Production, partial purification, and characterization of a glucoamylase from Epicoccum nigrum

Starch processing industries use amylases, accounting for approximately 30% of the world's enzyme market. Previously, an amylase-producing strain of Epicoccum nigrumwas isolated from maize grains. Although E. nigrumamylase production is already reported in the literature, no published data on production optimization or characterization of the produced enzyme exists. The objectives of this work were to improve the amylase production by theE. nigrumPG 16 strain and to purify and characterize the produced enzyme. The E. nigrumPG 16 amylase production best conditions in submerged culture were: inoculum of 4% (v v-1) of a five-days-old stationary culture homogenate, agitation at 100 rpm, 25°C, natural light, 72 hours of incubation, starch as thecarbon source, and an initial medium pH of 7.0. A molecular exclusion chromatography profile has shown the production of only one amylase, which was partially purified with ammonium precipitation and dialysis. The enzyme optima pH and temperature are 6.0 and 50°C, respectively. The partially purified enzyme lostits activity when incubated for 30 min in temperatures above 40°C, presenting a T50of 46.25°C. The KMand Vmaxof the partially purified enzyme are 1.72 mg mL-1of starch and 0.15 mgmin-1of degraded starch, respectively. The ion Ca2+slightly activated the studied enzyme. The ions Cu2+, Zn2+, and Fe3+and the detergents SDS and Tween 80 acted as inhibitors of the studied enzyme. The partially purified enzyme released glucose from p-nitrophenyl α-D-glucopyranoside (p-NPG). Glucose was the enzyme's main product from starch hydrolysis, as evidenced by thin-layer chromatography. The E. nigrumPG 16 studied enzyme is a glucoamylase and represents an alternative for enzymatic starch hydrolysis.(AU)

Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

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.

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.

Escherichia coli expression and characterization of alfa-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

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.

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.

Escherichia coli expression and characterization of alfa-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

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)

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)

Eurytrema coelomaticum infection: correlation between parasite burden and impairment of pancreatic exocrine enzyme secretion / Eurytrema coelomaticum: correlação entre carga parasitária e insuficiência pancreática exócrina

Eurytrema coelomaticum is a trematode reported in the pancreatic ducts of ruminants. It is conjectured that may cause disorders in the pancreas, as well as digestive and metabolic processes dependent on them. This study, determined if there is an impairment of exocrine pancreatic function, and correlated it with parasite burden. Pancreas, blood, and fecal samples were collected from 119 bovines at a abattoir. Stool samples were subjected to the gelatin and x-ray film digestion tests (to detect the presence of trypsin in feces). Using blood samples, the following biochemical tests were performed: amylase, lipase, glucose, fructosamine, cholesterol, triglycerides, total protein, albumin, and globulins. Analyses were correlated with pancreatic parasite burden. Cattle with a high parasitic load presented higher incidence of negative tests in both gelatin digestion and x-ray film digestion tests (P < 0.001) when compared to non-parasitized animals and those with a low parasitic load. Changes in those tests only occurred if the parasitemia was moderate or severe. The activity of the amylase and lipase enzymes was significantly higher in animals with low parasitemia (P < 0.05), compared to non-parasitized animals and with a high parasitic burden. In this study, in cases of high parasitemia, negative results were observed in both gelatin and x-ray film in the feces digestion tests. However, the low infection of E. coelomaticum, higher levels of serum amylase and lipase that also indicated loss of pancreatic exocrine functions were reported.

Eurytrema coelomaticum, um trematódeo de ductos pancreáticos de ruminantes. Conjectura-se que possa ocasionar transtornos nas funções pancreáticas, mais especificamente nos processos digestivos e metabólicos dependentes destas. Neste estudo, o objetivo foi determinar se há comprometimento da função pancreática exócrina, correlacionado-a a carga parasitária. Foram utilizados pâncreas e respectivas amostras de sangue e fezes de 119 bovinos. As amostras de fezes foram submetidas aos testes de digestão da gelatina em tubo e digestão de filme radiográfico, ambos para detecção de tripsina nas fezes. Foram realizados os seguintes exames bioquímicos em amostras de sangue: amilase, lipase, glicemia, frutosamina, colesterol, triglicerídeos, proteínas totais, albumina e globulinas. Após isto, as análises bioquímicas foram correlacionadas com a quantidade numérica de parasitas encontrados no pâncreas (post-mortem). Houve maior quantidade de testes negativos (digestão do filme radiográfico e prova de digestão da gelatina) nos animais com alta carga parasitária (P < 0.001), quando comparados aos animais não parasitados e com baixa carga parasitária. Portanto, os exames supracitados se alteram somente se a quantidade de parasitas for moderada ou severa. As atividades das enzimas amilase e lipase foram significativamente maiores nos animais que apresentavam baixa parasitemia (P < 0.05), em comparação com os animais com alta carga parasitária e não parasitados. Conclui-se que em quadros de alta parasitemia há alteração significativa nos testes de digestão nas fezes, e que em quadros de baixa parasitemia há alterações significativas nos valores de amilase e lipase séricas, ambos comprovando alterações pancreáticas importantes, de acordo com o quadro de parasitemia.

