Molecular Construction Of The New Sweet Protein Brazzein Mutant

Brazzein is a kind of natural plant sweet protein with high sweetness,low energy and good stability.However,the production of natural sweet protein is low,and the market price is high.Secondly,some sweet protein can not fold correctly or glycosylation in heterologous expression,which also limits the large-scale commercial production and application of sweet protein.In this study,the optimized brazzein gene sequence was cloned into the vector pET-SUMOand was introduced into Escherichia coli expression system.The results showed that the recombinant protein with solubilizing label had no sweet taste,and the protein not only had sweet taste,but also had a strong sweet taste after enzymatic digestion.The sweet threshold of wild type sweet protein brazzein was 1.5ugmL and denaturation temperature was 92 ℃,which indicated that synthetic sweet protein brazzein had the same thermal stability as natural sweet protein.Based on the surface charge of protein and previous research results,we designed the mutation.Through experiments,we found that the mutant protein(V7R 、 E9 K D29N、D40K、E41A、D50K and E53R),especially D50 K,was 5 times sweeter than that of wild type,and the sweetness of the mutant was 5 times higher than that of wild type.The sweetness threshold of E53 R was 0.3 ug/ mL and the sweetness threshold of mutant E53 R was 0.5 ug/mL.which indicated that the flexible C-terminal was also one of the main sites of interaction between brazzein and sweet receptor.In order to better understand the interaction between sweet protein and sweet receptor,five double mutations and two three mutations were constructed on the basis of single amino acid residues mutation.The sweetness of D29 N / D50 K was the most obvious,and the sweet threshold was 0.4 ug/mL.The sweetness of double mutation D29N/E53 R and D40 K / E53 R was not as good as that of single mutation,and the sweetness of D29 N / D40 K / E53 R was also lower than that of double mutation.The sweet protein brazzein may bind to several sites on the surface of sweet taste receptor,and the enhancement of multi-mutation sweetness is not a universal rule.