RESUMO
Every 1.2 s, a diabetic foot ulcer is developed, and every 20 s, one amputation is carried out in diabetic patients. Monitoring and controlling protease activity have been considered as a strategy for more efficient management of diabetic and other chronic wounds. This study aimed to develop a casein-based dressing that, by its disappearance, provides information about the activity of proteases and simultaneously harnesses proteolytic activity. Casein films were fabricated by using an aqueous solution, and heat treatment was successfully deployed as a green and clean approach to confer hydrolytic stability. Our results showed that casein-based films' mechanical characteristics, water absorption, and proteolytic stability could be controlled by the length of the heat treatment, which proved to be a useful tool. An increase in the treatment duration from 30 min to 3 h led to toleration of 2.4 times higher stress, 2 times lower water uptake, and 3.4 times higher proteolytic stability at examined conditions. Selected casein-based structures responded to Bacillus sp. bacteria's protease (BSP) and human neutrophil elastase (HNE) as representatives of bacterial and nonbacterial proteases found in the wounds at 10 and 200 ng mL-1 levels, respectively. The hydrolysis was accompanied by a 36% reduction in proteolytic activity measured by using a casein-based universal protease activity assay. The released casein fragments could scavenge 90% of the examined radicals. In-vitro cell culture studies showed that the hydrolysates were not cytotoxic, and the casein-based film had a favorable interaction with fibroblast cells, indicating its potential as a scaffold in the case that proteolytic activity would not be to the extent that causes its rapid disintegration. In general, these findings hold promise for applying the developed casein-based structure for detecting proteolytic activity without the need for any equipment, kits, or expertise and, more importantly, in a highly economical manner. In the case that the proteolytic activity would not be severe, it could also serve as a substrate for cell adhesion and growth; this would aid in the healing process.
Assuntos
Caseínas , Pé Diabético , Humanos , Peptídeo Hidrolases/metabolismo , Bandagens , Pé Diabético/terapia , Pé Diabético/diagnóstico , ÁguaRESUMO
Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.
Assuntos
Proteínas de Bactérias , Hidrolases de Éster Carboxílico , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Cinética , Simulação de Acoplamento Molecular , Polietilenotereftalatos/metabolismo , Especificidade por Substrato , Thermobifida/enzimologiaRESUMO
A novel strategy for improving wet resistance and bonding properties of starch-based adhesives using enzymatically polymerized lignosulfonates and carboxylic acids as additives was developed. Therefore, lignosulfonates were polymerized by laccase to a molecular weight of 750 kDa. Incorporation of low concentrations (up to 1% of the starch weight) of 1,2,3,4-butanetetracarboxylic acid (BTCA) led to further improvement on the properties of the adhesives, while addition of greater amounts of BTCA led to a decrease in the properties measured due to large viscosity increases. Great improvements in wet-resistance from 22 to 60 min and bonding times (from 30 to 20 s) were observed for an adhesive containing 8% enzymatically polymerized lignin and 1% BTCA. On the other hand, the addition of citric acid (CA) deteriorated the properties of the adhesives, especially when lignosulfonate was present. In conclusion, this study shows that the addition of the appropriate amount of enzymatically polymerized lignosulfonates together with carboxylic acids (namely BTCA) to starch-based adhesives is a robust strategy for improving their wet resistance and bonding times.
Assuntos
Adesivos , Lignina , Lignina/metabolismo , Amido , Ácidos CarboxílicosRESUMO
In the present work, different hydrolases were adsorbed onto polypropylene beads to investigate their activity both in short-esters and polyesters synthesis. The software MODDE® Pro 13 (Sartorius) was used to develop a full-factorial design of experiments (DoE) to analyse the thermostability and selectivity of the immobilized enzyme towards alcohols and acids with different chain lengths in short-esters synthesis reactions. The temperature optima of Candida antarctica lipase B (CaLB), Humicola insolens cutinase (HiC), and Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) were 85 °C, 70 °C, and 50 °C. CaLB and HiC preferred long-chain alcohols and acids as substrate in contrast to Thc_Cut1, which was more active on short-chain monomers. Polymerization of different esters as building blocks was carried out to confirm the applicability of the obtained model on larger macromolecules. The selectivity of both CaLB and HiC was investigated and best results were obtained for dimethyl sebacate (DMSe), leading to polyesters with a Mw of 18 kDa and 6 kDa. For the polymerization of dimethyl adipate (DMA) with BDO and ODO, higher molecular masses were obtained when using CaLB onto polypropylene beads (CaLB_PP) as compared with CaLB immobilized on macroporous acrylic resin beads (i.e., Novozym 435). Namely, for BDO the Mn were 7500 and 4300 Da and for ODO 8100 and 5000 Da for CaLB_PP and for the commercial enzymes, respectively. Thc_Cut1 led to polymers with lower molecular masses, with Mn < 1 kDa. This enzyme showed a temperature optimum of 50 °C with 63% of DMA and BDO when compared to 54% and 27%, at 70 °C and at 85 °C, respectively.
