Insights into the thermostability and product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04
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Analyze the thermostability, mode of action, and product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) from the biochemical and structural point of view.
Using three-dimensional co-crystal structure of Bst-MFA with acarbose as a guide, experiments were performed to analyze the thermostability, mode of action and product specificity of Bst-MFA. The results showed that the Ca2+–Ca2+–Ca2+ metal triad of Bst-MFA is responsible for its high thermostability. Multiple substrate binding modes, rather than one productive binding mode determined by non-reducing end recognition, are in accordance with an endo-type mode of action. Significant interactions between subsites − 5 and − 6 and glucosyl residues at the non-reducing end explain the maltopentaose (G5) and maltohexaose (G6) specificity of Bst-MFA.
Bst-MFA is a thermostable enzyme that preferentially produces G5 and G6, with an endo-type mode. The understanding of structure–function relationships provides the foundation for future efforts to the modification of Bst-MFA.
KeywordsAction mode Maltooligosaccharide-forming amylase Product specificity Structure Thermostability
This work was financially supported by the National Natural Science Foundation of China (No. 31722040, 31571882), the China Postdoctoral Science Foundation (No. 2018M632233), the Natural Science Foundation of Jiangsu Province (BK20180606), and the Jiangsu province “Collaborative Innovation Center of Food Safety and Quality Control” industry development program.
Experimental procedures, including construction of mutant and the effect of EDTA on the thermostability of Bst-MFA; Supplementary Table 1—optimal temperature of Bst-MFA and other MFAses; Supplementary Fig. 1—effect of EDTA on the thermostability of Bst-MFA; Supplementary Fig. 2—thermostability of mutant ΔIG181-182 at 80 °C.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.