Role of α-helical structure in organic solvent-activated homodimer of elastase strain K.
Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
Enzyme and Microbial Technology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia; E-Mails:  abubakar@biotech.upm.edu.my  (A.B.S.);  chee_fah_2000@yahoo.com  (C.F.W.).
Raja Noor Zaliha Raja Abd
RN
Rahman
Abu Bakar
AB
Salleh
Mahiran
M
Basri
Chee Fah
CF
Wong
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis / Q000187:drug effects; D004549:Elastin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000494:pharmacology; D008970:Molecular Weight; D009930:Organic Chemicals / Q000737:chemistry*; D010196:Pancreatic Elastase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D008670:Metals; D009930:Organic Chemicals; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012997:Solvents; D004549:Elastin; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa