Engineering of an efficient and enantioselective biocatalyst for the preparation of chiral pharmaceutical intermediates

Tang, Weng Lin

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https://hdl.handle.net/2142/26297 Copy

Description

Title

Engineering of an efficient and enantioselective biocatalyst for the preparation of chiral pharmaceutical intermediates

Author(s)

Tang, Weng Lin

Issue Date

2011-08-26T15:21:54Z

Director of Research (if dissertation) or Advisor (if thesis)

Zhao, Huimin

Doctoral Committee Chair(s)

Zhao, Huimin

Committee Member(s)

• Chen, Shing Bor
• Li, Zhi
• Rao, Christopher V.
• Gunawan, Rudiyanto

Department of Study

Chemical & Biomolecular Engr

Discipline

Chemical Engineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Directed Evolution
• P450
• High throughput screening
• protein engineering

Abstract

This Ph.D. thesis focuses on the engineering of an efficient and enantioselective biocatalyst via direct evolution and genetic engineering for the enantioselective hydroxylation of non-activated carbon atom, a useful but challenging reaction for the synthesis of chiral pharmaceutical intermediates. Our target enzyme is the novel P450pyr enzyme from Sphingomonas sp. HXN-200 that was found to catalyze the regio- and stereoselective hydroxylation of non-activated carbon atom with broad substrate range, high activity, excellent regioselectivity, and good to excellent enantioselectivity. Our target reaction is the enzymatic hydroxylation of N-benzyl pyrrolidine to its corresponding (R)- and (S)-N-benzyl-3-hydroxypyrrolidines which are important pharmaceutical intermediates. In this thesis, a two-enzyme-based colorimetric high-throughput ee screening assay and a mass spectrometry-based high-throughput ee screening assay were developed. The P450pyr monooxygenase was engineered by directed evolution for the enantioselective hydroxylation of N-benzyl pyrrolidine. Several mutants exhibiting increased and/or inverted enantioselectivity were identified, with product ee of 83% (R) and 65% (S) for mutants 1AF4A and 11BB12, respectively. The wild type P450pyr and its mutants were also purified and reconstituted with their auxiliary electron transport proteins, ferredoxin and ferredoxin reductase in vitro. The mutants were then used to catalyze the hydroxylations of a range of different substrates using whole-cell assays to investigate the changes in product ee. In addition, an efficient biocatalytic system with cofactor recycling was developed by co-expressing a glucose dehydrogenase from Bacillus substilis or a phosphite dehydrogenase from Pseudomonas stutzeri together with the P450pyr system in a recombinant Escherichia coli.  

Graduation Semester

2011-08

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http://hdl.handle.net/2142/26297

Copyright and License Information

Copyright 2011 Weng Lin Tang

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