•ATPS was used by partitioning and purification of PG.•PEG molar mass, phase concentrations and pH were evaluated on purification.•PEG molar mass and phosphate concentration have prominent effect on ...the response variables.•ATPS provides an efficient and attractive method for purifying the PG.
The partitioning and purification of polygalacturonases (PG) produced by Aspergillus niger URM 5162 were investigated in aqueous two-phase systems (ATPS), formed by polyethylene glycol and phosphate salts (PEG/phosphate). To evaluate the effect of the 4 independent variables – molar mass of polyethylene glycol (PEG) (400–8000g/mol – MPEG), PEG concentration (12.5–17.5%, w/w – CPEG), phosphate concentration (15–25%, w/w, CPHOS) and pH (6.0–8.0) – on the 4 response variables: partition coefficient (K), activity yield (Y), purification factor (PF) and selectivity (S), a factorial design (24) was used. The endo-polygalacturonases (endo-PG) and exo-polygalacturonase (exo-PG) were preferentially partitioned in the top phase. For endo- and exo-PG, the highest values for the response variables K (1.23 and 2.40), Y (74.04% and 17.97%), PF (8.18 and 1.98) and S (24.68 and 48.07), respectively, were obtained for a CPEG of 12.5% (w/w), MPEG of 8000g/mol, and CPHOS of 25% (w/w) at pH 6.0. These conditions were considered the most suitable for the purification of PG produced by A. niger URM 5162. Furthermore, the most important independent variables for endo- and exo-PG were CPHOS and MPEG, respectively. All independent variables studied and their interactions significantly influenced the response variables. According to these results, the PEG/phosphate system is a useful cost-effective alternative for purification of PG produced by A. niger URM 5162.
This is the first report of isolation of fungi present in fatty and defatted castor bean meal as well as the first of crop's selection to test the cellulolytic potential, in order to verify the ...diversity and potential of cellulolytic fungi in castor bean waste (Ricinus communis L.). For the screening on solid medium, it was used carboxymethylcellulose (CMC) as the sole carbon source. The microcrystalline cellulose (Avicel) was used as a substrate for submerged fermentation for production of cellobiohydrolase (FPase) and the CMC to produce endoglucanases (CMCase) and β-glycosidases (BG). 189 cultures of fungi were isolated, including 40 species of filamentous fungi and three yeasts. The Aspergillus was the most frequent found genus. Regarding the distribution of isolated species from defatted castor bean meal, the A. niger was the most frequent one; and within the fatty castor bean meal, the Emericela variecolor prevailed among other species. Among the 67 fungal cultures tested in the initial screening on solid media to assess the cellulolytic potential, 54 disclosed Cellulolytic Index (CI) ranging from 1.04 to 6.00 mm. The isolates were selected for enzyme production in liquid medium with values above 2.0 CI. They were obtained with A. japonicus URM5620 FPase activity (4.99 U/ml) and BG (0.05 U/ml), and Rhodotorula glutinis URM5724 activity of CMCase 3.58 U/ml. These cases occurred after 168 h of submersion for both species of fungi. In our study, we could conclude that the castor bean is a promising source of fungi capable of producing cellulolytic enzymes.
Aspergillus is a large tannase-producing genus able to provide enzymes which hydrolyse condensed tannins in waste coir residues. In this work, a tannase-acyl hydrolase (TAH) obtained from Aspergillus ...sydowii (SIS 25) was extracted and purified by PEG/citrate aqueous two-phase system (ATPS), biochemically characterized and applied for detannification of green tea. A statistical design (23) was used to evaluate the influence of the variables: molar mass of PEG (1000–6000 g/mol), PEG concentration (20.0–24.0% w/w) and sodium citrate concentration (15–20% w/w) at pH 6.0. ATPS efficiency was confirmed by obtaining two major chromatographic peaks (by size exclusion chromatography on a Superdex-G75 at 215 nm) with a unique peak responsible for the hydrolysis of 44% of the phenolic compounds in green tea. This study is focused on the obtention of a microbial tannase purified by ATPS, with potential for debittering and clarifying fruit juices.
•Aspergillus sydowii was able to produce tannase with biotechnological potential.•The optimum pH for tannase activity was observed between 5.0 and 6.0.•The results showed that, after hydrolysis, the sweet aftertaste and overall acceptability improved.•The removal of phenolic compounds in green tea by Tannase was achieved.•The results showed that the phenolic compounds of the green tea were significantly influenced by the amount of tannase added.
RESUMO: Os dermatófitos são fungos que podem causar infecções superficiais da pele, cabelo e unhas em humanos e animais. As espécies de dermatófitos mais frequentemente isoladas dos cães e gatos ...afetados por micoses são Microsporum gypseum e principalmente Microsporum canis. O papel crucial durante o processo de infecção é a produção de enzimas extracelulares essenciais para a invasão e estabelecimento do agente patogênico no tecido do hospedeiro. O objetivo deste trabalho foi isolar dermatófitos de cães e gatos e avaliar o perfil enzimático dos isolados obtidos. Amostras de pelos e escamas epidérmicas foram coletadas de cães e gatos em instalações veterinárias em Recife/PE, e os isolados foram identificados com base nas características macroscópicas e microscópicas. A análise qualitativa das enzimas urease, protease, lipase, colagenase e fosfolipase foi avaliada a partir dos dermatófitos isolados. Durante 10 meses, um total de 106 animais, que compreendeu de 99 cães e sete gatos com sinais clínicos, independentemente do sexo e raça foram avaliados. Apenas oito animais foram confirmados com dermatofitose, principalmente cães (n=7), sendo seis afetados por M. canis e um por M. gypseum, a raça mais afetada foi Yorkshire (n=3). No entanto, apenas um gato foi confirmado com M. canis. Não foi observada predisposição relacionada ao sexo quanto à ocorrência de dermatofitose nos cães e gatos avaliados. Os dermatófitos isolados apresentaram perfis semelhantes para as enzimas urease, lipase, protease, fosfolipase e colagenase, característica importante em infecções patogênicas. O diagnóstico clínico destas zoonoses com base na confirmação microbiológica e uma compreensão dos mecanismos subjacentes é de grande importância para o tratamento e prevenção de doenças fúngicas em animais.
