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Bioreactor Controls-III01:22

Bioreactor Controls-III

Strain improvement is a foundational strategy in industrial microbiology aimed at maximizing microbial productivity, particularly because natural isolates typically yield commercially valuable products in very low concentrations. Although optimizing the culture medium and environmental conditions can improve yields, these adjustments are inherently limited by the organism’s genetic potential. As a result, the focus shifts toward genetic modifications to enhance biosynthetic capacity. The...

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Engineering of β-Agarase with Enhanced Thermostability via Multitool Consensus Prediction and Structure-Guided Screening.

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Multienzyme Cascade Coimmobilization on ZIF-8-Coated Magnetic Nanoparticles for Efficient d-Allulose Synthesis.

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Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase Pedsa0632.

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Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues
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Interface-Targeted Rational Design Strategies for Enhancing the Thermostability of d-Allulose 3-Epimerase.

Kaifan Qiu1,2,3, Xingfei Li1,2,3, Yuxiang Bai1,2

  • 1The State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.

Journal of Agricultural and Food Chemistry
|July 14, 2026
PubMed
Summary

Engineers improved the heat stability of d-allulose 3-epimerase (DAEase) using a novel protein engineering strategy. This breakthrough enhances enzyme performance for industrial applications.

Keywords:
combinatorial mutagenesisd-allulose-3-epimeraseinterfacethermostability

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Area of Science:

  • Biochemistry
  • Protein Engineering
  • Enzyme Technology

Background:

  • d-Allulose 3-epimerase (DAEase) from *Clostridium cellulolyticum* H10 converts d-fructose to d-allulose.
  • Poor thermostability of native DAEase hinders its industrial use.

Purpose of the Study:

  • To enhance the thermostability of *Clostridium cellulolyticum* H10 DAEase.
  • To enable wider industrial applications of DAEase through improved heat resistance.

Main Methods:

  • Employed PROSS-guided combinatorial engineering and spatial clustering to target DAEase subunit interfaces.
  • Classified candidate mutations into interface core, adjacent, and distal regions.
  • Utilized stepwise iterative combination of mutations.

Main Results:

  • Developed two mutants, M5 and M6, with wild-type-like activity and significantly improved thermostability.
  • M5 and M6 showed increased Tm values (11.4 °C and 12.4 °C) and extended half-lives at 65 °C (3-fold and 12-fold).
  • Structural analysis revealed enhanced salt-bridge formation, monomer stabilization, and optimized assembly microenvironment.

Conclusions:

  • A spatially coordinated engineering strategy effectively enhances the thermostability of multimeric enzymes.
  • The developed mutants offer a promising solution for industrial d-allulose production.
  • This approach provides a blueprint for engineering other thermostable multimeric enzymes.