RESUMO
An actinobacteria strain was isolated from an olive waste mill and tested for protease production on skimmed milk media. The strain identification was achieved through both 16 S rDNA sequencing and phenotypic characterization. The enzyme was purified using the ammonium sulfate/t-butanol three-phase partitioning (TPP) method, followed by characterization to investigate the effect of pH, temperature, and various chemical agents. Subsequently, the enzyme was assessed for its milk coagulation activity. The strain belonging to the Streptomyces genera, exhibits significant phylogenetic and phenotypic differences from the aligned species, suggesting its novelty as a new strain. The enzyme was best separated in the TPP aqueous phase with a 5.35 fold and 56.25% yield. Optimal activity was observed at pH 9.0 and 60 °C, with more than half of the activity retained within the pH range of 7-10 over one hour. The protease demonstrated complete stability between 30 and 60 °C. While metallic ions enhanced enzyme activity, EDTA acted as an inhibitor. The enzyme displayed resistance to H2O2, SDS, Tween 80, and Triton X-100. Notably, it was activated in organic solvents (ethyl acetate, petroleum ether, and xylene), maintaining > 75% of its original activity in butanol, ethanol, and methanol. Additionally, the enzyme yielded high milk coagulant activity of 11,478 SU/mL. The new Streptomyces sp. protease revealed high activity and stability under a wide range of biochemical conditions. Its use in the dairy industry appears particularly promising. Further industrial process investigations will be valuable in determining potential uses for this enzyme.
Assuntos
Estabilidade Enzimática , Leite , Peptídeo Hidrolases , Filogenia , Streptomyces , Temperatura , Streptomyces/isolamento & purificação , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/classificação , Leite/microbiologia , Animais , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , RNA Ribossômico 16S/genéticaRESUMO
Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.
Assuntos
Carboxilesterase , Clonagem Molecular , Halobacterium salinarum , Proteínas Recombinantes , Carboxilesterase/genética , Carboxilesterase/metabolismo , Carboxilesterase/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Halobacterium salinarum/enzimologia , Halobacterium salinarum/genética , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Concentração de Íons de Hidrogênio , Cinética , Estabilidade Enzimática , Proteínas Arqueais/genética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , TemperaturaRESUMO
Glucoamylases are exo-enzymes that cleave the ends of the starch chain, releasing glucose units. In the current work, we described a novel 1,4-α-glucoamylase from an A. brasiliensis strain isolated from an environmental sample. The purified glucoamylase, GlaAb, has a molecular mass of 69 kDa and showed a starch binding domain. GlaAb showed a similar sequence to other fungal glucoamylases, and the molecular 3D model analysis of GlaAb suggests an overall structure as described in the literature, except by elongation in the loop connecting the 4th and 5th α-helices. The enzyme showed activity over a wide range of pH and temperature, with maximum activity at pH 4.5 and 60 °C. GlaAb was stable at 50 °C for 7 h, maintaining 67% residual activity, and it was not inhibited by glucose up to 0.1 M. The glucoamylase was 65% more active in the presence of Mn2+ and showed a Km of 2.21 mg mL-1, Vmax of 155 U mg-1, Kcat 179 s-1, and Kcat/Km 81.06 mg mL-1 s-1 using potato starch as substrate. The results obtained are promising and provide the basis for the development of applications of GlaAb in the industrial process.
RESUMO
Abstract Fish is the main source of animal protein for human diet. The aim of this study was to find out prevalence of pathogenic bacteria of two selected economically important fish of Pakistan namely Mahseer (Tor putitora) and Silver carp (Hypophthalmichthys molitrix). Live fish samples from hatcheries and dead fish samples from different markets of study area were randomly collected. The fish samples were analyzed for isolation, identification and prevalence of bacteria. The isolated bacteria from study fish were identified through biochemical test and about 10 species of pathogenic bacteria were identified including the pathogenic bacteria to human and fish namely, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. and Salmonella spp. The bacterial percentage frequency of occurrence in Silver carp and Mahseer fish showed Pseudomonas aeruginosa 21.42%, Staphylococcus epidermidis 17.85%, Escherichia coli 11.90%, Staphylococcus aureus 9.52%, Citrobacter spp. 9.52%, Serratia spp. 8.33%, Streptococcus iniae 7.14%, Stenotrophomonas spp. 5.95%, Bacillus spp. 4.76% and Salmonella spp. 3.57%. The study revealed that Fish samples of Mahseer and Silver carp that were collected from markets have found more isolates (10 bacterial species) than did the fresh fish pond samples (03 bacterial species) of hatcheries. The occurrence of pathogenic bacteria in study fish showed risk factor for public health consumers.