Uso do gérmen de milho desengordurado, com e sem adição de um complexo enzimático, em dietas para cães / [Use of defatted corngerm with and without the addition of an enzyme complex in dog diets]

Objetivou-se avaliar o coeficiente de digestibilidade aparente (CDA) dos nutrientes, a palatabilidade das dietas e as características fecais de cães alimentados com uma dieta controle e uma dieta contendo 20% de gérmen desengordurado (GD), com e sem adição de complexo enzimático (amilase, xilanase, betaglucanase e mananase). Para o experimento de digestibidade e das características fecais, foram utilizados 12 cães adultos, distribuídos em delineamento em blocos ao acaso, em esquema fatorial 2 x 2 (dieta x enzima). O segundo experimento avaliou a palatabilidade, por meio da primeira escolha e da razão de ingestão (RI) da dieta DC vs. 20% de GD, utilizando-se 16 cães. O teste de palatabilidade contou com três dias consecutivos, totalizando 48 repetições. A dieta com inclusão de 20% de GD teve os menores valores de CDA da MS, da EB e da EM (P<0,05). A inclusão do complexo enzimático melhorou o CDA da MS, da EB e da EM (P<0,05). Não foram observadas diferenças nas características fecais (P>0,05). Em relação à palatabilidade, os cães preferiram a dieta 20% de GD, tanto na primeira escolha como na RI (P<0,05). A inclusão de enzimas às dietas melhora a digestibilidade dos nutrientes e da EM, sendo um aditivo com potencial uso na alimentação de cães.(AU)

The objective was to evaluate the apparent digestibility coefficient (ADC) of nutrients, diet palatability and fecal characteristics of dogs fed diets containing degreased germ (DG), and a control diet (DC) - both with and without the addition of enzyme complex (amylase, xylanase, betaglucanase and mananase). For the digestibility and fecal characteristics experiment 12 adult dogs were used, distributed in a randomized block design, in a 2 x 2 factorial scheme (diet x enzyme). The second experiment evaluated palatability using the first choice and ingestion ratio (IR) of DC diet vs. 20%gD, using 16 dogs. The palatability test had three consecutive days, totaling 48 repetitions. The diet with inclusion of 20% DG had the lowest ADC values of DM, GE and ME (P <0.05). Inclusion of the enzyme complex improved ADC of DM, GE and ME (P <0.05). No differences in fecal characteristics were observed (P >0.05). Regarding palatability, dogs preferred the 20% DG diet in both first choice and IR (P <0.05). Inclusion of enzymes in diets improves nutrient digestibility and ME, being an additive with potential use in dog food.(AU)

Change in malate dehydrogenase and alpha amylase activities in Rubus fruticosus and Valeriana jatamansi treated granary weevil, Sitophilus granarius / Mudança nas atividades de malato desidrogenase e alfa amilase em Rubus fruticosus e gorgulho tratado com Valeriana jatamansi, Sitophilus granarius

Poor storage conditions provide favorable environment to stored grain pests for their growth. The bio-pesticides are the best alternatives to synthetic pesticides. Present study was conducted to compare toxicity of Rubus fruticosus and Valeriana jatamansi against granary weevil, Sitophilus granarius and subsequent changes in enzyme activity responsible for grain damage. In current research 5 g of R. fruticosus fruit and V. jatamansi rhizome powders were tested separately against S. granarius, in 50 g wheat whole grains for seven days in comparison with the control. The enzymatic activity of malate dehydrogenase and alpha-amylase was observed in the cellular extracts of S. granarius. The insects were crushed and homogenized in phosphate-buffer solution and centrifuged at 10000 rpm for 5 minutes. For the enzymatic measurement supernatant was tested; the spectrophotometer was adjusted at 340 nm. The reagents were mixed and incubated at 25 °C for five minutes. The cuvettes were placed in the experimental and reference sites of spectrophotometer and recorded the change in absorbance for 3-4 minutes. There was 5.60% and 14.92% reduction in the activity of malate dehydrogenase in R. fruticosus and V. jatamansi, treated insects, respectively. The alpha amylase enzyme activity was 6.82% reduced and 63.63% increase in R. fruticosus and V. jatamansi, treated insects, respectively. Present study addresses that both plant powders are effective against granary weevil by altering enzyme activities so both the plant powders can be used as bio-pesticides against the stored grains pests.(AU)

