Hydrolytic properties of two novel cellulases of Melanocarpus albomyces expressed in Trichoderma reesei

Nora Szijártó, Satu Hooman, Marika Alapuranen, Jarno Kallio, Maija Tenkanen, Matti Siika-aho, Kati Réczey, Jari Vehmaanperä, Liisa Viikari

Research output: Contribution to conferenceOther conference contributionScientific

Abstract

A key issue in the realization of fuel ethanol production from lignocellulosics is the utilization of hydrolytic enzymes with special characteristics (e.g. high cellulase activity at elevated operation temperatures) for efficient enzymatic hydrolysis of the technical substrate. Novel thermostable cellulases of the fungal ascomycete Melanocarpus albomyces are seen as promising candidates for this task. Prior to their introduction to the actual industrial process, hydrolytic properties of novel enzymes have to be characterised, preferably using model substrates as the first approach of investigation. In our present study, two of the previously identified M. albomyces cellulases, a 50-kDa endoglucanase (EG) Cel7A, and a 50-kDa cellobiohydrolase (CBH) Cel7B, produced in recombinant strains of the filamentous fungus Trichoderma reesei, were purified (DEAE-IEX) and tested in hydrolysis (60°C, pH 6.0) of crystalline and amorphous cellulose. Enzymes were studied both in their native forms, in which they do not bear a cellulose binding domain (CBD), and also with a modified structure, in which they harbour the CBD of T. reesei CBHI. Time courses of hydrolysis experiments were evaluated via monitoring the release of reducing sugars by the DNS method. Product profiles were analysed by high performance anion exchange chromatography (HPAEC). Both enzymes were active against tested substrates: Cel7B had greater activity than Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence for synergism was obtained when mixtures of the two enzymes were used with a constant total protein dosage. The presence of CBD enhanced hydrolysis rate in all experimental configurations. When individual enzymes were tested, CBD had a greater effect on the performance of CBH than that of EG, especially on crystalline substrate. In synergism, the positive role of CBD was even more evident. Comparison of Cel7B (+/-CBD) to corresponding enzymes of T. reesei (CBHI and CBHI core, respectively) indicated, that novel CBH of M. albomyces is more active on amorphous cellulose than the industrially relevant enzyme preparation from T. reesei under the circumstances applied hereby. Although using crystalline substrate a reverse order was observed, the higher T optima (approx. 70°C, at pH 7.0 with up to 60 min reaction time) of novel cellulases gives evidence for their potential to serve as attractive tools in high-temperature industrial hydrolysis processes.
Original languageEnglish
Publication statusPublished - 2005
MoE publication typeNot Eligible
EventRenewable Resources and Biorefineries - Ghent, Belgium
Duration: 19 Sep 200521 Sep 2005

Conference

ConferenceRenewable Resources and Biorefineries
CountryBelgium
CityGhent
Period19/09/0521/09/05

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