RESUMEN

Biochemical and kinetic properties are of special interest for the specific applications of α-amylases in industrial sectors such as textile industries, detergents, biofuels and food among others. Therefore, protein engineering is currently directed towards a continuous demand to improve the properties of amylases and thus meet the specific characteristics for various industrial sectors. In the present work, modular protein engineering was performed to improve the biochemical and kinetic properties of AmyJ33r an α-amylase isolated from Bacillus siamensis JJC33M consisting of five domains, A, B, C, D and E (SBD) (Montor-Antonio et al. in 3 Biotech 7:336, 2017. https://doi.org/10.1007/s13205-017-0954-8 ). AmyJ33r is not active on native starch, only showing activity on gelatinized starch. At the C-terminal, AmyJ33r has a starch binding domain (SBD, domain E) belonging to the CBM26 family. In this study, four truncated versions were constructed and expressed in E. coli (AmyJ33-AB, AmyJ33-ABC, AmyJ33-ABCD, and SBD) to determine the role of the A, B, C, D, and E domains in the biochemical behavior of AmyJ33r on starch. Biochemical and kinetic characterization of the truncated versions showed that domain C is essential for catalysis; domain D improved enzyme activity at alkaline pH values, is also involved negatively in thermostability at 40, 50, and 60 °C and its presence favored the production of maltooligosaccharides with a higher degree of polymerization (DP4). E domain have interaction with raw starch, also the deletion of E domain (SBD) favors the affinity for the substrate while the deletion of D domain increased enzyme kcat at the time of product release. In conclusion, AmyJ33-ABC has better kinetic parameters than AmyJ33-ABCD and AmyJ33r, but is less stable than these two enzymes.

Asunto(s)

Amilasas , Escherichia coli , alfa-Amilasas/genética , Amilasas/genética , Catálisis , Escherichia coli/genética , Almidón , Biocatálisis

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Eugenia dysenterica have been popularly used in Brazil for treating diabetes mellitus and its complications. The present study aimed to screen extracts from E. dysenterica fruit pulp, peel, seed and leaf for carbohydrate digestive enzymes inhibitors with antioxidant and anti-glycation capacities. MATERIALS AND METHODS: Ethanol extracts of E. dysenterica were subjected to a liquid-liquid fractionation and the fractions were used to evaluate their antioxidant properties and inhibitory potential against the formation of advanced glycation end-products (AGEs) and α-amylase and α-glucosidase. RESULTS: The ethyl acetate fraction (EtOAcF) from seed and the dichloromethane fraction (CH2Cl2F) and EtOAcF from leaf had high antioxidant capacities (ORAC >5500 µmol trolox eq g-1, FRAP >1500 µmol trolox eq g-1 and DPPH IC50 < 35 µg mL-1) and showed exceptional inhibitory activities against AGEs formation (glycation inhibition above 80% at 10 µg mL-1) and α-amylase and α-glucosidase (inhibition above 50% at 10 µg mL-1). The gallated B-types proanthocyanidins were the most active ingredients found in the leaf of E. dysenterica (CH2Cl2 and EtOAcF), being responsible for the notorious inhibitory effects against glycation and glycoside hydrolases due to their ortho-hydroxyl groups, which play role in scavenge and quench free radicals and glycated products, and may occupy the enzymes' substrate binding pocket. Furthermore, gallic acid, quercetin and its glycoside derivatives were detected by the first time in the E. dysenterica fruit seed (EtOAcF). CONCLUSIONS: The results strongly contribute to the understanding of the antidiabetic potential of seeds and leaves from E. dysenterica, a species from a global biodiversity hotspot, which appears to be linked to the prevention of oxidative stress, AGEs production and postprandial hyperglycemia.

Asunto(s)

Eugenia/química , Flavonoides/química , Frutas/química , Hojas de la Planta/química , Proantocianidinas/química , Animales , Antioxidantes/química , Antioxidantes/farmacología , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Productos Finales de Glicación Avanzada , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Polifenoles/química , Polifenoles/farmacología , alfa-Amilasas/genética , alfa-Amilasas/metabolismo , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismo

RESUMEN

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.(AU)

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A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.(AU)

Asunto(s)

alfa-Amilasas/genética , Geobacillus , Escherichia coli/genética , Vectores Genéticos

RESUMEN

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

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A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Asunto(s)

Escherichia coli/genética , alfa-Amilasas/genética , alfa-Amilasas/metabolismo , Temperatura , Estabilidad de Enzimas , Clonación Molecular , Geobacillus , Concentración de Iones de Hidrógeno

RESUMEN

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

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A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Asunto(s)

Escherichia coli/genética , Geobacillus , Vectores Genéticos , alfa-Amilasas/genética

RESUMEN

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

Asunto(s)

Escherichia coli , alfa-Amilasas , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Geobacillus , Concentración de Iones de Hidrógeno , Temperatura , alfa-Amilasas/genética , alfa-Amilasas/metabolismo

RESUMEN

Since the beginning of oil exploration, whole ecosystems have been affected by accidents and bad practices involving petroleum compounds. In this sense, bioremediation stands out as the cheapest and most eco-friendly alternatives to reverse the damage done in oil-impacted areas. However, more efforts must be made to engineer enzymes that could be used in the bioremediation process. Interestingly, a recent work described that α-amylase, one of the most evolutionary conserved enzymes, was able to promiscuously degrade n-alkanes, a class of molecules abundant in the petroleum admixture. Considering that α-amylase is expressed in almost all known organisms, and employed in numerous biotechnological processes, using it can be a great leap toward more efficient applications of enzyme or microorganism-consortia bioremediation approaches. In this work, we employed a strict computational approach to design new α-amylase mutants with potentially enhanced catalytic efficiency toward n-alkanes. Using in silico techniques, such as molecular docking, molecular dynamics, metadynamics, and residue-residue interaction networks, we generated mutants potentially more efficient for degrading n-alkanes, L183Y, and N314A. Our results indicate that the new mutants have an increased binding rate for tetradecane, the longest n-alkane previously tested, which can reside in the catalytic center for more extended periods. Additionally, molecular dynamics and network analysis showed that the new mutations have no negative impact on protein structure than the WT. Our results aid in solidifying this enzyme as one more tool in the petroleum bioremediation toolbox.

Asunto(s)

Alcanos/metabolismo , Simulación del Acoplamiento Molecular , alfa-Amilasas/metabolismo , Alcanos/química , Bacillus subtilis/enzimología , Biocatálisis , Biodegradación Ambiental , alfa-Amilasas/química , alfa-Amilasas/genética

RESUMEN

Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonusrhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.

Asunto(s)

Escarabajos/metabolismo , Tracto Gastrointestinal/metabolismo , Pinus/parasitología , Corteza de la Planta/parasitología , Almidón/metabolismo , alfa-Amilasas/metabolismo , Amilopectina/metabolismo , Amilosa/metabolismo , Animales , Unión Competitiva , Escarabajos/enzimología , Escarabajos/genética , Tracto Gastrointestinal/enzimología , Regulación Enzimológica de la Expresión Génica , Hidrólisis , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Unión Proteica , alfa-Amilasas/genética

RESUMEN

The subfamily GH13_1 of alpha-amylases is typical of Fungi, but it is also found in some unicellular eukaryotes (e.g., Amoebozoa, choanoflagellates) and non-bilaterian Metazoa. Since a previous study in 2007, GH13_1 amylases were considered ancestral to the Unikonts, including animals, except Bilateria, such that it was thought to have been lost in the ancestor of this clade. The only alpha-amylases known to be present in Bilateria so far belong to the GH13_15 and 24 subfamilies (commonly called bilaterian alpha-amylases) and were likely acquired by horizontal transfer from a proteobacterium. The taxonomic scope of Eukaryota genomes in databases has been greatly increased ever since 2007. We have surveyed GH13_1 sequences in recent data from ca. 1600 bilaterian species, 60 non-bilaterian animals and also in unicellular eukaryotes. As expected, we found a number of those sequences in non-bilaterians: Anthozoa (Cnidaria) and in sponges, confirming the previous observations, but none in jellyfishes and in Ctenophora. Our main and unexpected finding is that such fungal (also called Dictyo-type) amylases were also consistently retrieved in several bilaterian phyla: hemichordates (deuterostomes), brachiopods and related phyla, some molluscs and some annelids (protostomes). We discuss evolutionary hypotheses possibly explaining the scattered distribution of GH13_1 across bilaterians, namely, the retention of the ancestral gene in those phyla only and/or horizontal transfers from non-bilaterian donors.

