Engineering Candida antarctica lipase B by circular permutation and incremental truncation

The lipase B from Candida antarctica (CALB) is a versatile biocatalyst with broad applications in industry and in organic synthesis. In recent years, a growing interest has been put on CALB engineering to broaden its applications in asymmetric synthesis. In this dissertation a combined engineering strategy -- circular permutation and incremental truncation -- was employed, which yielded variants with greatly enhanced catalytic performance.;A random circularly permutated CALB library was generated and subsequent library screening identified 63 unique functional variants, whose termini constitutes approximately 20% positions of the whole polypeptide chain. Particularly interesting from the catalysis standpoint are permutations in the enzyme's putative lid and in the long helix alpha10 which flanks the active site pocket. Selected library members were characterized by kinetic, circular dichroism, and enantioselectivity analysis. The data demonstrated that circular permutation could greatly improve the catalytic performance of CALB, while the variants' overall three-dimensional structure and high enantioselectivity are retained.;Secondly, incremental truncation was employed to increase the thermostability of the most active permutant cp283 (circularly permuted CALB whose N-terminus starts at amino acid 283 of the wild-type sequence). An incremental truncation library was generated by truncating an extended loop formed by the original termini and the linker. Library screening identified functional variants with up to 11 amino acid truncation. Characterization of selected library members showed that the variants maintain their high activity as the parental cp283, and have improved thermostability. Both biochemical analysis and crystallographic study verified that thermostabilization is conferred by protein dimerization. Crystal structure of the best variant cp283-Delta7 was solved, which revealed a domain-swapped dimer structure with the N-terminal segment (Ala283∼Val315) swapped between two subunits, which to our knowledge is the first example of a dimeric alpha/beta hydrolase. The crystal structure also provides explanations to experimental results obtained on other circularly permuted CALB variants.;Overall, the results from this dissertation have shown that circular permutation could greatly improve the catalytic efficiency of CALB, and circular permutation in combination with incremental truncation has yielded better CALB variants with high activity, high enantioselectivity and moderate thermostability, which would be advantageous for future applications in organic synthesis.