High Level Expression of Myrosinase from <i>Citrobacter</i> sp. and Its Application in Removing Off-Flavor from Radish Red Pigment(柠檬酸杆菌黑芥子酶的高效表达及其对萝卜红色素异味的脱除作用)

Off-flavor” from radish red pigment has severely restricted its applications in food industry. In this study, the myrosinase gene (<i>Cmyr</i>) from <i>Citrobacter</i> sp. was cloned and expressed in <i>Komagataella phaffii</i> GS115. The purified enzyme was characterized and used for removing the “off-flavor” from radish red pigment. The transformant was incubated in a 5L fermenter for high cell density fermentation (132h), and produced extracellular myrosinase enzyme activity of 433.2U/mL and protein concentration of 6.8mg/mL. The recombinant enzyme was purified through Q-Sepharose anion exchange chromatography column to obtain the electrophpresis-grade pure enzyme. SDS-PAGE showed that the molecular weight of purified enzyme was about 70kDa. After purification, the optimal pH and temperature of recombinant myrosinase (Cmyr) were pH 7.0 and 35℃, respectively. The enzyme was stable up to 40℃ and within pH 6.0-8.0. It showed the highest specific activity (146U/mg) towards sinigrin. CmyR was used to remove the off-flavor from radish red pigment and effectively reduced “off-flavor” from radish red pigment at 0.3U/g (wet weight of radish), pH 6.0, and 40℃ for 8h. Compared with other radish red pigments, there was no significant “off-flavor” reversion in radish red pigment treated with Cmyr, after storage at 55℃ for 30d. Therefore, Cmyr has great potential for application in removing the off-flavor from radish red pigment.(萝卜红色素的“返味”问题,严重制约了其在食品工业中的应用。为解决“返味”问题,从柠檬酸杆菌(<i>Citrobacter</i> sp.)中克隆了黑芥子酶基因<i>Cmyr</i>,并在毕赤酵母(<i>Komagataella phaffii</i>)GS115中分泌表达,研究了该酶的酶学性质及其在脱除萝卜红色素异味方面的效果。结果表明,重组菌在5L发酵罐中高密度发酵132h后,测得该黑芥子酶酶活力为433.2U/mL,蛋白质质量浓度为6.8mg/mL。采用Q-Sepharose阴离子交换层析柱对该黑芥子酶进行纯化,得到电泳级纯酶,SDS-PAGE蛋白电泳分析表明,其分子质量为70kDa。测得该重组黑芥子酶(Cmyr)的最适pH值和温度分别为7.0、35℃,在pH值为6.0~8.0和40℃温度条件下稳定；该酶以Sinigrin为底物,比酶活为146U/mg；利用该酶对萝卜红色素中的异味进行脱除,发现该酶在添加量为0.3U/g(以萝卜湿重计)、pH值为6.0和40℃温度条件下反应8h后,能够很好地脱除萝卜红色素中的异味；与其他萝卜红色素相比,利用该酶制备的萝卜红色素,在55℃烘箱中放置30d后无明显“返味”现象产生。研究结果表明,黑芥子酶Cmyr在萝卜红色素异味的脱除中具有很大的应用潜力。)