Asunto(s)

Basidiomycota/genética , Evolución Molecular , Transferencia de Gen Horizontal , Transformación Genética , alfa-Amilasas/genética , Basidiomycota/metabolismo , Genes Fúngicos , Intrones , Filogenia

RESUMEN

Xylanase and α-amylase enzymes participate in the degradation of organic matter, acting in hemicellulose and starch mineralization, respectively, and are in high demand for industrial use. Mangroves represent a promising source for bioprospecting enzymes due to their unique characteristics, such as fluctuations in oxic/anoxic conditions and salinity. In this context, the present work aimed to bioprospect xylanases from mangrove soil using cultivation-dependent and cultivation-independent methods. Through screening from a metagenomic library, three potentially xylanolytic clones were obtained and sequenced, and reads were assembled into contigs and annotated. The contig MgrBr135 was affiliated with the Planctomycetaceae family and was one of 30 ORFs selected for subcloning that demonstrated only amylase activity. Through the cultivation method, 38 bacterial isolates with xylanolytic activity were isolated. Isolate 11 showed an enzymatic index of 10.9 using the plate assay method. Isolate 39 achieved an enzyme activity of 0.43 U/mL using the colorimetric method with 3,5-dinitrosalicylic acid. Isolate 39 produced xylanase on culture medium with salinity ranging from 1.25 to 5%. Partial 16S rRNA gene sequencing identified isolates in the Bacillus and Paenibacillus genera. The results of this study highlight the importance of mangroves as an enzyme source and show that bacterial groups can be used for starch and hemicellulose degradation.

Asunto(s)

Bacterias/aislamiento & purificación , Endo-1,4-beta Xilanasas/genética , Microbiología del Suelo , Humedales , alfa-Amilasas/genética , Bacillus/genética , Bacillus/aislamiento & purificación , Bacillus/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Celulosa/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Genes Bacterianos/genética , Metagenómica , Paenibacillus/genética , Paenibacillus/aislamiento & purificación , Paenibacillus/metabolismo , Planctomycetales/clasificación , Planctomycetales/genética , Planctomycetales/aislamiento & purificación , Planctomycetales/metabolismo , ARN Ribosómico 16S , Almidón/metabolismo , alfa-Amilasas/metabolismo

RESUMEN

The study aimed to evaluate nutrient digestibility and intestine gene expression in the progeny from cows supplemented during gestation and fed diets with or without rumen-protected fat (RPF) in the feedlot. Forty-eight Nellore steers, averaging 340 kg, were housed in individual pens and allotted in a completely randomized design using a 2 × 2 factorial arrangement (dams nutrition × RPF). Cows' supplementation started after 124 ± 21 days of gestation. The feedlot lasted 135 days and diets had the inclusion of zero or 6% of RPF. Digestibility was evaluated by total feces collection. Steers were slaughtered using the concussion technique and samples of pancreas and small intestine were collected immediately after the slaughter to analyze α-amylase activity, and the expression of SLC5A1, CD36, and CCK and villi morphometry. Feeding RPF increased nutrients digestibility (p < 0.01). There was no effect of maternal nutrition on digestibility and α-amylase activity in steers (p > 0.05). Duodenal expression of SLC5A1, CD36, and CCK increased in the progeny from restricted cows. In conclusion, protein restriction during mid to late gestation of dams has long-term effects on small-intestine length and on expression of membrane transporters genes in the duodenum of the progeny. However, maternal nutrition does not affect digestibility in the feedlot.

