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INTRODUCTION: Pectin-oligosaccharides (POS) serve diverse purposes as a food ingredient, antimicrobial and biostimulant in plants, and their functionality is linked to the degree of esterification. Grape and broccoli wastes emerge as environmentally friendly alternatives to obtaining pectin, serving as a sustainable source to producing POS. For example, microwaves have proven to be an effective and sustainable method to extract polysaccharides from plant matrices. OBJECTIVE: This work aims to use grape and broccoli wastes as alternative sources for obtaining pectin by microwave-assisted extraction and biotransformation into POS, which possess biological properties. MATERIAL AND METHODS: The extraction conditions were identified at a power of 400 W, 300 s for the extraction of pectin from grape pomace and broccoli waste. Biotransformation of pectins into POS, using commercial enzyme preparations (Viscozyme L and Pectinase). Characterisation was carried out by Fourier-transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. RESULTS: Physicochemical analysis indicated grape pomace and broccoli waste pectins had galacturonic acid content of 63.81 ± 1.67 and 40.83 ± 2.85 mg 100 mg-1, low degree of esterification of 34.89% and 16.22%, respectively. Biotransformation of pectins into POS resulted in a 20% hydrolysis rate. The main enzymatic activity was polygalacturonase for the degradation of the main structure of the pectin. CONCLUSION: Production of POS from agro-industrial wastes by emerging technologies, such as the combined use of microwave-assisted extraction and enzymatic processes, represents an alternative method for the generation of bioactive compounds with distinctive properties suitable for different applications of interest.
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In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6â¯U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ËC) with a 2.83â¯mg/mL and 0.063 µmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43â¯mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7â¯mg/mL, the time for the fabric to absorb water was 19.77â¯seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38â¯seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.
Assuntos
Proteínas Fúngicas , Poligalacturonase , Saccharomycetales , Biomassa , Eurotiales/enzimologia , Eurotiales/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrólise , Cinética , Poligalacturonase/metabolismo , Poligalacturonase/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Saccharomycetales/genética , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Indústria Têxtil , TêxteisRESUMO
AIM: To identify and analyse genes that encode pectinases in the genome of the fungus Colletotrichum lindemuthianum, evaluate the expression of these genes, and compare putative pectinases found in C. lindemuthianum with pectinases produced by other fungi and oomycetes with different lifestyles. METHODS AND RESULTS: Genes encoding pectinases in the genome of C. lindemuthianum were identified and analysed. The expression of these genes was analysed. Pectinases from C. lindemuthianum were compared with pectinases from other fungi that have different lifestyles, and the pectinase activity in some of these fungi was quantified. Fifty-eight genes encoding pectinases were identified in C. lindemuthianum. At least six types of enzymes involved in pectin degradation were identified, with pectate lyases and polygalacturonases being the most abundant. Twenty-seven genes encoding pectinases were differentially expressed at some point in C. lindemuthianum during their interactions with their host. For each type of pectinase, there were at least three isoenzyme groups. The number of pectinases present in fungi with different lifestyles seemed to be related more to the lifestyle than to the taxonomic relationship between them. Only phytopathogenic fungi showed pectate lyase activity. CONCLUSIONS: The collective results demonstrate the pectinolytic arsenal of C. lindemuthianum, with many and diverse genes encoding pectinases more than that found in other phytopathogens, which suggests that at least part of these pectinases must be important for the pathogenicity of the fungus C. lindemuthianum. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of these pectinases could further the understanding of the importance of this broad pectinolytic arsenal in the common bean infection and could be exploited for biotechnological purposes.
