RESUMEN
The present study aimed to explore the optimal zymogram of combination of 6 carbohydrases (glucoamylase, pullulanase, maltase, thermostable α-amylase, medium temperature α-amylase, and cold-active α-amylase) supplemented in corn-soybean based diet of broilers aged 1 to 3 wk for the maximum starch digestibility, by using in vitro simulated gastrointestinal digestion and response surface method. The third generation of simulated monogastric animal digestion system was used for in vitro digestion experiment. By using single factor completely random design, the optimal supplement levels of single carbohydras were determined by the reducing sugar release amount and improved dry matter digestibility, which were the parameters representing the starch digestibility of the diet. Additionally, Box-Behnken response surface method was used to predict the optimal combination of 6 carbohydrases. The results showed that the optimistic zymogram of 6 carbohydrases in corn-soybean based diet for broilers aged 1 to 3 wk were 297.39 U/g glucoamylase, 549.72 U/g pullulanase, 3.01 U/g maltase, 1,455.73 U/g thermostable α-amylase, 278.64 U/g medium temperature α-amylase, and 1,985.97 U/g cold-active α-amylase, and the associated reduced sugar release amount and improved dry matter digestibility were 215.98 mg/g, and 6.23%, respectively. Furthermore, we conducted in vitro digestion experiments with diets supplemented with the predicted optimistic zymogram and found that the experimental reduced sugar release amount and improved dry matter digestibility were 219.26 mg/g and 6.31% respectively, whose errors to the predicted optimistic reducing sugar release amount and the improved dry matter digestibility were 1.05% and 1.02%. To sum up, the predicted optimal zymogram of 6 carbohydrases in the present study were capable to improve the starch digestibility in diet for broilers aged 1 to 3 wk, which were represented by increased reduced sugar release amount and improved dry matter digestibility.
Asunto(s)
Glicósido Hidrolasas/administración & dosificación , Almidón/química , Alimentación Animal , Animales , Pollos , Suplementos Dietéticos , Glicósido Hidrolasas/farmacología , Técnicas In Vitro , Glycine max/química , Zea mays/químicaRESUMEN
(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni-NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0-50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20-30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn2+ and Co2+. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.
Asunto(s)
Quitosano/química , Glicósido Hidrolasas/farmacología , Oligosacáridos/química , Renibacterium , Animales , Organismos Acuáticos , Frío , Glicósido Hidrolasas/química , HumanosRESUMEN
Biofilm formation is often attributed to postharvest bacterial persistence on fresh produce and food handling surfaces. In this study, a predicted glycosyl hydrolase enzyme was expressed, purified, and validated for the removal of microbial biofilms from biotic and abiotic surfaces under conditions used for chemical cleaning agents. Crystal violet biofilm staining assays revealed that 0.1 mg/ml of enzyme inhibited up to 41% of biofilm formation by Escherichia coli O157:H7, E. coli 25922, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Furthermore, the enzyme was effective at removing mature biofilms, providing a 35% improvement over rinsing with a saline solution alone. Additionally, a parallel-plate flow cell was used to directly observe and quantify the impact of enzyme rinses on E. coli O157:H7 cells adhering to spinach leaf surfaces. The presence of 1 mg/liter enzyme resulted in nearly 6-times-higher detachment rate coefficients than a deionized (DI) water rinse, while the total cells removed from the surface increased from 10% to 25% over the 30-min rinse time, reversing the initial phases of biofilm formation. Enzyme treatment of all 4 cell types resulted in significantly reduced cell surface hydrophobicity and collapse of negatively stained E. coli 25922 cells imaged by electron microscopy, suggesting potential polysaccharide surface modification of enzyme-treated bacteria. Collectively, these results point to the broad substrate specificity and robustness of the enzyme for different types of biofilm stages, solution conditions, and pathogen biofilm types and may be useful as a method for the removal or inhibition of bacterial biofilm formation. IMPORTANCE In this study, the ability of an engineered enzyme to reduce bacterial adhesion and biofilm formation of several foodborne pathogens was demonstrated, representing a promising option for enhancing or replacing chlorine and other chemical sanitizers in food processing applications. Specifically, significant reductions of biofilms of the pathogens Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes are observed, as are reductions in initial adhesion. Enzymes have the added benefits of being green, sustainable alternatives to chemical sanitizers, as well as having a minimal impact on food properties, in contrast to many alternative antimicrobial options such as bleach that aim to minimize food safety risks.