Assuntos
Ésteres/síntese química , Aromatizantes/síntese química , Poliésteres/síntese química , Biocatálise , Candida/enzimologia , Hidrolases de Éster Carboxílico/química , Enzimas Imobilizadas/química , Gênero de Fungos Humicola/enzimologia , Proteínas Fúngicas/química , Lipase/química , Polimerização , Thermobifida/enzimologiaRESUMO
Modification of kraft lignin (KL), traditionally uses harsh and energy-demanding physical and chemical processes. In this study, the potential of the bacterial laccase CotA (spore coating protein A) for oxidation of KL under mild conditions was assessed. Thereby, the efficiency of CotA to oxidize both softwood and hardwood KL of varying purity at alkaline conditions was examined. For the respective type of wood, the highest oxidation activity by CotA was determined for the medium ash content softwood KL (MA_S) and the medium ash content hardwood KL (MA_H), respectively. By an up to 95% decrease in fluorescence and up to 65% in phenol content coupling of the structural lignin units was indicated. These results correlated with an increase in viscosity and molecular weight, which increased nearly 2 and 20-fold for MA_H and about 1.3 and 6.0-fold for MA_S, respectively. Thus, this study confirms that the CotA laccase can oxidize a variety of KL at alkaline conditions, while the origin and purity of KL were found to have a major impact on the efficiency of oxidation. Under the herein tested conditions, it was observed that the MA_H KL showed the highest susceptibility to CotA oxidation when compared to the other hardwood KLs and the softwood KLs. Therefore, this could be a viable method to produce sustainable resins and adhesives.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Lacase/metabolismo , Lignina/química , Proteínas de Bactérias/genética , Lacase/genética , Peso Molecular , OxirreduçãoRESUMO
Hazardous waste disposal via incineration generates a substantial amount of ashes and slags which pose an environmental risk due to their toxicity. Currently, these residues are deposited in landfills with loss of potentially recyclable raw material. In this study, the use of acidophilic bioleaching bacteria (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, and Leptospirillum ferrooxidans) as an environmentally friendly, efficient strategy for the recovery of valuable metals from incineration residues was investigated. Zinc, Cobalt, Copper, and Manganese from three different incineration residues were bio-extracted up to 100% using A. ferrooxidans under ferrous iron oxidation. The other metals showed lower leaching efficiencies based on the type of culture used. Sulfur-oxidizing cultures A. ferrooxidans and A. thiooxidans, containing sulfur as the sole substrate, expressed a significantly lower leaching efficiency (up to 50%). According to ICP-MS, ashes and slags contained Fe, Zn, Cu, Mn, Cr, Cd, and Ni in economically attractive concentrations between 0.2 and 75 mg g-1. Compared to conventional hydrometallurgical and pyrometallurgical processes, our biological approach provides many advantages such as: the use of a limited amount of used strong acids (H2SO4 or HCl), recycling operations at lower temperatures (~30 °C) and no emission of toxic gases during combustion (i.e., dioxins and furans).
Assuntos
Acidithiobacillus , Incineração , Bactérias , Ferro , Oxirredução , EnxofreRESUMO
Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.
