• Journal of Microbiology and Biotechnology
• /
• v.15 no.3
• /
• pp.537-543
• /
• 2005

Subtilisin (EC 3.4.21.14) is the major extracellular alkaline serine protease of Bacillus species. Previously, we found that subtilisins did not migrate in the electrophoretic field in the Laemmili buffer system due to their high pI values (over 8.8); however, it formed a 'binding mode' at the top of the separating gel [5]. Utilizing this characteristic, four subtilisins from Bacillus sp. strains (e.g., B. subtilis 168, B. subtilis KCTC 1021, B. amyloliquefaciens KCTC 3002, and Bacillus sp. DJ-1 and DJ-4) were easily and quickly identified by an over-running electrophoretic technique with a miniscale culture supernatant (less than 20 ml) without any column chromatographic steps. Two subtilisins (DJ-l and a recombinant version) from Bacillus sp. DJ-l were characterized, and the enzymatic properties were determined by SDS-fibrin zymography and densitometric analysis. Based on this observation, the recombinant pro-subtilisin DJ-l showed the same 'binding mode,' similar to native subtilisin DJ-l. On the other hand, mature subtilisin DJ -1 without pro-peptide showed no enzymatic activity.

• Archives of Pharmacal Research
• /
• v.24 no.6
• /
• pp.601-606
• /
• 2001

A simple assay method of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation was studied and validated by capillary electrophoresis (CE) using micellar electrokinetic chromatography (MEKC) techniques. Factors affecting the migration behavior and separation performances of the peptide; type of buffers pH, butler concentration, and concentration of sodium dodecyl sulfates (SDS) were investigated to optimize the analytical performance. CE was performed using running buffers 50.0 mM borate (pH 8.5) containing 12.5 mM SDS at 20 $mutextrm{V}$ of the applied voltage. Calibration curves for the rhEGF showed good linearity (r>0.999) over the wide dynamic range from 1.25 to $100{\mu\textrm{g}}/ml$. Sample analysis was performed by using standard addition method to eliminate the matrix effects of dosage vehicle. This method is assumed to be useful for quality control (QC) of various forms of pharmaceutical products of the peptide.