Assuntos
Colletotrichum , Fabaceae , Colletotrichum/genética , Fabaceae/microbiologia , Fungos/metabolismo , Poligalacturonase/genética , Poligalacturonase/metabolismoRESUMO
Pectin is a major constituent of the plant cell wall, comprising compounds with important industrial applications such as homogalacturonan, rhamnogalacturonan and xylogalacturonan. A large array of enzymes is involved in the degradation of this amorphous substrate. The Glycoside Hydrolase 28 (GH28) family includes polygalacturonases (PG), rhamnogalacturonases (RG) and xylogalacturonases (XG) that share a structure of three to four pleated ß-sheets that form a rod with the catalytic site amidst a long, narrow groove. Although these enzymes have been studied for many years, there has been no systematic analysis. We have collected a comprehensive set of GH28 encoding sequences to study their evolution in fungi, directed at obtaining a functional classification, as well as at the identification of substrate specificity as functional constraint. Computational tools such as Alphafold, Consurf and MEME were used to identify the subfamilies' characteristics. A hierarchic classification defines the major classes of endoPG, endoRG and endoXG as well as three exoPG classes. Ascomycete endoPGs are further classified in two subclasses whereas we identify four exoRG subclasses. Diversification towards exomode is explained by loops that appear inserted in a number of turns. Substrate-driven diversification can be identified by various specificity determining positions that appear to surround the binding groove.
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Corn and common bean have been cultivated together in Mesoamerica for thousands of years in an intercropping system called "milpa," where the roots are intermingled, favoring the exchange of their microbiota, including symbionts such as rhizobia. In this work, we studied the genomic expression of Rhizobium phaseoli Ch24-10 (by RNA-seq) after a 2-h treatment in the presence of root exudates of maize and bean grown in monoculture and milpa system under hydroponic conditions. In bean exudates, rhizobial genes for nodulation and degradation of aromatic compounds were induced; while in maize, a response of genes for degradation of mucilage and ferulic acid was observed, as well as those for the transport of sugars, dicarboxylic acids and iron. Ch24-10 transcriptomes in milpa resembled those of beans because they both showed high expression of nodulation genes; some genes that were expressed in corn exudates were also induced by the intercropping system, especially those for the degradation of ferulic acid and pectin. Beans grown in milpa system formed nitrogen-fixing nodules similar to monocultured beans; therefore, the presence of maize did not interfere with Rhizobium-bean symbiosis. Genes for the metabolism of sugars and amino acids, flavonoid and phytoalexin tolerance, and a T3SS were expressed in both monocultures and milpa system, which reveals the adaptive capacity of rhizobia to colonize both legumes and cereals. Transcriptional fusions of the putA gene, which participates in proline metabolism, and of a gene encoding a polygalacturonase were used to validate their participation in plant-microbe interactions. We determined the enzymatic activity of carbonic anhydrase whose gene was also overexpressed in response to root exudates.
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Processes for the enzymatic hydrolysis of polysaccharides in biorefineries are becoming increasingly important. The complex network of reactions involved in polysaccharide hydrolysis can be described by stochastic models that advance in steps of time. Such models have the potential to be important tools for guiding process design and operation, and several have been developed over the last two decades. We evaluate these models. Many of the current stochastic models for the hydrolysis of colloidal polysaccharides use empirical parameters that have no recognized biological meaning. Only one model uses classical parameters of enzyme kinetics, namely specificity constants and saturation constants. Recent stochastic models for the hydrolysis of insoluble cellulose give valuable insights into the molecular-level phenomenon that limit hydrolysis rates. We conclude that, if stochastic models of enzymatic polysaccharide hydrolysis are to become widely used tools for guiding process development, then further improvements are required.
Assuntos
Celulase , Polissacarídeos , Celulase/metabolismo , Celulose , Hidrólise , CinéticaRESUMO
Fungi are capable of sensing light from ultraviolet to far-red and they use light as a source of information about the environment anticipating stress and adverse conditions. Lentinus crinitus is a lignin-degrading fungus which produces laccase and other enzymes of biotechnological interest. The effect of blue light on fungal enzymatic activity has been studied; however, it has not been found studies on the effect of the blue light on carbohydrate-active enzymes and on mycelial biomass production of L. crinitus. We aimed to investigate carbohydrate-active enzymes activity and mycelial biomass production of L. crinitus cultivated under continuous illumination with blue light. L. crinitus was cultivated in malt extract medium in the dark, without agitation, and under continuous illumination with blue light-emitting diodes. The blue light reduced the total cellulase, pectinase and xylanase activities but increased the endoglucanase activity. Blue light reduced the mycelial growth of L. crinitus in 26% and the enzymatic activity-to-mycelial biomass ratio (U mg-1 dry basis) increased in 10% total cellulase, 33% endoglucanase, and 16% pectinase activities. Also, it is suggested that L. crinitus has a photosensory system and it could lead to new process of obtaining enzymes of biotechnological interest.