Asunto(s)
Escherichia coli/efectos de los fármacos , Glicósido Hidrolasas/farmacología , Listeria monocytogenes/efectos de los fármacos , Salmonella typhimurium/efectos de los fármacos , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Escherichia coli/fisiología , Escherichia coli/ultraestructura , Manipulación de Alimentos/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Listeria monocytogenes/fisiología , Hojas de la Planta/microbiología , Salmonella typhimurium/fisiología , Spinacia oleracea/microbiologíaRESUMEN
Multi-Drug Resistant (MDR) uropathogenic bacteria have increased in number in recent years and the development of new treatment options for the corresponding infections has become a major challenge in the field of medicine. In this respect, recent studies have proposed bacteriophage (phage) therapy as a potential alternative against MDR Urinary Tract Infections (UTI) because the resistance mechanism of phages differs from that of antibiotics and few side effects have been reported for them. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are the most common uropathogenic bacteria against which phage therapy has been used. Phages, in addition to lysing bacterial pathogens, can prevent the formation of biofilms. Besides, by inducing or producing polysaccharide depolymerase, phages can easily penetrate into deeper layers of the biofilm and degrade it. Notably, phage therapy has shown good results in inhibiting multiple-species biofilm and this may be an efficient weapon against catheter-associated UTI. However, the narrow range of hosts limits the use of phage therapy. Therefore, the use of phage cocktail and combination therapy can form a highly attractive strategy. However, despite the positive use of these treatments, various studies have reported phage-resistant strains, indicating that phage-host interactions are more complicated and need further research. Furthermore, these investigations are limited and further clinical trials are required to make this treatment widely available for human use. This review highlights phage therapy in the context of treating UTIs and the specific considerations for this application.
Asunto(s)
Bacterias/virología , Bacteriófagos/fisiología , Terapia de Fagos , Infecciones Urinarias/microbiología , Infecciones Urinarias/terapia , Animales , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Farmacorresistencia Bacteriana Múltiple , Glicósido Hidrolasas/farmacología , Especificidad del Huésped , Humanos , Klebsiella pneumoniae/virología , Proteus mirabilis/virología , Escherichia coli Uropatógena/virologíaRESUMEN
Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC-MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.
Asunto(s)
Antifúngicos , Bacillus subtilis/enzimología , Proteínas Bacterianas , Fusarium/crecimiento & desarrollo , Glicósido Hidrolasas , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/farmacologíaRESUMEN
Cyclodextrinase (CDase) and cyclodextrin glucosyltransferase (CGTase) were synergistically used to provide a novel enzymatic method in lowing in vitro digestibility of waxy maize starch. The molecular structure, malto-oligosaccharide composition, and digestibility properties of the generated products were investigated. The molecular weight was reduced to 0.3 × 105 g/mol and 0.2 × 105 g/mol by simultaneous and sequential treatment with CDase and CGTase, while the highest proportion of chains with degree of polymerization (DP) < 13 was obtained by simultaneous treatment. The resistant starch contents were increased to 27.5% and 36.9% by simultaneous and sequential treatments respectively. Dual-enzyme treatment significantly promoted the content of malto-oligosaccharides (MOSs) by hydrolyzing cyclodextrins from CGTase with CDase. However, the replacement of cyclodextrins by MOSs did not obviously influence the digestibility of the products. The starch digestion kinetics further revealed the hydrolysis pattern of these two enzymes on the starch hydrolysate. It was proved that the starch digestibility could be lowered by modulating the molecular structure and beneficial MOSs content by this dual-enzyme treatment.
