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

Summary The nucleotide sequence of a 2.314 kb DNA segment containing a gene (cedl) 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. cellulose endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Cedl showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Cedl enzyme released cellobiose from p-nitrophenyl--d-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Cedl 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 M_r of approximately 61000. The calculated M_r of the ced1 gene product was 61023.Abbreviations Ap ampicillin - ced1 gene coding for Ced1 - Ced1 cellodextrinase from B. fibrisolvens - CMC carboxymethylcellulose - LB Luria Bertani - ORF open reading frame - pNPC p-nitrophenyl--d-cellobioside - PC phosphate citrate - HCA hydrophobic cluster analysis