Fungos são capazes de sentir a luz com comprimentos de onda que variam do ultravioleta ao infravermelho e usam a luz como fonte de informação sobre o ambiente, antecipando condições adversas e de estresse. Lentinus crinitus é um fungo ligninolítico que produz lacase e outras enzimas de interesse biotecnológico. O efeito da luz azul na atividade enzimática de fungos já foi estudado, contudo, ainda não há estudos sobre o efeito da luz azul na produção de enzimas ativas sobre carboidratos (CAZymes, carbohydrate-active enzymes) e de biomassa micelial de L. crinitus. O objetivo deste estudo foi investigar a avitivade de CAZymes e a produção de biomassa micelial de L. crinitus cultivado sob iluminação continua com luz azul. L. crinitus foi cultivado em meio extrato de malte, sem agitação, na ausência de luz e sob luz continua fornecida por diodos emissores de luz azul. A luz azul reduziu a atividade de cellulase total, pectinase e xilanase, mas aumentou a atividade de endoglucanase. A luz azul reduziu o crescimento micelial de L. crinitus em 26% e aumentou a razão atividade enzimática/biomassa micelial (U mg-1 em base seca) de cellulase total em 10%, endoglucanase em 33% e pectinase em 16%. Além disso, sugere-se que L. crinitus possua um sistema fotossensorial que poderia ser explorado para a otimização de bioprocessos que visam a obtenção de enzimas de interesse biotecnológico.
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Poligalacturonase , Lentinula , Celulases , LuzRESUMO
Abstract The objective of the present study was to develop a cost-effective medium, using agro-industrial wastes for the production of a polygalacturonase by Wickerhanomyces anomalus of interest in cassava starch industries. The effect of several raw agro-industrial wastes and others nutrients on polygalacturonase production by W. anomalus, were evaluated, in a reference fermentation medium, using statistical designs, by batch culture. The ability of the cell-free supernatant to extract cassava starch was evaluated. Lemon peel was the best inducer for the production of PGase. Statistical analysis of the data showed that lemon peel, Mg+2 and PO4HK2 had significant effect on PGase production, and the others variables (yeast extract, Ca+2, Fe+2, amino acid and trace element solution) were no significant. PGase synthesis reached ~31 EUmL-1, in the OFM (glucose, lemon peel, urea, vitamins, KH2PO4 and MgSO4), after 12 h of culture, at a lab scale bioreactor. PGase of W. anomalus, was able to disintegrate cassava tuber tissue, and the starch granules contained within the cells were released into the reaction medium. Lemon peel can be used as inducer for PGase production by W. anomalus, in a low cost culture medium, appropriate for the production of the enzyme at large scale.
