Shuji Kawai

Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures.

Abstract The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detegents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, α-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed.

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.

Molecular Cloning ofRuminococcus albusCellulase Gene

The gene directing the synthesis of the β-1,4-endoglucanase (1,4-d-glucan glucohydrase) (EC 3.2.1.4) of Ruminococcus albus F-40 was cloned in Escherichia coli C600. The recombinant plasmid pRA1 contained a 3.6 kilobase pair(kb)-Hind III fragment derived from R. albus. Forty-five percent of the cellulase activity encoded by pRA 1 was detected in the periplasmic space and the remaining 55% was in the inner cellular fraction. The optimum pH and temperature of the cloned enzyme were pH 5.7 and 37°C, respectively. The enzyme could digest carboxymethyl cellulose (CMC) and lichenan, but it was inactive for laminarin, xylan, and cellobiose.