Studies on Molecular Construction of the Sweet Protein monellin Mutant with Intense Sweetness and High Thermal Stability and Its Properties
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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