A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium
sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose
chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic
fragments of the purified SapHM were obtained by electrospray ionization quadrupole timeof-
flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM
contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and
Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a
signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino
acid sequences and mass spectrometry. Pure SapHM was optimally active at 80°C in 50 mM
glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50°C and pH 4.0-9.0. The protease
relative activity was increased in the presence of Ni2+, Mn2+, and Cu2+ to 112%, 113%, and 147%
of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl
sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited
by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The Km and Vmax
values were estimated to be 35.7 mg/ml, and 5 × 104 U/mg for casein. Substrate specificity
analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum
fibrin.

Keywords: Characterization, Cloning, Protease, Purification, Streptomyces sp.