The orientation of the enzyme molecular on the interface of the carrier affects its activity. Therefore, it is very important to controllably induce the orientation of the enzyme on the surface to improve the performance of the immobilized enzyme . Magnetic nanoparticles were used to construct microenvironments with the different surface hydrophobicity and charge characteristics by controlled modification, and those particles with various microenvironments were further used to study their interaction with the lipase . The amount and activity of immobilized enzyme on different magnetic nanoparticles surfaces were studied by physical adsorption and covalent binding. Through the enzyme surface and particle surface characteristics analysis, the possible preferred orientation of enzyme and enzyme conformation on different surfaces were inferred, which well explained the effect of surface induction on enzyme loading and activity. The methods of surface microenvironment regulation and the strategy of controllable induction of enzyme orientation adopted in this study are enlightening for the rational design of immobilized enzyme methods.
Despite directed evolution being a practical and efficient method of improving the properties of enzymes, a trade-off between the targeted property and other essential properties often exists which hinders the efficiency of directed evolution. In our previous work, mutant CVH of an esterase from Rhodobacter sphaeroides with high enantioselectivity was obtained by directed evolution, unfortunately its activity cannot catch another mutant YH. To compensate the trade-off of mutant CVH, site-directed saturation mutagenesis was conducted on four residues, three (Asn62, Met121, and Leu145) were hot spots determined from directed evolution, and one (Tyr27) was introduced to make up the large distance between a mutation (Asn62) and the substrate. A new mutant (HMVY) with high enantioselectivity and comparable activity to YH was obtained. According to the kinetic analysis and molecular dynamics simulations, it was understood that the high enantioselectivity and poor activity of mutant CVH was caused by different decrement of efficiency constants to two isomers, ( R )-, ( S )-methyl mandelate, and the high enantioselectivity and activity of mutant HWVY was caused by improved activity towards the preferred substrate (( S )-methyl mandelate), which provided the interpretation of the trade-off compensation. This work could provide a way to compensate the trade-off of enantioselectivity and activity in the process of enzyme evolution.
危险品化学品经营许可证（带存储）