Asunto(s)
Amilopectina/química , Amilopectina/metabolismo , Glucosiltransferasas/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Glicósido Hidrolasas/metabolismo , Oligosacáridos/análisis , Zea mays/química , Ciclodextrinas/metabolismo , Digestión , Sinergismo Farmacológico , Glucosiltransferasas/farmacología , Sistema de la Enzima Desramificadora del Glucógeno/farmacología , Glicósido Hidrolasas/farmacología , Hidrólisis/efectos de los fármacos , Cinética , Estructura Molecular , Peso Molecular , PolimerizacionRESUMEN
Hospital-acquired infection is a great challenge for clinical treatment due to pathogens' biofilm formation and their antibiotic resistance. Here, we investigate the effect of antiseptic agent polyhexamethylene biguanide (PHMB) and undecylenamidopropyl betaine (UB) against biofilms of four pathogens that are often found in hospitals, including Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, Gram-positive bacteria Staphylococcus aureus, and pathogenic fungus, Candida albicans. We show that 0.02% PHMB, which is 10-fold lower than the concentration of commercial products, has a strong inhibitory effect on the growth, initial attachment, and biofilm formation of all tested pathogens. PHMB can also disrupt the preformed biofilms of these pathogens. In contrast, 0.1% UB exhibits a mild inhibitory effect on biofilm formation of the four pathogens. This concentration inhibits the growth of S. aureus and C. albicans yet has no growth effect on P. aeruginosa or E. coli. UB only slightly enhances the anti-biofilm efficacy of PHMB on P. aeruginosa biofilms. However, pretreatment with PslG, a glycosyl hydrolase that can efficiently inhibit and disrupt P. aeruginosa biofilm, highly enhances the clearance effect of PHMB on P. aeruginosa biofilms. Meanwhile, PslG can also disassemble the preformed biofilms of the other three pathogens within 30 min to a similar extent as UB treatment for 24 h.
Asunto(s)
Betaína/farmacología , Biguanidas/farmacología , Biopelículas/efectos de los fármacos , Desinfectantes/farmacología , Glicósido Hidrolasas/farmacología , Pseudomonas aeruginosa/enzimología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/prevención & control , Betaína/análogos & derivados , Candida albicans/efectos de los fármacos , Candida albicans/fisiología , Candidiasis/prevención & control , Infección Hospitalaria/prevención & control , Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Ácidos Undecilénicos/química , Ácidos Undecilénicos/farmacologíaRESUMEN
Two methods, HCl and enzymatic treatments, were evaluated for diversification of morphological and functional properties of cellulose nanofibers (CNF) from two- stage-alkaline pre-treated wheat straw (WS). The extraction conditions were optimized by a central composite designed experimental approach varying time (4-8 h) and temperature (80-120 °C) for the HCl-based treatment and time (4-8 h), and FiberCare dosage (50-100 endo-1,4-ß-glucanase unit/g) and Viscozyme (10-20 fungal ß-glucanase units/g) for the enzyme-based treatment. The CNF yields, morphological (polydispersity index (PdI), length and diameter), and functional (crystallinity and thermal degradation) properties were compared. The CNF produced by the HCl (HCN) and enzymatically (ECN) attained diameters ~17 nm had PdI, length, and crystallinity of 0.53, 514 nm & 70%, and 0.92, 1.0 µm & 48%, respectively. Thus, the HCN morphology suits homogenous nano-applications, whereas that of the ECN, would suit heterogenous nano-applications. The HCN and ECN yields were similar (~20%) with optimal production time of 7.41 and 4.64 h, respectively. Both the HCN & ECN can be classified as thermally stable nanocolloids with maximum thermal degradation temperatures of ~380 °C and Zeta potential ~-16 mV. The two CNF production methods have potential synergetic effects on CNF production, morphological, and functional properties.
Asunto(s)
Celulosa/aislamiento & purificación , Nanofibras/química , Celulasas/farmacología , Celulosa/química , Coloides/química , Cristalización , Proteínas Fúngicas/farmacología , Glicósido Hidrolasas/farmacología , Calor , Ácido Clorhídrico/farmacología , Complejos Multienzimáticos/farmacología , Tallos de la Planta/química , Tallos de la Planta/efectos de los fármacos , Electricidad Estática , Triticum/químicaRESUMEN
Implanted medical devices such as central venous catheters are highly susceptible to microbial colonization and biofilm formation and are a major risk factor for nosocomial infections. The opportunistic pathogen Pseudomonas aeruginosa uses exopolysaccharides, such as Psl, for both initial surface attachment and biofilm formation. We have previously shown that chemically immobilizing the Psl-specific glycoside hydrolase, PslGh, to a material surface can inhibit P. aeruginosa biofilm formation. Herein, we show that PslGh can be uniformly immobilized on the lumen surface of medical-grade, commercial polyethylene, polyurethane, and polydimethylsiloxane (silicone) catheter tubing. We confirmed that the surface-bound PslGh was uniformly distributed along the catheter length and remained active even after storage for 30 days at 4 °C. P. aeruginosa colonization and biofilm formation under dynamic flow culture conditions in vitro showed a 3-log reduction in the number of bacteria during the first 11 days, and a 2-log reduction by day 14 for PslGh-modified PE-100 catheters, compared to untreated catheter controls. In an in vivo rat infection model, PslGh-modified PE-100 catheters showed a â¼1.5-log reduction in the colonization of the clinical P. aeruginosa ATCC 27853 strain after 24 h. These results demonstrate the robust ability of surface-bound glycoside hydrolase enzymes to inhibit biofilm formation and their potential to reduce rates of device-associated infections.