Assuntos
Poligalacturonase/biossíntese , Reatores Biológicos , Amidos e Féculas , Resíduos Industriais , Análise Custo-Benefício , Agricultura , FermentaçãoRESUMO
Mango (Mangifera indica L.) is an important commercial fruit that shows a noticeable loss of firmness during ripening. Polygalacturonase (PG, E.C. 3.2.1.15) is a crucial enzyme for cell wall loosening during fruit ripening since it solubilizes pectin and its activity correlates with fruit softening. Mango PGs were mapped to a genome draft using seventeen PGs found in mango transcriptomes and 48 bonafide PGs were identified. The phylogenetic analysis suggests that they are related to Citrus sinensis, which may indicate a recent evolutive divergence and related functions with orthologs in the tree. Gene expression analysis for nine PGs showed differential expression for them during post-harvest fruit ripening, MiPG21-1, MiPG14, MiPG69-1, MiPG17, MiPG49, MiPG23-3, MiPG22-7, and MiPG16 were highly up-regulated. PG enzymatic activity also increased during maturation and these results correlate with the loss of firmness observed in mango during post-harvest ripening, between the ethylene production burst and the climacteric peak. The analysis of PGs promoter regions identified regulatory sequences associated to ripening such as MADS-box, ethylene regulation like ethylene insensitive 3 (EIN3) factors, APETALA2-like and ethylene response element factors. During mango fruit ripening the action of at least these nine PGs contribute to softening, and their expression is regulated at the transcriptional level. The prediction of the tridimensional structure of some PGs showed a conserved parallel beta-helical fold related to polysaccharide hydrolysis and a modular architecture, where exons correspond to structural elements. Further biotechnological approaches could target specific softening-related PGs to extend mango post-harvest shelf life.
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AIMS: The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. METHODS AND RESULTS: The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent Km , Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56 mg ml-1 , 163·1 U mg-1 and 277 s-1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. CONCLUSIONS: The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.
Assuntos
Sucos de Frutas e Vegetais , Penicillium/enzimologia , Poligalacturonase/química , Poligalacturonase/metabolismo , Biotecnologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Pectinas/metabolismo , Penicillium/metabolismo , Poligalacturonase/isolamento & purificação , TemperaturaRESUMO
Background: Burdock (Arctium lappa L.) is a fructan-rich plant with prebiotic potential. The aim of this study was to develop an efficient enzymatic route to prepare fructooligosaccharides (FOS)-rich and highly antioxidative syrup using burdock root as a raw material. Results: Endo-inulinase significantly improved the yield of FOS 2.4-fold while tannase pretreatment further increased the yield of FOS 2.8-fold. Other enzymes, including endo-polygalacturonase, endo-glucanase and endo-xylanase, were able to increase the yield of total soluble sugar by 11.1% (w/w). By this process, a new enzymatic process for burdock syrup was developed and the yield of burdock syrup increased by 25% (w/w), whereas with FOS, total soluble sugars, total soluble protein and total soluble polyphenols were enhanced to 28.8%, 53.3%, 8.9% and 3.3% (w/w), respectively. Additionally, the scavenging abilities of DPPH and hydroxyl radicals, and total antioxidant capacity of the syrup were increased by 23.7%, 51.8% and 35.4%, respectively. Conclusions: Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods.
Assuntos
Oligossacarídeos/química , Raízes de Plantas/química , Frutose/química , Glicosídeo Hidrolases/metabolismo , Antioxidantes/química , Oligossacarídeos/metabolismo , Poligalacturonase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Cromatografia Líquida de Alta Pressão , Radical Hidroxila , Arctium , Alimento Funcional , Polifenóis , Frutose/metabolismo , Antioxidantes/metabolismoRESUMO
The kinetics and thermodynamics of pectin hydrolysis in cashew apple juice by polygalacturonase (PG) from Aspergillus aculeatus URM4953 covalently-immobilized on calcium alginate beads were investigated. Immobilized-PG activity in cashew apple juice was the highest at 20⯰C, showing a maximum hydrolysis rate of 58.2â¯mg/mL·min, a catalytic constant of 166.2â¯s-1 and an affinity constant of 113.0â¯mg/mL. Since the enzyme exhibited an allosteric behavior, the hydrolysis rate was modeled, with excellent accuracy, by the Hill Equation as function of pectin concentration. The Hill coefficient increased from 3 to 5 with increasing temperature from 20 to 50⯰C, evidencing a positive cooperativity mechanism. The reaction activation energy and the standard enthalpy variation of enzyme unfolding were 80.3 and 16.6â¯kJ/mol, respectively. Consistently with the kinetic results, PG-catalyzed pectin hydrolysis proceeded with maximum spontaneity at 20⯰C, showing activation Gibbs free energy, enthalpy and entropy of 59.3â¯kJ/mol, 77.9â¯kJ/mol and 63.4â¯J/mol·K, respectively. Immobilized PG was successful in the hydrolysis of cashew apple juice pectin, requiring a low temperature to act optimally.