Resumo O peixe é a principal fonte de proteína animal para a alimentação humana. O objetivo deste estudo foi descobrir a prevalência de bactérias patogênicas de dois peixes economicamente importantes selecionados do Paquistão, nomeadamente Mahseer (Tor putitora) e carpa prateada (Hypophthalmichthys molitrix). Amostras de peixes vivos de incubatórios e amostras de peixes mortos de diferentes mercados da área de estudo foram coletadas aleatoriamente. As amostras de peixes foram analisadas quanto ao isolamento, identificação e prevalência de bactérias. As bactérias isoladas dos peixes do estudo foram identificadas através de testes bioquímicos e cerca de 10 espécies de bactérias patogênicas foram identificadas incluindo as bactérias patogênicas para humanos e peixes, nomeadamente, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. e Salmonella spp. A porcentagem de freqüência de ocorrência bacteriana em carpa prateada e peixes Mahseer mostrou Pseudomonas aeruginosa 21,42%, Staphylococcus epidermidis 17,85%, Escherichia coli 11,90%, Staphylococcus aureus 9,52%, Citrobacter spp. 9,52%, Serratia spp. 8,33%, Streptococcus iniae 7,14%, Stenotrophomonas spp. 5,95%, Bacillus spp. 4,76% e Salmonella spp. 3,57%. O estudo revelou que as amostras de peixes de Mahseer e carpa prateada coletadas nos mercados encontraram mais isolados (10 espécies bacterianas) do que as amostras de peixes frescos (03 espécies bacterianas) de incubatórios. A ocorrência de bactérias patogênicas nos peixes do estudo apresentou fator de risco para consumidores de saúde pública.
Assuntos
Humanos , Animais , Carpas , Paquistão , Bactérias , Lagoas , IncidênciaRESUMO
Abstract Fish is the main source of animal protein for human diet. The aim of this study was to find out prevalence of pathogenic bacteria of two selected economically important fish of Pakistan namely Mahseer (Tor putitora) and Silver carp (Hypophthalmichthys molitrix). Live fish samples from hatcheries and dead fish samples from different markets of study area were randomly collected. The fish samples were analyzed for isolation, identification and prevalence of bacteria. The isolated bacteria from study fish were identified through biochemical test and about 10 species of pathogenic bacteria were identified including the pathogenic bacteria to human and fish namely, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. and Salmonella spp. The bacterial percentage frequency of occurrence in Silver carp and Mahseer fish showed Pseudomonas aeruginosa 21.42%, Staphylococcus epidermidis 17.85%, Escherichia coli 11.90%, Staphylococcus aureus 9.52%, Citrobacter spp. 9.52%, Serratia spp. 8.33%, Streptococcus iniae 7.14%, Stenotrophomonas spp. 5.95%, Bacillus spp. 4.76% and Salmonella spp. 3.57%. The study revealed that Fish samples of Mahseer and Silver carp that were collected from markets have found more isolates (10 bacterial species) than did the fresh fish pond samples (03 bacterial species) of hatcheries. The occurrence of pathogenic bacteria in study fish showed risk factor for public health consumers.
