Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of T
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Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of TIM Barrel Cheng-Hua Wang 1 & Liang-Hua Lu 1 & Cheng Huang 1 & Bing-Fang He 2 & Ri-Bo Huang 3,4 Received: 17 September 2019 / Accepted: 12 March 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
This study reported simultaneously improved thermostability and hydrolytic pattern of αamylase 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. Keywords Alpha-amylase . (β/α)8 barrel . Hydrolytic pattern . Rational design . Thermostability
Introduction The TIM barrel (also called (β/α)8 barrel) is the most frequently occurring folding motif in proteins [1, 2] and is adopted by versatile enzymes in all the E. C. enzyme classes except ligase [3, 4]. The canonical TIM barrel fold consists of eight α-helices and eight parallel β strands that alternate along the peptide backbone [5]. The catalytic and substrate-binding residues
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003308-8) contains supplementary material, which is available to authorized users.
* Cheng-Hua Wang [email protected] Extended author information available on the last page of the article
Applied Biochemistry and Biotechnology
locate at the “catalytic face,” which includes the C-terminal ends of β strands and the loops that extend from these strands. While the stabilizing residues are of the “stability face,” that includes the hydrophobic core, N-terminal ends of the barrel, and the αβ-loops linking the αhelices with the subsequent β strands [5]. The “division of labor” between catalytic activity and stability and the “diversity in function but similar structure” properties have made the TIM barrel an ideal object for protein engineering [4, 6]. Although various factors contribute to the formation and stability of the TIM barrel fold [7, 8], such as packing of the β strand residues in the barrel core [7, 9], amino acid clustering patte
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