Enzyme Engineering Open Access

Yu, Ying (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/2r36tx76s?locale=en
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Abstract

Enzyme Engineering by Circular Permutation: Functional and Structural Investigation of Permutants
By Ying Yu

Enzyme engineering is an important tool to overcome the limitation of natural enzymes as biocatalysts. Although a number of significant achievements have been accomplished from evaluating large combinatorial libraries, more and more scientists and engineers have turned their attentions to small and high quality libraries. Circular permutation (CP) is a sequence rearrangement technique that has long been used for studying protein folding and sequence-function relationship. As an engineering tool, random CP does not require massive libraries due to its small theoretical size and thus provides opportunities for enzymes limited by the absence of high-throughput screening methods.

Previously, the engineering of Candida antarctica lipase B (CALB) by random CP yielded permutants with significant rate enhancements. In this dissertation, we extend the technique to three other industrially important enzymes including epoxide hydrolase (EchA), cutinase and xylanase (BcX). Screening of the Bcx library identified 35 unique functional variants with new termini distributed across the whole sequence and subsequent characterization revealed permutants with up to 4-fold increased activity. In contrast, CP of EchA and cutinase only yielded permutants highly similar to wild-type enzymes. Our attempts to address this problem by optimizing the linker using combinatorial approaches were not successful.

To follow up our previous studies on permuted CALB, we evaluated the performance of our best permutant cp283 with a series of esters, as well as pure and complex triglycerides. In comparison with the wild-type enzyme, cp283 showed consistently higher catalytic activity (2.6- to 9-fold) for transesterification and interesterification when using 1-butanol and ethyl acetate as acyl acceptors, indicating the potential application of our cpCALB in biodiesel production. Furthermore, in order to better understand the consequences resulted from CP, we investigated the function and structure of the new termini of cp283Δ7, a variant of cp283 with truncated linker. Although the17 residues at the C-terminus are not visible in the crystal structure, our results suggest that they are important for enzyme function and likely exist as a dynamic helical arrangement rather than being highly flexible and unstructured.

Table of Contents

Table of Contents
1 Introduction Enzyme Engineering and Circular Permutation...1

1.1 Achievements of enzyme engineering...1
1.2 Enzyme engineering methods...4
1.3 Circular permutation (CP) of proteins...5

1.3.1 Circular permutation in nature...5
1.3.2 Laboratory circular permutation...7
1.3.3 Linker design in circular permutation...11

1.4 Functional and structural consequences of circular permutation...12

1.4.1 Influence of circular permutation on protein function...12
1.4.2 Influence of circular permutation on protein structure...13

1.5 Applying circular permutation in enzyme design...15
1.6 Circular permutation of CALB...17

2 Engineering Biocatalysts by Circular Permutation...20

2.1 Introduction...20

2.1.1 Agrobacterium radiobacter epoxide hydrolase (EchA)...21
2.1.2 Fusarium solani cutinase...22
2.1.3 Bacillus circulans xylanase (Bcx)...24

2.2 Materials and methods...25

2.2.1 Materials...25
2.2.2 Random circular permutation of echA...26
2.2.3 Preparation of CP vector for echA...29
2.2.4 Library Construction and selection...29
2.2.5 Construction of rational designed EchA permutants...30
2.2.6 Construction of EchA library comprising varying linkers...30
2.2.7 Screening of EchA library comprising varying linkers...32
2.2.8 Random circular permutation of cutinase...32
2.2.9 Screening of CP cutinase library...33
2.2.10 Construction and screening of random linker cutinase library...35
2.2.11 Circular permutation of cutinase with random linker...35
2.2.12 Random circular permutation of BcX...36
2.2.13 Screening of cpBcx library...37

2.3 Results and discussions...38

2.3.1 Random circular permutation of EchA...38
2.3.2 Characterization of rational designed EchA permutants...40
2.3.3 Optimization of EchA linker with combinatorial approaches...43
2.3.4 Random circular permutation of cutinase...46
2.3.5 Optimization of cutinase linker with combinatorial approaches...48
2.3.6 CP of cutinase with random linkers...49
2.3.7 Random circular permutation of Bcx...50

2.4 Conclusions...52

3 Improved Triglyceride Transesterification by Circular Permuted Candida antarctica Lipase B...53

3.1 Introduction...53
3.2 Materials and methods...55

3.2.1 Materials...55
3.2.2 Kinetic analysis of lipases with chromogenic and fluorescence substrates...55
3.2.3 Kinetic analysis of transesterification reaction...56
3.2.4 Trans and interesterification of vegetable oil...56

3.3 Results and Discussions...57

3.3.1 Impact of acyl and alcohol moieties of substrates on the catalytic performance of cp283...57
3.3.2 Transesterification of triglyceride...60
3.3.3 Alcoholysis of complex triglyceride mixture and oils...62
3.3.4 Interesterification with ethyl acetate...64
3.3.5 Recycling of immobilized enzyme...65

3.4 Conclusions...65

4 Functional and Structural Investigation of the New Termini of Permuted CALB...67

4.1 Introduction...67
4.2 Materials and Methods...70

4.2.1 Materials...70
4.2.2 Construction of termini truncation and site-directed mutagenesis variants...72
4.2.3 Protein expression in P. pastoris...72
4.2.4 Protein purification by HIC and SE chromatography...72
4.2.5 Kinetic analysis of lipases activity with chromogenic and fluorescence substrates...73
4.2.6 Circular dichroism analysis...73
4.2.7 Peptide synthesis and purification...74
4.2.8 Cloning and purification of sortase A...75
4.2.9 Peptide labeling and sortase-mediated protein ligation...76
4.2.10 Labeling of Cys mutants...76

4.3 Results and Discussions...77

4.3.1 Functional consequence of termini deletion...77
4.3.2 CD characterization of termini deletion variants...79
4.3.3 Role of the two leucine residues in the invisible C-terminus...80
4.3.4 Labeling of new C-terminus by protein-peptide ligation...82
4.3.5 Secondary structure of C-terminal peptide...85
4.3.6 Characterization and labeling of Cys mutants...87

4.4 Conclusions...89

5 Conclusions and Perspectives...90

5.1 Conclusions...90
5.2 Perspectives...92

References...93

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