Assuntos
Ascorbato Oxidase/metabolismo , Aspergillus flavus/enzimologia , Lacase/metabolismo , Sequência de Aminoácidos , Ascorbato Oxidase/química , Cobre/metabolismo , Cinética , Lacase/química , Modelos Moleculares , Oxirredução , Especificidade por SubstratoRESUMO
The folate antagonist methotrexate is a cytotoxic drug used in the treatment of several cancer types. The entry of methotrexate into the cell is mediated by two main transport systems: the reduced folate carrier and membrane-associated folate receptors. These transporters differ considerably in their mechanism of (anti)folate uptake, substrate specificity, and tissue specificity. Although the mechanism of action of the reduced folate carrier is fairly well-established, that of the folate receptor has remained unknown. The development of specific folate receptor-targeted antifolates would be accelerated if additional mechanistic data were to become available. In this work, we used two fluorescently labeled conjugates of methotrexate, differently linked at the terminal groups, to clarify the uptake mechanism by folate receptor-α. The results demonstrate the importance of methotrexate amino groups in the interaction with folate receptor-α.
Assuntos
Receptor 1 de Folato/metabolismo , Antagonistas do Ácido Fólico/metabolismo , Metotrexato/análogos & derivados , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/metabolismo , Transporte Biológico Ativo , Linhagem Celular Tumoral , Endocitose , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Receptor 1 de Folato/química , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/química , Humanos , Metotrexato/química , Metotrexato/metabolismo , Modelos Biológicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteína Carregadora de Folato Reduzido/química , Proteína Carregadora de Folato Reduzido/metabolismoRESUMO
Synthetic polyesters are today the second-largest class of ingredients in household products and are entering wastewater treatment plants (WWTPs) after product utilization. One approach to improve polymer biodegradation in wastewater would be to complement current processes with polyester-hydrolyzing enzymes and their microbial producers. In this study, the hydrolysis of poly(oxyethylene terephthalate) polymer by hydrolases from wastewater microorganisms was investigated in vitro and under realistic WWTP conditions. An esterase and a cutinase from Pseudomonas pseudoalcaligenes and a lipase from Pseudomonas pelagia were heterologously expressed in Escherichia coli BL21-Gold(DE3) and were purified by a C-terminal His6 tag. The hydrolases were proven to hydrolyze the polymer effectively, which is a prerequisite for further biodegradation. The hydrolases maintained high activity up to 50 % upon lowering the temperature from 28 to 15 °C to mimic WWTP conditions. The hydrolases were also not inhibited by the wastewater matrix. Polyester-hydrolyzing enzymes active under WWTP conditions and their microbial producers thus have the potential to improve biological treatment of wastewater rich in synthetic polymers.
Assuntos
Esterases/metabolismo , Lipase/metabolismo , Poliésteres/síntese química , Poliésteres/metabolismo , Águas Residuárias/química , Biodegradação Ambiental , Esterases/química , Lipase/química , Estrutura Molecular , Poliésteres/química , Pseudomonas/enzimologia , Pseudomonas/isolamento & purificação , Pseudomonas pseudoalcaligenes/enzimologia , Pseudomonas pseudoalcaligenes/isolamento & purificação , TemperaturaRESUMO
The question remains whether wound swabs yield similar culture results to the traditional gold standard, biopsies. Swabs are not invasive and easy to perform. However, they are believed to capture microorganisms from the surface rather than microorganisms that have invaded tissue. Several studies compared swabs and biopsies using different populations and sampling methods, complicating the ability to draw conclusions for clinical practice. This study aimed to compare swab and biopsy in clinical practice, by including a variety of wounds and using standard sampling and culture procedures. Swabs (Levine technique) and biopsies were taken for microbiological culture in a standardized manner from the same location of one wound for each patient. Statistical analyses were performed to determine overall agreement, and observed agreement and kappa for specific microorganisms. A variety of wounds of 180 patients from different healthcare facilities in The Netherlands were included. Skin flora was more frequently cultured from swabs, resulting in similar recovery rates when excluding skin flora (1.34 vs 1.35). Swabs were able to identify all microorganisms cultured from biopsies in 131 wounds (72.8%) wounds. Most frequently identified organisms were Staphylococcus aureus, Pseudomonas aeruginosa, and beta-haemolytic streptococci species. Observed agreement and kappa for these organisms varied between 87.2 and 97.8% and 0.73 and 0.85, respectively. This study demonstrates that swabs and biopsies tend to yield the same culture results when taken from the same location. For frequently occurring microorganisms, agreement between the two methods was even higher. Therefore, there seems to be no direct need for invasive biopsy in clinical practice.
