甜味蛋白monellin高甜度、强热稳定性突变体的分子构建及性质研究

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

本文采用PCR技术对单链monellin(MNEI)进行定向突变,将构建好的重组质粒pET15b-MNEI双位点突变体转化到大肠杆菌BL21-codonPlus(DE3)-RIL中进行异源蛋白质的重组表达。通过镍柱亲和层析和分子筛收集纯化后的目的蛋白,使用蒸馏水在截留分子质量为3.5 ku的透析袋中进行蛋白透析,将透析得到的目的蛋白采用双盲法感官品评测定甜味阈值,蛋白突变体的二级结构和热稳定性采用圆二色谱仪测定。结果表明:成功构建了该蛋白3个双位点突变体E2M/E50N、E2Q/E50N及E2A/E50N,其中突变体E2Q/E50N成功表达与纯化。与野生型MNEI对照相比,测得的突变体E2Q/E50N的甜味阈值为0.64μg/mL,甜度提升近1倍;Tm值为78℃,热稳定性提高4℃。以上研究结果可为甜味蛋白monellin在食品、饮料和医药行业的生产与应用提供技术参考。

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 column 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 dialysis was determined by double-blind sensory evaluation.The secondary structure and thermal stability of the protein mutants 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.Compared with wild-type MNEI control,the sweet threshold of the mutant E2Q/E50N was 0.64μg/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.