MOLECULAR-CLONING OF NUCLEOTIDE-SEQUENCE OF A NEUTRAL CELLULASE GENE OF THERMOPHILIC CLOSTRIDIUM SP F3

A 1.7-kb HindIII fragment of Clostridium DNA cloned in Escherichia coli was shown to direct the synthesis of a neutral cellulase. The optimum pH was 6.5 for the enzyme encoded on the 1.7 kb DNA fragment and 7.5 for the enzyme produced by transformant was slightly decreased from 70 C to 65 C. The neutral cellulase gene was located on a 1.2-kb DNA fragment as a functional state, derived from the 1.7-kb DNA fragment, and the nucleotide sequence was determined. The coding sequence showed an open reading frame encoding 552 bp corresponding to 184 amino acid residues with molecular weight of 20, 378 daltons. However, the regulatory sequences and/or N-terminal region of the encoded polypeptide were not detected in the sequence encoded the neutral cellulase gene. The enzyme encoded on the 1.7 kb fragment might consist of a half region (C-terminal region) of the mature enzyme, and be expressed by lacZ promoter of vector, pUC9.