RESUMO A trombose é uma doença patofisiológica causada pelo acúmulo de fibrina no sangue. Proteases fibrinolíticas com potente atividade trombolítica são produzidas por diversas fontes microbianas. Considerando a biodiversidade microbiana da região amazônica, o presente estudo teve como objetivo a seleção, produção e caracterização bioquímica da enzima fibrinolítica de Streptomyces sp. isolado de líquens da Amazônia. Streptomyces DPUA1576 foi a melhor produtora com atividade fibrinolítica de 283 mm2. Três variáveis em dois níveis foram utilizadas para determinar as variáveis mais relevantes na produção da enzima fibrinolítica (FA). Os parâmetros estudados foram agitação (0.28 - 1.12 g), temperatura (28 - 36 ºC) e pH (6.0 - 8.0) e todos obtiveram efeitos significativos na produção fibrinolítica. A maior atividade fibrinolítica (304 mm2) foi obtida a 1.12 g, 28 ºC e pH 8.0. O extrato bruto da fermentação foi usado para determinar as propriedades bioquímicas da enzima. Atividades proteásica e fibrinolítica foram estáveis durante 6 horas no intervalo de pH entre 6.8 - 8.4 e 5.8 - 9.2, respectivamente. Temperatura ótima para a atividade proteásica foi entre 35 - 55 °C, enquanto que para a atividade fibrinolítica foi de 45 ºC. Atividade proteásica foi inibida por íons Cu2+ e Co2+, fluoreto de fenilmetilsulfonil e pepstatina A, na qual sugere que a enzima é uma serino-protease. O extrato enzimático degradou o fibrinogênio nas subunidades Aα, Aβ e γ. Os resultados apresentados indicam que Streptomyces sp. DPUA 1576 produz enzimas com atividade fibrinolítica e fibrinogenolítica, enzimas com aplicações importantes na indústria farmacêutica.

ABSTRACT Thrombosis is a pathophysiological disorder caused by accumulation of fibrin in the blood. Fibrinolytic proteases with potent thrombolytic activity have been produced by diverse microbial sources. Considering the microbial biodiversity of the Amazon region, this study aimed at the screening, production and biochemical characterization of a fibrinolytic enzyme produced by Streptomyces sp. isolated from Amazonian lichens. The strain Streptomyces DPUA1576 showed the highest fibrinolytic activity, which was 283 mm2. Three variables at two levels were used to assess their effects on the fibrinolytic production. The parameters studied were agitation (0.28 - 1.12 g), temperature (28 - 36 ºC) and pH (6.0 - 8.0); all of them had significant effects on the fibrinolytic production. The maximum fibrinolytic activity (304 mm2) was observed at 1.12 g, 28 ºC, and pH of 8.0. The crude extract of the fermentation broth was used to assess the biochemical properties of the enzyme. Protease and fibrinolytic activities were stable during 6 h, at a pH ranging from 6.8 to 8.4 and 5.8 to 9.2, respectively. Optimum temperature for protease activity ranged between 35 and 55 °C, while the highest fibrinolytic activity was observed at 45 ºC. Proteolytic activity was inhibited by Cu2+ and Co2+ ions, phenylmethylsulfonyl fluoride (PMSF) and pepstatin A, which suggests that the enzyme is a serine protease. Enzymatic extract cleaved fibrinogen at the subunits Aα-chain, Aβ-chain, and γ-chain. The results indicated that Streptomyces sp. DPUA 1576 produces enzymes with fibrinolytic and fibrinogenolytic activity, enzymes with an important application in the pharmaceutical industry.

This study aimed to identify antioxidant peptides from caprine casein hydrolysates by papain application using MALDI-TOF mass spectrometer, and a 2² full factorial design, with 4 axial points, in order to evaluate kinetic parameters (time and pH) effects on the degree of hydrolysis as well as the antioxidant activity of Moxotó goat milk casein peptides. Degree of hydrolysis was determined by total and soluble protein ratio in casein. Antioxidant activity was measured by ABTS method with 2, 2-cation-azinobis (3-ethylbenzothiazoline-6-sulfonic acid). TROLOX was used as standard. Peptide pattern and sequence of antioxidant amino acids were obtained using MALDI-TOF/MS. The highest degree of hydrolysis (28.5%) and antioxidant activity (2329.6 mmol.L TROLOX. mg- 1 peptide) were observed in the permeate. NENLL, NPWDQVK and LLYQEPVLGPV peptides, detected in the permeate, were pointed as the responsible for antioxidant activity, suggesting their potential application as food supplement and pharmaceutical products.

Enzymes obtained by fermentation processes offer a number of advantages and have been widely researched and used throughout the world. This study aimed to partially characterise an inulinase produced from palm and cassava peel. The enzyme was produced via the solid-state fermentation of Aspergillus japonicus URM5633. The optimal temperatures were 50ºC and 55ºC, and the optimal pH values were 5.2 and 3.4 for inulinase fermentatively produced from palm and cassava peel, respectively. The thermostability measurements for inulinase produced in palm showed that the relative activity remained below 100% until 30 minutes of stability for all temperatures, but reached 106.8% at a temperature of 50ºC after 60 minutes. Inulinase from the crude extract of cassava peel was pH stable and only decreased to 55% of the maximal activity over the course of the assay, suggesting that this enzyme can be used in inulinase production and can be utilized in food industries.

