title: Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling.
abstract: 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 degrees C, which was 10 degrees 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 degrees 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 degrees C. The melting temperatures of III-1 and III-2, as determined by differential scanning calorimetry, increased by 6.1 degrees C and 11.4 degrees 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 with the mutations.
authors.affiliation: Center for Agricultural Biomaterials, and Department of Food Science and Biotechnology, School of Agricultural Biotechnology, Seoul National University, South Korea.
authors.forename: Shuang-Yan
authors.initials: SY
authors.lastname: Tang
authors.forename: Quang-Tri
authors.initials: QT
authors.lastname: Le
authors.forename: Jae-Hoon
authors.initials: JH
authors.lastname: Shim
authors.forename: Sung-Jae
authors.initials: SJ
authors.lastname: Yang
authors.forename: Joong-Huck
authors.initials: JH
authors.lastname: Auh
authors.forename: Cheonseok
authors.initials: C
authors.lastname: Park
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D032621:DNA Shuffling; D004269:DNA, Bacterial / Q000235:genetics*; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006207:Half-Life; D006358:Hot Temperature; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011995:Recombination, Genetic; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase; D002118:Calcium