University of Illinois Urbana-Champaign

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

Tang, Weng Lin

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Tang_WengLin.pdf

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

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
Date of Ingest
2011-08-26T15:21:54Z
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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