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KMID : 0545120150250111944
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Journal of Microbiology and Biotechnology
2015 Volume.25 No. 11 p.1944 ~ p.1953
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Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30
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Xin Yan
Sun Zhi Bin
Chen Qiong Zhen
Wang Jue
Wang Yi Cheng
Luogong Lin Feng
Li Shu Huan
Dong Wei Liang
Cui Zhong Li
Huang Yan
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Abstract
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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.
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KEYWORD
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Characterization, Cloning, Protease, Purification, Streptomyces sp.
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