Abstract
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.
Highlights
Enzymology in aqueous media has led to a molecular understanding of function of proteins over years
Homogeneous non-aqueous enzymology emerges as an attractive alternative to other non-aqueous systems in order to overcome some inherent drawbacks associated with heterogeneous non-aqueous biocatalysis [7]
It was surprising that preliminary observation of non-trichloroacetic acid (TCA) precipitated sample, which represented the native form of the protein, on the SDS-PAGE had unveiled a size of approximately 66 kDa (Figure 1A) It is twice as large as the size of wild-type elastase strain K [14]
Summary
Enzymology in aqueous media has led to a molecular understanding of function of proteins over years. The use of circular dichroism (CD) spectroscopy, on the other hand, has directed to a better understanding of protein conformation in these media in regards to its secondary structures changes [8,9] Along with this development, an extracellular organic solvent tolerant protease producer, Pseudomonas aeruginosa strain K, was isolated from benzene-toluene-ethylbenzene-xylene (BTEX). Due to an overwhelming interest in exploring the organic solvent tolerant property possessed by the bacterium, its wild-type proteases were successfully purified and well characterized to withstand in numerous organic solvents of both hydrophilic and hydrophobic nature [13,14] In this communication, our effort is focused on the characterization of recombinant elastase strain K which attributed hugely on the effect of organic solvents on the protein elastinolytic activity in relation to its structure
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