|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1998 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1997 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Subtilisin YaB is biologicaly synthesized via a pre-pro type precursor as is the case of subtilisin BPN'. The pre-sequence is processed off in the course of secretion across cytoplasmic membrane and the pro-form is secreted into culture media. Then the pro-form is autocatalytically processed to the active enzyme (mature form), that is subtilisin YaB.No one, however, has succeeded in the isolation of secreted pro-form and the structural elucidation of pro-form among several subtilisins. In the case of subtilisin BPN', there are several indications that the pro-sequence is acting as the guide sequence for the correct folding of mature form enzyme. In the 1997 fiscal year, we constructed an experimental system to obtain the pro-form of subtilisin YaB.When an active center mutant subtilisin YaB (Ser2l4Cys) encoding plasmid was introduced in several strains of Bacillus subtlis, only WB600 strain (6 extracellular Protease deficient mutant) secreted a putative pro-form of 52kDa as estimated in SDS-gel electrophresis. The putative pro-form must be in an unsusual shape in the course of gel electorphoresis, because the detected value is in keen contrast to the estimated molecular weight of 36 kDa. Unfortunately the amount of the putative pro-form was scarce. In the 1998 fiscal year, we tried to improve the yield and to obtain the more confident evidence that the putaitve pro-form is the real pro-form. In spite of several trials to vary the cultural condition the yield of the putative pro-form was not elevated. The stuctural characteristics of the putative pro-form has been remained to be elucidated.