Assuntos
Alginatos/química , Anacardium/química , Aspergillus/enzimologia , Enzimas Imobilizadas/metabolismo , Sucos de Frutas e Vegetais , Microesferas , Pectinas/metabolismo , Poligalacturonase/metabolismo , Hidrólise , Cinética , Termodinâmica , Fatores de TempoRESUMO
Among the structural polymers present in the plant cell wall, pectin is the main component of the middle lamella. This heterogeneous polysaccharide has an α-1,4 galacturonic acid backbone, which can be broken by the enzymatic action of pectinases, such as exo-polygalacturonases, that sequentially cleave pectin from the non-reducing ends, releasing mono or di-galacturonic acid residues. Constant demand for pectinases that better suit industrial requirements has motivated identification and characterization of novel enzymes from diverse sources. Bacillus licheniformis has been used as an important source for bioprospection of several industrial biomolecules, such as surfactants and enzymes, including pectate lyases. Here we cloned, expressed, purified, and biochemically and structurally characterized an exo-polygalacturonase from B. licheniformis (BlExoPG). Its low-resolution molecular envelope was derived from experimental small-angle scattering data (SAXS). Our experimental data revealed that BlExoPG is a monomeric enzyme with optimum pH at 6.5 and optimal temperature of approximately 60°C, at which it has considerable stability over the broad pH range from 5 to 10. After incubation of the enzyme for 30min at pH ranging from 5 to 10, no significant loss of the original enzyme activity was observed. Furthermore, the enzyme maintained residual activity of greater than 80% at 50°C after 15h of incubation. BlExoPG is more active against polygalacturonic acid as compared to methylated pectin, liberating mono galacturonic acid as a unique product. Its enzymatic parameters are Vmax=4.18µM.s-1,Km=3.25mgmL-1 and kcat=2.58s-1.
Assuntos
Bacillus licheniformis/enzimologia , Poligalacturonase/química , Espalhamento a Baixo Ângulo , Concentração de Íons de Hidrogênio , Poligalacturonase/isolamento & purificação , Poligalacturonase/metabolismo , Temperatura , Difração de Raios XRESUMO
The Chilean strawberry (Fragaria chiloensis) fruit has interesting organoleptic properties, but its postharvest life is affected by gray mold decay caused by Botrytis cinerea. The effect of preharvest applications of methyl jasmonate (MeJA) or chitosan on the molecular defense-related responses and protection against gray mold decay were investigated in Chilean strawberry fruit during postharvest storage. Specifically, we inoculated harvested fruit with B. cinerea spores and studied the expression of genes encoding for the pathogenesis-related (PR) proteins ß-1,3-glucanases (FcBG2-1, FcBG2-2 and FcBG2-3) and chitinases (FcCHI2-2 and FcCHI3-1), and for polygalacturonase inhibiting proteins (FcPGIP1 and FcPGIP2) at 0, 2, 24, 48, and 72 h post inoculation (hpi). Remarkably, MeJA- and chitosan-treated fruit exhibited a lower incidence of B. cinerea infection than the control-treated at 48 and 72 hpi. At the molecular level, both are efficient elicitors for priming in F. chiloensis fruit since we observed an upregulation of the FcBG2-1, FcBG2-3, FcPGIP1, and FcPGIP2 at 0 hpi. Moreover, a chitosan-mediated upregulation of FcPGIPs at early times post inoculation (2-24 hpi) and MeJA upregulated FcBGs (24-72 hpi) and FcPGIP1 at later times could contribute to reduce B. cinerea incidence by differential upregulation of defense genes. We concluded that preharvest applications of MeJA or chitosan had a long-lasting effect on the reduction of B. cinerea incidence during postharvest as well as an enhancer effect on the induction of PR and PGIP gene expression.
