title: Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30.
abstract: 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 time-of- 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 Ni(2+), Mn(2+), and Cu(2+) 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 × 10(4) U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xin
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Qiongzhen
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Jue
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Yicheng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Linfeng
authors.initials: L
authors.lastname: Luogong
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Shuhuan
authors.initials: S
authors.lastname: Li
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Weiliang
authors.initials: W
authors.lastname: Dong
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D016000:Cluster Analysis; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D020536:Enzyme Activators / Q000032:analysis; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D010450:Endopeptidases; D057057:Serine Proteases; C087822:alkaline protease
keywords: Characterization; Cloning; Protease; Purification; Streptomyces sp.