Improving The Thermostability Of Cholesterol Oxidase By Targeted Mutagenesis Guided By Computational Model For The Protein Unfolding Free Energy

Improving structural stability of Cholesterol oxidase(COD) would be meaningful since the enzyme has been used for detecting blood cholesterol and could be used for reducing cholesterol in food. This paper describes how the COD mutation sites were predicted based on bioinformatics method in order to improve thermal stability of cholesterol oxidase, and the results and mechanisms from the directed mutagenesis. The main results include:1) Sequences and structures alignment of cholesterol oxidases lid region from Streptomyces SA-COO(CODs) and Rhodococcus sp.(CODr) were analyzed. Two mutation strategies were designed, and CODr-M1(four amino acids removed) and CODr-M2(Ala inserted) were constructed. The results showed that both the thermal stability of two mutants had been weakened, but CODr-M1 has displayed higher substrate specificity to pregnenolone, which indicated that the lid region in COD is related to the substrate specificity of this enzyme.2) Rosetta was used to predict amino acid substitutions of all the 506 residues in CODr based on calculating the unfolding free energy and total energy of the mutations, and the amino acid substitutions which might cause the improving of COD thermostability were sorted out according to the calculation output. Point mutagenesis were conducted, and 6 substitutions(D71G, N86 H, P149 D, P354 D, N459 G and P464E) with positive effect on the protein thermostability have been screened out. It was found that all the positive mutations occurred on the CODr molecular surface. Among these mutants, the stability of N86 H had a 2.6-fold higher than that of wild-type.3) Sub-mutagenesis and duel-mutagenesis were carried out in order to construct submutants with improved thermostability. Since N86 looks like a key site on COD thermostability, seven site mutations were further conducted on the site of N86. The N86 H mutant showing highest thermostability was chosen to run combinatory mutagenesis with D71 G,N86H,P149 D,P354D and Q108 E respectively. The dual-mutant N86H/D71 G was found to have better thermostability, as a half-life of 26.6 min, and about 3.9-fold of the wild-type. The study of enzymatic properties was found catalytic activity of cholesterol are reduced in combination mutatnts.4) The molecular mechanism of COD thermostability improving was interpreted by protein structure interpretation. The CD spectra research suggests increasing α-helix or β-sheet content was the result why the thermal stability of mutatnts increased. By comparing COD molecular structures between the mutants and wild type, it showed that point mutagenesis changed not the protein skeleton structure, but the interactions between the neighborhoods or nearby amino acid residues. The simulated modeling shows that all the intermolecular hydrogen bonding become strengthened and hydrophobic interaction on CODr protein surface become weakened in the positive COD mutants, the mechanisms of CODr thermostability improving could be reasonably explained.