Assuntos
Acetatos/farmacologia , Botrytis/fisiologia , Quitosana/farmacologia , Ciclopentanos/farmacologia , Fragaria/genética , Fragaria/microbiologia , Frutas/microbiologia , Oxilipinas/farmacologia , Doenças das Plantas/microbiologia , Regulação para Cima/efeitos dos fármacos , Botrytis/efeitos dos fármacos , Fragaria/efeitos dos fármacos , Fragaria/imunologia , Frutas/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de TempoRESUMO
An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.
Assuntos
Aspergillus/enzimologia , Poligalacturonase/química , Poligalacturonase/isolamento & purificação , Poligalacturonase/metabolismo , Carbono/metabolismo , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia em Camada Fina/métodos , Citrus sinensis/química , Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Estabilidade Enzimática/efeitos dos fármacos , Etanol/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ácidos Hexurônicos/metabolismo , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/métodos , Cinética , Metais/metabolismo , Peso Molecular , Pectinas/metabolismo , Dióxido de Enxofre/metabolismo , Temperatura , Fatores de TempoRESUMO
En el presente trabajo se verificó la presencia de algunas enzimas relacionadas con la pared celular vegetal (poligalacturonasa, pectato liasa, proteasa y xilanasa) en raíces de clavel (Dianthus caryophyllus L.). Así mismo, se determinaron los niveles de actividad de las mismas. Estos niveles se analizaron en diferentes espacios celulares: en el fluido intercelular que hace parte del apoplasto, en el simplasto y en el tejido total de las raíces de clavel (apoplasto y simplasto). Para extraer el fluido intercelular, se ensayaron dos metodologías. Para obtener el contenido intracelular (simplasto) y el extracto total (apoplasto y simplasto) en raíces de clavel se ensayaron tres metodologías que utilizaban como solución i) extractante buffer fosfato, ii) buffer fosfato con PVPP y iii) lavados con acetona a las raíces de clavel, antes de la extracción con buffer fosfatos. Los resultados mostraron el efecto de las diferentes soluciones en las actividades enzimáticas y en el contenido de proteína. Se propuso una de estas metodologías para extraer las cuatro enzimas en un único paso y realizar análisis comparativo de actividad enzimática.
The presence of some enzymes related to cell wall (polygalacturonase, the pectate lyase, protease and xylanase) in carnation (Dianthus caryophyllus L.) roots as well as the activity levels were determined. These levels were analyzed in different cellular places: the intercellular fluid that is part of the apoplast, the symplast, and the total level (apoplast and symplast) in carnation roots. Two methods were tested to extract the intercellular fluid. To obtain the intracellular content (symplast) and total extract (apoplast+symplast), three methods were tested, using as extracting solution i) phosphate buffer, ii) phosphate buffer + PVPP, iii) before the extraction with phosphate buffer, the carnation roots were washed with acetone. The results showed the effect of different extracting solutions in the enzymatic activities and in the protein content. A new only one step method is proposed to extract the four enzymes and make the comparative analysis of enzymatic activity.
No presente trabalho foi evidenciada a presença de algumas enzimas relacionadas com a parede celular vegetal: poligalacturonasa, pectato liasa, proteasa e xilanasa e se determinaram seus níveis de atividade, em raízes de cravo (Dianthus caryophyllus L.). Os níveis se analisaram em diferentes espaços celulares: no fluido intercelular que faz parte do apoplasto, no simplasto e no tecido total das raízes de cravo (apoplasto e simplasto). Foram avaliadas duas metodologias para extrair o fluido intercelular. Para obter o conteúdo intracelular (simplasto) e o extrato total (apoplasto e simplasto) se avaliaram três metodologias que utilizavam como solução extratora i) buffer fosfato, ii) buffer fosfato com PVPP e iii) lavados com acetona às raízes de cravo, antes da extração com buffer fosfato. Os resultados mostraram o efeito das diferentes soluciones nas atividades enzimáticas e no conteúdo de proteína. Se propõem uma de estas metodologias para extrair as quatro enzimas num único passo e realizar a analise comparativa da atividade enzimática.
