Role of α-Helical Structure in Organic Solvent-Activated Homodimer of Elastase Strain K

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.

Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease

“…Elastase strain K is an organic solvent tolerant protease that is isolated from Pseudomonas aeruginosa strain K, wherein the organic solvent tolerant protease exhibits stability and enhanced activity in a wide range of polar organic solvents. In Rahman et al, recombinant elastase strain K had previously shown stability in 25% (v/v) polar organic solvents such as ethanol, methanol, 1-propanol, and DMSO [14][15][16]. The ability of elastase strain K to withstand a broad range of organic solvents allows it to be applied in the enzyme industry and to be run through extreme processes.…”

Characterization of thermostable aminoacylase from Geobacillus sp. strain SZN

“…The inhibitory effect of divalent ions, such as Zn 2+ and Ni 2+ , was clearly observed on aminoacylase SZN. These ions have been reported to inhibit the proteolytic activities of elastase strain K (Rahman et al, 2011), ME-4 (Cheng et al, 2009) and PseA (Gupta et al, 2005) which also happened to be metalloproteases. The highest half-life of aminoacylase was reported by Hollingsworth (2002), who found that the stability of aminoacylase from Thermococcus litoralis was 25 h at 70°C.…”

“…Our results showed that 1 mM metal ion reduced the aminoacylase activity, whereas the 10 mM Cu 2+ Mg 2+ , Mn 2+ and Na + increased the aminoacylase activity to 107, 153, 160 and 136%, respectively (Supplementary Material 2c). We postulate that increased concentration of divalent ions, such as Mg 2+ and Mn 2+ , may result in enhancement of activity, probably due to increased stability in enzymesubstrate and product complexes (Knape et al, 2017;Rahman et al, 2011).…”

Capillary-Seeding Crystallization and Preliminary Crystallographic Analysis of a Solvent-Tolerant Elastase from Pseudomonas aeruginosa Strain K

“…The recombinant elastase strain K was overexpressed in E. coli KRX/pCon2(3) and purified to homogeneity as described by Rahman et al [11] with slight modifications. Three liters of E. coli KRX/pCon2(3) culture was harvested through centrifugation and the precipitated cells were dissolved in reaction buffer (50 mM Tris-Cl, pH 8.5) before sonication to release the enzyme.…”

“…Exploring the stability of this enzyme (elastase from P. aeruginosa strain K) and using molecular genetics for gene overexpression were well documented by Wong et al [10]. Further investigations to characterize the purified recombinant elastase strain K showed its capability for activity in hydrophilic organic solvent media [11]. Given the previous investigations, our research will focus on recombinant elastase strain K crystallization and structure determination to discern its structural features as an organic solvent-stable enzyme.…”