Assuntos
Infecções por Acinetobacter/microbiologia , Biópsia , Contagem de Colônia Microbiana/métodos , Técnicas Microbiológicas/métodos , Infecções por Pseudomonas/microbiologia , Infecções Estafilocócicas/microbiologia , Procedimentos Desnecessários , Infecção dos Ferimentos/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Contagem de Colônia Microbiana/instrumentação , Feminino , Humanos , Masculino , Técnicas Microbiológicas/instrumentação , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Manejo de EspécimesRESUMO
Certain members of the carboxylesterase superfamily can act at the interface between water and water-insoluble substrates. However, nonnatural bulky polyesters usually are not efficiently hydrolyzed. In the recent years, the potential of enzyme engineering to improve hydrolysis of synthetic polyesters has been demonstrated. Regions on the enzyme surface have been modified by using site-directed mutagenesis in order to tune sorption processes through increased hydrophobicity of the enzyme surface. Such modifications can involve specific amino acid substitutions, addition of binding modules, or truncation of entire domains improving sorption properties and/or dynamics of the enzyme. In this review, we provide a comprehensive overview on different strategies developed in the recent years for enzyme surface engineering to improve the activity of polyester-hydrolyzing enzymes.
Assuntos
Bactérias/enzimologia , Bactérias/genética , Carboxilesterase/genética , Carboxilesterase/metabolismo , Bioengenharia , Hidrólise , Mutagênese Sítio-Dirigida , Poliésteres/metabolismoRESUMO
The growing pollution of the environment with plastic debris is a global threat which urgently requires biotechnological solutions. Enzymatic recycling not only prevents pollution but also would allow recovery of valuable building blocks. Therefore, we explored the existence of microbial polyesterases in microbial communities associated with the Sphagnum magellanicum moss, a key species within unexploited bog ecosystems. This resulted in the identification of six novel esterases, which were isolated, cloned, and heterologously expressed in Escherichia coli The esterases were found to hydrolyze the copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT) and the oligomeric model substrate bis[4-(benzoyloxy)butyl] terephthalate (BaBTaBBa). Two promising polyesterase candidates, EstB3 and EstC7, which clustered in family VIII of bacterial lipolytic enzymes, were purified and characterized using the soluble esterase substrate p-nitrophenyl butyrate (Km values of 46.5 and 3.4 µM, temperature optima of 48°C and 50°C, and pH optima of 7.0 and 8.5, respectively). In particular, EstC7 showed outstanding activity and a strong preference for hydrolysis of the aromatic ester bond in PBAT. Our study highlights the potential of plant-associated microbiomes from extreme natural ecosystems as a source for novel hydrolytic enzymes hydrolyzing polymeric compounds. IMPORTANCE: In this study, we describe the discovery and analysis of new enzymes from microbial communities associated with plants (moss). The recovered enzymes show the ability to hydrolyze not only common esterase substrates but also the synthetic polyester poly(butylene adipate-co-butylene terephthalate), which is a common material employed in biodegradable plastics. The widespread use of such synthetic polyesters in industry and society requires the development of new sustainable technological solutions for their recycling. The discovered enzymes have the potential to be used as catalysts for selective recovery of valuable building blocks from this material.
Assuntos
Bactérias/enzimologia , Esterases/genética , Esterases/metabolismo , Poliésteres/metabolismo , Sphagnopsida/microbiologia , Butiratos/metabolismo , Hidrólise , Microbiota/genética , Microbiota/fisiologiaRESUMO
The treatment of wound infection still constitutes a major threat in health care due to the increasing number of bacterial resistances and the difficulty of timely infection detection. Here, we present a smart antimicrobial system that is activated in case of infection based on elevated lysozyme activities. N-acetyl chitosan (degree of N-acetylation: 40%) was synthesized and hydrolysis by lysozyme in artificial wound fluid (AWF) was demonstrated. This resulted in the formation of N-acetylated chito oligosaccharides (COS) with a degree of polymerization of 2-5 units. The COS were shown to serve as substrate for cellobiose dehydrogenase (CDH) leading to the production of 1 mM antimicrobial hydrogen peroxide (H2 O2 ) after 24 h incubation at 37°C in AWF. Growth inhibition was seen upon incubation of Escherichia coli and Staphylococcus aureus with this chitosan-CDH system over 8 h. This approach represents the first self-regulating system for the infection responsive inhibition of bacterial growth in response to lysozyme as infection biomarker. Biotechnol. Bioeng. 2017;114: 416-422. © 2016 Wiley Periodicals, Inc.