As más condições de armazenamento proporcionam um ambiente favorável às pragas armazenadas para o crescimento. Os biopesticidas são as melhores alternativas aos pesticidas sintéticos. O presente estudo foi conduzido para comparar a toxicidade de Rubus fruticosus e Valeriana jatamansi contra gorgulhos, Sitophilus granarius e subsequentes alterações na atividade enzimática responsáveis por danos aos grãos. Na pesquisa atual, 5 g de frutos de R. fruticosus e pós de rizoma de V. jatamansi foram testados separadamente contra S. granarius, em 50 g de grãos integrais de trigo por sete dias, em comparação com o controle. A atividade enzimática da malato desidrogenase e alfa-amilase foi observada nos extratos celulares de S. granarius. Os insetos foram esmagados e homogeneizados em solução tampão fosfato e centrifugados a 10000 rpm por 5 minutos. Para a medição enzimática, o sobrenadante foi testado; o espectrofotômetro foi ajustado a 340 nm. Os reagentes foram misturados e incubados a 25 °C por cinco minutos. As cubetas foram colocadas nos locais experimentais e de referência do espectrofotômetro e registradas as alterações na absorbância por 3-4 minutos. Houve redução de 5,60% e 14,92% na atividade da malato desidrogenase em R. fruticosus e V. jatamansi, insetos tratados, respectivamente. A atividade da enzima alfa amilase foi reduzida em 6,82% e aumento de 63,63% em R. fruticosus e V. jatamansi, insetos tratados, respectivamente. O presente estudo aborda que ambos os pós de plantas são eficazes contra o gorgulho do celeiro, alterando as atividades enzimáticas, de modo que ambos os pós de plantas possam ser usados como biopesticidas contra pragas de grãos armazenados.(AU)

Change in malate dehydrogenase and alpha amylase activities in Rubus fruticosus and Valeriana jatamansi treated granary weevil, Sitophilus granarius / Mudança nas atividades de malato desidrogenase e alfa amilase em Rubus fruticosus e gorgulho tratado com Valeriana jatamansi, Sitophilus granarius

Poor storage conditions provide favorable environment to stored grain pests for their growth. The bio-pesticides are the best alternatives to synthetic pesticides. Present study was conducted to compare toxicity of Rubus fruticosus and Valeriana jatamansi against granary weevil, Sitophilus granarius and subsequent changes in enzyme activity responsible for grain damage. In current research 5 g of R. fruticosus fruit and V. jatamansi rhizome powders were tested separately against S. granarius, in 50 g wheat whole grains for seven days in comparison with the control. The enzymatic activity of malate dehydrogenase and α-amylase was observed in the cellular extracts of S. granarius. The insects were crushed and homogenized in phosphate-buffer solution and centrifuged at 10000 rpm for 5 minutes. For the enzymatic measurement supernatant was tested; the spectrophotometer was adjusted at 340 nm. The reagents were mixed and incubated at 25 °C for five minutes. The cuvettes were placed in the experimental and reference sites of spectrophotometer and recorded the change in absorbance for 3-4 minutes. There was 5.60% and 14.92% reduction in the activity of malate dehydrogenase in R. fruticosus and V. jatamansi, treated insects, respectively. The alpha amylase enzyme activity was 6.82% reduced and 63.63% increase in R. fruticosus and V. jatamansi, treated insects, respectively. Present study addresses that both plant powders are effective against granary weevil by altering enzyme activities so both the plant powders can be used as bio-pesticides against the stored grains pests.

As más condições de armazenamento proporcionam um ambiente favorável às pragas armazenadas para o crescimento. Os biopesticidas são as melhores alternativas aos pesticidas sintéticos. O presente estudo foi conduzido para comparar a toxicidade de Rubus fruticosus e Valeriana jatamansi contra gorgulhos, Sitophilus granarius e subsequentes alterações na atividade enzimática responsáveis ​​por danos aos grãos. Na pesquisa atual, 5 g de frutos de R. fruticosus e pós de rizoma de V. jatamansi foram testados separadamente contra S. granarius, em 50 g de grãos integrais de trigo por sete dias, em comparação com o controle. A atividade enzimática da malato desidrogenase e α-amilase foi observada nos extratos celulares de S. granarius. Os insetos foram esmagados e homogeneizados em solução tampão fosfato e centrifugados a 10000 rpm por 5 minutos. Para a medição enzimática, o sobrenadante foi testado; o espectrofotômetro foi ajustado a 340 nm. Os reagentes foram misturados e incubados a 25 °C por cinco minutos. As cubetas foram colocadas nos locais experimentais e de referência do espectrofotômetro e registradas as alterações na absorbância por 3-4 minutos. Houve redução de 5,60% e 14,92% na atividade da malato desidrogenase em R. fruticosus e V. jatamansi, insetos tratados, respectivamente. A atividade da enzima alfa amilase foi reduzida em 6,82% e aumento de 63,63% em R. fruticosus e V. jatamansi, insetos tratados, respectivamente. O presente estudo aborda que ambos os pós de plantas são eficazes contra o gorgulho do celeiro, alterando as atividades enzimáticas, de modo que ambos os pós de plantas possam ser usados ​​como biopesticidas contra pragas de grãos armazenados.

