Paper Title
Nanoimmobilization of Cholesterol Oxidase and Applications In Biotransformation and Protein Refolding

Abstract
Cholesterol oxidases (ChOx) are bacterial flavo-oxidoreductases with varied industrial and medical applications. The present study describes the immobilization of ChOx from Pseudomonas aeruginosaPseA(ChOxP), Rhodococcuserythropolis MTCC 3951(ChOxR) and Streptomyces sp.(ChOxS) on functionalized Iron (II, III) Oxide magnetic nanoparticles(MNP) and silica nanoparticles(SNP) to improve their biochemical/structural properties and facilitate novel applications. The maximum immobilization efficiency achieved was 68%, 84% and 78% for ChOxP,ChOxR and ChOxSin case of MNP-bioconjugates and 65%, 80% and 71% for ChOxP,ChOxR and ChOxSin case of SNP-bioconjugates respectively. The catalytic efficiency Vmax/Km, was found to be double in the case of nanobioconjugates as compared to that of the native enzyme. The immobilized enzymes retained 55% of their activity even after 10 consecutive reaction cycles. The optimum temperature and pH remained unchanged at 30°C and 7.5 respectively. The heat stability of ChOx was enhanced significantly till 70ºC while the range of pH and storage stability was also broadened. The structural characterization of the nanobioconjugates was carried out through Scanning Electron Microscopy and Transmission Electron Microscopy. The immobilized preparations were used for the biotransformation of cholesterol and 7-ketocholesterol to Cholest-4-en-3-one and Cholest-4-en-3,7-dione respectively, which are industrially important steroid precursors. Proteins can be denatured by changes in temperature, pH or due to the presence of denaturants. Protein misfolding is also a major cause of many neurodegenerative diseases. Nanoparticles act as artificial ‘chaperones’, due to favorable orientation of the proteins on their scaffold which prevents aggregation and reconfigures denatured proteins into their native functional state. In this study, thermal denaturation of cholesterol oxidasesfrom Pseudomonas aeruginosa, Rhodococcuserythropolis and Streptomyces sp. was studied and functionalized magnetic Iron (II, III) oxide nanoparticles(MNPs) were used for refolding of the denatured protein. The thermal inactivation kinetics of native ChOx was studied at temperatures 50-70ºC for different time intervals. Maximum denaturation was obtained at 70ºC and was taken up for consequent studies. Further these thermally denatured proteins were refolded using functionalized magnetic Iron (II, III) oxide nanoparticles.The thermally denatured enzymes were incubated with nanoparticles at 25 °C with constant shaking. Overnight incubation of the denatured ChOx at 70° C with functionalized MNPs resulted in 47, 79 and 29.4% regain in enzyme activity in case of ChOxP, ChOxS and ChOxS respectively.Protein conjugation to the MNPswas confirmed using FTIR, DLS, Zeta Potential Measurements, fluorescence and CD spectroscopy. The CD spectra of the renatured enzyme was almost similar to that of the native one. With thermal denaturation, the protein structure underwent unfolding leading to an increase in fluorescence intensity. In the presence of MNPs, the protein refolded back to its original structure as evident from reduction in fluorescence intensity.This study proves that magnetic nanoparticles are effective in the refolding of heat denatured ChOx which regain their structure and restore activity to a great extent.