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  • Title: Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of TIM Barrel.
    Author: Wang CH, Lu LH, Huang C, He BF, Huang RB.
    Journal: Appl Biochem Biotechnol; 2020 Sep; 192(1):57-70. PubMed ID: 32219624.
    Abstract:
    This study reported simultaneously improved thermostability and hydrolytic pattern of α-amylase from Bacillus subtilis CN7 by rationally engineering the mostly conserved central beta strands in TIM barrel fold. Nine single point mutations and a double mutation were introduced at the 2nd site of the β7 strand and 3rd site of the β5 strand to rationalize the weak interactions in the beta strands of the TIM barrel of α-amylase. All the five active mutants changed the compositions and percentages of maltooligosaccharides in final hydrolytic products compared to the product spectrum of the wild-type. A mutant Y204V produced only maltose, maltotriose, and maltopentaose without any glucose and maltotetraose, indicating a conversion from typical endo-amylase to novel maltooligosaccharide-producing amylase. A mutant V260I enhanced the thermal stability by 7.1 °C. To our best knowledge, this is the first report on the simultaneous improvement of thermostability and hydrolytic pattern of α-amylase by engineering central beta strands of TIM barrel and the novel "beta strands" strategy proposed here may be useful for the protein engineering of other TIM barrel proteins.
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