Asunto(s)

Fenómenos Fisiológicos Nutricionales de los Animales , Dieta Alta en Grasa/veterinaria , Dieta con Restricción de Proteínas/veterinaria , Dieta/veterinaria , Fenómenos Fisiologicos Nutricionales Maternos , Preñez , Animales , Bovinos , Digestión/fisiología , Proteínas de Transporte de Ácidos Grasos/genética , Proteínas de Transporte de Ácidos Grasos/metabolismo , Femenino , Expresión Génica , Intestino Delgado/anatomía & histología , Intestino Delgado/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Embarazo , Proteínas de Transporte de Sodio-Glucosa/genética , Proteínas de Transporte de Sodio-Glucosa/metabolismo , alfa-Amilasas/genética , alfa-Amilasas/metabolismo

RESUMEN

BACKGROUND: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). RESULTS: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS: We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

Asunto(s)

Inhibidores Enzimáticos/metabolismo , Expresión Génica , Ingeniería Genética/métodos , Nicotiana/genética , alfa-Amilasas/antagonistas & inhibidores , Animales , Evolución Molecular Dirigida , Inhibidores Enzimáticos/química , Silenciador del Gen , Control de Insectos/métodos , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Gorgojos , alfa-Amilasas/genética , alfa-Amilasas/metabolismo

RESUMEN

Background: α-Amylase is widely used in the starch processing, food and paper industries, hydrolyzing starch, glycogen and other polysaccharides into glucose, maltose and oligosaccharides. An α-amylase gene family from Aspergillus niger CBS513.88 encode eight putative α-amylases. The differences and similarities, biochemical properties and functional diversity among these eight α-amylases remain unknown. Results: The eight genes were cloned and expressed in Pichia pastoris GS115 by shaking-flask fermentation under the induction of methanol. The sequence alignment, biochemical characterizations and product analysis of starch hydrolysis by these α-amylases were investigated. It is found that the eight α-amylases belonged to three different groups with the typical structure of fungal α-amylase. They exhibited maximal activities at 30­40°C except AmyG and were all stable at acidic pH. Ca2+ and EDTA had no effects on the activities of α-amylases except AmyF and AmyH, indicating that the six amylases were Ca2+ independent. Two novel α-amylases of AmyE and AmyF were found. AmyE hydrolyzed starch into maltose, maltotriose and a small amount of glucose, while AmyF hydrolyzed starch into mainly glucose. The excellent physical and chemical properties including high acidic stability, Ca2+-independent and high maltotriose-forming capacity make AmyE suitable in food and sugar syrup industries. Conclusions: This study illustrates that a gene family can encode multiple enzymes members having remarkable differences in biochemical properties. It provides not only new insights into evolution and functional divergence among different members of an α-amylase family, but the development of new enzymes for industrial application.

Asunto(s)

Aspergillus niger/enzimología , alfa-Amilasas/genética , alfa-Amilasas/química , Pichia/metabolismo , Almidón , Temperatura , Industria de Alimentos , Clonación Molecular , Fermentación , Concentración de Iones de Hidrógeno , Hidrólisis

RESUMEN

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Asunto(s)

Semillas/enzimología , Hordeum/enzimología , Thermococcus/metabolismo , alfa-Amilasas/metabolismo , Semillas/genética , Semillas/microbiología , Transformación Genética , Hordeum/genética , Hordeum/microbiología , Cerveza , Estabilidad de Enzimas , Plantas Modificadas Genéticamente/enzimología , Clonación Molecular , Técnicas de Transferencia de Gen , alfa-Amilasas/genética , Fermentación , Termotolerancia , Calor , Concentración de Iones de Hidrógeno

RESUMEN

The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter. The active TvLEGU-1 recombinant protein (rTvLEGU-1) was secreted into the medium when tvlegu-1-opt was fused to the Aspergillus niger alpha-amylase signal peptide. The rTvLEGU-1 secretion was influenced by the gene copy number and induction temperature. Data indicate that increasing tvlegu-1-opt gene copy number was detrimental for heterologous expression of the enzymatically active TvLEGU-1. Indeed, expression of TvLEGU-1 had a greater impact on cell viability for those clones with 26 or 29 gene copy number, and cell lysis was observed when the induction was carried out at 30 °C. The enzyme activity in the medium was higher when the induction was carried out at 16 °C and in P. pastoris clones with lower gene copy number. The results presented here suggest that both copy number and induction temperature affect the rTvLEGU-1 expression in its native-like and active conformation.

