Quantitative assay system for specific enzyme activity using antibody: the case of protease, subtilisin BPN'.
Assay on a high-quality microtiter plate was found to allow for quantitative analysis of bacterial serine protease, subtilisin BPN', and its mutant enzymes which had been genetically engineered to be adapted to low-temperatures (Taguchi et al., 1998. Appl. Environ. Microbiol. 64, 492-495), by using polyclonal antibody against subtilisin BPN'. The use of polyclonal antibody was crucial in normalizing the number of various different enzyme molecules in culture supernatant samples of recombinant strains of Bacillus subtilis, giving rise to the performance of specific activity assay of the enzymes. Relative activity of each mutant subtilisin BPN' to wild-type enzyme was estimated by monitoring the increased value of absorbance caused by enzymatic hydrolysis of a chromogenic substrate. The relative activity in each enzyme estimated by this method showed good coincidence with that estimated by kinetic parameters, kcat/K(m) of the purified enzymes. We termed the system as 'ABEA' (antibody-bound enzyme assay). The efficient ABEA system developed here would be useful for the determination of specific activity of other enzymes of interest and provide us versatile applications in the field of evolutionary engineering.
Miyota
Y
Y
Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.
Komada
S
S
Momose
H
H
Taguchi
S
S
D000818:Animals; D000906:Antibodies; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013381:Subtilisins / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D011994:Recombinant Proteins; D013381:Subtilisins