Abstract
α-Amylases (1,4-α-D-glucanohydrolases) are widely used in starch liquefaction, but their acid stability needs to be continuously explored to reduce the costs of raw material and operation. In this study, to better meet the industrial requirements, the acid stability of Bacillus licheniformis α-amylase (BLA) was further improved by directed evolution using error prone polymerase chain reaction (PCR). The mutant BLA (G81R) was selected with the improved acid stability based on a high-throughput activity assay. After incubating at pH 4.5 for 40 min, G81R still retained 10% of its initial activity, but the wild-type (WT) was already inactive. The kcat/Km value of G81R at pH 4.5 was 1.4-fold higher than that of WT. Combined with the three-dimensional structural modeling analysis, the improved stability of G81R under low pH condition might be due to the interactions of electrostatic, hydrophilicity, and helix propensity. Therefore, these findings would be beneficial for developing BLA with properties suitable for applications in industrial starch processing.
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Abbreviations
- BAA:
-
Bacillus amyloliquefaciens α-amylase
- BLA:
-
Bacillus licheniformis α-amylase
- CAZy:
-
Carbohydrate-Active enZymes α-amylase
- GSA:
-
Geobacillus stearothermophilus α-amylase
- GTA:
-
Geobacillus thermoleovorans α-amylase
- IPTG:
-
isopropyl β-D-1-thiogalactopyranoside
- LB:
-
Luria-Bertani
- PCR:
-
polymerase chain reaction
- PFA:
-
Pyrococcus furiosus α-amylase
- SDS-PAGE:
-
sodiumdodecyl sulfate-polyacrylamide gel electrophoresis α-amylase
- THA:
-
Thermococcus hydrothermalis α-amylase
- WT:
-
wild-type
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Acknowledgments
This work was supported by the National Key R&D Program of China (2017YFD0201405-04); the China Postdoctoral Science Foundation (2018 M641660); the Tianjin Natural Science Fund of China (17JCYBJC23700); and the Public Service Platform Program of Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process Control (17PTGCCX00190).
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Huang, L., Shan, M., Ma, J. et al. Directed evolution of α-amylase from Bacillus licheniformis to enhance its acid-stable performance. Biologia 74, 1363–1372 (2019). https://doi.org/10.2478/s11756-019-00262-7
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DOI: https://doi.org/10.2478/s11756-019-00262-7