Characterization of a Thermostable Extracellular β-Glucosidase with Activities of Exoglucanase and Transglycosylation from Paecilomyces thermophila

Abstract

The purification and characterization of a novel extracellular beta-glucosidase from Paecilomyces thermophila J18 was studied. The beta-glucosidase was purified to 105-fold apparent homogeneity with a recovery yield of 21.7% by DEAE 52 and Sephacryl S-200 chromatographies. Its molecular masses were 116 and 197 kDa when detected by SDS-PAGE and gel filtration, respectively. It was a homodimeric glycoprotein with a carbohydrate content of 82.3%. The purified enzyme exhibited an optimal activity at 75 degrees C and pH 6.2. It was stable up to 65 degrees C and in the pH range of 5.0-8.5. The enzyme exhibited a broad substrate specificity and significantly hydrolyzed p-nitrophenyl-beta- d-glucopyranoside ( pNPG), cellobiose, gentiobiose, sophorose, amygdalin, salicin, daidzin, and genistin. Moreover, it displayed substantial activity on beta-glucans such as laminarin and lichenan, indicating that the enzyme has some exoglucanase activity. The rate of glucose released by the purified enzyme from cellooligosaccharides with a degree of polymerization (DP) ranging between 2 and 5 decreased with increasing chain length. Glucose and glucono-delta-lactone inhibited the beta-glucosidase competitively with Ki values of 73 and 0.49 mM, respectively. The beta-glucosidase hydrolyzed pNPG, cellobiose, gentiobiose, sophorose, salicin, and amygdalin, exhibiting apparent Km values of 0.26, 0.65, 0.77, 1.06, 1.39, and 1.45 mM, respectively. Besides, the enzyme showed transglycosylation activity, producing oligosaccharides with higher DP than the substrates when cellooligosaccharides were hydrolyzed. These properties make this beta-glucosidase useful for various biotechnological applications.

An Overview on Fungal Cellulases with an Industrial Perspective

“…Similarly, the activity of cellulase produced by A. terreus M11 was increased in the presence of Mn 2+ ; but the presence of Hg 2+ , Cu 2+ , Pb 2+ and detergents slightly decreased or completely abolished the activity of cellulase [72]. Yang et al [80] demonstrated that the cellulase produced by Paecilomyces thermophila was strongly inhibited by Hg 2+ ; while the activity was highly enhanced in the presence Zn 2+ .…”

Caracterização da atividade β-glucosidásica de Humicola insolens

“…Por outro lado, é bem conhecido que fungos termófilos normalmente produzem β-glucosidases altamente glicosiladas, particularmente aquelas que são secretadas para o meio extracelular Venturi et al, 2002;Yang et al, 2008 A diferença no teor de carboidratos de BGH I e BGH II pode estar relacionada à diferença de localização celular das enzimas. A maior percentagem de BGH I encontra-se no espaço periplasmático e na parede celular, sugerindo que o organismo produza esta enzima para agir fora da célula.…”

“…Já as β-glucosidases da maioria dos fungos termófilos mostram temperatura ótima entre 55 e 75°C, e geralmente são mais termoestáveis que as enzimas dos fungos mesófilos (Mashewari et al, 2000;Yang et al, 2008;Kaur et al, 2007). As enzimas purificadas de H. grisea e também de outros isolados de H. insolens (Yoshioka & Hayashida, 1980;Rao & Murthy, 1988) apresentaram temperatura ótima de 50°C.…”

“…Analogamente ao observado neste estudo, a maioria das β-glucosidases fúngicas são estáveis numa ampla faixa de pH Yang et al, 2008;Bhiri et al, 2008).…”

“…Portanto, as β-glicosidases não são responsáveis apenas pela produção de glicose a partir de celobiose, mas permitem também a ação eficiente de exoglucanases e endoglucanases (Kaur et al, 2007). Por outro lado, grande parte das β-glucosidases conhecidas são inibidas por glicose Lynd et al, 2002;Yang et al, 2008;Kaur et al, 2007;Karnchanatat et al, 2007;Bhiri et al, 2008). Assim, existe um interesse crescente na busca por enzimas tolerantes ao produto final, as quais poderiam melhorar acentuadamente a eficiência do processo de "sacarificação" da celulose.…”