sp. LD5, which was predicted to have excellent industrial properties. Bioinformatics analysis and enzymatic properties of recombinant TsAly7A (rTsAly7A) were investigated. TsAly7A belonged to the fifth subfamily of polysaccharide lyase family 7 (PL7). The optimal temperature and pH of rTsAly7A was 30 °C and 9.1 in Glycine-NaOH buffer, respectively. The pH stability of rTsAly7A under alkaline conditions was pretty good and it can remain at above 90% of the initial activity at pH 8.9 in Glycine-NaOH buffer for 12 h. In the presence of 100 mM NaCl, rTsAly7A showed the highest activity, while in the absence of NaCl, 50% of the highest activity was observed. The rTsAly7A was an endo-type alginate lyase, and its end-products of alginate degradation were unsaturated oligosaccharides (degree of polymerization 2-6). Collectively, the rTsAly7A may be a good industrial production tool for producing AOS with high degree of polymerization.
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Alginatos , Gammaproteobacteria , Polimerização , Alginatos/metabolismo , Cloreto de Sódio , Hidróxido de Sódio , Concentração de Íons de Hidrogênio , Oligossacarídeos/química , Polissacarídeo-Liases/metabolismo , Gammaproteobacteria/metabolismo , Especificidade por Substrato , Proteínas de Bactérias/metabolismoRESUMO
Quorum sensing (QS) is employed by the opportunistic pathogen Pseudomonas aeruginosa to regulate physiological behaviors and virulence. QS inhibitors (QSIs) are potential anti-virulence agents for the therapy of P. aeruginosa infection. During the screening for QSIs from Chinese herbal medicines, falcarindiol (the major constituent of Notopterygium incisum) exhibited QS inhibitory activity. The subinhibitory concentration of falcarindiol exerted significant inhibitory effects on the formation of biofilm and the production of virulence factors such as elastase, pyocyanin, and rhamnolipid. The mRNA expression of QS-related genes (lasB, phzH, rhlA, lasI, rhlI, pqsA, and rhlR) was downregulated by falcarindiol while that of lasR was not affected by falcarindiol. The transcriptional activation of the lasI promoter was inhibited by falcarindiol in the P. aeruginosa QSIS-lasI selector. Further experiments confirmed that falcarindiol inhibited the las system using the reporter strain Escherichia coli MG4/pKDT17. Electrophoretic mobility shift assay (EMSA) showed that falcarindiol inhibited the binding of the transcription factor LasR and the lasI promoter region. Molecular docking showed that falcarindiol interacted with the Tyr47 residue, leading to LasR instability. The decrease of LasR-mediated transcriptional activation was responsible for the reduction of downstream gene expression, which further inhibited virulence production. The inhibition mechanism of falcarindiol to LasR provides a theoretical basis for its medicinal application.
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Apiaceae/química , Di-Inos/farmacologia , Álcoois Graxos/farmacologia , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum , Di-Inos/isolamento & purificação , Álcoois Graxos/isolamento & purificação , Compostos Fitoquímicos/isolamento & purificaçãoRESUMO
Brown algae is a kind of renewable resource for biofuels production. As the major component of carbohydrate in the cell walls of brown algae, alginate can be degraded into unsaturated monosaccharide by exo-type alginate lyases, then converted into 4-deoxy-L-erythro-5-hexoseulose uronate (DEH) by a non-enzyme reaction, which is an important raw material for the preparation of bioethanol. In our research, a novel exo-type alginate lyase, VsAly7D, belonging to the PL7 family was isolated from marine bacterium Vibrio sp. QY108 and recombinantly expressed in Escherichia coli. The purified VsAly7D demonstrated the highest activity at 35 °C, whereas it still maintained 46.5% and 83.1% of its initial activity at 20 °C and 30 °C, respectively. In addition, VsAly7D exhibited the maximum activity under alkaline conditions (pH 8.0), with the simultaneously remaining stability between pH 8.0 and 10.0. Compared with other reported exo-type enzymes, VsAly7D could efficiently degrade alginate, poly-ß-D-mannuronate (polyM) and poly-α-L-guluronate (polyG) with highest specific activities (663.0 U/mg, 913.6 U/mg and 894.4 U/mg, respectively). These results showed that recombinant VsAly7D is a suitable tool enzyme for unsaturated alginate monosaccharide preparation and holds great promise for producing bioethanol from brown algae.
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Alginatos/metabolismo , Polissacarídeo-Liases/metabolismo , Vibrio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Ácido Glucurônico/metabolismo , Concentração de Íons de Hidrogênio , Monossacarídeos/metabolismo , Phaeophyceae/microbiologiaRESUMO
Chondroitinases, catalyzing the degradation of chondroitin sulfate (CS) into oligosaccharides, not only play a crucial role in understanding the structure and function of CS, but also have been reported as a potential candidate drug for the treatment of high CS-related diseases. Here, a marine bacterium Vibrio hyugaensis LWW-1 was isolated, and its genome was sequenced and annotated. A chondroitinase, VhChlABC, was found to belong to the second subfamily of polysaccharide lyase (PL) family 8. VhChlABC was recombinant expressed and characterized. It could specifically degrade CS-A, CS-B, and CS-C, and reached the maximum activity at pH 7.0 and 40 °C in the presence of 0.25 M NaCl. VhChlABC showed high stability within 8 h under 37 °C and within 2 h under 40 °C. VhChlABC was stable in a wide range of pH (5.0~10.6) at 4 °C. Unlike most chondroitinases, VhChlABC showed high surfactant tolerance, which might provide a good tool for removing extracellular CS proteoglycans (CSPGs) of lung cancer under the stress of pulmonary surfactant. VhChlABC completely degraded CS to disaccharide by the exolytic mode. This research expanded the research and application system of chondroitinases.