Asunto(s)
Catéteres Venosos Centrales , Pseudomonas aeruginosa , Animales , Biopelículas , Catéteres de Permanencia/microbiología , Glicósido Hidrolasas/farmacología , RatasRESUMEN
We recently identified a ß-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. Gaa16B, belonging to the glycoside hydrolase 16 family of ß-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. Recombinant Gaa16B lacking the carbohydrate-binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 ∘C and pH 6-7, respectively, and the protein was highly stable at 55 ∘C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl2. The Km and Vmax of rGaa16Bc for agarose were 6.4 mg/mL and 953 U/mg, respectively. Thin-layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose and neoagarobiose. Partial hydrolysis products (PHPs) of rGaa16Bc had an average molecular weight of 88-102 kDa and exhibited > 60% hyaluronidase inhibition activity at a concentration of 1 mg/mL, whereas the completely hydrolyzed product (CHP) showed no hyaluronidase at the same concentration. The biochemical properties of Gaa16B suggest that it could be useful for producing functional neoagaro-oligosaccharides. Additionally, the PHP of rGaa16Bc may be useful in promoting its utilization, which is limited due to the gel strength of agar.
Asunto(s)
Gammaproteobacteria , Glicósido Hidrolasas/farmacología , Animales , Organismos Acuáticos , Cosmecéuticos , Glicósido Hidrolasas/química , Concentración de Iones de Hidrógeno , HidrólisisRESUMEN
One of the potential antibiofilm strategies is to use lytic phages and phage-derived polysaccharide depolymerases. The idea is to uncover bacteria embedded in the biofilm matrix making them accessible and vulnerable to antibacterials and the immune system. Here we present the antibiofilm efficiency of lytic phage KP34 equipped with virion-associated capsule degrading enzyme (depolymerase) and its recombinant depolymerase KP34p57, depolymerase-non-bearing phage KP15, and ciprofloxacin, separately and in combination, using a multidrug-resistant K. pneumoniae biofilm model. The most effective antibiofilm agents were (1) phage KP34 alone or in combination with ciprofloxacin/phage KP15, and (2) depolymerase KP34p57 with phage KP15 and ciprofloxacin. Secondly, applying the commonly used biofilm microtiter assays: (1) colony count, (2) LIVE/DEAD BacLight Bacterial Viability Kit, and (3) crystal violet (CV) biofilm staining, we unravelled the main advantages and limitations of the above methods in antibiofilm testing. The diverse mode of action of selected antimicrobials strongly influenced obtained results, including a false positive enlargement of biofilm mass (CV staining) while applying polysaccharide degrading agents. We suggest that to get a proper picture of antimicrobials' effectiveness, multiple examination methods should be used and the results must be read considering the principle of each technique and the antibacterial mechanism.
Asunto(s)
Bacteriófagos/enzimología , Biopelículas/efectos de los fármacos , Descubrimiento de Drogas/métodos , Glicósido Hidrolasas/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/fisiología , Proteínas Virales/farmacología , Antibacterianos/farmacología , Cápsulas Bacterianas/efectos de los fármacos , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Klebsiella pneumoniae/virología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Virión/enzimologíaRESUMEN
PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.
Asunto(s)
Proteínas Bacterianas/farmacología , Glicósido Hidrolasas/farmacología , Paenibacillus/enzimología , Infecciones Neumocócicas/tratamiento farmacológico , Streptococcus pneumoniae/efectos de los fármacos , Animales , Cápsulas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/uso terapéutico , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Infecciones Neumocócicas/microbiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
Bacterial cellulose (BC) is recognized as a wound dressing material well-suited for chronic wounds; however, it has no intrinsic antimicrobial activity. Further, the formation of biofilms can limit the effectiveness of the pre-saturation of BC with antimicrobial agents. Here, to hinder biofilm formation by P. aeruginosa, we immobilized the hydrolytic domain of PelA (a glycohydrolase involved in the synthesis of biofilm polysaccharide Pel) on the surface of BC. The immobilization of 32.35⯱â¯1.05â¯mg PelAh per g BC membrane resulted in an eight-fold higher P. aeruginosa cell detachment from BC membrane, indicating reduced biofilm matrix stability. Further, 1D and 2D infrared spectroscopy analysis indicated systematic reduction of polysaccharide biofilm elements, confirming the specificity of immobilized PelAh. Importantly, BC-PelAh was not cytotoxic towards L929 fibroblast cells. Thus, we conclude that PelAh can be used in BC wound dressings for safe and specific protection against biofilm formation by P. aeruginosa.
