Lipase Specificity and Selectivity : Engineering, Kinetics and Applied Catalysis (2010)

The specificity and selectivity of the enzyme Candida antarctica lipase B (CALB) were studiedfor several substrates and applications.With help of molecular modeling, the active site of CALB was redesigned for the ring openingpolymerization of D,D‐lactide. Two mutants, with about 90‐fold increase in activity ascompared to the wild‐type enzyme, were created. Changing a glutamine into alanineaccounted for this increase in both mutants by creating a larger space in the acyl donorpocket. The new space made it possible to accommodate the bulky substrate and improvethe transition state‐active site complementarity during polymer chain propagation.The enantioselectivity of CALB towards secondary alcohols was engineered by rationalredesign of the stereoselectivity pocket in the enzyme active site. A larger space created by asingle point mutation resulted in an 8’300’000 times change in enantioselectivity towards 1‐phenylethanol and the enantiopreference was inverted into S‐preference. The activitytowards the S‐enantiomer increased 64’000 times in the mutant as related to the wild‐type.The solvent and temperature effects on the enantioselectivity were studied for severalsubstrates and revealed the importance of entropy in the change in enantioselectivity.Substrate selectivity is of great importance for the outcome of enzyme catalyzed polymersynthesis. Ring opening polymerization (ROP) of γ‐acyloxy‐ε‐caprolactones will result in apolyester chain with pendant functional groups. CALB was found to have activity not onlytowards the lactone but also towards the γ‐ester leading to rearrangement of the monomersyielding γ‐acetyloxyethyl‐γ‐butyrolactone. This selectivity between the lactone and the γ‐ester was dependent on the type of group in the γ position and determined the ratio ofpolymerization and rearrangement of the monomers. Molecular dynamics simulations wereused to gain molecular understanding of the selectivity between the lactone and γ‐ester.In order to obtain (meth)acrylate functional polyesters we investigated the use of 2‐hydroxyethyl (meth)acrylate (HEA and HEMA) as initiators for ring opening polymerization.We found that, in addition to the ring opening polymerization activity, CALB catalyzed thetransacylation of the acid moiety of the initiators. The selectivity of CALB towards thedifferent acyl donors in the reaction resulted in a mixture of polymers with different endgroups. A kinetic investigation of the reaction showed the product distribution with timewhen using HEA or HEMA with ε‐caprolactone or ω‐pentadecalactone.The high selectivity of CALB towards lactones over (meth)acrylate esters such as ethyleneglycol di(meth)acrylate was used to design a single‐step route for the synthesis ofdi(meth)acrylated polymers. By mixing ω‐pentadecalactone with the ethylene glycoldi(meth)acrylate and the enzyme in solvent free conditions, we obtained &gt;95 % ofdi(meth)acrylated polypentadecalactone.Taking advantage of the high chemoselectivity of CALB, it was possible to synthesizepolyesters with thiol and/or acrylate functional ends. When using a thioalcohol as initiatorCALB showed high selectivity towards the alcohol group over the thiol group as acyl acceptorfor the ROP reaction. The enzymatic ability of catalyzing simultaneous reactions (ROP andtransacylation) it was possible to develop a single‐step route for the synthesis ofdifunctionalized polyesters with two thiol ends or one thiol and one acrylate end by mixingthe initiator, lactone and a terminator.