Asunto(s)

Proteasas de Cisteína , Expresión Génica , Pichia/metabolismo , Proteínas Protozoarias , Proteínas Recombinantes de Fusión , Trichomonas vaginalis/genética , Aspergillus niger/enzimología , Aspergillus niger/genética , Proteasas de Cisteína/biosíntesis , Proteasas de Cisteína/química , Proteasas de Cisteína/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Pichia/genética , Señales de Clasificación de Proteína/genética , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Trichomonas vaginalis/enzimología , alfa-Amilasas/biosíntesis , alfa-Amilasas/química , alfa-Amilasas/genética

RESUMEN

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.

Asunto(s)

Proteínas de Artrópodos , Carnivoría/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Palinuridae , alfa-Amilasas , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , ADN Complementario/genética , ADN Complementario/metabolismo , Glicosilación , Isoenzimas/biosíntesis , Isoenzimas/genética , Palinuridae/genética , Palinuridae/metabolismo , Proteolisis , alfa-Amilasas/biosíntesis , alfa-Amilasas/genética

RESUMEN

The study of digestive physiology is an important issue in species that have been introduced in aquaculture like the spotted rose snapper (Lutjanus guttatus). The aims of this study were to describe the expression of digestive enzymes (trypsinogen, chymotrypsinogen, α-amylase, lipoprotein lipase, phospholipase A and pepsinogen) and their relation with orexigenic (neuropeptide Y, NPY) and anorexigenic (cholecystokinin, CCK) factors during the larval development and to evaluate the effect of weaning in their expression. The results showed that the transcripts of all the assayed digestive enzymes, with the exception of pepsinogen, and NPY and CCK were already present in L. guttatus from the hatching stage. The expression of all the enzymes was low during the yolk-sac stage (0-2 days after hatching, DAH), whereas after the onset of exogenous feeding at 2 DAH, their expression increased and fluctuated throughout larval development, which followed a similar pattern as in other marine fish species and reflected changes in different types of food items and the progressive maturation of the digestive system. On the other hand, weaning of L. guttatus larvae from live prey onto a microdiet between 25 and 35 DAH significantly affected the relative expression of most pancreatic digestive enzymes during the first weaning days, whereas chymotrypsinogen 2 and lipoprotein lipase remained stable during this period. At the end of co-feeding, larvae showed similar levels of gene expression regardless of the diet (live prey vs. microdiet), which indicated that larvae of L. guttatus were able to adapt their digestive capacities to the microdiet. In contrast, feeding L. guttatus larvae with live feed or microdiet did not affect the expression of CCK and NPY. The relevance of these findings with regard to current larval rearing procedures of L. guttatus is discussed.

Asunto(s)

Digestión/genética , Perciformes/genética , Animales , Colecistoquinina/genética , Quimotripsinógeno/genética , Femenino , Expresión Génica , Larva/genética , Larva/crecimiento & desarrollo , Lipoproteína Lipasa/genética , Masculino , Neuropéptido Y/genética , Pepsinógeno A/genética , Perciformes/crecimiento & desarrollo , Perciformes/metabolismo , Fosfolipasas A2/genética , ARN Mensajero/metabolismo , Tripsinógeno/genética , alfa-Amilasas/genética

RESUMEN

Phytohormones have different characteristics and functions, and they may be subject to changes in their gene expression and synthesis during seed development. In this study, we evaluated the physiological qualities of habanero peppers (Capsicum chinense Jacquin) during seed development and the expression of genes involved in germination. Seeds were obtained from fruits harvested at different stages of development [i.e., 14, 21, 28, 35, 42, 49, 56, 63, and 70 days after anthesis (DAA)]. Immediately after harvesting, the seeds were subjected to various tests to determine moisture content, germination, first count germination, and seedling emergence. Real-time polymerase chain reaction was used to evaluate the expression of various genes, including MAN2, NCED, B73, ICL6, and GA3ox. Electrophoresis was used to assess the expression of various enzymes, including α-amylase, isocitrate-lyase, and endo-ß-mannanase. Habanero peppers harvested at 70 DAA and subjected to 7 days of rest exhibited higher germination rates and vigor compared to those harvested at all other developmental stages. Peppers harvested at 63 DAA without drying exhibited higher α amylase and AmyB73 gene expression levels. Peppers harvested at 70 DAA with 7 days of rest exhibited higher endo-ß-mannanase expression levels. MAN2 gene expression increased during the development of non-dried seeds until 70 DAA. Peppers harvested at 42 DAA exhibited the highest isocitrate-lyase and ICL6 gene activity levels in comparison to those at all other developmental stages.

