Construction Of Novel Mutants Of The Sweet-tasting Protein Monellin And Optimization Of Their Sweetness And Thermal Stability

Sweet-tasting protein monellin has many advantages,such as high sweetness and low calories,and can be degraded into amino acids which can be absorbed and utilized by human body.It can be used as a sweet substitute for some sugar lovers or diabetic patients who need to strictly control sugar intake.However,the weak high-temperature resistance of the sweet-tasting protein monellin greatly limits its applications in the food,beverage and pharmaceutical industries.Although sweet-tasting protein monellin has been successfully heterologously expressed in the yeast,Escherichia coli or other hosts,it is difficult to achieve the industrial production due to its poor heat and acid resistance and low yield.In this study,the recombinant plasmid with the monellin gene(p ET15b-MNEI)was transformed into Escherichia coli BL21 for heterologous expression,and the total protein solution was purified by centrifugation,ultrasonication and nickel column affinity chromatography to collect the target protein.The sweet taste threshold of target protein was determined by the professional team members by double-blind method,and the threshold of wild-type monellin was 1.12μg/m L.The variation of circular dichroism spectra at 190-260 nm was measured by circular dichroism spectrometer.The melting point temperature of protein(T_m)was measured by circular dichroism at wavelength222 nm from 50℃to 95℃and was calculated.The T_m value of wild-type monellin was 74℃.In order to improve the sweetness and thermal stability of sweet-tasting protein monellin,we designed a series of mutation sites based on the electricity of amino acids on the protein surface,hydrophobicity and van der Waals forces in the protein,and many mutants of this protein were constructed successfully.By testing the sweetness and thermal stability of the successfully expressed mutated proteins,two excellent mutants D21N and E2Q/E50N were successfully selected.Compared with the wild-type monellin,the sweetness of the two mutants was significantly enhanced,and the sweetness thresholds were 0.70μg/m L and 0.64μg/m L,respectively.Furthermore,the thermal stability of the mutant E2Q/E50N was significantly improved,and the melting point temperature was increased to 78℃.In this study,the activity-enhanced mutants D21N and E2Q/E50N were structurally modeled,and their surface charge distributions was studied.The results showed that the sweetness of the mutants was significantly enhanced than wild-type monellin by mutating the negatively charged amino acids on the protein surface to polar uncharged amino acids,probably due to that reduction of the negative charge on the protein surface enhances binding of the protein to the sweet taste receptor T1R2/T1R3.The stability of the mutant E2Q/E50N was significantly improved.It was found that some changes could occurred in the three-dimensional structure of the protein,resulting in enhanced hydrophilicity and increased stability.In this study,the sweetness and stability of sweet-tasting protein monellin were improved by site-directed mutagenesis,and the mutants with better properties could promote the industrial production of sweet protein monellin,which provides good materials for the further commercial production of sweet-tasting protein monellin in the future.