TAILIEUCHUNG - Báo cáo khoa học: Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution

The tyrosine phenol-lyase fromSymbiobacterium toebiiwas engineered to improve both its stability and catalytic activity by the application of ran-dom 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 C. | Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution Eugene Rha1 Sujin Kim1 Su-Lim Choi1 Seung-Pyo Hong2 Moon-Hee Sung2 Jae J. Song3 and Seung-Goo Lee1 1 IndustrialBiotechnology Bioenergy Research Center KRIBB Daejeon South Korea 2 BioLeaders Corp. Daejeon South Korea 3 Molecular Bioprocess Research Center KRIBB JungUp South Korea Keywords N-terminalarm protein engineering structuralrelevance Symbiobacterium toebii tyrosine phenol-lyase Correspondence . Lee and J. J. Song 111 Gwahangno Yuseong Daejeon 305-806 South Korea Fax 82 42 860 4379 Tel 82 42 860 4373 E-mail sglee@ jjsong@ Received 17 April2009 revised 11 August 2009 accepted 24 August 2009 doi 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 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 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 reassemblyscreening 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 .

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