Satoru Mizutani

On-line control of glucose concentration using an automatic glucose analyzer

Abstract The fundamental characteristics of an automatic glucose analyzer which consists of sampling, sensor, and operation units were examined. The glucose sensor is a dual cathode type which has an immobilized glucose oxidase membrane coupled with an oxygen sensor. Using this glucose sensor combined with an automatic sampling device, on-off control of the glucose concentration in fed-batch cultures of Saccharomyces cerevisiae was carried out. When the glucose concentration to be controlled was set at 0.3 and 10 g/l, the concentration was well maintained within the range of 0.08−0.54 g/l in the former, and within 9.2–11.1 g/l in the latter. In the former experiment, 1.67 g/l of ethanol was produced at the end of cultivation (OD570=34). On the other hand, 12.9 g/l of ethanol was accumulated at the end of cultivation (OD570=43) in the latter experiment. Fed-batch cultures of Micrococcus ruteus were also carried out. The glucose concentration was set at 2.5 g/l. The microorganism grew up to OD610=264 and the glucose concentration was maintained within 2.0 and 3.1 g/l.

On-off regulation of gene expression from tryptophan promoter with cross-flow filtration

SummaryRecombinant plasmid containing β-galactosidase gene fused to trp promoter (pMCT98) and that containing cloned trp repressor gene (pRLK13) were introduced into Escherichia coli C600. The bacterium was cultivated in a jar-fermetor equipped with a cross-flow filtration apparatus to attain the on-off regulation of the gene expression by controlling tryptophan concentration in the medium. In logarithmic growth phase, the cross-flow filtration was started. Tryptophan concentration dropped to a low level within 1 h and an efficient expression of β-galactosidase gene was started. By this twostage cultivation, very high biomass was achieved (final OD570: 150) and the amount of produced β-galactosidase was about 10% of total cellular proteins.

On-off regulation of the tryptophan promoter in fed-batch culture

Abstract Fundamental properties of the trp promoter were investigated in fed-batch culture using a recombinant containing the lacZ gene controlled by this promoter. In tryptophan-deficient conditions, the amount of β-galactosidase accumulated in the cell was 10% of total cellular proteins. In the presence of the amino acid, it was repressed at a lower level, but considerable expression was observed in the later stages of cultivation. Although increasing concentration of tryptophan seemed to repress the promoter more completely, it strongly inhibited the bacterial growth. On-off regulation of the promoter was achieved by controlling the tryptophan level during fed-batch culture.