Date of Award

1986

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

A (beta)-glucosidase gene was cloned from a cellulolytic bacterium, which was obtained by hybrid-formation between Bacillus cereus and a Cellulomonas sp. through protoplast fusion, into Escherichia coli plasmid pBR322 using recombinant DNA techniques. E. coli strain transformants of JM83 harboring this cloned plasmid were able to utilize cellobiose as a carbon source. (beta)-glucosidase activity was assayed in these cells by P-nitrophenyl (beta)-D-glucopyranoside (PNPG) hydrolysis. Hybridization of the cloned DNA fragment with DNA from the hybrid organism was performed by Southern transfer (92) and DNA/DNA dot blot procedures. The hybridization data strongly suggested that the DNA cloned into pBR322 was from the genome of the hybrid organism. A restriction map was constructed by analysis of the cleavage sites of restriction endonucleases that recognize specific hexanucleotide sequences. The size of the cloned DNA fragment was 1.2 kb as determined by agarose gel electrophoresis. The nucleotide sequence was determined by subcloning partial fragment of 1.2 kb DNA into M13 phage and using the dideoxy sequencing method developed by Sanger et al. (85). The DNA sequence showed that the cloned DNA contained an open reading frame probably coding for (beta)-glucosidase activity, though not ending with a translation stop codon. This presumably incomplete open reading frame has a coding capacity for 319 amino acids, that corresponds to a protein with a molecular weight of 38,000 daltons.

Pages

138

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