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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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

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

Examples

Experimental program
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Example Embodiment

[0023] Example 1

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

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

[0026] The wild-type single-stranded monellin gene was synthesized by Zhongmeitaihe Biotechnology Co., Ltd. and the two restriction enzyme sites of XhoI and NotI were primered at both ends. The target gene fragment and the empty plasmid of PGAPZαA were digested with T4DNA ligase. Ligated overnight at 37°C, transformed into DH5α Escherichia coli competent cells, spread it on LLB solid medium containing 25μg / ml Zeocin, cultivated overnight to pick out positive transformants, extracted plasmids with a plasmid extraction kit and verified by sequencing, the wild type was successfully constructed 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 enzymes to cut the target gene fragment, and the reflection system is as fol...

Example Embodiment

[0042] Example 2

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

[0044] (1) Identification of protein thermal stability

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

[0046] (2) Determination of protein sweetness threshold

[0047] The control group used 10000μg / ml sucrose and diluted the protein into a gradient from 0.25 to 150μg / ml. Let the samples be tasted by 5 males, 5 females and 10 healthy adu...

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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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