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The aim of this research was the partial characterization of polygalacturonase (PG) extracts produced by a newly isolated Penicillium brasilianum and Aspergillus niger in submerged fermentation. The partial characterization of the crude enzymatic extracts showed optimum activity at pH 5.5 and 37 °C for both extracts. The results of temperature stability showed that PG from both microorganisms were more stable at 55 °C. However, the enzyme obtained by P. brasilianum presents a half-life time (t 1/2 = 693.10 h), about one order of magnitude higher than those observed in for A. niger at 55 °C. In terms of pH stability, the PG produced by P. brasilianum presented higher stability at pH 4.0 and 5.0, while the PG from A. niger showed higher stability at pH 5.0.
Assuntos
Aspergillus niger/enzimologia , Penicillium/enzimologia , Poligalacturonase/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
The cell wall in plants offers protection against invading organisms and is mainly composed of the polysaccharides pectin, cellulose, and hemicellulose, which can be degraded by plant cell wall degrading enzymes (PCWDEs). Such enzymes are often synthesized by free living microorganisms or endosymbionts that live in the gut of some animals, including certain phytophagous insects. Thus, the ability of an insect to degrade the cell wall was once thought to be related to endosymbiont enzyme activity. However, recent studies have revealed that some phytophagous insects are able to synthesize their own PCWDEs by endogenous genes, although questions regarding the origin of these genes remain unclear. This study describes two pectinases from the sugarcane weevil, Sphenophorus levis Vaurie, 1978 (Sl-pectinases), which is considered one of the most serious agricultural pests in Brazil. Two cDNA sequences identified in a cDNA library of the insect larvae coding for a pectin methylesterase (PME) and an endo-polygalacturonase (endo-PG)-denominated Sl-PME and Sl-endoPG, respectively-were isolated and characterized. The quantitative real-time reverse transcriptase polymerase chain reaction expression profile for both Sl-pectinases showed mRNA production mainly in the insect feeding stages and exclusively in midgut tissue of the larvae. This analysis, together Western blotting data, suggests that Sl-pectinases have a digestive role. Phylogenetic analyses indicate that Sl-PME and Sl-endoPG sequences are closely related to bacteria and fungi, respectively. Moreover, the partial genomic sequences of the pectinases were amplified from insect fat body DNA, which was certified to be free of endosymbiotic DNA. The analysis of genomic sequences revealed the existence of two small introns with 53 and 166 bp in Sl-endoPG, which is similar to the common pattern in fungal introns. In contrast, no intron was identified in the Sl-PME genomic sequence, as generally observed in bacteria. These data support the theory of horizontal gene transfer proposed for the origin of insect pectinases, reinforcing the acquisition of PME genes from bacteria and endo-PG genes from fungi.
Assuntos
Hidrolases de Éster Carboxílico/genética , Trato Gastrointestinal/enzimologia , Poligalacturonase/genética , Gorgulhos/enzimologia , Gorgulhos/genética , Sequência de Aminoácidos , Animais , Bactérias , Sequência de Bases , Parede Celular/metabolismo , DNA Complementar , Fungos , Larva/enzimologia , Larva/genética , Pecten/metabolismo , Filogenia , RNA MensageiroRESUMO
BACKGROUND: In the food industry, the use of pectinase preparations with high pectin esterase (PE) activity leads to the release of methanol, which is strictly regulated in food products. Herein, a pectin-degrading enzyme (PDE) complex exhibiting low PE activity of three Aspergillus sojae ATCC 20235 mutants (M3, DH56 and Guserbiot 2.230) was investigated. Production of exo-/endo-polygalacturonase (PG), exo-polymethylgalacturonase (PMG) and pectin lyase (PL) by mutant M3 and A. sojae using two different carbon sources was evaluated in solid-state fermentation. Finally, experimental preparations obtained from the mutants and commercial pectinases standardized to the same potency were screened for PDEs. RESULTS: Mutant M3 grown on sugar beet was found to be the best producer of exo-PG, endo-PG, exo-PMG and PL, with maximum yields of 1111, 449, 130 and 123 U g(-1), respectively. All experimental preparations exhibited low PE activity, at least 21.5 times less than commercial pectinases, and higher endo-PG (40 U mL(-1)). CONCLUSION: Mutant M3 was the best PDE producer using sugar beet. Mutant strains presented a PDE complex featuring high endo-PG and very low PE activities. This novel complex with low de-esterifying activity can be exploited in the food industry to degrade pectin without releasing methanol.
