Rational Design And Modification Of Sweet Protein Monellin

Natural sweet protein monellin has a high sweetness(about 3000 times sweeter than sucrose)and low calorie,suggesting its potential in food applications.However,due to its low stability at the heat and acid conditions,the extensive applications of monellin in food industry are unpractical now.According to the amino acid sequence of the single strand monellin sweet protein(GenBank: AFF58925.1),the monellin gene was synthesized with the optimized codon usage in E.coli.The recombinant plasmid was introduced into E.coli to express the protein,and the protein was purified by metal chelating affinity chromatography and gel filtration chromatography.The sweetness threshold and thermostability of the sweet protein were studied by experiments.To investigate the probable effect of His-tag sequence on the properties of the sweet protein,we used the enzyme thrombin to cleavage and remove the His-tag sequence of the sweet protein.Considering the low stability of protein monellin,we change amino acid in monellin by means of sequence,structure analysis,and site-directed mutagenesis,which can achieve an objective that the thermostability of monellin is improved with no sweetness decrease.Of the mutants investigated,the effects of E23 A and C41 A mutants weremost remarkable.The former displayed significantly improved thermal stability,while its sweetness was not changed.The mutated protein was stable after 30 min incubation at 85 °C.The latter showed increased sweetness and slight improvement of thermostability.Furthermore,we found that most mutants enhancing the thermostability of the protein were distributed at the two ends of ?-helix.Through this study,we demonstrate that we can improve the stability of monellin protein by means of gene mutagenesis,which can realize the industrialization production of monellin and provide a new kind of sugar substitute for human.