D-mannose isomerase can catalyze the mutual conversion between D-fructose and D-mannose, which has application prospects in the biological enzymatic preparation of D-mannose. However, most of the reported D-mannose isomerases have poor thermal stability that affects its application in industry. In this study, D-mannose isomerase from Escherichia coli Dh5α was heterologously expressed and its enzymatic properties were studied. Then, its thermostability was modified. The results indicated that the optimum reaction temperature of D-mannose isomerase was 30 ℃, the optimum reaction pH was 6.0-7.0, Km, kcat and kcat/Km were 963.4 mmol/L, 1.38 s-1, and 1.4×10-3 L/(mmol·s), respectively. Through rational modification, a mutant A241P/A116V/G253A/Q379P with significantly improved thermal stability was obtained. Its optimum reaction temperature was increased by 15 ℃. At 50 ℃, its half-life was 33 times that of the wild type, and its relative enzyme activity was 60% higher than that of the wild type. Molecular dynamics simulation analysis showed that the introduction of proline and the enhancement of hydrophobic improve the rigidity of the protein, thus improving the thermal stability. In this study, the enzymatic properties and thermal stability of D-mannose isomerase from mainstream sources were investigated, which provides a theoretical basis for the efficient preparation of D-mannose by bio-enzyme method. It also lays a foundation for the study of the structure-stability of D-mannose isomerase.
SHEN Ling
,
ZHAO Liting
,
SHEN Yu
,
CHEN Lei
,
LI Junxun
,
GU Zhenghua
,
LI Youran
,
SHI Guiyang
,
DING Zhongyang
. Characterization and thermostability modification of D-mannose isomerase[J]. Food and Fermentation Industries, 2023
, 49(24)
: 103
-110
.
DOI: 10.13995/j.cnki.11-1802/ts.035239
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