Journal of the science of food and agriculture · 2026
Rational engineering of ribitol dehydrogenase for enhanced thermostability and efficient biocatalytic production of allitol.
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Shang Tianwen, Yang Jiangang, Men Yan, Luo Xuegang, Zhu Yueming, Sun Yuanxia
Аннотация
BACKGROUND: Allitol is a rare sugar alcohol with potential as a low-calorie sweetener and food ingredient; however, its biocatalytic production from d-allulose is limited by the thermostability and cofactor efficiency of the ribitol dehydrogenases (RDH).
RESULTS: In this study, the RDH from Klebsiella oxytoca was engineered for enhanced thermostability using consensus-guided mutagenesis targeting non-conserved, non-active-site residues, followed by stepwise combination. The resulting M4 mutant showed markedly improved thermostability, with its melting temperature (Tm) elevated by 19.6 °C relative to the wild type, accompanied by a 10 °C increase in the optimum reaction temperature and a substantially broader pH activity range. Molecular dynamics simulations indicated that the four substitutions clustered at subunit interfaces and reinforced hydrophobic packing, stabilizing the tetrameric assembly and contributing to enhanced thermostability. An enzyme complex combining M4 with formate dehydrogenase (FDH) was constructed to enable efficient cofactor regeneration. This increased the conversion by 28% compared with the free enzyme combination and achieved nearly complete substrate conversion under optimized conditions.
CONCLUSION: This study significantly improved the thermostability of RDH using consensus-guided interfacial design and constructed a cofactor-regenerating enzyme complex for the efficient biocatalytic production of allitol. © 2026 Society of Chemical Industry.
RESULTS: In this study, the RDH from Klebsiella oxytoca was engineered for enhanced thermostability using consensus-guided mutagenesis targeting non-conserved, non-active-site residues, followed by stepwise combination. The resulting M4 mutant showed markedly improved thermostability, with its melting temperature (Tm) elevated by 19.6 °C relative to the wild type, accompanied by a 10 °C increase in the optimum reaction temperature and a substantially broader pH activity range. Molecular dynamics simulations indicated that the four substitutions clustered at subunit interfaces and reinforced hydrophobic packing, stabilizing the tetrameric assembly and contributing to enhanced thermostability. An enzyme complex combining M4 with formate dehydrogenase (FDH) was constructed to enable efficient cofactor regeneration. This increased the conversion by 28% compared with the free enzyme combination and achieved nearly complete substrate conversion under optimized conditions.
CONCLUSION: This study significantly improved the thermostability of RDH using consensus-guided interfacial design and constructed a cofactor-regenerating enzyme complex for the efficient biocatalytic production of allitol. © 2026 Society of Chemical Industry.