RESUMO
BACKGROUND: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. OBJECTIVES: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. METHODS: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. RESULTS: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. CONCLUSION: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.
Assuntos
Sucos de Frutas e Vegetais/análise , Proteínas Fúngicas/química , Malus/química , Pectinas/química , Poligalacturonase/química , Sporothrix/química , Cátions Bivalentes , Clonagem Molecular , Cobre/química , Estabilidade Enzimática , Tecnologia de Alimentos/métodos , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ferro/química , Cinética , Peso Molecular , Pichia/genética , Pichia/metabolismo , Poligalacturonase/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Prata/química , Sporothrix/enzimologia , TemperaturaRESUMO
De-oiled Jatropha curcas seed cake, a plentiful by-product of biodiesel industry was used as substrate for the production of a useful xylanase from Sporotrichum thermophile in solid state fermentation. Under the optimized conditions, 1025U xylanase/g (deoiled seed cake) was produced. The xylanase exhibited half life of 4h at 45°C and 71.44min at 50°C respectively. It was stable in a broad pH range of 7.0-11.0. Km and Vmax were 12.54mg/ml and 454.5U/ml/min respectively. S. thermophile xylanase is an endoxylanase free of exoxylanase activity, hence advantageous for xylan hydrolysis to produce xylooligosachharides. Hydrolysis of oat spelt xylan by S. thermophile xylanase yielded 73% xylotetraose, 15.4% xylotriose and 10% xylobiose. The S. thermophile endoxylanase thus seem potentially useful in the food industries.
Assuntos
Biotecnologia/métodos , Endo-1,4-beta-Xilanases/biossíntese , Fermentação , Glucuronatos/biossíntese , Jatropha/química , Oligossacarídeos/biossíntese , Óleos de Plantas/isolamento & purificação , Sementes/química , Sporothrix/enzimologia , Metabolismo dos Carboidratos/efeitos dos fármacos , Carbono/farmacologia , Fermentação/efeitos dos fármacos , Umidade , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Cinética , Sporothrix/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Resíduos , Xilanos/metabolismoRESUMO
Phytase of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorganic phosphate from Ca(+2), Mg(+2), and Co(+2) phytates more efficiently than those of Al(3+), Fe(2+), Fe(3+), and Zn(2+). The hydrolysis rate was higher at 60 degrees C as compared to 26 degrees Celsius. Among all the organic acids tested, citrate was more effective in enhancing solubilization of insoluble phytate salts by phytase than others. The dry weight and inorganic phosphate contents of the wheat plants were high when supplemented with phytase or fungal spores. The plants provided with 5 mg phytate per plant exhibited enhanced growth and inorganic phosphate. With increase in the dosage of phytase, there was increase in growth and inorganic phosphate of plants, the highest being at 20 U per plant. The compost made employing the combined native microflora of the wheat straw and S. thermophile promoted growth of the plants. The plant-growth-promoting effect was also higher with the compost made using S. thermophile than that from only the native microflora.
Assuntos
6-Fitase/farmacologia , Espaço Extracelular/enzimologia , Sporothrix/enzimologia , Temperatura , Triticum/efeitos dos fármacos , Triticum/crescimento & desenvolvimento , Biomassa , Ácidos Carboxílicos/farmacologia , Hidrólise/efeitos dos fármacos , Ácido Fítico/metabolismo , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Solo , Solubilidade/efeitos dos fármacosRESUMO
Phytase production by a thermophilic mould Sporotrichum thermophile Apinis was investigated in solid state fermentation (SSF) using sesame oil cake as the substrate. Scanning electron microscopy of the fermented sesame oil cake revealed a dense growth of the mould with abundant conidia. Glucose, ammonium sulphate and incubation period were identified as the most significant factors by Plackett-Burman design. The optimum values of the critical components determined by central composite design of response surface methodology for the maximum phytase production were glucose 3%, ammonium sulphate 0.5% and incubation period 120 h. An overall 2.6-fold improvement in phytase production was achieved due to optimization. Highest enzyme production (348.76 U/g DMR) was attained at a substrate bed depth of 1.5 cm in enamel coated metallic trays. The enzyme liberated inorganic phosphate from wheat flour and soymilk with concomitant dephytinization and liberation of soluble inorganic phosphate.
Assuntos
6-Fitase/metabolismo , Eliminação de Resíduos/métodos , Sporothrix/enzimologia , Biotecnologia/métodos , Fermentação , Cinética , Óleo de Gergelim/metabolismo , Sporothrix/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
The phytase production by Sporotrichum thermophile TLR50 was recorded on all the commonly used animal feed ingredients tested to varying degrees in solid-state fermentation. Enzyme production increased to 180 U/g of dry moldy residue (DMR) in sesame oil cake at 120 h and 45 degrees C at the initial substrate-to-moisture ratio of 1:2.5 and aw of 0.95. Supplementation of sesame oil cake with glucose and ammonium sulfate further enhanced phytase titer (282 U/g of DMR). An overall 76% enhancement in phytase production was achieved owing to optimization. The mold secreted acid phosphatase, amylase, xylanase, and lipase along with phytase. By the action of phytase, inorganic phosphate was liberated efficiently, leading to dephytinization of sesame oil cake.
Assuntos
6-Fitase/biossíntese , Fermentação , Ácido Fítico/metabolismo , Óleo de Gergelim/metabolismo , Sporothrix/enzimologia , 6-Fitase/isolamento & purificação , Fosfatase Ácida/biossíntese , Sulfato de Amônio/farmacologia , Amilases/biossíntese , Ração Animal , Animais , Endo-1,4-beta-Xilanases/biossíntese , Ativação Enzimática , Glucose/farmacologia , Microbiologia Industrial , Lipase/biossíntese , Ácido Fítico/química , Especificidade por Substrato , Temperatura , Fatores de TempoRESUMO
Cellulase production and growth of a strain of Sporotrichum thermophile were studied by using a mineral salts medium supplemented with yeast extract and insoluble cellulose. The effects of cultural conditions, such as pH, nitrogen source, substrate concentration, and temperature, were examined. Maximum production of C1 and CX cellulases occurred at 45 C in 2 to 4 days, in the presence of 1% Solka/Floc as substrate, when NaNO3 or urea used as sources of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appeared to be similar to that reported for Trichoderma viride grown in a favorable environment. However, comparable yields of cellulase were produced by S. thermophile in less than one-quarter the time required by mesophilic fungi.