RESUMO: Os dermatófitos são fungos que podem causar infecções superficiais da pele, cabelo e unhas em humanos e animais. As espécies de dermatófitos mais frequentemente isoladas dos cães e gatos ...afetados por micoses são Microsporum gypseum e principalmente Microsporum canis. O papel crucial durante o processo de infecção é a produção de enzimas extracelulares essenciais para a invasão e estabelecimento do agente patogênico no tecido do hospedeiro. O objetivo deste trabalho foi isolar dermatófitos de cães e gatos e avaliar o perfil enzimático dos isolados obtidos. Amostras de pelos e escamas epidérmicas foram coletadas de cães e gatos em instalações veterinárias em Recife/PE, e os isolados foram identificados com base nas características macroscópicas e microscópicas. A análise qualitativa das enzimas urease, protease, lipase, colagenase e fosfolipase foi avaliada a partir dos dermatófitos isolados. Durante 10 meses, um total de 106 animais, que compreendeu de 99 cães e sete gatos com sinais clínicos, independentemente do sexo e raça foram avaliados. Apenas oito animais foram confirmados com dermatofitose, principalmente cães (n=7), sendo seis afetados por M. canis e um por M. gypseum, a raça mais afetada foi Yorkshire (n=3). No entanto, apenas um gato foi confirmado com M. canis. Não foi observada predisposição relacionada ao sexo quanto à ocorrência de dermatofitose nos cães e gatos avaliados. Os dermatófitos isolados apresentaram perfis semelhantes para as enzimas urease, lipase, protease, fosfolipase e colagenase, característica importante em infecções patogênicas. O diagnóstico clínico destas zoonoses com base na confirmação microbiológica e uma compreensão dos mecanismos subjacentes é de grande importância para o tratamento e prevenção de doenças fúngicas em animais.
ABSTRACT: Dermatophytes are fungi that can cause superficial infections of the skin, hair and nails in man and animals. The most frequent dermatophyte species isolated from dogs and cats are Microsporum gypseum, most notably Microsporum canis. The crucial role during the infection process is the production of extracellular enzymes essential for the invasion and establishment of the pathogen in the host tissue. The objective of this research was to isolate dermatophytes from dogs and cats and evaluate the enzymatic profile of the isolates obtained. Hair samples and epidermal scales were collected from dogs and cats in veterinary facilities in Recife-PE, and the isolates were identified based on macroscopic and microscopic characteristics. The qualitative analysis of the enzymes urease, protease, lipase, collagenase and phospholipase was evaluated from the isolated dermatophytes. During 10 months, a total of 106 animals, comprising of 99 dogs and seven cats with clinical signs, regardless of sex and race were evaluated. Only eight animals were confirmed with dermatophytosis, mostly dogs (n=7), being six affected by M. canis and one by M. gypseum, the race most affected was Yorkshire (n=3). However, only one cat was confirmed with M. canis. No sex-related predisposition was observed regarding the occurrence of dermatophytosis in dogs and cats evaluated. Isolated dermatophytes showed similar profiles for the enzymes urease, lipase, protease, phospholipase and collagenase, important characteristic for pathogenic infections. The diagnosis of this zoonosis based on microbiological confirmation and a better understanding of the underlying mechanisms is of great importance for the treatment and prevention of fungal diseases in animals.
► Aqueous two-phase system was used by partitioning and purification of cellulases. ► PEG molar mass, phase concentrations and pH were evaluated on purification. ► PEG molar mass has prominent effect ...on partition coefficient, yield and purity. ► ATPS provides an efficient and attractive method for purifying the cellulases.
The partitioning and purification of cellulases from the cellulolytic complexes produced by Aspergillus japonicus URM5620 were investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1000–8000g/mol), PEG concentration (20.0–24.0%, w/w), sodium citrate (15–20%, w/w) concentration and pH (6.0–8.0) on the differential partitioning and purification of the cellulolytic complex composed of β-glucosidase (βG), endoglucanase (CMCase) and total cellulase (FPase). The βG preferentially partitioned to the salt phase; however, when the molar mass and concentration of PEG were simultaneously increased, the partition coefficient increased (K=3.0). For βG, 20% (w/w) PEG 8000 (g/mol) and 10% (w/w) sodium citrate at pH 8.0 produced the best results; the partition coefficient was approximately 2.9, the activity yield was 77%, and the purification factor was 1.9. The FPase and CMCase preferentially partitioned to the top phase. The highest K values (2.9 and 3.0) were obtained when the top phase consisted of PEG 1000 (g/mol). The best activity yield for FPase (2.67%) was obtained with 24% (w/w) PEG 1000 (g/mol) and 15% (w/w) sodium citrate at pH 8.0. However, the highest purification factor (64.8) was observed with 20% (w/w) PEG 8000 (g/mol). For CMCase, the highest activity yield (1.64%) and purification factor (45.9) were obtained with 22% (w/w) PEG 3350 (g/mol) and 17.5% (w/w) sodium citrate at pH 7.0. The PEG/sodium citrate system resulted in separations characteristic of the cellulases of cellulolytic complexes, where βG partitions to the salt phase and FPase and CMCase partition to the top phase. This process provides an efficient and attractive increase in the purification factor.
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