D-allulose-3-epimerase mutants, host cells and their use in the synthesis of allulose

By performing multi-point mutations on D-allulose-3-epimerase, a mutant with high activity and high stability was constructed, which solved the problem of insufficient catalytic activity and stability of the existing enzyme, met the needs of industrial production, and improved the conversion rate and enzyme lifespan.

CN121065159BActive Publication Date: 2026-07-10BINZHOU SANYUAN BIOLOGICAL TECH
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
BINZHOU SANYUAN BIOLOGICAL TECH
Filing Date
2025-08-05
Publication Date
2026-07-10

AI Technical Summary

Technical Problem

Existing D-allulose-3-epimerases have limited catalytic activity, low conversion efficiency, and poor stability, making them unsuitable for the high-temperature, high-sugar, and high-ionic-strength environments of large-scale industrial production.

Method used

By performing multiple point mutations at sites such as F47A, N72E, N114G, and C221S on the wild-type D-allulose-3-epimerase derived from Ruminococcus CAG55, a highly active and stable D-allulose-3-epimerase mutant was constructed and expressed and applied in Escherichia coli and Bacillus subtilis.

Benefits of technology

It significantly improved the catalytic activity and stability of D-allulose-3-epimerase, with a conversion rate of 36.3% when the host bacterium was Escherichia coli and 35.5% when the host bacterium was Bacillus subtilis. Furthermore, the enzyme activity could be maintained at 80% even after 30 repeated uses after immobilization.

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Abstract

The application discloses D-allulose-3-epimerase mutants, host cells and application thereof in synthesis of allulose, and belongs to the technical field of genetic engineering. Ruminococcus sp. A series of D-allulose-3-epimerase mutants are obtained by simultaneously mutating any one or more of the 47th, 72nd, 114th and 221st sites of wild-type D-allulose-3-epimerase derived from Ruminococcus CAG55 (CAG55), and the catalytic activity and stability of the mutants are significantly improved. The catalytic activity of the mutant obtained by simultaneously mutating the 47th, 72nd, 114th and 221st sites is the highest. The D-allulose-3-epimerase mutant provided by the application has great application value in the field of D-allulose production.
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