Molecular Characterization of a Mesophilic Cellobiose 2-Epimerase That Maintains a High Catalytic Efficiency at Low Temperatures

Chen, Qiuming; Wu, Yue; Huang, Zhaolin; Zhang, Wenli; Mu, Wanmeng

doi:10.1021/acs.jafc.1c02025

Cited by 6 publications

(1 citation statement)

References 42 publications

(63 reference statements)

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“…The free energy could be divided into enthalpic and entropic components. Since our previous study showed that the entropy term did not significantly affect the prediction accuracy on the relative binding energies of ligands to CEs, 34 we ranked the binding affinities by comparing the enthalpic energy instead of absolute energy. The entropy term was not considered in this study.…”

Section: Enzyme Assay and Analyticalmentioning

confidence: 99%

Rational Design of Loop Dynamics for a Barrel-Shaped Enzyme by Introducing Disulfide Bonds

Chen,

Wu,

et al. 2024

J. Agric. Food Chem.

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Loop dynamics redesign is an important strategy to manipulate protein function. Cellobiose 2-epimerase (CE) and other members of its superfamily are widely used for diverse industrial applications. The structural feature of the loops connecting barrel helices contributes greatly to the differences in their functional characteristics. Inspired by the in-silico mutation with molecular dynamics (MD) simulation analysis, we propose a strategy for identifying disulfide bond mutation candidates based on the prediction of protein flexibility and residue−residue interaction. The most beneficial mutant with the newly introduced disulfide bond would simultaneously improve both its thermostability and its reaction propensity to the targeting isomerization product. The ratio of the isomerization/epimerization catalytic rate was improved from 4:103 to 9:22. MD simulation and binding free energy calculations were applied to provide insights into molecular recognition upon mutations. The comparative analysis of enzyme/ substrate binding modes indicates that the altered catalytic reaction pathway is due to less efficient binding of the native product.The key residue responsible for the observed phenotype was identified by energy decomposition and was further confirmed by the mutation experiment. The rational design of the key loop region might be a promising strategy to alter the catalytic behavior of all (α/α)6-barrel-like proteins.

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Section: Enzyme Assay and Analyticalmentioning

confidence: 99%

Rational Design of Loop Dynamics for a Barrel-Shaped Enzyme by Introducing Disulfide Bonds

Chen,

Wu,

et al. 2024

J. Agric. Food Chem.

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A novel cellobiose 2‐epimerase from anaerobic halophilic Iocasia fonsfrigidae and its ability to convert lactose in fresh goat milk into epilactose

Eat,

Wulansari,

Ketbot

et al. 2024

J Sci Food Agric

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BACKGROUNDCellobiose 2‐epimerase (CE) has received great attention due to its potential applications in the food and pharmaceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3‐1 (IfCE) was successfully expressed in Escherichia coli and characterized.RESULTSUnlike other CEs, the purified IfCE shows only epimerization activity toward β‐1,4‐glycosidic linkages of disaccharides, including mannobiose, cellobiose and lactose, but not for monosaccharides, β‐1,4‐glycosidic linkages of trisaccharides and α‐1,4‐glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low‐calorie prebiotic sweetener with medicinal and pharmacological properties, was obtained from 10 mg mL−1 lactose. IfCE was highly active against lactose under NaCl concentrations up to 500 mmol L−1, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues may protect the enzyme from Cl− and Na+ ions from the environment, respectively. Under normal conditions, IfCE was able to convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been investigated as a safe enzyme for human allergen.CONCLUSIONThe results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy products by converting lactose that causes digestive problems in people with lactose intolerance into epilactose. © 2024 Society of Chemical Industry.

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Computer-aided mining of a psychrophilic cellobiose 2-epimerase from the Qinghai-Tibet Plateau gene catalogue

Xu,

Ji,

et al. 2024

International Journal of Biological Macromolecules

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Molecular Characterization of a Mesophilic Cellobiose 2-Epimerase That Maintains a High Catalytic Efficiency at Low Temperatures

Cited by 6 publications

References 42 publications

Rational Design of Loop Dynamics for a Barrel-Shaped Enzyme by Introducing Disulfide Bonds

Rational Design of Loop Dynamics for a Barrel-Shaped Enzyme by Introducing Disulfide Bonds

A novel cellobiose 2‐epimerase from anaerobic halophilic Iocasia fonsfrigidae and its ability to convert lactose in fresh goat milk into epilactose

Computer-aided mining of a psychrophilic cellobiose 2-epimerase from the Qinghai-Tibet Plateau gene catalogue

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