Assuntos
Aspergillus niger/enzimologia , Beta vulgaris , Fermentação , Complexos Multienzimáticos/metabolismo , Mutação , Pectinas/metabolismo , Poligalacturonase/metabolismo , Aspergillus niger/genética , Aspergillus niger/crescimento & desenvolvimento , Meios de Cultura , Esterases/metabolismo , Esterificação , Humanos , Liases/biossíntese , Liases/metabolismo , Metanol/metabolismoRESUMO
Wickerhamomyces anomalus, una levadura aislada de frutas cítricas en la provincia de Misiones, Argentina, produce una poligalacturonasa (endo-PG) con capacidad macerante de tejidos vegetales. El objetivo del presente trabajo fue determinai los parámetros cinéticos y estequiométricos del crecimiento de W. anomalus y la producción de la enzima poligalacturonasa en un medio de cultivo sintético, operado en sistema por lote, en un biorreactor a escala laboratorio. Los cultivos se realizaron en un biorreactor de 4 l que contenía 3 l de un medio sintético compuesto por glucosa, pectina de citrus, vitaminas, aminoácidos, sulfato de amonio y sales, y se incubaron con agitación y aireación, a 30 °C durante 12 h. El transcurso de proceso fermentativo se siguió por medidas de biomasa, glucosa residual, actividad poligalacturonasa y contenido de O2 y CO2 de los gases a la salida del reactor. La velocidad específica de crecimiento máxima (-im) de W. anomalus fue de 0,337 h-1 y el rendimiento de biomasa producida (Yx/s) de 0,401 gx/gs. Al finalizar el cultivo, la actividad PG en el sobrenadante fue de PG de ~ 83,7 UE/ml. La actividad específica y la productividad obtenidas fueron de ~ 1,91. 10(4) UE/gx y ~ 9.301 UE/l.h, respectivamente. El cociente respiratorio fue cercano a 1 durante el proceso fermentativo. No se formó ningún otro producto, además de biomasa y CO 2 . El cultivo por lote resultó ser una buena alternativa para la producción de PG a partir de W. anomalus, obteniéndose un extracto con elevada actividad enzimática, en un medio de cultivo sintético y de bajo costo.
Wickerhamomyces anomalus, a yeast isolated from citrus fruit peels in the province of Misiones, Argentina, produces a polygalacturonase (endo-PG) with maceration activity of vegetable tissues. The objective of the present work was to determine kinetic and stoichiometric parameters of W. anomalus growth and polygalacturonase production in a synthetic culture medium, operating in a batch-type bioreactor at laboratory scale. Cultures were performed in a bioreactor of 4 l, containing 3 l of a synthetic medium composed of glucose, citrus pectin, vitamins, amino acids, ammonium sulfate and salts, and were incubated with agitation (450 rpm) and aeration at 30 °C, during 12 h. The course of the fermentation process was followed by measuring biomass, residual glucose, polygalacturonase activity and O2 and CO2 content of outlet gases from the reactor. The maximum specific growth rate (Um) of W. anomalus was 0.337 h-1 and the biomass yield (Yx/s) was 0.40 gx/gs. At the end of the culture, PG activity in the supernatant was ~84 UE/ml. The specific activity and the productivity obtained were ~1.91 104 UE/gx and ~9,301 UE/l.h, respectively. Respiratory quotient was approximately 1.0 throughout the fermentation process. No other product different from biomass and CO2 was detected. Batch culture could be an adequate alternative for the production of polygalacturonase from W. anomalus and an extract with high enzymatic activity using a synthetic and economic culture medium could be obtained.