Asunto(s)
Acetobacteraceae/química , Vendajes , Biopelículas/efectos de los fármacos , Celulosa/química , Glicósido Hidrolasas/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Acetobacteraceae/fisiología , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Biopelículas/crecimiento & desarrollo , Línea Celular , Celulosa/biosíntesis , Celulosa/aislamiento & purificación , Clonación Molecular , Enzimas Inmovilizadas/biosíntesis , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/genética , Ratones , Dominios Proteicos , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Brain and other nervous system cancers are the 10th leading cause of death worldwide. Genome instability, cell cycle deregulation, epigenetic mechanisms, cytoarchitecture disassembly, redox homeostasis as well as apoptosis are involved in carcinogenesis. A diet rich in fruits and vegetables is inversely related with the risk of developing cancer. Several studies report that cruciferous vegetables exhibited antiproliferative effects due to the multi-pharmacological functions of their secondary metabolites such as isothiocyanate sulforaphane deriving from the enzymatic hydrolysis of glucosinolates. We treated human astrocytoma 1321N1 cells for 24 h with different concentrations (0.5, 1.25 and 2.5% v/v) of sulforaphane plus active myrosinase (Rapha Myr®) aqueous extract (10 mg/mL). Cell viability, DNA fragmentation, PARP-1 and γH2AX expression were examined to evaluate genotoxic effects of the treatment. Cell cycle progression, p53 and p21 expression, apoptosis, cytoskeleton morphology and cell migration were also investigated. In addition, global DNA methylation, DNMT1 mRNA levels and nuclear/mitochondrial sirtuins were studied as epigenetic biomarkers. Rapha Myr® exhibited low antioxidant capability and exerted antiproliferative and genotoxic effects on 1321N1 cells by blocking the cell cycle, disarranging cytoskeleton structure and focal adhesions, decreasing the integrin α5 expression, renewing anoikis and modulating some important epigenetic pathways independently of the cellular p53 status. In addition, Rapha Myr® suppresses the expression of the oncogenic p53 mutant protein. These findings promote Rapha Myr® as a promising chemotherapeutic agent for integrated cancer therapy of human astrocytoma.
Asunto(s)
Anoicis/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Astrocitoma/metabolismo , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Proteínas de Neoplasias/metabolismo , Sirtuinas/metabolismo , Astrocitoma/tratamiento farmacológico , Astrocitoma/patología , Línea Celular Tumoral , Glicósido Hidrolasas/farmacología , Humanos , Isotiocianatos/farmacología , SulfóxidosRESUMEN
Xylosidases with tolerance to high concentration of salts, organic solvents, and enzyme hydrolytic products are preferential for industrial application but were rarely reported. In this study, a novel xylosidase XYL21 belong to glycoside hydrolase 39 was characterized with optimal temperature of 45 °C and optimal pH of 5.50. Different to other GH39 xylosidases, XYL21 had excellent tolerance to salts, the activity of which is not inhibited but slightly increased in 0.50-1.50 M NaCl. It is also tolerant to organic solvents, especially retaining 105.18% relative activity even in the presence of 15.00% (v/v) ethanol. Moreover, XYL21 was insensitive to the final lignocellulose hydrolysis products including glucose, xylose, arabinose, mannose and galactose, which retains 111.36% and 53.49% relative activity in 0.30 and 0.90 M xylose, respectively. Further structural modeling analysis indicated that its excellent tolerance may be attributed to its high structural flexibility caused by the high proportion of random coils. Furthermore, XYL21 had a wide substrate specificity to catalyze xylan and xylo-oligosaccharides, and it significantly cooperated with xylanase to improve the hydrolysis efficiency with 1.52-fold. Considering these unique properties, XYL21 is a good candidate for both basic research and various potential industrial applications such as seafood processing and bioethanol production.
