Two different proteases from Streptomyces hygroscopicus are involved in transglutaminase activation.

Transglutaminase (TGase), the only commercial enzyme in the food industry capable of introducing covalent bonds to proteins, is secreted as a zymogen (Pro-TGase) in several Streptomyces species. In previous studies, only a metalloprotease has been isolated from Streptomyces mobaraensis as an endogenous TGase-activating protease (TAP). In this study, not only an endogenous metalloprotease but also an endogenous serine protease is found to be involved in TGase activation in Streptomyces hygroscopicus. In a cell-free system, the TAP inhibitor was first precipitated with cetyltrimethyl ammonium bromide (CTAB) to maintain TAP activity. Subsequently, different types of protease inhibitors were added to identify the TAP involved in TGase activation in S. hygroscopicus. TGase activation was inhibited by 1 mM phenylmethanesulfonyl fluoride (PMSF) and 10 mM ethylenediaminetetraacetic acid (EDTA), indicating the involvement of serine protease and metalloprotease in the TGase activation process. Furthermore, the TAP purified from a liquid culture of S. hygroscopicus was identified as a serine protease, which is different from the TAP isolated from S. mobaraensis. In addition, Streptomyces Pro-TGases were found to have a conserved amino acid sequence preceding the N-terminal of TGase, which contained cleavage sites for both serine protease and metalloprotease. These results indicate that endogenous serine and metalloproteases are both involved in TGase activation in S. hygroscopicus. To the authors' knowledge, this is the first report that an endogenous serine protease is involved in Streptomyces TGase activation.