Recombinant expression, activity screening and functional characterization identifies three novel endo-1,4-β-glucanases that efficiently hydrolyse cellulosic substrates

Abstract

The hydrolysis of cellulose into fermentable sugars is a costly and rate-limiting step in the production of biofuels from renewable feedstocks. Developing new cellulase systems capable of increased cellulose hydrolysis rates would reduce biofuel production costs. With this in mind, we screened 55 fungal endoglucanases for their abilities to be expressed at high levels by Aspergillus niger and to hydrolyze amorphous cellulose at rates significantly greater than that obtained with TrCel5A, one of the major endoglucanases in the Trichoderma reesei cellulase system. This screen identified three endoglucanases, Aureobasidium pullulans ApCel5A, Gloeophyllum trabeum GtCel12A and Sporotrichum thermophile StCel5A. We determined that A. niger expressed the three endoglucanases at relatively high levels (≥0.3 g/l) and that the hydrolysis rate of ApCel5A and StCel5A with carboxymethylcellulose 4M as substrate was five and two times greater than the T. reesei Cel5A. The ApCel5A, GtCel12A and StCel5A enzymes also demonstrated significant synergy with Cel7A/CbhI, the major exoglucanase in the T. reesei cellulase system. The three endoglucanases characterized in this study are, therefore, promising candidate endoglucanases for developing new cellulase systems with increased rates of cellulose saccharification.

The Characterization of the Endoglucanase Cel12A from Gloeophyllum trabeum Reveals an Enzyme Highly Active on β-Glucan

“…These results differ from previous thermal stability results on testing of G. trabeum Cel12A against CMC in similar conditions, which showed a much shorter half-life of 2 hours [24].…”

“…We speculate that similar molecular organization can facilitate interactions of G. trabeum Cel12A with none-crystalline polysaccharides. Furthermore, we and others [24] showed that GtCel12A displays activity, albeit low, towards the commercial cellulose Avicel. It should be noted that the commercial microcrystalline cellulose, Avicel (Fluka, St Louis, USA) and Sigmacell (Sigma-Aldrich, St Louis, USA) contains amorphous regions and are heterogeneous substrates.…”

“…The GH12 enzyme from Aspergillus japonicus shows highest activity on β-glucan from barley and lichenan [20]. Previously Cel12A from G. trabeum was shown to be active on CMC with minor activities on birchwood xylan and PASC [10] and is also active on filter paper and Avicel (FGSC A1513) [24]. Using a broader range of substrates to determine substrate specificity, we showed that G. trabeum Cel12A has its highest activity on β-glucan under optimal conditions of pH 4.5 and 50°C.…”

Recombinant Cellulase and Cellulosome Systems

“…Both activity and level of expression were compared to that of TrCel5A, one of the major endoglucanases from T. reesei. This screen identified three EGLs, from species Aureobasidium pullulans, Gloeophyllum trabeum and Sporotrichum thermophile with expression levels and hydrolysis performances superior to those of the Trichoderma enzyme [87].…”

“…The cellobiohydrolase contained six times more Nlinked glycans when expressed in A. niger, and its activity was reduced, underlining the critical effect of post-translation modifications on recombinant cellulases [86]. Recently, a library of EGLs from various fungi were cloned and expressed in A. niger [87]. Both activity and level of expression were compared to that of TrCel5A, one of the major endoglucanases from T. reesei.…”