Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction
✍ Scribed by Hamamoto, Tetsuo ;Foong, Frances ;Shoseyov, Oded ;Doi, Roy H.
- Publisher
- Springer
- Year
- 1992
- Tongue
- English
- Weight
- 889 KB
- Volume
- 231
- Category
- Article
- ISSN
- 0026-8925
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✦ Synopsis
The nucleotide sequence of engD, an endo-beta-1,4-glucanase gene from Clostridium cellulovorans was determined (Genbank Accession No. M37434). The COOH-terminal part of the gene product, EngD, contained a Thr-Thr-Pro repeated sequence followed by a region that has homology to the exoglucanase of Cellulomonas fimi. EngD and EngB, another C. cellulovorans endoglucanase, show 75% amino acid sequence homology at their NH2-termini, in contrast to their carboxyterminal domains which show no homology. EngD had endoglucanase activity on carboxymethylcellulose (CMC), cellobiosidase activity on p-nitrophenyl-cellobioside (p-NPC), and partial hydrolytic activity on crystalline cellulose (Avicel), while EngB showed hydrolytic activity against only CMC. Chimeric proteins between EngB and EngD were constructed by exchanging the non-homologous COOH-terminal regions. Chimeric proteins that contained the NH2-terminus of EngD retained cellobiosidase activity but chimeras with the EngB NH2-terminus showed no cellobiosidase activity. Hydrolysis of crystalline cellulose (Avicelase activity) was observed only with the enzyme containing the EngD NH2-terminus and EngD COOH-terminus.