Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli

Abstract

The nucleotide sequence of a 2.314 kb DNA segment containing a gene (ced1) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulosa endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Ced1 showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Ced1 enzyme released cellobiose from p-nitrophenyl-beta-D-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Ced1 enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent Mr of approximately 61,000. The calculated Mr of the ced1 gene product was 61,023.