Effect of different levels of supplementary alpha-amylase in finishing broilers

The objective of this study was to investigate the effects of alpha-amylase supplementation on performance and carcass and cuts yield in broilers during finishing phase (21 to 42 days). A completely randomized experimental design with six treatments and eight replications was used: Positive Control (PC): no enzyme; Negative control (NC): without enzyme formulated with 6.34% less energy (- 6.34% ME); NC1: with 250 g ton-1 of alpha-amylase; NC2: with 500 g ton-1 of alpha-amylase; NC3: with 750 g ton-1 of alpha-amylase; NC4: with 1000 g ton-1 of alpha-amylase. Significant differences in weight gain, feed intake and feed conversion were reported with the inclusion of alpha-amylase. The NC treatment with inclusion of amylase provided improvement values of weight gain, feed conversion and feed intake, but do not was similarly in comparison to those obtained in broilers fed a diet that completely met the requirements during this phase. There was no dietary influence on carcass parameters. The use of alphaamylase was effective in improving performance in broilers fed a diet of 200 kcal kg-1 of reduced metabolizable energy.(AU)

Effect of different levels of supplementary alpha-amylase in finishing broilers

The objective of this study was to investigate the effects of alpha-amylase supplementation on performance and carcass and cuts yield in broilers during finishing phase (21 to 42 days). A completely randomized experimental design with six treatments and eight replications was used: Positive Control (PC): no enzyme; Negative control (NC): without enzyme formulated with 6.34% less energy (- 6.34% ME); NC1: with 250 g ton-1 of alpha-amylase; NC2: with 500 g ton-1 of alpha-amylase; NC3: with 750 g ton-1 of alpha-amylase; NC4: with 1000 g ton-1 of alpha-amylase. Significant differences in weight gain, feed intake and feed conversion were reported with the inclusion of alpha-amylase. The NC treatment with inclusion of amylase provided improvement values of weight gain, feed conversion and feed intake, but do not was similarly in comparison to those obtained in broilers fed a diet that completely met the requirements during this phase. There was no dietary influence on carcass parameters. The use of alphaamylase was effective in improving performance in broilers fed a diet of 200 kcal kg-1 of reduced metabolizable energy.

Citral as food additive for common snook - zootechnical parameters and digestive enzymes / Citral como aditivo na ração para robalo flecha - parâmetros zootécnicos e enzimas digestivas

Essential oils of plants whose main compound is citral showed beneficial effects when added to fish feed. The objective of the present study was to evaluate the dietary effect of the addition of citral on zootechnical parameters and digestive enzyme activities of Centropomus undecimalis. Juveniles were fed for 45 days with diets containing different amounts of citral (0.0 - control, 0.5, 1.0, and 2.0 mL per kg of diet). The water quality parameters were kept stable during the experiment and no mortality was observed. At the end of the experimental period, the treatment 0.5 mL citral per kg of diet had the lowest weight gain and specific growth rate, and the highest feed conversion, while the same parameters did not differ between the other treatments. Pepsin activity was higher in the stomach of fish fed with 0.5 mL citral per kg of diet and amylase activity was higher in the intestine of fish fed with 0.5 and 2.0 mL citral per kg of diet compared to the control group. Intestinal lipase activity was higher in all groups that were fed with citral compared to the control group. Chymotrypsin and trypsin activities showed no difference between groups. Consequently, dietary addition of citral at any of the levels tested is not recommended for common snook.(AU)

Óleos essenciais de plantas, cujo composto majoritário é o citral, mostraram efeitos benéficos quando adicionados à ração para peixes. O objetivo do presente estudo foi avaliar o efeito da adição de citral sobre os parâmetros zootécnicos e atividades das enzimas digestivas de Centropomus undecimalis. Os juvenis foram alimentados por 45 dias com dietas contendo diferentes quantidades de citral (0,0 - controle, 0,5, 1,0 e 2,0 mL por kg de ração). Os parâmetros de qualidade da água foram mantidos estáveis ​​durante o experimento e nenhuma mortalidade foi observada. Ao final do período experimental, o tratamento 0,5 mL citral por kg de ração teve o menor ganho de peso e taxa de crescimento específico e a maior conversão alimentar, enquanto os mesmos parâmetros não diferiram entre os demais tratamentos. A atividade da pepsina foi maior no estômago de peixes alimentados com 0,5 mL de citral por kg de ração e a atividade de amilase foi maior no intestino de peixes alimentados com 0,5 e 2,0 mL citral por kg de ração comparado ao grupo controle. A atividade da lipase intestinal foi maior em todos os grupos que foram alimentados com citral, em comparação ao grupo controle. As atividades de quimotripsina e tripsina não mostraram diferença entre os grupos. Consequentemente, a adição de citral na ração em qualquer um dos níveis testados não é recomendada para robalo flecha.(AU)

Changes in digestive enzymes activities during the initial ontogeny of wolf cichlid, Parachromis dovii (Perciformes: Cichlidae)

Wolf cichlid, Parachromis dovii, is a species with a high potential for aquaculture in Central America; however, the knowledge of the digestive physiology in larvae period is limited. For these reason, this study evaluated the changes on digestive enzymes (alkaline and acid proteases, trypsin, chymotrypsin, aminopeptidase, carboxypeptidase, lipases, amylases, and phosphatases) during early ontogeny by biochemical analysis. All digestive enzymes were detected at first feeding (6 days after hatching, DAH, 9.49 mm, 168 degree-days DD). Afterwards all enzymes reached two main peaks in activity at 14 or 22 DAH (15.10 mm, 364 DD and 20.83 mm, 550 DD, respectively). Later, there was a gradual decrease in activity for trypsin and acid and alkaline phosphatases until reach the lowest values at 41 DAH. In the case of acid proteases, chymotrypsin, aminopeptidase, carboxypeptidase, lipase and amylase, all activities reached their maximum values at the end of the larval period, except for alkaline proteases, which showed the maximum value at 14 DAH (15.10 mm, 364 DD). Parachromis dovii larvae have an early capability to hydrolyze exogenous food, agreeing with other carnivorous neotropical cichlid species, for this reason we proposed that the weaning process could begin at 14 DAH.(AU)

