Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

Abstract

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t (50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).

Purification and Characterization of Amyloglucosidase Enzyme from the Thermophilic <i>Endomycopsis fibuligera</i> Using Sago Starch as a Substrate

“…It was found a single band protein with molecular weight of 86.5 kDa [11]. The molecular weight of amyloglucosidase in different organism sources have been reported in ranging from 49 to 112 kDa [12].…”

Purificação de amiloglicosidase de Aspergillus niger

“…A literatura destaca o emprego de algumas técnicas de purificação para a amiloglicosidase, como fracionamento com sulfato de amônio; cromatografia de troca iônica, de interação hidrofóbica e por afinidade; e extração por sistema aquoso bifásico (CHEN; ZHANG; ZHOU, 2005;MICHELIN et al, 2008). Os processos cromatográficos são extensamente usados com alto desempenho na etapa de purificação de produtos biotecnológicos.…”

“…das indústrias de processamento de amido, por amiloglicosidases destas fontes se deve ao fato de que estes microrganismos são reconhecidos como seguros sendo aceitos pela Food and Drug Administration (FDA) (MICHELIN et al, 2008).…”

“…Observa-se que os resultados obtidos neste trabalho são superiores aos encontrados na literatura para purificação da amiloglicosidase empregando cromatografia de troca iônica. Vale ressaltar que vários trabalhos (MICHELIN et al 2008;CHEN;ZHANG;ZHOU, 2005) empregaram várias etapas para purificação da enzima, obtendo valores finais de fator de purificação e recuperação inferiores aos obtidos neste trabalho, onde purificou-se a enzima obtida diretamente do extrato bruto, através de uma única etapa de purificação.…”

Purification and characterization of a novel α-glucosidase from an Antarctic yeast Dioszegia fristingensis isolate

“…For its part, several α-glucosidases and glucoamylases from mesophilic bacteria, fungi and yeasts have been described, which, in general, display optimal activity at temperatures up to 45°C and at acidic pH [21][22][23]. The mostly used α-glucosidases and glucoamylases in industrial processes have been obtained from Aspergillus species with major activity displayed at temperatures between 45-60°C [13,24,25]. Currently, the search for novel microbial sources of amylases with high activity at low temperatures, for energy-saving, gains importance.…”