Thermostable organic solvent tolerant protease from gram-positive bacteria

A purified protease obtained from Bacillus subtilis isolate Rand, a representative sample of which has been deposited as CCTCC accession number M2013058, having the following properties:

• a) an apparent molecular weight of 28 kD determined by SDS-PAGE;
• b) a pH stability with buffers ranging from pH 5 to pH 11 at 25° C.;
• c) a working temperature in the range between 37° C. and 70° C. at pH 7.0;
• d) organic solvents stability of 25% (v/v) of organic solvents ranging from a log P of 0.8 to a log P of 8.8 for 30 min and at 37° C.;
• e) working inhibitors including ethylenediaminetetraacetic acid (EDTA), bestatin, pepstatin A, phenylmethanesulfonyl fluoride (PMSF), 1,4-Dithio-DL-threitol (DTT), and 2-Mercaptoethanol for 30 min at 37° C.;
• f) working metal ions such as K+, Na+, Mg2+, Ca2+, Mg2+, Fe2+, Co2+, Cu2+, Zn2+ and Li2+ and
• g) substrate specificity with natural substrate including casein, albumin, haemoglobin and azocasein, the stability of the purified protease is substantially 100%;
• with azocasein after 30 minutes incubation at 37° C.