El guapote lagunero (Parachromis dovii) es una especie con un alto potencial para la acuicultura en la región de América Central; sin embargo, existe un conocimiento limitado sobre la capacidad digestiva en el periodo larval. Por este motivo, este estudio evaluó los cambios de las enzimas digestivas (proteasas alcalinas y ácidas, tripsina, quimotripsina, aminopeptidasa, carboxipeptidasa, lipasas, amilasas y fosfatasas) durante la ontogenia temprana mediante análisis bioquímico. Todas las enzimas digestivas analizadas se detectaron en la primera alimentación (6 días después de la eclosión, DAH, 9.49 mm, 168 día-grados DD). Después, todas las enzimas alcanzaron dos picos máximos a los 14 o 22 DAH (15.10 mm, 364 DD and 20.83 mm, 550 DD, respectivamente). Después las actividades tripsina, fosfatasas ácidas y alcalina disminuyeron a sus valores más bajos a los 41 DAH. En el caso de las proteasas ácidas y alcalinas, quimotripsina, aminopeptidasa, carboxipeptidasa, lipasa y amilasa, los niveles de actividad aumentaron y alcanzaron su máximo valor al final del período larvario, excepto las proteasas alcalinas, que mostraron su máximo valor a los 14 DAH (15.10 mm, 364 DD). Las larvas de P. dovii tienen una capacidad temprana para hidrolizar alimentos exógenos, lo que concuerda con otras especies de cíclidos neotropicales carnívoros, por lo que proponemos que el proceso de destete inicie a los 14 DAH.(AU)

Changes in digestive enzymes activities during the initial ontogeny of wolf cichlid, Parachromis dovii (Perciformes: Cichlidae)

Wolf cichlid, Parachromis dovii, is a species with a high potential for aquaculture in Central America; however, the knowledge of the digestive physiology in larvae period is limited. For these reason, this study evaluated the changes on digestive enzymes (alkaline and acid proteases, trypsin, chymotrypsin, aminopeptidase, carboxypeptidase, lipases, amylases, and phosphatases) during early ontogeny by biochemical analysis. All digestive enzymes were detected at first feeding (6 days after hatching, DAH, 9.49 mm, 168 degree-days DD). Afterwards all enzymes reached two main peaks in activity at 14 or 22 DAH (15.10 mm, 364 DD and 20.83 mm, 550 DD, respectively). Later, there was a gradual decrease in activity for trypsin and acid and alkaline phosphatases until reach the lowest values at 41 DAH. In the case of acid proteases, chymotrypsin, aminopeptidase, carboxypeptidase, lipase and amylase, all activities reached their maximum values at the end of the larval period, except for alkaline proteases, which showed the maximum value at 14 DAH (15.10 mm, 364 DD). Parachromis dovii larvae have an early capability to hydrolyze exogenous food, agreeing with other carnivorous neotropical cichlid species, for this reason we proposed that the weaning process could begin at 14 DAH.(AU)

El guapote lagunero (Parachromis dovii) es una especie con un alto potencial para la acuicultura en la región de América Central; sin embargo, existe un conocimiento limitado sobre la capacidad digestiva en el periodo larval. Por este motivo, este estudio evaluó los cambios de las enzimas digestivas (proteasas alcalinas y ácidas, tripsina, quimotripsina, aminopeptidasa, carboxipeptidasa, lipasas, amilasas y fosfatasas) durante la ontogenia temprana mediante análisis bioquímico. Todas las enzimas digestivas analizadas se detectaron en la primera alimentación (6 días después de la eclosión, DAH, 9.49 mm, 168 día-grados DD). Después, todas las enzimas alcanzaron dos picos máximos a los 14 o 22 DAH (15.10 mm, 364 DD and 20.83 mm, 550 DD, respectivamente). Después las actividades tripsina, fosfatasas ácidas y alcalina disminuyeron a sus valores más bajos a los 41 DAH. En el caso de las proteasas ácidas y alcalinas, quimotripsina, aminopeptidasa, carboxipeptidasa, lipasa y amilasa, los niveles de actividad aumentaron y alcanzaron su máximo valor al final del período larvario, excepto las proteasas alcalinas, que mostraron su máximo valor a los 14 DAH (15.10 mm, 364 DD). Las larvas de P. dovii tienen una capacidad temprana para hidrolizar alimentos exógenos, lo que concuerda con otras especies de cíclidos neotropicales carnívoros, por lo que proponemos que el proceso de destete inicie a los 14 DAH.(AU)

