Construction Of The New Sweet-tasting Protein Monellin Mutants With High Sweetness And Thermal Stability

The sweet protein monellin has a high sweetness and low calorie.Compared with other chemical sweeteners,monellin has no toxic side effects and can be regarded as a sweet additive for cardiovascular,diabetic and hyperlipidemia patients who are related to the consumption of sugars.Nevertheless,due to its low thermal stability and tedious production process,the extensive applications of monellin are limited now.The recombinant plasmid pET15b-MNEI which we constructed in previous study was introduced into E.coli BL21-CodonPlus?DE3?-R-IL for protein expression.Wild-type monellin protein was purified by affinity chromatography and molecular sieve chromatography.The sweetness threshold of wild-type monellin protein was determined to be 1.1?g/ml by a double-blind method.The melting point of the protein was evaluated by circular dichroism spectrophotometer through a stepwise improvement of temperature,and the calculated T_m value of wild-type monellin was74.2°C.In order to improve the sweetness and thermal stability of the monellin protein,we analyzed the protein surface charge,the role of internal hydrophobic and vander Waals force based on the structure of MNEI protein?PDB:2O9U?,and then designed some mutants accordingly.We found that three mutants E2N,E23A and Y65R exhibited improved properties,while six double-sites mutants showed to be insoluble.After the combination of the three mutated sites,the double-sites mutant E2N/E23A showed high swteeness and strong thermostability,with a sweetness threshold 0.38?g/ml and T_m value 84.9°C.These results suggested that the ionization state of the key amino acid at the protein core was critical for the thermal stability of the sweet protein.In this study,site-directed mutagenesis was performed to improve the sweetness and thermostability of sweet-tasting protein monellin.The mutant E2N/E23A can facilitate the large-scale production of the sweet-tasting protein.Moreover,the amino acid sites maintaining the protein structure revealed in this study can provide a basis to further investigate the monellin protein aggregation.