Engineering Candida antarctica lipase B by circular permutation and incremental truncation Open Access

Qian, Zhen (2007)

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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 α10 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-Δ7 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 α/β 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.

Table of Contents

Chapter 1 Introduction 1 1.1 A general introduction to lipases 1 1.1.1 Reactions catalyzed by lipases 1 1.1.2 Applications of lipases 1 1.1.3 The fold of lipases 3 1.2 Candida antarctica lipase B 4 1.2.1 Structural features and catalytic mechanism of CALB 4 1.2.2 Thermostability, substrate specificity, and enantioselectivity of CALB 11 1.2.3 Overexpression of CALB 14 1.2.4 CALB engineering 15 1.3 Circular permutation of proteins 19 1.3.1 Naturally occurring circular permutations in proteins 19 Mechanisms of circular permutation in nature 19 Functional importance of circular permutation in nature 19 Circularly permuted α/β hydrolase-fold protein in nature 22 1.3.2 Circular permutation as a genetic engineering approach 25 Rational design of circularly permuted proteins 25 Random circular permutation 27 Chapter 2 Improving the catalytic activity of Candida antarctica lipase B by circular permutation 30 2.1 Introduction 30 2.2 Materials and methods 31 2.2.1 Construction of wt-CALB expression vector 31 2.2.2 Random circulation permutation of CALB gene 32 2.2.3 Creation of the pPIC9-cp-CALB library 33 Direct library cloning 33 Two-step library cloning 35 2.2.4 Creation of the (His)6-tag free pPIC9-cp-CALB library 37 2.2.5 Library screening 38 2.2.6 Protein expression, purification, and activity assays 40 2.3 Results and discussions 40 2.3.1 Creation of the cp-CALB library 40 2.3.2 Library screening and analysis 41 2.3.3 Selected library members for kinetic characterization 45 2.4 Conclusions 48 Chapter 3 Characterization of circularly permutated CALB variants 49 3.1 Introduction 49 3.2 Materials and methods 50 3.2.1 Construction of His-free protein expression vector 50 3.2.2 Protein expression, purification, and activity assays 52 3.2.3 Circular dichroism analysis 52 3.2.4 P. pastoris fermentation 52 3.2.5 Protein immobilization 52 3.2.6 Active site titration of immobilized lipase 53 3.2.7 Kinetic analysis of lipase catalyzed esterification reactions 53 3.3 Results and discussions 55 3.3.1 Kinetic analysis of (His)6-tag and C-terminal extension free circularly permuted CALB variants 59 3.3.2 Circular dichroism analysis of selected variants 62 3.3.3 Enantioselectivity comparison of cp283 with wt-CALB 64 3.4 Conclusions 67 Chapter 4 Secondary engineering and thermostabilization of circularly permuted CALB 68 4.1 Introduction 68 4.2 Materials and methods 71 4.2.1 Creation of the cp283 incremental truncation library 71 4.2.2 Creation of the C-terminal incremental truncation library 72 4.2.3 Protein expression, purification, and activity assays 73 4.2.4 Circular dichroism analysis and gel filtration 73 4.3 Results and Discussions 74 4.3.1 Creation of the incremental truncation libraries 74 Creation of the cp283 incremental truncation library 74 Analysis of the cp283 incremental truncation library 76 Creation and analysis of the C-terminal incremental truncation library 79 4.3.2 Characterization of selected variants 80 Kinetic analysis on selected variants 83 Circular dichroism analysis on selected variants 83 Gel filtration analysis on selected variants 84 4.3.3 Stabilization mechanism of the cp283 truncation variants 89 4.4 Conclusions 91 Chapter 5 The crystal structure of cp283-Δ7: circular permutation and domain swapping 92 5.1 Introduction 92 5.2 Materials and methods 96 5.2.1 Crystallization of cp283-Δ7 96 cp283-Δ7 expression and purification 96 cp283-Δ7 crystallization 96 Crystal data collection 97 5.2.2 Construction and characterization of the truncation variants 99 Construction of the truncation variants 99 Protein expression, purification and activity assays 99 Circular dichroism analysis of the variants 99 5.3 Results and discussions 100 5.3.1 The crystal structure of cp283-Δ7 100 Crystal structure of cp283-Δ7 100 Energetic contributions to domain swapping and thermostabilization 102 Comparison of structures of wt-CALB with cp283-∆7 104 Explanations for experimental results on circularly permuted variants 104 5.3.2 Truncation study on cp283-Δ7 and cp283 107 Creation of the truncation variants 107 Kinetic analysis of the truncation variants 109 Circular dichroism analysis of the truncation variants 109 5.4 Conclusions 112 Chapter 6 Conclusions and perspectives 113 Appendices 116 A Materials and media 116 A1 Chemicals 116 A2 Enzymes 117 A3 Strains and plasmids 118 A4 Buffers and Media 118 B General Methods 119 B1 Protein expression in P. pastoris 119 B2 Protein purification by Ni-NTA column 119 B3 Protein purification by HIC and gel filtration 120 B4 Activity assays 121 B5 P. pastoris fermentation 121 B6 Circular dichroism analysis 123 References 124

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