Hemosuccus = pancreaticus in a dog: A rare case of gastrointestinal hemorrhage

A 9-year-old, 5.5-kg (12.12-lb) spayed female Poodle was evaluated due to recurrent seizures and possible diabetes. The dog also had a history of anorexia, polyuria and polydipsia. Serum biochemistry results revealed increases in pancreatic amylase and glucose associated with decreased protein levels. Abdominal ultrasound suggested acute pancreatitis and/or a pancreatic tumor with intense peripancreatic vascularization. The clinical condition of the dog worsened despite medical treatment, and death occurred shortly thereafter. At necropsy, blood clots were found in the stomach and in the intestinal lumen, which was filled with undigested blood. The pancreas was moderately increased in size with an irregular surface and multifocal yellow firm areas. In the cranial portion of the pancreas, a single nodule, approximately 2 cm in diameter, was found. Histologic evaluation of the pancreas revealed chronic and acute lesions characterized by lymphohistiocytic pancreatitis, periductal fibrosis, degeneration of pancreatic islets and necrotizing and fibrinopurulent pancreatitis. The nodule was diagnosed as pancreatic adenocarcinoma. These clinical, imaging, and histopathologic findings were compatible with Hemosuccus pancreaticus. To the authors’ knowledge, this is the first report of Hemosuccus pancreaticusin a dog and it should be considered as a differential diagnosis for dogs with pancreatic disease and gastrointestinal bleeding.

Hemosuccus = pancreaticus in a dog: A rare case of gastrointestinal hemorrhage

A 9-year-old, 5.5-kg (12.12-lb) spayed female Poodle was evaluated due to recurrent seizures and possible diabetes. The dog also had a history of anorexia, polyuria and polydipsia. Serum biochemistry results revealed increases in pancreatic amylase and glucose associated with decreased protein levels. Abdominal ultrasound suggested acute pancreatitis and/or a pancreatic tumor with intense peripancreatic vascularization. The clinical condition of the dog worsened despite medical treatment, and death occurred shortly thereafter. At necropsy, blood clots were found in the stomach and in the intestinal lumen, which was filled with undigested blood. The pancreas was moderately increased in size with an irregular surface and multifocal yellow firm areas. In the cranial portion of the pancreas, a single nodule, approximately 2 cm in diameter, was found. Histologic evaluation of the pancreas revealed chronic and acute lesions characterized by lymphohistiocytic pancreatitis, periductal fibrosis, degeneration of pancreatic islets and necrotizing and fibrinopurulent pancreatitis. The nodule was diagnosed as pancreatic adenocarcinoma. These clinical, imaging, and histopathologic findings were compatible with Hemosuccus pancreaticus. To the authors knowledge, this is the first report of Hemosuccus pancreaticusin a dog and it should be considered as a differential diagnosis for dogs with pancreatic disease and gastrointestinal bleeding.(AU)

Outstanding impact of soil tillage on the abundance of soil hydrolases revealed by a metagenomic approach

The soil represents the main source of novel biocatalysts and biomolecules of industrial relevance. We searched for hydrolases in silico in four shotgun metagenomes (4,079,223 sequences) obtained in a 13-year field trial carried out in southern Brazil, under the no-tillage (NT), or conventional tillage (CT) managements, with crop succession (CS, soybean/wheat), or crop rotation (CR, soybean/maize/wheat/lupine/oat). We identified 42,631 hydrolases belonging to five classes by comparing with the KEGG database, and 44,928 sequences by comparing with the NCBI-NR database. The abundance followed the order: lipases > laccases > cellulases > proteases > amylases > pectinases. Statistically significant differences were attributed to the tillage system, with the NT showing about five times more hydrolases than the CT system. The outstanding differences can be attributed to the management of crop residues, left on the soil surface in the NT, and mechanically broken and incorporated into the soil in the CT. Differences between the CS and the CR were slighter, 10% higher for the CS, but not statistically different. Most of the sequences belonged to fungi (Verticillium, and Colletotrichum for lipases and laccases, and Aspergillus for proteases), and to the archaea Sulfolobus acidocaldarius for amylases. Our results indicate that agricultural soils under conservative managements may represent a hotspot for bioprospection of hydrolases.(AU)

Filamentous fungi producing enzymes under fermentation in cassava liquid waste / Fungos filamentosos produtores de enzimas sob fermentação em manipueira

The conversion of agroindustrial residues by microorganisms has been explored from fermentative processes to obtain several bioactive molecules. The objective of this work was to isolate and select filamentous fungi present in cassava liquid waste for the production of amylase, carboxymethylcellulose (CMCase), pectinase and xylanase using the same residue as induction substrate in fermentative processes. A total of 65 filamentous fungi were isolated and qualitative tests indicated that approximately 86% of these strains were able to produce at least one of the enzymes and 32% capable of producing the four enzymes. Fermentation assays in cassava liquid residue-containing medium showed 6 fungal lines as potential enzyme producers. The maximum activities of pectinase, xylanase, amylase and CMCase were respectively observed at 96 hours of fermentation by the strain by the strain Aspergillus sp. B5C; at 120 hours (163.6 ± 0.13 nKat mL-1), by Aspergillus sp. B4I; at 144 hours (99.8 ± 0.24 nKat mL-1), by Penicillium sp. B3A; and at 48 hours (55.5 ± 0.21 nKat mL-1), by Aspergillus sp. B4O. These results suggest that cassava liquid waste was source of filamentous fungi producing amylase, CMCase, pectinase and xylanase, as well as a promising alternative substrate for bioprocesses aiming the production of enzymes.

