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Project ID: ENK6-CT-2002-00604
Finanziato nell'ambito di: FP5-EESD
Paese: Finland

Novel thermostable enzyme systems

Superior cellulases operating at high hydrolysis temperature were screened from various microbial strains. Two cellobiohydrolases, three endoglucanases, two xylanases and three beta-glucosidases were purified to homogeneity. The specific activities of the purified thermostable cellobiohydrolases against synthetic substrates and Avicel were generally higher than those of Trichoderma. Cellobiose inhibition of all purified enzymes was less severe than with Trichoderma CBHI. During the project thirteen genes (genomic copies) were cloned, sequenced and transferred into T.reesei for production. Eleven were expressed in T. reesei. The genes were transformed into a host strain, which has all the major cellulase genes deleted. In addition, CBDs (cellulose binding domains) were attached to some of the thermophilic enzymes, which naturally lack the CBD. The cloned enzymes were produced in T. reesei in laboratory scale and preliminarily characterized using the culture supernatants.

Most of the enzymes were 10-15 ºC more thermostable than their T. reesei counterparts. The most interesting cellobiohydrolases were purified from crude culture filtrates using affinity chromatography. The specific activities of the purified enzymes against synthetic substrates and Avicel cellulose were generally higher than those of Trichoderma. Cellobiose inhibition of all purified enzymes was less severe than with Trichoderma CBHI. The thermal stability of the enzymes was about 10 °C higher than that of T. reesei CBHI. Based on kinetic measurements, the new cellobiohydrolases were clearly more efficient in hydrolysis than the state-of-art enzyme T. reesei CBHI.

The kinetic constants of the beta-glucosidase preparations were determined. Substrate and product inhibition pattern analysis showed that the novel thermostable ß-glucosidase was less affected by end-product inhibition. However, similarly to the reference enzymes, the activity of the thermostable ß-glucosidase proved to be more influenced by glucose at 70°C than at 50°C.

Thermophilic cellulase preparations were manufactured in pilot scale for designing the developmental cellulase preparation for hydrolysis experiments in the project. The T. reesei strains in the project were constructed to produce essentially monocomponent enzyme preparations. Three thermophilic cellulases, identified as most promising enzymes in their categories (CBH, EG and ß-glucosidase) were produced in T. reesei and mixed to compose a novel mixture of thermophilic cellulases. For the xylan containing substrates, thermophilic xylanase was added.

A T. reesei strain was constructed to be used for evaluating the production of the cloned cellulases using various production conditions and raw materials. To test the utilization of various side fractions of the process, hydrolysed hemicellulose both from wet-oxidation and steam pretreatment using corn stover as raw material was examined. These hemicellulose hydrolysates could be used to replace some of the defined medium.

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