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KMID : 0545120040140010153
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 1 p.153 ~ p.157
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Enhanced Stability of Tyrosine Phenol-Lyase from Symbiobacterium toebii by DNA Shuffling
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KIM, JIN-HO
SONG, JAE JUN/KIM, BONG-GYUN/SUNG, MOON-HEE/LEE, SANG CHUL
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Abstract
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Tyrosine phenol-lyase(TPL) is a useful enzyme for the synthesis of pharmaceutical aromatic amino acids. In the current study, sequential DNA shuffling and screening were used to enhance the stability of TPL. Twenty-thousand mutants were screened, and several improved variants were isolated. one variant named A14V, in which the 13th amino acid alanine was substituted by valin, exhibited a higher temperature and denaturant stability than the wild-type TPL. The purified mulant TPL, A13V, retained about 60% of its activity at 76℃, whereas the activity of the wild-type TPL decreased to less than 20% at the same temperature. Plus, A13V exhibited about 50% activity with 3M urea, while the wild-type TPL lost almost all its catalytic activity, indicating an increased denaturant tolerance in the mutant A13V. It is speculated that the substitution of Val for the Ala in the β-strand of the N-terminal arm was responsible for the heightened stabilization, and that the current results will contribute to further research on the structural stability of TPL.
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KEYWORD
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