Resumo O peixe é a principal fonte de proteína animal para a alimentação humana. O objetivo deste estudo foi descobrir a prevalência de bactérias patogênicas de dois peixes economicamente importantes selecionados do Paquistão, nomeadamente Mahseer (Tor putitora) e carpa prateada (Hypophthalmichthys molitrix). Amostras de peixes vivos de incubatórios e amostras de peixes mortos de diferentes mercados da área de estudo foram coletadas aleatoriamente. As amostras de peixes foram analisadas quanto ao isolamento, identificação e prevalência de bactérias. As bactérias isoladas dos peixes do estudo foram identificadas através de testes bioquímicos e cerca de 10 espécies de bactérias patogênicas foram identificadas incluindo as bactérias patogênicas para humanos e peixes, nomeadamente, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. e Salmonella spp. A porcentagem de freqüência de ocorrência bacteriana em carpa prateada e peixes Mahseer mostrou Pseudomonas aeruginosa 21,42%, Staphylococcus epidermidis 17,85%, Escherichia coli 11,90%, Staphylococcus aureus 9,52%, Citrobacter spp. 9,52%, Serratia spp. 8,33%, Streptococcus iniae 7,14%, Stenotrophomonas spp. 5,95%, Bacillus spp. 4,76% e Salmonella spp. 3,57%. O estudo revelou que as amostras de peixes de Mahseer e carpa prateada coletadas nos mercados encontraram mais isolados (10 espécies bacterianas) do que as amostras de peixes frescos (03 espécies bacterianas) de incubatórios. A ocorrência de bactérias patogênicas nos peixes do estudo apresentou fator de risco para consumidores de saúde pública.
RESUMO
Lignin, a complex heteropolymer present in plant cell walls, is now recognized as a valuable renewable resource with potential applications in various industries. The lignin biorefinery concept, which aims to convert lignin into value-added products, has gained significant attention in recent years. ß-etherases, enzymes that selectively cleave ß-O-4 aryl ether bonds in lignin, have shown promise in lignin depolymerization. In this study, the ß-etherase LigF from Altererythrobacter sp. B11 was cloned, expressed, purified, and biochemically characterized. The LigF-AB11 enzyme exhibited optimal activity at 32 °C and pH 8.5 when catalyzing the substrate PNP-AV. The enzyme displayed mesophilic behavior and demonstrated higher activity at moderate temperatures. Stability analysis revealed that LigF-AB11 was not thermostable, with a complete loss of activity at 60 °C within an hour. Moreover, LigF-AB11 exhibited excellent pH stability, retaining over 50 % of its activity after 1 h under pH conditions ranging from 3.0 to 11.0. Metal ions and surface impregnation agents were found to affect the enzyme's activity, highlighting the importance of considering these factors in enzymatic processes for lignin depolymerization. This study provides valuable insights into the biochemical properties of LigF-AB11 and contributes to the development of efficient enzymatic processes for lignin biorefineries. Further optimization and understanding of ß-etherases will facilitate their practical application in the valorization of lignin.
RESUMO
The use of chicken waste can contribute to the development of new processes and obtaining molecules with high added value. An experimental design was applied to evaluate the effect of moisture, temperature, and inoculum size on the production of antioxidant peptides and proteases by A. oryzae IOC3999 through solid-state fermentation (SSF) of chicken viscera meal. As a result, the process conditions strongly influenced protease production and antioxidant activity of the fermented products. A global analysis of the results indicated that the most adequate conditions for SSF were (assay 9): 40% initial moisture, 30 °C as the incubation temperature, 5.05 × 106 spores/g as the inoculum size, and 48-h fermentation as the fermentation time. Under this condition, the antioxidant activities for the ABTS- and DPPH-radicals inhibition and ferric reducing antioxidant power (FRAP) methods were 376.16, 153.29, and 300.47 (µmol TE/g), respectively, and the protease production reached 428.22 U/g. Ultrafiltration of the crude extract obtained under optimized fermentation conditions was performed, and the fraction containing peptides with molecular mass lower than 3 kDa showed the highest antioxidant activity. The proteases were biochemically characterized and showed maximal activity at pH values ranging from 5.0 to 6.0 and a temperature of 50 °C. The thermodynamic parameters indicated that the process of thermal protease inactivation is not spontaneous (ΔG*d > 88.78 kJ/mol), increasing with temperature (ΔH*d 27.01-26.88 kJ/mol), and with reduced disorder in the system (ΔS*d < - 197.74 kJ/mol) probably caused by agglomeration of partially denatured enzymes.
Assuntos
Aspergillus oryzae , Animais , Aspergillus oryzae/metabolismo , Peptídeo Hidrolases , Antioxidantes , Galinhas/metabolismo , Vísceras/metabolismo , Temperatura , Endopeptidases , Peptídeos , FermentaçãoRESUMO
L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg-1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the Vmax and Km were 145.2 µmol mL-1 min-1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.
