RESUMO
This study presents data on the production, purification, and properties of a thermostable ß-xylanase produced by an Aspergillus awamori 2B.361 U2/1 submerged culture using wheat bran as carbon source. Fractionation of the culture filtrate by membrane ultrafiltration followed by Sephacryl S-200 and Q-Sepharose chromatography allowed for the isolation of a homogeneous xylanase (PXII-1), which was 32.87 kDa according to MS analysis. The enzyme-specific activity towards soluble oat spelt xylan, which was found to be 490 IU/mg under optimum reaction conditions (50°C and pH 5.0-5.5), was 17-fold higher than that measured in the culture supernatant. Xylan reaction products were identified as xylobiose, xylotriose, and xylotetraose. K (m) values (mg ml(-1)) for soluble oat spelt and birchwood xylan were 11.8 and 9.45, respectively. Although PXII-1 showed 85% activity retention upon incubation at 50 °C and pH 5.0 for 20 days, incubation at pH 7.0 resulted in 50% activity loss within 3 days. PXII-1 stability at pH 7.0 was improved in the presence of 20 mM cysteine, which allowed for 85% activity retention for 25 days. This study on the production in high yields of a remarkably thermostable xylanase is of significance due to the central role that this class of biocatalyst shares, along with cellulases, for the much needed enzymatic hydrolysis of biomass. Furthermore, stable xylanases are important for the manufacture of paper, animal feed, and xylooligosaccharides.
Assuntos
Aspergillus/enzimologia , Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Animais , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Cisteína/metabolismo , Fibras na Dieta/metabolismo , Endo-1,4-beta-Xilanases/química , Ativadores de Enzimas/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Peso Molecular , Especificidade por Substrato , Fatores de Tempo , Ultrafiltração/métodos , Xilanos/metabolismoRESUMO
Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53â¯kDa monomer and 124 and 178â¯kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44â¯kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60⯰C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9⯵g/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.