TAILIEUCHUNG - Báo cáo khoa học: Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling

DNA shuffling was used to improve the thermostability of maltogenic amylase from Bacillus thermoalkalophilusET2. Two highly thermostable mutants, III-1 and III-2, were generated after three rounds of shuffling and recombination of mutations. Their optimal reaction temperatures were all 80 C, which was 10 C higher than that of the wild-type. | ềFEBS Journal Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling Shuang-Yan Tang1 Quang-Tri Le1 Jae-Hoon Shim1 Sung-Jae Yang1 Joong-Huck Auh1 Cheonseok Park2 and Kwan-Hwa Park1 1 Center for AgriculturalBiomaterials and Department of Food Science and Biotechnology Schoolof AgriculturalBiotechnology Seoul National university South Korea 2 Department of Food Science and Biotechnology and Institute of Life Sciences and Resources Kyunghee University Yongin South Korea Keywords DNA shuffling maltogenic amylase MAase neopullulanase site-directed mutagenesis thermostability Correspondence . Park Department of Food Science and Biotechnology SeoulNationalUniversity Shillim-dong Kwanak-gu Seoul151-742 Korea Fax 82 2 8735095 Tel 82 2 8804854 8804852 E-mail parkkh@ Received 28 March 2006 revised 22 May 2006 accepted 24 May 2006 doi DNA shuffling was used to improve the thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2. Two highly thermostable mutants III-1 and III-2 were generated after three rounds of shuffling and recombination of mutations. Their optimal reaction temperatures were all 80 C which was 10 C higher than that of the wild-type. The mutant enzyme III-1 carried seven mutations N147D F195L N263S D311G A344V F397S and N508D. The half-life of III-1 was about 20 times greater than that of the wild-type at 78 C. The mutant enzyme III-2 carried M375T in addition to the mutations in III-1 which was responsible for the decrease in specific activity. The half-life of III-2 was 568 min while that of the wild-type was 1 min at 80 C. The melting temperatures of III-1 and III-2 as determined by differential scanning calorimetry increased by C and C respectively. Hydrogen bonding hydrophobic interaction electrostatic interaction proper packing and deamidation were predicted as the mechanisms for the enhancement of thermostability in the enzymes

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