Abstract
A novel alkylsulfatase gene, sdsAP, was cloned from a newly isolated bacterium Pseudomonas sp. S9. It encoded a protein of 675 amino acids with a calculated molecular mass of 74.9 kDa. The protein contained a typical N-terminal signal peptide of 41 amino acid residues, followed by a metallo-β-lactamase like domain at the N-terminus and a SCP-2-like domain at the C-terminus. This domain organization mode suggested that it belonged to the type III sulfatase. The mature alkylsulfatase was overexpressed in Escherichia coli. The optimal temperature and pH of the recombinant SdsAP were 70°C and 9.0, respectively. Notably, at optimal conditions, the purified recombinant SdsAP had a high specific activity of 23.25 μmol min−1 mg−1, a K m (app) of 264.3 μmol, and a V max (app) of 33.8 μmol min−1 mg−1 for SDS. Additionally, it still retained more than 90% activity after incubation at 65°C for 1 h, which was much different from other alkylsulfatases reported. The recombinant enzyme hydrolyzed the primary alkyl sulfate such as sodium octyl sulfate and sodium dodecyl sulfate (SDS). It was a Zn2+-containing and Ca2+ activated alkylsulfatase. This is the first report to explore the various characteristics of the heterologous recombinant alkylsulfatase in details. These favorable properties could make SdsAP attractive to be useful in the degradation of SDS-containing waste.
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This work was supported by the grant of No. 200805050 from the Marine Scientific Research Special Foundation for Public Sector Program.
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Communicated by T. Matsunaga.
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792_2011_357_MOESM1_ESM.doc
Supplementary Fig. S1 Determination of the molecular weight of SdsAP. Standard proteins: (1) Catalase (206 kDa), (2) Adolase (177 kDa), (3) Albumin (63.5 kDa), (4) Ovalbumin (48 kDa). The arrow indicates the elution volume of SdsAP. Gel filtration was performed on a column (1 cm × 100 cm) of Sepharose G-150. 0.1 M NaH2PO4-Na2HPO4 buffer (pH 7.0) was used as the elution buffer. (DOC 39 kb)
792_2011_357_MOESM2_ESM.doc
Supplementary Fig. S2 Effect of temperature and pH on stability of SDS. (A) Temperature effect on the stability of SDS. (B) pH effect on the stability of SDS. Temperature stability was determined by measuring the residual amount of 500 μl of 50 mM Tris–HCl buffer (pH 9.0) containing SDS with a final concentration of 0.01% (w/v) at different temperatures (30–90°C) for 1 h. pH stability was determined by measuring the residual amount of 500 μl of different pHs (pH 4.0-11.0) containing SDS with a final concentration of 0.01% (w/v) at 70°C for 1 h. pH profiles were mearsured in 50 mM of different buffers: acetic acid buffer (pH 4.0-5.0), sodium phosphate buffer (pH 6.0-7.0), Tris–HCl buffer (pH 8.0 and pH 9.0), and glycine-NaOH buffer (pH 10.0-11.0). The relative activity was defined as the percentage of amount determined with respect to that measured 500 μl distilled water containing SDS with a final concentration of 0.01% (w/v) at room temperature for 1 h. (DOC 122 kb)
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Long, M., Ruan, L., Li, F. et al. Heterologous expression and characterization of a recombinant thermostable alkylsulfatase (sdsAP). Extremophiles 15, 293–301 (2011). https://doi.org/10.1007/s00792-011-0357-4
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DOI: https://doi.org/10.1007/s00792-011-0357-4