Immobilization of cholesterol oxidases on functionalized Silica Nanoparticles for biotransformation of cholesterol and 7-ketocholesterol.

Abstract

Cholesterol oxidation leads to the development of several oxysterols such as 7-ketocholesterol (7KC), which are linked to various age-related conditions. An approach to reduce their toxicity is proposed using enzymes from microbial sources to degrade them. Our earlier studies identified Pseudomonas aeruginosa PseA and Rhodococcus erythropolis MTCC 3951 as potential strains capable of using 7KC as their sole carbon source. These strains produced cholesterol oxidase as the primary enzyme in the degradation pathway. To enhance applicability, cholesterol oxidase (ChOx) enzymes from P. aeruginosa PseA (ChOxP), R. erythropolis MTCC 3951 (ChOxR), and a commercial variant from Streptomyces sp. (ChOxS) were immobilized on silane functionalized silica nanoparticles (SNP) using covalent-coupling methods. The immobilization efficiency was 68 %, 86 %, and 83 % for ChOxP, ChOxR, and ChOxS respectively. The catalytic efficiency of the immobilized enzyme was nearly twice that of the free enzyme, with increased stability across a wide range of temperatures (10-70°C) and pH levels (4.0-9.0), although the optimum pH (7.5) and temperature (30°C) remained unchanged. The nano-immobilized cholesterol oxidases were reusable up to 10 cycles. Further, enzyme immobilization on nanoparticles was confirmed by FTIR, SEM, and TEM. Biotransformation of cholesterol and 7KC using the nanobioconjugates produced pharmaceutically important molecules 4-cholesten-3-one and 4-cholesten-3,7-dione respectively.