A full two-level factorial design was employed to study the influence of PEG molar mass (MM PEG), PEG concentration (C PEG) and phosphate concentration (C PHOSPH) on proteases partition by Lentinus citrinus DPUA 1535 in a PEG/phosphate aqueous two-phase system (ATPS). For all ATPS studied, proteases partitioned for the top phase and the best proteases extraction condition was obtained with MM PEG = 6000 g mol-1, C PEG = 17.5% (w/w) and C PHOSPH = 25% (w/w) with (1.1) purification factor and (151%) activity yield. Findings reported here demonstrate a practical strategy that serves as a first step for proteases purification from crude extract by L. citrinus.

O objetivo deste trabalho foi determinar as variáveis que influenciam a produção simultânea de celulases e xilanase por Aspergillus aculeatus URM 4953 e A. phoenicis URM 4924, com uso de bagaço de cana-de-açúcar como substrato. As variáveis avaliadas foram pH inicial, tempo de cultivo, concentração do substrato, agitação, concentração do inóculo, temperatura, tipo de bagaço de cana-de-açúcar e espécie de Aspergillus. Foi utilizado o planejamento fatorial fracionário 2(8-4), com três repetições no ponto central. Todas as variáveis analisadas influenciaram a produção de pelo menos uma das enzimas. As maiores atividades enzimáticas observadas foram: celulases totais, 0,45 UI mL-1; endoglucanase, 0,60 UI mL-1; exoglucanase, 0,17 U mL-1 ; celobiase 6,42 UI mL-1; e xilanase, 30,05 U mL-1. As melhores condições para produção das enzimas foram: pH 6,0; tempo de cultivo de 168 horas; bagaço de cana-de-açúcar residual como substrato; e A. aculeatus URM 4953, que produziu as celulases e a xilanase simultaneamente. As variáveis que influenciam a produção simultânea das celulases e xilanase são pH, tempo de cultivo, tipo de bagaço de cana-de-açúcar e espécie de Aspergillus.

The objective of this work was to determine the variables that influence the simultaneous production of cellulases and xylanase by Aspergillus aculeatus URM 4953 and A. phoenicis URM 4924, using sugarcane bagasse as substrate. Initial pH, cultivation time, substrate concentration, agitation, inoculum concentration, temperature, sugarcane bagasse type and Aspergillus species were the variables evaluated. It was used the 2(8-4) fractional factorial design, with three replicates in the central point. All analysed variables influenced at least one of the enzyme production. The highest enzymatic activities were: total cellulases, 0.45 UI mL-1; endoglucanase, 0.60 UI mL-1; exoglucanase, 0.17 U mL-1; cellobiase, 6.42 UI mL-1; and xylanase, 30.05 U mL-1. The best conditions for enzyme production were: pH 6.0; cultivation time of 168 hours; residual sugarcane bagasse as substrate; and A. aculeatus URM 4953, which produced simultaneously all the cellulases and xylanase. The variables that influence the simultaneous production of cellulases and xylanase are pH, cultivation time, sugarcane bagasse type, and Aspergillus species.

O objetivo deste trabalho foi avaliar os efeitos da temperatura, pH, relação enzima:substrato (E:S) e tempo de reação sobre a hidrólise enzimática da caseína do leite de cabra Moxotó, com uso de diferentes enzimas proteolíticas. A hidrólise enzimática da caseína caprina foi realizada com uso de tripsina, pepsina e papaína. Para cada enzima, foram utilizados dois níveis de cada variável, na avaliação de seus efeitos sobre a hidrólise da caseína, em um fatorial completo 2(4). Os produtos da hidrólise foram visualizados em eletroforese SDS-PAGE. O melhor valor do grau de hidrólise com a enzima papaína foi obtido em pH 6,5, E:S de 1:150 e 5 horas de hidrólise a 50ºC (28,17%); com a tripsina, em pH 8,5, E:S de 1:150 e 5 horas a 40ºC (29,55%); e com a pepsina, em pH 3,0, E:S de 1:100 e 5 horas a 40ºC (38,27%). A hidrólise da caseína caprina é influenciada positivamente pelo pH e tempo de hidrólise, com o uso da pepsina. Foram observadas interações significativas antagônicas entre pH e relação E:S, com o uso da tripsina; e entre pH, temperatura e tempo de hidrólise, com o uso da papaína. A pepsina apresenta elevada eficiência na hidrólise de αs1, β e κ-caseína, evidenciada por hidrolisados com massa molar abaixo de 14,4 kDa.

The aim of this work was to evaluate the effects of temperature, pH, enzyme:substrate ratio (E:S), and reaction time on the enzymatic hydrolysis of Moxotó breed goat's milk casein, using different proteolytic enzymes. Enzymatic hydrolysis of the capric casein was performed using trypsin, pepsin and papain. For each enzyme, two levels of the variables were used to assess their effects on the hydrolysis of casein, using a full factorial design (2(4)). The hydrolysis products were visualized on SDS-PAGE. The best hydrolysis degree using papain were at pH 6.5, E:S of 1:150 and 5 hours of hydrolysis at 50°C (28.17%); for trypsin, at pH 8.5, E:S of 1:150 and 5 hours at 40°C (29.55%); and for pepsin, at pH 3.0, E:S of 1:100 and 5 hours at 40°C (38.27%). Capric casein hydrolysis is affected positively by pH and reaction time, using pepsin. Significant antagonistic interactions were observed between pH and E:S, with trypsine; and between pH, temperature and reaction time, with papain. Pepsin has high αs1, β e κ-casein hydrolysis efficiency, evidenced by the molar masses below 14.4 kDa of its products.