Rahman Raja Noor Zaliha Raja Abd, Salleh Abu Bakar, Basri Mahiran, Wong Chee Fah
Enzyme and Microbial Technology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia; E-Mails:
Int J Mol Sci. 2011;12(9):5797-814. doi: 10.3390/ijms12095797. Epub 2011 Sep 9.
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.
从大肠杆菌KRX/pCon2(3)中过表达的重组弹性蛋白酶菌株K，通过疏水相互作用色谱和离子交换色谱相结合的方法纯化至同质，最终产率为48%，比活性提高了25倍。通过SDS-PAGE和MALDI-TOF分析，纯化后的蛋白质呈现出首次报道的65 kDa同型二聚体大小，这一大小与铜绿假单胞菌通常报道的33 kDa单体完全不同。有机溶剂稳定性实验表明，其稳定性模式与先前报道的规律完全相反，先前报道中在亲水性有机溶剂如二甲基亚砜、甲醇、乙醇和1-丙醇中观察到活性稳定性和增强。从二级结构变化的角度解释了该酶在亲水性溶剂中的高稳定性和增强作用。尽管添加溶剂后α-螺旋结构略有减少，但在25%和50%的甲醇中分别观察到弹性蛋白水解激活和稳定性。进一步的表征实验推测弹性蛋白酶菌株K在广泛的温度、pH值、金属离子、表面活性剂、变性剂和底物特异性范围内具有很高的稳定性和增强作用，表明其在洗涤剂配方中的潜在应用。
