In this paper,PCR technology was used to carry out targeted mutation of single-stranded monellin(MNEI),and the constructed recombinant plasmid pET15b-MNEI double-site mutant was transformed into Escherichia coli BL21-codonPlus(DE3)-RIL for recombinant expression of heterologous protein.The target proteins were purified by nickel col-umn affinity chromatography and collected by molecular sieve.The dialysis of the proteins was carried out using distilled water in a dialysis bag with a interception volume of 3.5 ku.The sweet threshold of the target proteins obtained by dialy-sis was determined by double-blind sensory evaluation.The secondary structure and thermal stability of the protein mu-tants were determined by circular dichrometry.The results showed that three double-site mutants E2M/E50N,E2Q/E50N and E2A/E50N were successfully constructed,and the mutant E2Q/E50N was successfully expressed and purified.Com-pared with wild-type MNEI control,the sweet threshold of the mutant E2Q/E50N was 0.64 µg/mL,and the sweetness was nearly doubled;the Tm value was 78 ℃,the thermal stability was improved by 4 ℃.These results could provide theoretical basis and technical support for the production and application of sweet protein monellin in food,beverage and pharmaceutical industries.
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