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Monellin protein mutant and preparation method thereof

A protein mutant and protein technology, applied in the field of protein mutants, can solve the problems such as the inability of large-scale fermentation production of monellin protein, hidden dangers of fermented products, production and transportation restrictions, etc., to save the cost of later purification, reduce the probability of contamination, The effect of increasing the sweetness of the protein

Inactive Publication Date: 2016-05-11
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The single-chain monellin protein loses its activity when treated at 65°C, which limits its production and transportation
[0003] Single-chain monellin protein has also been successfully expressed in other organisms by genetic engineering methods, such as E. coli expression system, plant expression system, and Bacillus subtilis expression system. However, due to the problems of yield and toxic metabolites in these expression systems, monellin protein cannot be scaled Fermentation production
The use of methanol to induce the fermentation of Pichia pastoris has greatly improved the yield of monellin protein, but due to the addition of toxic substance methanol, the safety of fermentation products has become a hidden danger

Method used

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  • Monellin protein mutant and preparation method thereof
  • Monellin protein mutant and preparation method thereof
  • Monellin protein mutant and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0024] Preparation of single-chain monellin mutant gene protein

[0025] (1) Construction of wild-type single-chain monellin protein carrier

[0026] The wild-type single-stranded monellin whole gene was synthesized by Zhongmei Taihe Biotechnology Co., Ltd., and two restriction endonuclease sites of XhoI and NotI were primers at both ends, and the target gene fragment and PGAPZαA empty plasmid were digested with T4 DNA ligase Ligate overnight at 37°C, transform into DH5α Escherichia coli competent cells, spread on LLB solid medium containing 25 μg / ml Zeocin, culture overnight to pick positive transformants, use plasmid extraction kit to extract plasmids for sequencing verification, and successfully construct wild type The monellin protein carrier PGAPZαA-SCM.

[0027] (2) Site-directed mutation of wild-type single-chain monellin protein

[0028] Use XhoI and NotI two restriction endonucleases to cut out the target gene fragment, and the reaction system is as follows:

[002...

Embodiment 2

[0043] Determination of thermal stability and sweetness threshold of single-chain monellin mutant proteins

[0044] (1) Identification of protein thermal stability

[0045] The same batch of purified proteins were treated at 65°C, 70°C, 75°C, 80°C, and 85°C for 2h, 4h, 6h, 8h, and 10h respectively. The treated samples were detected by SDS-PAGE, and the strips of the gel were observed. With brightness, initially determine the denaturation temperature and maximum tolerance temperature of the protein. It has been detected that the mutant protein E2N still exists after being treated at 75°C for 6 hours and at 80°C for 2 hours (the attached figure figure 1 ), the thermal stability of the wild-type single-chain monellin protein was increased by 10°C.

[0046] (2) Determination of protein sweetness threshold

[0047] In the control group, 10000 μg / ml sucrose was used to dilute the protein into a gradient from 0.25 to 150 μg / ml. Let 10 healthy adults, 5 males and 5 females taste ...

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Abstract

The invention discloses a monellin protein mutant and a preparation method thereof. Site-specific mutagenesis is performed on a single-chain monellin, a thermostable mutant is obtained and successfully expressed in pichia pastoris, and the technical problem that sweet protein is easy to degrade in the production and transportation process is solved; meanwhile, protein sweetness is improved, the good sweet effect can be achieved on the condition of a small amount of monellin protein mutant, and a solid foundation is laid for large-scale industrial marketization of the sweet protein.

Description

technical field [0001] The invention relates to the technical field of protein mutants, in particular to a monellin protein mutant and a preparation method thereof. Background technique [0002] The sweet protein monellin is a natural sweetener extracted from the West African plant Dioscoreophyllumcumminsii. It has a strong sweet taste, and its sweetness is about 3000 times that of sucrose with the same quality under the same conditions. The natural monellin protein is composed of two different peptides The chains are bound together by covalent bonds and are composed of chain A and chain B. Studies have found that the natural monellin protein is prone to lose activity at high temperatures. In 1989, Kim et al combined the two peptide chains into one protein through genetic engineering. chain, which greatly improves its thermal stability and remains stable in a wide range of PH, while its sweetness does not change much. Since it does not contain sugar, it can be used as the b...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/81
CPCC07K14/415C12N15/815C12N2800/102
Inventor 刘波蔡成固
Owner QILU UNIV OF TECH
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