RESUMO
Bacillus sp. CL14 crude protease was partially characterized and applied to obtain antioxidant whey protein isolate (WPI) hydrolysates. Optimal activity occurred at pH 9.0 and 60 °C. Ca2+, Mg2+, and Mn2+ (5 mM) enhanced activity (12-26%), whereas Co2+, Cu2+, Fe2+, and Zn2+ inhibited it (50-94%). At 1% (v/v), Tween 20 and Triton X-100 enhanced activities (21-27%), ß-mercaptoethanol decreased it (15%), and dimethyl sulfoxide (DMSO) had no effect. Sodium dodecyl sulfate (SDS; 0.1%, w/v) increased activity by 36%. Complete inhibition by phenylmethylsulfonyl fluoride (PMSF), and 85% inhibition by ethylenediaminotetraacetic acid, indicates its serine protease character and the importance of cations for activity/stability. With 5 mM Ca2+, protease was optimally active at 65 °C and completely stable after 20 min at 40-55 °C. Crude protease preferentially hydrolyzed WPI and soy protein, followed by casein. WPI hydrolysis was then performed (55 °C, pH 9.0, 5 mM Ca2+) for 0-180 min. Contents of trichloroacetic acid (TCA)-soluble proteins in WPI hydrolysates (HWPI) increased from 29% (0 min) to 50-52% (60-180 min), accompanied by enhanced radical scavenging activity (14%, 0 min; â¼34%, 60-180 min) and Fe2+-chelating ability (56%, 0 min; â¼74%, 45-180 min). CL14 protease might represent an alternative biocatalyst to obtain antioxidant hydrolysates from WPI and, potentially, from other food proteins.
Assuntos
Antioxidantes , Endopeptidases , Proteínas do Soro do Leite , Antioxidantes/farmacologia , Antioxidantes/química , Endopeptidases/química , Serina Proteases , Hidrólise , Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Hidrolisados de Proteína/químicaRESUMO
Abstract There is a paucity of research conducted on microbial prevalence in pheasants. The microbiota of captive birds has zoonotic significance and must be characterize. Present study is therefore planned to assess the microbiota from oral, fecal and gut content of captive avian species. It will be helpful in characterization of harmful microbes. Different samples taken from oral, gut and feces of ring-necked pheasants (Phasianus colchicus), green pheasants (Phasianus versicolor), golden pheasant (Chrysolophus pictus) and silver pheasant (Lophura nycthemera). Samples were collected, diluted, and inoculated onto different agar plates (MacConkey, SS agar, MSA and nutrient agar) for cultivation of bacterial species. Colonies of E.coli, Staphylococcus spp. Brachyspira spp. and Campylobacter spp were observed based on colony morphology. Colony forming unit showed E. coli as frequently found bacteria in fecal, oral and gut contents of all the above pheasants. The overall significance difference was found among bacterial species of golden pheasants, green pheasant, ring-necked pheasant, and silver pheasants. It was concluded that E.coli is predominant isolated from heathy pheasants followed by Campylobacter, Staphylococcus and Brachyspira.
Resumo Há uma escassez de pesquisas realizadas sobre a prevalência microbiana em faisões. A microbiota de aves em cativeiro tem significado zoonótico e deve ser caracterizada. O presente estudo está, portanto, planejado para avaliar a microbiota do conteúdo oral, fecal e intestinal de espécies aviárias em cativeiro. Será útil na caracterização de micróbios nocivos. Diferentes amostras retiradas da boca, intestino e fezes de faisões de pescoço redondo (Phasianus colchicus), faisões verdes (Phasianus versicolor), faisões dourados (Chrysolophus pictus) e faisão prateado (Lophura nycthemera). As amostras foram coletadas, diluídas e inoculadas em diferentes placas de ágar (MacConkey, ágar SS, MSA e ágar nutriente) para o cultivo de espécies bacterianas. Colônias de E. coli, Staphylococcus spp., Brachyspira spp. e Campylobacter spp foram observados com base na morfologia da colônia. A unidade formadora de colônia mostrou E. coli como bactéria frequentemente encontrada no conteúdo fecal, oral e intestinal de todos os faisões acima. A diferença de significância geral foi encontrada entre as espécies bacterianas de faisões dourados, faisões verdes, faisões de pescoço anelado e faisões prateados. Verificou-se que a E.coli é predominantemente isolada de faisões saudáveis, seguida por Campylobacter, Staphylococcus e Brachyspira.