A conversão de resíduos agroindustriais por micro-organismos tem sido explorada a partir de processos fermentativos para obtenção de diversas moléculas bioativas. O objetivo deste trabalho foi isolar e selecionar fungos filamentosos presentes em manipueira para produção de amilase, carboximetilcelulase (CMCase), pectinase e xilanase utilizando o próprio resíduo como substrato indutor. Um total de 65 fungos filamentosos foi isolado e testes qualitativos indicaram que, aproximadamente, 86% dessas linhagens foram hábeis em produzir pelo menos uma das enzimas e 32% capazes de produzir as quatro enzimas. Ensaios fermentativos em meio contendo manipueira apontaram 6 linhagens fúngicas como potenciais produtoras de enzimas. As atividades máximas de pectinase, xilanase, amilase e CMCase foram observadas, respectivamente, às 96 horas de fermentação (67.4 ± 0,6 nKat mL-1), pela linhagem Aspergillus sp. B5C; às 120 horas (163.6 ± 0,13 nKat mL-1), por Aspergillus sp. B4I; às 144 horas (99.8 ± 0,24 nKat mL-1), por Penicillium sp. B3A; e às 48 horas (55.5 ± 0,21 nKat mL-1), por Aspergillus sp. B4O. Estes resultados sugerem a manipueira como fonte de fungos filamentosos produtores de amilase, CMCase, pectinase e xilanase, além de um promissor substrato alternativo para bioprocessos visando a produção dessas enzimas.

Filamentous fungi producing enzymes under fermentation in cassava liquid waste / Fungos filamentosos produtores de enzimas sob fermentação em manipueira

The conversion of agroindustrial residues by microorganisms has been explored from fermentative processes to obtain several bioactive molecules. The objective of this work was to isolate and select filamentous fungi present in cassava liquid waste for the production of amylase, carboxymethylcellulose (CMCase), pectinase and xylanase using the same residue as induction substrate in fermentative processes. A total of 65 filamentous fungi were isolated and qualitative tests indicated that approximately 86% of these strains were able to produce at least one of the enzymes and 32% capable of producing the four enzymes. Fermentation assays in cassava liquid residue-containing medium showed 6 fungal lines as potential enzyme producers. The maximum activities of pectinase, xylanase, amylase and CMCase were respectively observed at 96 hours of fermentation by the strain by the strain Aspergillus sp. B5C; at 120 hours (163.6 ± 0.13 nKat mL-1), by Aspergillus sp. B4I; at 144 hours (99.8 ± 0.24 nKat mL-1), by Penicillium sp. B3A; and at 48 hours (55.5 ± 0.21 nKat mL-1), by Aspergillus sp. B4O. These results suggest that cassava liquid waste was source of filamentous fungi producing amylase, CMCase, pectinase and xylanase, as well as a promising alternative substrate for bioprocesses aiming the production of enzymes.(AU)

A conversão de resíduos agroindustriais por micro-organismos tem sido explorada a partir de processos fermentativos para obtenção de diversas moléculas bioativas. O objetivo deste trabalho foi isolar e selecionar fungos filamentosos presentes em manipueira para produção de amilase, carboximetilcelulase (CMCase), pectinase e xilanase utilizando o próprio resíduo como substrato indutor. Um total de 65 fungos filamentosos foi isolado e testes qualitativos indicaram que, aproximadamente, 86% dessas linhagens foram hábeis em produzir pelo menos uma das enzimas e 32% capazes de produzir as quatro enzimas. Ensaios fermentativos em meio contendo manipueira apontaram 6 linhagens fúngicas como potenciais produtoras de enzimas. As atividades máximas de pectinase, xilanase, amilase e CMCase foram observadas, respectivamente, às 96 horas de fermentação (67.4 ± 0,6 nKat mL-1), pela linhagem Aspergillus sp. B5C; às 120 horas (163.6 ± 0,13 nKat mL-1), por Aspergillus sp. B4I; às 144 horas (99.8 ± 0,24 nKat mL-1), por Penicillium sp. B3A; e às 48 horas (55.5 ± 0,21 nKat mL-1), por Aspergillus sp. B4O. Estes resultados sugerem a manipueira como fonte de fungos filamentosos produtores de amilase, CMCase, pectinase e xilanase, além de um promissor substrato alternativo para bioprocessos visando a produção dessas enzimas.(AU)

The effect of exogenous enzymes on the performance and digestibility of nutrients in broiler / O efeito de enzimas exógenas no desempenho e digestibilidade de nutrientes em frangos de corte

