Kinetic Effects of Circular Permutation and Lid Swapping Techniques on Candida antarctica Lipase B 公开

Unvala, Cydney Cade (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/ng451j17c?locale=zh
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Abstract

Candida antarctica lipase B (CALB) is an enzyme of the α/β-hydrolase-fold family known to catalyze a wide variety of reactions including hydrolysis, esterification, amination, transesterification, and polymerization. This catalytic versatility, along with broad substrate specificity and enantioselective properties, make CALB an attractive target for a number of industrial applications particularly in detergents, dairy processing, and pharmaceutical synthesis. Its high industrial potential has prompted increased interest in methods of enhancing CALB's versatile functions. One means of accomplishing this has been through lid swapping. Lid swapping is a protein engineering technique that identifies the lid region on one protein and replaces it with the corresponding lid region from a homologous protein in order to alter access to the protein's active site. This technique has far-reaching implications including heightened catalytic activity and improved enantioselectivity. Similar enhancements have been established through circular permutation, an alternative technique that relocates the N- and C-terminus within a protein, preserving the protein's structure while changing its local flexibility and access to its active site. I hypothesize that performing both techniques on CALB conjointly will synergistically improve its activity and enantioselective preferences without significantly compromising its structural integrity. Using the circularly permuted variant cp283Δ7 (constructed by the Lutz group) as a base, the lid region was replaced with the lid of the CALB homologue from Gibberella zeae. The resulting variant, cp283Δ7-GZ, has exhibited a substantial 4-fold increase in hydrolytic activity with p-nitrophenol butyrate in comparison to wild-type CALB. However, the variant's activity compared to its base structure, cp283Δ7, shows decreased activity. Nevertheless, results suggest that CALB responds reasonably well to the structural alterations introduced by both techniques and encourage further investigation of their effects on the structural stability and enantioselective properties of CALB.

Table of Contents

Table of Contents

1. Introduction ........................................................................................................................... 1
a. Lipases .............................................................................................................................. 1
b. Candida antarctica Lipase B .................................................................................................. 5
c. Protein Engineering .............................................................................................................. 5
2. Materials and Methods ............................................................................................................. 11
a. Materials ............................................................................................................................ 11
b. Protein Engineering ............................................................................................................. 11
c. Protein Expression ............................................................................................................... 11
d. Protein Purification .............................................................................................................. 12
e. Activity Assay ..................................................................................................................... 13
f. Protein Immobilization .......................................................................................................... 13
g. Active-Site Titration of Immobilized Lipase .............................................................................. 14
3. Results .................................................................................................................................. 16
a. Protein Expression ............................................................................................................... 16
b. Protein Purification and Concentration .................................................................................... 16
c. Activity Assay ...................................................................................................................... 17
d. Protein Immobilization .......................................................................................................... 18
e. Active-Site Titration .............................................................................................................. 20
4. Discussion ............................................................................................................................... 21
5. References .............................................................................................................................. 23

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