A cyclodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at -108 to -67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (Mr) and enzymatic properties as follows: Mr, 80,000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55 degrees C; and stability below 45 degrees C. The yields of the products from starch as the substrate were 25% for beta- and 5% for gamma-cyclodextrin.

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A novel mitogen, E-15, which induced blastogenesis of mouse splenocytes, was purified from the culture filtrate of an actinomycete through ethanol precipitation, anion and cation exchange column chromatography, and gel filtration.The producing organism was identified as Nocardia asteroides. Spectronic study demonstrated that it was a polysaccharide. Acid hydrolysis of E-15 yielded glucose and glucosamine. The active substance was eluted at the molecular mass between 90 kDa and 750 kDa on gel filtration. E-15 induced a mitogenic response of mouse splenocytes above 1 /ig/ml and its potency of induction was superior to a lipopolysaccharide that is knownto be a polyclonal B cell-specific mitogen. It showed no toxicity up to lOOjig/ml. The cell surface phenotypes of the blast cells induced by E-15 were analyzed by flow cytometry; they had surface immunoglobulins but no Lyt-2 antigen. Thus it was suggested that E-15 was a B-cell-specific mitogen.