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Proteínas Bacterianas/aislamiento & purificación , Glicósido Hidrolasas/aislamiento & purificación , Suelo/química , Alcoholes/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Clonación Molecular , Sinergismo Farmacológico , Genes Bacterianos , Inhibidores de Glicósido Hidrolasas/farmacología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/farmacología , Concentración de Iones de Hidrógeno , Hidrólisis , Lignina/metabolismo , Modelos Moleculares , Monosacáridos/farmacología , Conformación Proteica , Tolerancia a la Sal , Sales (Química)/farmacología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Cloruro de Sodio/farmacología , Microbiología del Suelo , Solventes/farmacología , Especificidad por Sustrato , Temperatura , Xilanos/metabolismoRESUMEN
Inulinases are inulin catalyzing enzymes which belongs to glycoside hydrolases (GH) family 32. Bacteria, fungi and yeasts are the potential sources of inulinases. In the present biotechnological era, inulinases are gaining considerable attention, due to their wide range of applications which includes the production of high fructose syrup, fructooligosaccharides and many other important metabolites like bioethanol, organic acids, single cell oil, 2,3-butanediol, single cell proteins, etc. These applications of inulinases have attracted the researchers world-wide to understand the inulin-inulinase interactions for polyfructan hydrolysis. To understand these interactions, the information on structural organization of inulinases is very important which is scarce in literature. The current review highlights the structural and functional properties of inulinases, and difference in their structural organization. The biotechnological potential of inulinases for the production of different bio-products from inulin/inulin-rich raw materials using different bioprocessing strategies has also been elaborated.
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Biotecnología/métodos , Glicósido Hidrolasas , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Biocombustibles , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/farmacología , Jarabe de Maíz Alto en Fructosa/metabolismo , Microbiología Industrial , Inulina/metabolismo , Modelos Moleculares , Oligosacáridos/metabolismo , Compuestos Orgánicos/metabolismo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The genes encoding α-1,3-glucanases (Agls; AglST1 and AglST2) from Streptomyces thermodiastaticus HF3-3 were cloned and were then expressed in Escherichia coli Rosetta-gami B (DE3). We purified the resultant histidine (His)-tagged α-1,3-glucanases (recombinant enzymes, rAglST1 and rAglST2). Both the recombinant enzymes were similar to the wild-type enzymes. We examined the effects of rAglST1 and rAglST2 on the formation and degradation of biofilms on glass plates with Streptococcus mutans NRBC 13955 by evaluating the biofilm content (%), release of reducing sugar (mM), release of S. mutans (log CFU/mL), and the biofilm structure using laser scanning microscopy (LSM). The results showed that after incubation for 16 h, rAglST1 and rAglST2 reduced the formation of biofilm to 52% and 49% of the control, respectively. The result may reflect the fact that the concentration of the reducing sugar and the number of S. mutans cells in the rAglATs-added medium were higher than in the control medium. After an 8-h treatment with rAglST1 and rAglST2, biofilms decreased to less than 60% of the control. The number of S. mutans cells in the reaction mixture gradually increased during the incubation period. The enzymes can degrade the biofilms that were pre-formed on the glass plate by more than 50% after a 30-min incubation in the presence of toothpaste ingredients (1% w/v of sodium fluoride, benzethonium chloride, and sodium dodecyl sulfate) at 50°C. Our study showed that rAglST1 and rAglST2 have advantageous properties for dental care applications.
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Proteínas Bacterianas/farmacología , Biopelículas/efectos de los fármacos , Glicósido Hidrolasas/farmacología , Streptomyces/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/crecimiento & desarrollo , Streptococcus mutans/metabolismo , Streptomyces/genética , Temperatura , Pastas de Dientes/químicaRESUMEN
BACKGROUND Laminaria japonica polysaccharide (LJP), a fucose enriched sulfated polysaccharide has been demonstrated to have excellent anticoagulant and antithrombotic activities. However, the antithrombotic effect of low molecular weight polysaccharide from enzymatically modified of LJP (LMWEP) remains unknown. MATERIAL AND METHODS LMWEP was prepared by fucoidanase enzymatic hydrolysis, and the antithrombotic and anticoagulant activities, and the underlying mechanism were investigated thoroughly. Rats were randomly divided into 6 groups (8 rats in each group): the blank control group, the blank control group treated with LMWEP (20 mg/kg), the model group, the model group treated with heparin (2 mg/kg), the model group treated with LJP (20 mg/kg), and the model group treated with LMWEP (20 mg/kg). After 7 days of intravenous administration, blood was collected for biochemical parameters examinations. RESULTS LMWEP increased the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), 6-keto prostaglandin F1alpha (6-Keto-PGF1alpha), and endothelial nitric oxide synthase (eNOS). In addition, LMWEP decreased fibrinogen (FIB), endothelin-1 (ET-1), thromboxane B2 (TXB2), erythrocyte sedimentation rate (ESR), and hematocrit (HCT). CONCLUSIONS LMWEP, an enzymatically modified fragment with a molecular weight of 25.8 kDa, is a potential antithrombotic candidate for treatment of thrombosis related diseases.