Assuntos
Anti-Infecciosos , Desidrogenases de Carboidrato , Quitosana/química , Modelos Biológicos , Muramidase , Infecção dos Ferimentos , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/farmacologia , Escherichia coli/efeitos dos fármacos , Humanos , Muramidase/química , Muramidase/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/prevenção & controleRESUMO
We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. © 2017 Wiley Periodicals, Inc.
Assuntos
Actinobacteria/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/ultraestrutura , Simulação de Acoplamento Molecular/métodos , Poliésteres/química , Ativação Enzimática , Estabilidade Enzimática , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Water-soluble polyesters are used in a range of applications today and enter wastewater treatment plants after product utilization. However, little is known about extracellular enzymes and aquatic microorganisms involved in polyester biodegradation and mineralization. In this study, structurally different ionic phthalic acid based polyesters (the number-average molecular weights (Mn) 1770 to 10â¯000 g/mol and semi crystalline with crystallinity below 1%) were synthesized in various combinations. Typical wastewater microorganisms like Pseudomonas sp. were chosen for in-silico screening toward polyester hydrolyzing enzymes. Based on the in-silico search, a cutinase from Pseudomonas pseudoalcaligenes (PpCutA) and a putative lipase from Pseudomonas pelagia (PpelaLip) were identified. The enzymes PpCutA and PpelaLip were demonstrated to hydrolyze all structurally different polyesters. Activities on all the polyesters were also confirmed with the strains P. pseudoalcaligenes and P. pelagia. Parameters identified to enhance hydrolysis included increased water solubility and polyester hydrophilicity as well as shorter diol chain lengths. For example, polyesters containing 1,2-ethanediol were hydrolyzed faster than polyesters containing 1,8-octanediol. Interestingly, the same trend was observed in biodegradation experiments. This information is important to gain a better mechanistic understanding of biodegradation processes of polyesters in WWTPs where the extracellular enzymatic hydrolysis seems to be the limiting step.
Assuntos
Poliésteres/metabolismo , Águas Residuárias , Hidrólise , Ácidos Ftálicos/metabolismo , Pseudomonas/metabolismoRESUMO
Biodegradable polyesters have a large potential to replace persistent polymers in numerous applications and to thereby reduce the accumulation of plastics in the environment. Ester hydrolysis by extracellular carboxylesterases is considered the rate-limiting step in polyester biodegradation. In this work, we systematically investigated the effects of polyester and carboxylesterase structure on the hydrolysis of nanometer-thin polyester films using a quartz-crystal microbalance with dissipation monitoring. Hydrolyzability increased with increasing polyester-chain flexibility as evidenced from differences in the hydrolysis rates and extents of aliphatic polyesters varying in the length of their dicarboxylic acid unit and of poly(butylene adipate-co-terephthalate) (PBAT) polyesters varying in their terephthalate-to-adipate ratio by Rhizopus oryzae lipase and Fusarium solani cutinase. Nanoscale nonuniformities in the PBAT films affected enzymatic hydrolysis and were likely caused by domains with elevated terephthalate contents that impaired enzymatic hydrolysis. Yet, the cutinase completely hydrolyzed all PBAT films, including films with a terephthalate-to-adipate molar ratio of one, under environmentally relevant conditions (pH 6, 20 °C). A comparative analysis of the hydrolysis of two model polyesters by eight different carboxylesterases revealed increasing hydrolysis with increasing accessibility of the enzyme active site. Therefore, this work highlights the importance of both polyester and carboxylesterase structure to enzymatic polyester hydrolysis.
Assuntos
Poliésteres , Biodegradação Ambiental , Domínio Catalítico , Hidrólise , Lipase , PolímerosRESUMO
A novel esterase, PpEst, that hydrolyses the co-aromatic-aliphatic polyester poly(1,4-butylene adipate-co-terephthalate) (PBAT) was identified by proteomic screening of the Pseudomonas pseudoalcaligenes secretome. PpEst was induced by the presence of PBAT in the growth media and had predicted arylesterase (EC 3.1.1.2) activity. PpEst showed polyesterase activity on both whole and milled PBAT film releasing terephthalic acid and 4-(4-hydroxybutoxycarbonyl)benzoic acid while end product inhibition by 4-(4-hydroxybutoxycarbonyl)benzoic acid was observed. Modelling of an aromatic polyester mimicking oligomer into the PpEst active site indicated that the binding pocket could be big enough to accommodate large polymers. This is the first report of a PBAT degrading enzyme being identified by proteomic screening and shows that this approach can contribute to the discovery of new polymer hydrolysing enzymes. Moreover, these results indicate that arylesterases could be an interesting enzyme class for identifications of polyesterases.
