Rational design of enantioselective enzymes requires considerations of entropy.

Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta(R-S)DeltaH(++)) and entropy (Delta(R-S)DeltaS(++)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta(R-S)DeltaS(++) was in all cases equally significant as Delta(R-S)DeltaH(++) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++). Although the changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta(R-S)DeltaH(++) than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta(R-S)DeltaS(++).

Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain.

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.

Improved enantioselectivity of a lipase by rational protein engineering.

A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity. 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutations gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity. The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.