[39] Bacillus subtilis neutral protease

Molecular cloning and nucleotide sequence of a DNA fragment from Bacillus natto that enhances production of extracellular proteases and levansucrase in Bacillus subtilis

“…1). Since the molecular weights of alkaline and neutral proteases are about 27,000 and 45,000, respectively (28,58) 16 h. The culture supernatants (1 ml) were assayed as described in Materials and Methods. Protease activity shows the total activity of both the alkaline and neutral proteases.…”

“…One is an alkaline protease known as subtilisin, a serine protease sensitive to diisopropylfluorophosphate with an alkaline pH optimum (28), and the other is a neutral protease, a metaloenzyme sensitive to EDTA with a neutral pH optimum (58). Recently, the structural genes of the two proteases from both B. subtilis and Bacillus amyloliquefaciens have been cloned and determined for their nucleotide sequences (40,51,53,55,57).…”

Macromolecular and enzymatic abnormalities induced by a synthetic pyrethroid, Ripcord (Cypermethrin), in adult beetles of a stored grain pest,Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae)

“…The homogenate was centrifuged at 4,900g for 10 min at 4°C. The supernatant thus obtained was used for the estimation of acid phosphatase (AcP*), orthophosphoric-monoester phosphohydrolase, acid optimum, (EC:3.1.3.2), and alkaline phosphatase (AkP, EC: 3.1.3.1) activities according to Kind and King (1954); lactate dehydrogenase (LDH, L-lactate: NAD oxidoreductase, EC:1.1.1.27) activity by a method based on Cabaud and Wroblewski (1958); isocitrate dehydrogenase (ICDH, threo-Ds isocitrate: NADP oxidoreductase, EC:1.1.1.42) activity by a procedure described by Bell and Baron (1960); aspartate aminotransferase (ASAT, Laspartate: 2-oxoglutarate aminotransferase, EC:2.6.1.1) and alanine aminotransferase (ALAT, L-alanine: 2-oxoglutarate aminotransferase, EC: 2.6.1.2) activities according to Reitman and Frankel (1959); cholinesterase (ChE, acetylcholine acetylhydrolase, EC:3.1.1.7) activity accord-ing to Rappaport et al (1959); amylase (1,4-D glucan glucanohydrolase, EC: 3.2.1.1) activity according to the procedure described in Wootton (1964); protease activity by the method of Yasunobu and McConn (1970); trehalase, invertase, maltase, and lactase activities according to the procedure described by Dahlqvist (1966).…”

Further studies on an asporogenous mutant of Bacillus subtilis lacking in vivo proteolytic activity.

“…A portion (5 ml) of the culture fluid centrifuged at 8,000 x g for 2 min was dialyzed against 0.05 M Tris-HC1 buffer (pH 7.3) for 24 hr. The protease activity of the dialyzed fluid was measured by the method of YASUNOBU et al (4). To the supernatant (0.5 ml) was added 0.05 M Tris-HCI buffer (1 ml, pH 7.3) and 0.6% milk casein solution (1.5 ml, pH 7.3) and the reaction mixture was incubated for 10 min at 30°.…”