Here we tested how feeding with exogenous enzymes affects the performance, carcass yield, and ileal digestibility in broilers from 21- to 42-days-old. The study included 1008 male broilers randomly distributed into six diets: positive control (PC), corn and soybean meal containing all the nutritional requirements; negative control (NC), with a 120 kcal.kg-1 reduction in metabolizable energy relative to the PC; enzyme complex one (NC + 100 ppm of endo-1,4-beta-xylanase); enzyme complex two (NC + 200 ppm of xylanase, amylase and protease); enzyme blend one (NC + 100 FXU kg-1 of xylanase and 200 FXU kg-1 of amylase); and enzyme blend two (NC + 100 FXU kg-1 of xylanase and 300 FXU kg-1 of amylase). The enzyme complex two (CN + 100 FXU.kg de xilanase e 300 FXU.kg de amilase) and enzyme blend two (NC + 100 FXU kg-1 of xylanase and 300 FXU kg-1 of amylase) had greater weight gain, feed conversion, carcass yield, and digestibility of nutrients than the NC (P < 0,05). We found that enzyme complex two and enzyme blend two valued their nutritional matrix promoting better performance and digestibility of broilers.

O trabalho foi realizado para avaliar o uso de enzimas sobre o desempenho, rendimento de carcaça e digestibilidade ileal em frangos de corte no período de 21 a 42 dias de idade. Foram utilizados 1008 frangos de corte, machos, distribuídos em seis dietas: controle positivo (CP) a base de milho e farelo de soja, contendo todas as exigências nutricionais, controle negativo (CN) com redução de 120 kcal.kg-1de energia metabolizável, complexo enzimático um (CN + 100 ppm de endo-1,4-beta-xilanase); complexo enzimático dois ( CN + 200 ppm de xilanase, amilase e protease); blend enzimático um (CN + 100 FXU. kg de xilanase e 200 FXU.kg de amilase e blend enzimático dois (CN + 100 FXU.kg de xilanase e 300 FXU.kg de amilase). O complexo enzimático dois (CN + 200 ppm de xilanase, amilase e protease) e blend enzimático dois (CN + 100 FXU.kg de xilanase e 300 FXU.kg de amilase) proporcionaram ganho de peso, conversão alimentar, rendimento de carcaça e digestibilidade de nutrientes superior ao controle negativo (P < 0,05). Concluiu-se que o complexo enzimático dois e blend enzimático dois valorizaram sua matriz nutricional promovendo melhor desempenho e digestibilidade em frangos de corte.

The effect of exogenous enzymes on the performance and digestibility of nutrients in broiler / O efeito de enzimas exógenas no desempenho e digestibilidade de nutrientes em frangos de corte

Here we tested how feeding with exogenous enzymes affects the performance, carcass yield, and ileal digestibility in broilers from 21- to 42-days-old. The study included 1008 male broilers randomly distributed into six diets: positive control (PC), corn and soybean meal containing all the nutritional requirements; negative control (NC), with a 120 kcal.kg-1 reduction in metabolizable energy relative to the PC; enzyme complex one (NC + 100 ppm of endo-1,4-beta-xylanase); enzyme complex two (NC + 200 ppm of xylanase, amylase and protease); enzyme blend one (NC + 100 FXU kg-1 of xylanase and 200 FXU kg-1 of amylase); and enzyme blend two (NC + 100 FXU kg-1 of xylanase and 300 FXU kg-1 of amylase). The enzyme complex two (CN + 100 FXU.kg de xilanase e 300 FXU.kg de amilase) and enzyme blend two (NC + 100 FXU kg-1 of xylanase and 300 FXU kg-1 of amylase) had greater weight gain, feed conversion, carcass yield, and digestibility of nutrients than the NC (P < 0,05). We found that enzyme complex two and enzyme blend two valued their nutritional matrix promoting better performance and digestibility of broilers.(AU)

O trabalho foi realizado para avaliar o uso de enzimas sobre o desempenho, rendimento de carcaça e digestibilidade ileal em frangos de corte no período de 21 a 42 dias de idade. Foram utilizados 1008 frangos de corte, machos, distribuídos em seis dietas: controle positivo (CP) a base de milho e farelo de soja, contendo todas as exigências nutricionais, controle negativo (CN) com redução de 120 kcal.kg-1de energia metabolizável, complexo enzimático um (CN + 100 ppm de endo-1,4-beta-xilanase); complexo enzimático dois ( CN + 200 ppm de xilanase, amilase e protease); blend enzimático um (CN + 100 FXU. kg de xilanase e 200 FXU.kg de amilase e blend enzimático dois (CN + 100 FXU.kg de xilanase e 300 FXU.kg de amilase). O complexo enzimático dois (CN + 200 ppm de xilanase, amilase e protease) e blend enzimático dois (CN + 100 FXU.kg de xilanase e 300 FXU.kg de amilase) proporcionaram ganho de peso, conversão alimentar, rendimento de carcaça e digestibilidade de nutrientes superior ao controle negativo (P < 0,05). Concluiu-se que o complexo enzimático dois e blend enzimático dois valorizaram sua matriz nutricional promovendo melhor desempenho e digestibilidade em frangos de corte.(AU)