Brazilian sweet protein mutant and application thereof

By modifying brassinolide through site-directed mutagenesis and Pichia pastoris expression system, the problems of insufficient sweetness and industrialization of brassinolide have been solved, and the efficient expression and large-scale production of high-sweetness brassinolide have been achieved, breaking through the limitations of existing technologies.

CN122444846APending Publication Date: 2026-07-24WUHAN POLYTECHNIC UNIVERSITY
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Patent Information

Application Number
CN202610916187.2
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2026-06-24
Publication Date
2026-07-24

AI Technical Summary

Technical Problem

The industrialization of Brazilian sweet protein currently faces challenges such as limited natural fruit resources, low extraction efficiency, high costs, and low yield and weak activity of microbial heterologous expression systems, making it difficult to meet the needs of industrial applications.

Method used

The amino acid sequence of Brazil sweet protein was modified by site-directed mutagenesis to construct a high-sweetness mutant, which was then expressed efficiently using a Pichia pastoris expression system. The mutant was then fermented and produced using the pPIC9K recombinant expression vector and the Pichia pastoris strain.

Benefits of technology

It significantly improves the sweetness of brassinoprotein by 60% to 340%, simplifies the large-scale production process, solves the technical pain points of insufficient sweetness and high difficulty in industrialization, and provides industrial application value for high-sweetness brassinoprotein mutants.

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Abstract

The invention discloses a Brazilian sweet protein mutant and application thereof, and belongs to the technical field of protein modification. Aiming at the defects that the sweetness of wild type brazilian sweet protein is insufficient, the industrial sugar substitute application requirement is difficult to meet, and the yield of a microbial heterologous expression system needs to be improved, the invention adopts a protein engineering directed evolution technology to screen and obtain single mutants P13E, D26K and I49G, and further constructs and obtains combined mutants P13E / D26K, P13E / I49G, D26K / I49G and P13E / D26K / I49G. The mutants are subjected to heterologous efficient expression in a pichia pastoris expression system, the sweetness performance of the mutants is systematically evaluated, and the result shows that compared with wild-type brazitame protein, the sweetness of the mutants can be improved by 60%-340%, and the mutants have the characteristics of excellent stability and pure sweetness, can be used as a novel sweetening agent, are applied to the fields of food processing and the like, and have broad application prospects. The method has important industrial application value.
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