Asunto(s)

Capsicum/genética , Frutas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Semillas/genética , Capsicum/crecimiento & desarrollo , Capsicum/metabolismo , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Germinación , Isocitratoliasa/genética , Isocitratoliasa/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/metabolismo , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Semillas/crecimiento & desarrollo , Semillas/metabolismo , alfa-Amilasas/genética , alfa-Amilasas/metabolismo , beta-Manosidasa/genética , beta-Manosidasa/metabolismo

RESUMEN

α-Amylases are common enzymes responsible for hydrolyzing starch. Insect-pests, whose larvae develop in seeds, rely obligatorily on α-amylase activity to digest starch, as their major food source. Considering the relevance of insect α-amylases and the natural α-amylase inhibitors present in seeds to protect from insect damage, we report here the molecular cloning and nucleotide sequence of the full-length AmyHha cDNA of the coffee berry borer, Hypothenemus hampei, a major insect-pest of coffee crops. The AmyHha sequence has 1879 bp, containing a 1458 bp open reading frame, which encodes a predicted protein with 485 amino acid residues, with a predicted molecular mass of 51.2 kDa. The deduced protein showed 55-79% identity to other insect α-amylases, including Anthonomus grandis, Ips typographus and Sitophilus oryzae α-amylases. In depth analysis revealed that the highly conserved three amino acid residues (Asp184, Glu220, and Asp285), which compose the catalytic site are also presented in AmyHha amylase. The AmyHha gene seems to be a single copy in the haploid genome and AmyHha transcription levels were found higher in L2 larvae and adult insects, both corresponding to major feeding phases. Modeling of the AmyHha predicted protein uncovered striking structural similarities to the Tenebrio molitor α-amylase also displaying the same amino acid residues involved in enzyme catalysis (Asp184, Glu220 and Asp285). Since AmyHha gene was mostly transcribed in the intestinal tract of H. hampei larvae, the cognate α-amylase could be considered a high valuable target to coffee bean insect control by biotechnological strategies.

Asunto(s)

Escarabajos/fisiología , ADN Complementario/genética , Conducta Alimentaria , alfa-Amilasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Escarabajos/clasificación , Escarabajos/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido , alfa-Amilasas/química

RESUMEN

The freshwater pearl mussel Hyriopsis cumingii is of commercial importance because it produces the freshwater pearl; however, knowledge about the molecular characterization and regulation mechanisms of α-amylase remains unknown for this species. In this study, the full-length cDNA of the α-amylase gene (HcAmy) was isolated from H. cumingii by the rapid amplification of cDNA ends. Tissue-specific expression analysis showed that HcAmy mRNA was mainly expressed in the hepatopancreas; although, the gene was also expressed in the adductor muscle, intestine, gill, and crystalline style. After 2 weeks starvation, the expression of HcAmy mRNA in the hepatopancreas was upregulated at 24 h after re-feeding or when exposed to algal concentration of 32 µg/L chlorophyll-a, indicating that the HcAmy mRNA expression in H. cumingii is regulated by algal availability. The results of this study confirm that the HcAmy gene is an important component of the carbohydrate metabolism of H. cumingii fed phytoplankton. In addition, this study demonstrates that the modulation of this gene is dependent on environmental food availability, including starvation, re-feeding time following a period of starvation, and algal concentrations during re-feeding.

Asunto(s)

Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hepatopáncreas/metabolismo , Unionidae/genética , alfa-Amilasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlorella/fisiología , Chlorophyta/fisiología , ADN Complementario/química , ADN Complementario/genética , Conducta Alimentaria/fisiología , Agua Dulce , Branquias/enzimología , Branquias/metabolismo , Hepatopáncreas/enzimología , Mucosa Intestinal/metabolismo , Intestinos/enzimología , Datos de Secuencia Molecular , Filogenia , Fitoplancton/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Unionidae/enzimología , alfa-Amilasas/clasificación