Assuntos
Proteínas de Bactérias/química , Plásticos Biodegradáveis/metabolismo , Hidrolases de Éster Carboxílico/química , Poliésteres/metabolismo , Pseudomonas pseudoalcaligenes/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plásticos Biodegradáveis/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Expressão Gênica , Modelos Moleculares , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Poliésteres/química , Ligação Proteica , Proteômica , Pseudomonas pseudoalcaligenes/genéticaRESUMO
Grass silage was evaluated as a possible substrate in anaerobic digestion for generation of biogas in mesophilic and thermophilic long-term operation. Furthermore, the molecular biological parameter Metabolic Quotient (MQ) was evaluated as early warning system to predict process disturbance. Since this substrate is rich in nitrogen, high ammonia concentration of up to 2.2 g * kgFM-1 emerged. The high buffer capacity of the ammonium/ammonia system can disguise upcoming process acidification. At organic loading rates (OLR) below 1.0 kgVS * m-3 * d-1 (VS: volatile solids) for thermophilic and below 1.5 kgVS * m-3 * d-1 for mesophilic reactors, stable processes were established. With increasing OLR, the process was stressed until it broke down in the thermophilic reactors at an OLR of 3.5 kgVS * m-3 * d-1 or was stopped at an OLR of 4.5 kgVS * m-3 * d-1 in the mesophilic reactors. Mainly propionic acid accumulated in concentrations of up to 6.5 g * kgFM-1. Due to the high buffer capacity of the reactor sludge, the chemical parameter TVA/TIC (ratio of total volatile acids to total inorganic carbon) did not clearly indicate process disturbance in advance. In contrast, the MQ indicated metabolic stress of the methanogens before process breakdown and thus showed its potential as early warning system for process breakdown. During the whole experiment, hydrogenotrophic methanogens dominated. In the thermophilic reactors, Methanoculleus IIA-2 sp. 2 and Methanothermobacter wolfeii were dominant during stable process conditions and were displaced by Methanobacterium III sp. 4, a possible new bioindicator for disturbances at these conditions. In the mesophilic reactors, mainly Methanobacterium III sp. 4 was dominant at stable, stressed and acidified processes. A hitherto uncultivated genospecies, Methanobacteriaceae genus IV(B) sp. 3 was determined as possible new bioindicator for mesophilic process disturbance.
Assuntos
Biocombustíveis , Fermentação , Metano/biossíntese , Nitrogênio/metabolismo , Anaerobiose , Reatores Biológicos , Concentração de Íons de Hidrogênio , Poaceae , Esgotos , TemperaturaRESUMO
There is a strong need for simple and fast diagnostic tools for the detection of wound infection. Immune system-derived enzymes like myeloperoxidase are efficient biomarkers for wound infection that emerge in the early stage infection process. In this study, 5-amino-2-methoxyphenol was functionalized with alkoxysilane to allow visual detection of MPO on carrier materials, for example, in test strips. Indeed, MPO activity was visually detectable in short time in wound background. Oxidation of the substrate was followed spectrophotometrically and proved via HPLC. LC-ESI TOF and NMR analyses unveiled the reaction mechanism and a dimeric reaction product responsible for the visualization of MPO activity. The substrate specificity and sensitivity toward MPO detection was proved and tests with infected wound fluids were successfully performed. The study demonstrates the suitability of the novel MPO substrate for the detection of wound infection and the covalent immobilization on diagnostic carrier materials. Biotechnol. Bioeng. 2016;113: 2553-2560. © 2016 Wiley Periodicals, Inc.
Assuntos
Biomarcadores/análise , Colorimetria/métodos , Guaiacol/química , Peroxidase/análise , Infecção dos Ferimentos/diagnóstico , Infecção dos Ferimentos/metabolismo , Adsorção , Materiais Biocompatíveis/química , Técnicas Biossensoriais/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.