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Purification of a High Molecular Mass Protein in Streptococcus mutans
Published on: September 14, 2019
Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30
Yan Xin1, Zhibin Sun1, Qiongzhen Chen1
1Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, P.R. China.
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.
Insights
A novel alkaline protease, SapHM, from Streptomyces sp. M30 was purified and characterized. This serine protease exhibits optimal activity at 80°C and pH 9.0, with broad stability and activation by metal ions and certain chemicals.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Alkaline proteases are crucial enzymes with diverse industrial applications.
- Streptomyces species are known producers of various bioactive compounds, including proteases.
- Characterization of novel proteases can lead to new biotechnological tools.
Purpose of the Study:
- To purify and characterize a novel alkaline protease, SapHM, from Streptomyces sp. M30.
- To determine the enzyme's biochemical properties, including optimal conditions, stability, and substrate specificity.
- To elucidate the genetic basis and structural features of the alkaline protease.
Main Methods:
- Enzyme purification using ammonium sulfate precipitation and chromatography (hydrophobic interaction, DEAE-Sepharose).
- Protein identification and characterization using mass spectrometry and nucleotide sequencing.
- Enzyme activity assays, stability tests, and substrate specificity analysis.
Main Results:
- Purified SapHM exhibited a specific activity of 29,070 U/mg with a yield of 15.5%.
- The gene sapHM encodes a 392-amino acid protein, with the mature peptide mass of 37.1 kDa.
- SapHM demonstrated optimal activity at 80°C and pH 9.0, broad pH/temperature stability, and activation by specific metal ions and chemicals. It was identified as a serine protease.
- The enzyme showed activity on casein, bovine serum albumin, and bovine serum fibrin.
Conclusions:
- SapHM is a novel, thermostable serine alkaline protease from Streptomyces sp. M30 with potential industrial applications.
- The enzyme's characteristics suggest its utility in various biotechnological processes requiring high temperatures and alkaline conditions.
- Further studies could explore its specific applications and potential for protein engineering.
