RESUMO
An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.
Assuntos
Aspergillus/enzimologia , Poligalacturonase/química , Poligalacturonase/isolamento & purificação , Poligalacturonase/metabolismo , Carbono/metabolismo , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia em Camada Fina/métodos , Citrus sinensis/química , Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Estabilidade Enzimática/efeitos dos fármacos , Etanol/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ácidos Hexurônicos/metabolismo , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/métodos , Cinética , Metais/metabolismo , Peso Molecular , Pectinas/metabolismo , Dióxido de Enxofre/metabolismo , Temperatura , Fatores de TempoRESUMO
BACKGROUND: In the food industry, the use of pectinase preparations with high pectin esterase (PE) activity leads to the release of methanol, which is strictly regulated in food products. Herein, a pectin-degrading enzyme (PDE) complex exhibiting low PE activity of three Aspergillus sojae ATCC 20235 mutants (M3, DH56 and Guserbiot 2.230) was investigated. Production of exo-/endo-polygalacturonase (PG), exo-polymethylgalacturonase (PMG) and pectin lyase (PL) by mutant M3 and A. sojae using two different carbon sources was evaluated in solid-state fermentation. Finally, experimental preparations obtained from the mutants and commercial pectinases standardized to the same potency were screened for PDEs. RESULTS: Mutant M3 grown on sugar beet was found to be the best producer of exo-PG, endo-PG, exo-PMG and PL, with maximum yields of 1111, 449, 130 and 123 U g(-1), respectively. All experimental preparations exhibited low PE activity, at least 21.5 times less than commercial pectinases, and higher endo-PG (40 U mL(-1)). CONCLUSION: Mutant M3 was the best PDE producer using sugar beet. Mutant strains presented a PDE complex featuring high endo-PG and very low PE activities. This novel complex with low de-esterifying activity can be exploited in the food industry to degrade pectin without releasing methanol.