ENHANCED PRODUCTION OF α-AMYLASE AND PLASMID STABILITY IN BATCH AND/OR CONTINUOUS CULTURES OF BACILLUS STEAROTHERMOPHILUS (pAT9)

A mutant strain of Bacillus stearothermophilus GR2 whose ff-amylase had been derepressed from glucose was obtained from a parental strain of B. stearothermophilus CU21 by a mutagenic treatment. Both strains of B. stearothermophilus CU21 and GR2 were transformed with a recombinant plasmid pAT9 wherein the structural gene of α-amylase of the parent strain had been cloned. Performance of both transformants, i.e. the production yield of ff-amylase, the growth rate, the stability as well as content of the plasmid were studied in batch and/or continuous cultures in comparison with the parent and the mutant as host strains. The yield of a-amylase production by the transformant B. stearothermophilus GR2(pAT9) was enhanced to about 55 times larger than the parent strain B. stearothermophilus CU21 when compared in batch, whereas the enhancement was more than 100 times in continuous cultures. The growth rate of the transformant became remarkably lower than that of the host, respectively and in addition, the mutant B. stearothermophilus GR2 exhibited a growth-rate deterioration as compared to the parent B. stearothermophilus CU21. Regardless of the transformant, plasmid stability decreased with the decrease of dilution rate in continuous cultures. Plasmid content in B. stearothermophilus GR2(pAT9) remained almost unchanged despite the change in dilution rate. The particular behavior of the transformant whose plasmid stability decreased following the decrease of dilution rate without the change in plasmid content was discussed with reference to the similar but contradictory trend of decrease in the plasmid stability of pLPll in B. stearothermophilus CU21 in the previous paper (Aiba and Koizumi, 1984; Koizumi et al., 1985). An excellent yield of α-amylase production by B. stearolhermophilus GR2(pAT9) in continuous culture was considered to be commensurate with the most powerful strain of Bacillus subtilis in the literature that has been prepared by multiple mutageneses.