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Fibrinolíticos/farmacología , Laminaria/química , Medicina Tradicional China/métodos , Animales , Anticoagulantes/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Pruebas de Coagulación Sanguínea/métodos , Glicósido Hidrolasas/farmacología , Laminaria/efectos de los fármacos , Laminaria/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo III/sangre , Tiempo de Tromboplastina Parcial/métodos , Polisacáridos/farmacología , Tiempo de Protrombina/métodos , Ratas , Ratas Sprague-Dawley , Trombosis/sangreRESUMEN
Bacteria forming biofilms on surgical implants is a problem that might be alleviated by the use of antibacterial coatings. In this article, recombinant spider silk was functionalized with the peptidoglycan degrading endolysin SAL-1 from the staphylococcal bacteriophage SAP-1 and the biofilm-matrix-degrading enzyme Dispersin B from Aggregatibacter actinomycetemcomitans using direct genetic fusion and/or covalent protein-protein fusion catalyzed by Sortase A. Spider silk assembly and enzyme immobilization was monitored using quartz crystal microbalance analysis. Enzyme activity was investigated both with a biochemical assay using cleavage of fluorescent substrate analogues and bacterial assays for biofilm degradation and turbidity reduction. Spider silk coatings functionalized with SAL-1 and Disperin B were found to exhibit bacteriolytic effect and inhibit biofilm formation, respectively. The strategy to immobilize antibacterial enzymes to spider silk presented herein show potential to be used as surface coatings of surgical implants and other medical equipment to avoid bacterial colonization.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Proteínas Bacterianas/farmacología , Biopelículas/efectos de los fármacos , Glicósido Hidrolasas/farmacología , Seda/farmacología , Bacterias/crecimiento & desarrollo , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/genética , Bacteriófagos/metabolismo , Biopelículas/crecimiento & desarrollo , Glicósido Hidrolasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Seda/genética , Proteínas Virales/genéticaRESUMEN
The aim of this study was to investigate the effect on growth performance and nutrient utilisation when supplementing diets deficient in energy and protein with carbohydrase enzymes or xylo-oligosaccharide in broilers challenged with coccidia. 960 Ross 308 broilers were used in this 21-day study. The treatments were arranged into a 2×4 factorial with 2 challenge states (challenged and non-challenged) and 4 different additive types (control, xylanase alone, xylanase and ß-glucanase mixture and xylo-oligosaccharide). On day 14, the challenged group received 12× the recommended dose of coccidiosis vaccine while the non-challenged group received a sham treatment of water only. The birds and feed were weighed on days 0, 14 and 21. On day 21, two birds per pen were euthanized, the caeca were removed and the contents collected for short chain fatty acid analysis. Six more birds per pen were euthanized and ileal digesta were collected and pooled per pen for nutrient digestibility analysis. Feed intake was greater (P < 0.05) on days 14 and 21 when xylo-oligosaccharide was included in the diet compared to the xylanase and ß-glucanase mixture in birds challenged with coccidiosis. Including xylo-oligosaccharide in the diet improved (P < 0.05) the digestibility of nitrogen and supplementing diets with the xylanase and ß-glucanase mixture improved (P < 0.05) the digestibility of several amino acids. The concentration of arabinose and xylose was (P < 0.001) greater when broiler diets were supplemented with carbohydrase enzymes or xylo-oligosaccharide compared to the control. Although there was an increase in short chain fatty acid production due to the addition of carbohydrase enzymes or xylo-oligosaccharide, there was no additive effect on the %G+C profile of caecal bacteria however there was a negative effect of coccidiosis. In conclusion, the similarity in the response to carbohydrase enzymes or xylo-oligosaccharide supplementation illustrates that the hydrolysis products from carbohydrase activity may have prebiotic like effects.