Ancestral Sequence Reconstruction Enhances Thermal Stability of D-Allulose 3-Epimerase

GUAN Lijun; ZHU Ling; WANG Kunlun; LI Jialei; GAO Yang; YAN Song; ZHANG Xindi; CHEN Qing; JI Nina; LI Bo

doi:10.13386/j.issn1002-0306.2024010227

Volume 45 Issue 21

Oct. 2024

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Science and Technology of Food Industry > 2024 > 45(21): 121-128. > DOI: 10.13386/j.issn1002-0306.2024010227

GUAN Lijun, ZHU Ling, WANG Kunlun, et al. Ancestral Sequence Reconstruction Enhances Thermal Stability of D-Allulose 3-Epimerase[J]. Science and Technology of Food Industry, 2024, 45(21): 121−128. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2024010227.

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Ancestral Sequence Reconstruction Enhances Thermal Stability of D-Allulose 3-Epimerase

GUAN Lijun^{1, 2,},
ZHU Ling^{1, 2},
WANG Kunlun^{1, 2},
LI Jialei^{1, 2},
GAO Yang^{1, 2},
YAN Song^{1, 2},
ZHANG Xindi^{1, 2},
CHEN Qing^{1, 2},
JI Nina³,
LI Bo^{1, 2, ,}

1.
Food Processing Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
2.
Heilongjiang Province Key Laboratory of Food Processing, Harbin 150086, China
3.
Soybean Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China

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Received Date: January 23, 2024
Available Online: August 23, 2024

Graphical Abstract

Abstract

Abstract

To solve the problem of poor thermal stability of the current D-allulose 3-epimerase (DAEase), the ancestor sequences of DAEase with different catalytic domains were reconstructed by big data mining, reasonable modification and ancestor sequence reconstruction (ASR) strategy under the guidance of phylogenetic information. The expression vectors of the ancestor sequences were constructed, and DAEase A13 with significantly enhanced thermal stability was screened by recombinant expression and molecular docking, and its enzymatic properties were characterized. In addition, the molecular mechanism of thermal stability enhancement of DAEase A13 was revealed based on structural analysis and molecular dynamics. The results showed that the half-life of A13 constructed based on ASR strategy could reach 8.4 h at 70 ℃, indicating that its thermal stability was significantly enhanced compared with that of wild-type (WT) enzyme. The maximum conversion rate of A13 reached 31%, indicating that the catalytic activity of A13 was slightly higher than that of WT enzyme. The structural and molecular dynamics analysis revealed that the increase in hydrogen bonding and hydrophobic interaction in ASR A13 was the main factor responsible for maintaining the stability of the enzyme's molecular structure at high temperatures. The results showed that ASR strategy could modify DAEases to enhance the stability, activity or hybridity, which could provide superior biocatalyst sources for various industrial applications of functional sugars.
- ancestral sequence reconstruction,
- D-allulose,
- D-allulose 3-epimerase,
- thermostability

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References

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