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Simultaneously improving the specific activity and thermostability of α-amylase BLA by rational design

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Abstract

Higher activity and alkaline α-amylases are desired for textile desizing and detergent additive. Here, rational design was used to improve the specific activity and thermostability of the α-amylase BLA from Bacillus licheniformis. Seventeen mutants of BLA were designed based on sequence consensus analysis and folding free energy calculation, and characterized by measuring their respective activity and thermostability at pH 8.5. Among them, mutant Q360C exhibited nearly threefold improved activity than that of wild-type and retained a higher residual activity (75% vs 59% for wild-type) after preincubation at 70 ℃ for 30 min. The modeled structures and molecular dynamics simulations analysis demonstrated that the enhanced hydrophobic interaction near residue 360 and reduced disturbance to the conformation of catalytic residues are the possible reasons for the improved thermostability and activity of Q360C. The results suggest that 360th of BLA may act as a hotspot for engineering other enzymes in the GH13 superfamily.

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Abbreviations

PCR:

Polymerase chain reaction

IPTG:

Isopropyl-β-d-thiogalactopyranoside

OD:

Optical density

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Acknowledgements

This work was supported by National Key R and D Program of China (NO.2018YFA0901100), National Natural Science Foundation of China (31970059) and Fundamental Research Funds for the Central Universities (buctrc202131).

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Authors and Affiliations

  1. College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China

    Xin Cui & Guimin Zhang

  2. State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, 430062, Hubei, China

    Xin Cui, Xin Yuan, Shunyi Li, Xinlin Hu, Jing Zhao & Guimin Zhang

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Correspondence to Jing Zhao or Guimin Zhang.

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Cui, X., Yuan, X., Li, S. et al. Simultaneously improving the specific activity and thermostability of α-amylase BLA by rational design. Bioprocess Biosyst Eng 45, 1839–1848 (2022). https://doi.org/10.1007/s00449-022-02790-0

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