Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution = 분자진화를 이용하여 TPL 활성과 안정성 동시 개량

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Title
Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution = 분자진화를 이용하여 TPL 활성과 안정성 동시 개량
Author(s)
Eugene Rha; Su Jin Kim; Su Lim Choi; S P Hong; M H Sung; Jae Jun SongSeung Goo Lee
Bibliographic Citation
FEBS Journal, vol. 276, no. 21, pp. 6187-6194
Publication Year
2009
Abstract
The tyrosine phenol-lyase from Symbiobacterium toebii was engineered to improve both its stability and catalytic activity by the application of random mutagenesis and subsequent reassembly of the acquired mutations. Activity screening of the random library produced four mutants with a two-fold improved activity, whereas parallel screening after heat treatment at 65 °C identified three mutants with half-inactivation temperatures improved by up to 5.6 °C. The selected mutants were then reassembled using the staggered extension PCR method, and subsequent screening of the library produced seven mutants with up to three-fold improved activity and half-inactivation temperatures improved by up to 11.2 °C. Sequence analyses revealed that the stability-improved hits included A13V, E83K and T407A mutations, whereas the activity-improved hits included the additional T129I or T451A mutation. In particular, the A13V mutation was propagated in the hits with improved stability during the reassembly-screening process, indicating the critical nature of the N-terminal moiety for enzyme stability. Furthermore, homology modeling of the enzyme structure revealed that most of the stability mutations were located around the dimer-dimer interface, including the N-terminus, whereas the activity-improving mutations were located further away, thereby minimizing any interference that would be detrimental to the co-improvement of the stability and catalytic activity of the enzyme.
Keyword
N-terminal armProtein engineeringStructural relevanceSymbiobacterium toebiiTyrosine phenol-lyase
ISSN
1742-464X
Publisher
Wiley
DOI
http://dx.doi.org/10.1111/j.1742-4658.2009.07322.x
Type
Article
Appears in Collections:
Synthetic Biology and Bioengineering Research Institute > Synthetic Biology Research Center > 1. Journal Articles
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
Synthetic Biology and Bioengineering Research Institute > 1. Journal Articles
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