Synthesis and Characterization of Semisynthetic Metallothermolysin
Khalid, Syarajatul Erma (2008) Synthesis and Characterization of Semisynthetic Metallothermolysin. Masters thesis, Universiti Putra Malaysia.
Modification of native enzyme has gone through various strategies and evolution of the process itself. Thermolysin (TLN) from Bacillus thermoproteolyticus rokko was modified as a semisynthetic metallothermolysin which comprises enzyme, ligand and metal ion. Ligands used were benzamidine (BEN), 1,10-phenanthroline (PHN), p-aminobenzamidine (PBZ) and ethanolamine (ETA). The metal ions chosen were magnesium (Mg2+), zinc (Zn2+), calcium (Ca2+) and nickel (Ni2+). The semisynthetic metallothermolysin activities were evaluated on hydrolysis reaction of azocasein. Among the four ligands, complex of TLN-PBZ showed the highest specific activity (2219.5 Unit per mg (U/mg)) at optimum PBZ concentration of 0.6 mM. The study followed by the attachment of Mg2+ to TLN-PBZ complex which gave the best specific activity compared to other metal ions (39406.4 U/mg). The optimum concentration of Mg2+ was found best at 0.08 mM. Several parameters were also investigated such as studies on effect of pH, temperature, time course and thermostability. As a result, the semisynthetic metallothermolysin maintained at pH surrounding of 7.0 in tris-HCl buffer and found optimum at 80oC for reaction up to 3 hours (96.7% of relative activity). For thermostability test, the semisynthetic metallothermolysin can retain its activity up to 90% at pre-heated temperature of 80oC. Electronic absorption like the UV/Visible (UV/Vis) and UV/Fluorescence spectrophotometer and Circular Dichroism (CD) spectropolarimetry method were used to characterize the optical properties of metallothermolysin. In UV/Vis spectrophotometer, the binding of PBZ to TLN curve caused a bathocromic shift (λmax from 279 nm to 274 nm) and became hypsochromicsm (λmax from 274 nm to 272 nm) with the additional of Mg2+. Changes in UV/Vis were also supported by UV/Fluorescence, when changes happened to the emission characteristic of TLN-PBZ spectrum (373.2 nm) and the spectrum continues to shift (374.0 nm) for TLN-PBZ-Mg. The CD spectropolarimetry suggested some changes of α helix and β sheet at far UV molar ellipticity readings with decreased of α helix from 37% (TLN) to 20.6% (TLN-PBZ) and then to 19.8% (TLN-PBZ-Mg). Meanwhile, a further decrease of β sheet from 32.6% (TLN) to 18.7% (TLN-PBZ) and then to 11.0% (TLN-PBZ-Mg) was also observed.
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