| The natural sweet protein brazzein is the smallest sweet protein that was isolated from the red fruit of the West African plant Pentadinplandra brazzeana Baillon by Ming and Hellekant in 1994.It has the advantages of strong thermal stability,low calorie,high sweetness and rich nutritional value.However,due to its low yield,the natural brazzein protein can not meet the market demand.Furthermore,the heterogenous expression of this sweet protein in prokaryotes expression system(e.g.E.coli)often lead to the unfolding of recombinant protein or formation of inclusion bodies,which limit its commercial production and application.In this study,the optimized brazzein gene sequence based on Pichia pastoris preference was cloned into the pGAPZαA vector,and then transformed into the pichia pastoris cells by electroporation.Subsequently,the recombinant protein was obtained by heterogenous expression,concentrating,purification and dialysis.The results showed that the recombinant protein had obvious sweetness with a sweet threshold of 125μg/mL.Furthermore,the yield of the recombinant protein was 60 mg/L in this expression system and its denaturation temperature was 86.7 ℃,indicating that the recombinant protein in Pichia pastoris has the same thermostability as the natural sweet protein.With this successfully constructed expression system,we designed 6 single mutants based on the charge properties of amino acids and protein structure.The results showed that the sweetness of the 6 single mutants were unevenly increased,suggesting that the sweet protein interacts with the sweet receptor via the electrostatic interaction of multiple sites.Resultantly,3 mutated proteins D40 K 、 E41 A and D50 K with significantly improved sweetness were obtained,of which D50 K was 6-fold sweeter than the wild type,and its sweetness threshold reached to 20 μg/mL.In order to further understand the relationship between the structure and function of these mutants,the secondary structure of wild-type and its mutants were determined.The results showed that the mutants retained the secondary structure of wild-type.The melting temperatures of D40 K,E41A and D50 K were higher than that of wild-type brazzein according to the circular dichroism gradient temperature increasement method results.Moreover,the heat treatment experiments indicated that the wild-type and mutants tasted sweet after 6 h incubation at 85℃,and E41 A had a slight sweetness after6 h incubation at 90 ℃.These results sugget the good application potential of the mutants obtained in this study. |
