Applied Biochemistry and Biotechnology, Vol.192, No.1, 57-70, 2020
Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of TIM Barrel
This study reported simultaneously improved thermostability and hydrolytic pattern of alpha-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 beta 7 strand and 3rd site of the beta 5 strand to rationalize the weak interactions in the beta strands of the TIM barrel of alpha-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 degrees C. To our best knowledge, this is the first report on the simultaneous improvement of thermostability and hydrolytic pattern of alpha-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.