Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance.
The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75 degrees C, which was 15 degrees C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9 degrees C. The half-life of ThMA-DM was 172 min at 80 degrees C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.
Kim
Young-Wan
YW
National Laboratory for Functional Food Carbohydrates, Center for Agricultural Biomaterials, and Department of Food Science and Technology, Seoul National University, Suwon 441-744, Korea.
Choi
Ji-Hye
JH
Kim
Jung-Wan
JW
Park
Cheonseok
C
Kim
Jung-Woo
JW
Cha
Hyunju
H
Lee
Soo-Bok
SB
Oh
Byoung-Ha
BH
Moon
Tae-Wha
TW
Park
Kwan-Hwa
KH
D001426:Bacterial Proteins / Q000737:chemistry*; D005075:Biological Evolution; D002384:Catalysis; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D009154:Mutation; D011487:Protein Conformation; D013824:Thermus / Q000201:enzymology*
D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase