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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

Authors 

You Jin NaSu Jin KimSu Lim ChoiS P HongM H SungJae Jun SongSeung Goo Lee

Publisher 

Wiley-Blackwell

Issue Date 

2009

Citation 

FEBS Journal, vol. 276, no. 21, pp. 6187-6194

Keywords 

N-terminal armProtein engineeringStructural relevanceSymbiobacterium toebiiTyrosine phenol-lyase

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.

ISSN 

1742-464X

Link 

http://dx.doi.org/10.1111/j.1742-4658.2009.07322.x

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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