萝卜硫素是一种具有抗氧化、防癌、抗癌等生物活性的异硫氰酸酯类化合物,但因产量低限制了其应用。黑芥子酶可水解硫代葡萄糖苷生成异硫氰酸酯类化合物。该研究首次在毕赤酵母中分别表达了源于西兰花的黑芥子酶BoTGG1和甘蓝蚜虫的黑芥子酶BMYR,结果重组BMYR活性更高,且宿主毕赤酵母X33比GS115、载体pPICZα比pPIC9 K更利于表达黑芥子酶。构建的重组菌株X33/pPICZα-BMYR在3 L发酵罐中高密度培养,酶活力达到0.64 U/mL,为摇瓶水平的8倍。在催化底物质量浓度3 g/L,反应时间60 min,pH 4.5,反应温度45 ℃ 的条件下,生成的萝卜硫素质量浓度达到1.21 g/L,对萝卜硫苷的摩尔转化率为99.6%,为目前萝卜硫素制备的最高水平。该研究为黑芥子酶的生产及萝卜硫素的制备提供了策略参考。
Sulforaphane is a kind of isothiocyanate with antioxidant, anti-cancer and anticancer activities, but its application is limited due to its low yield. Myrosinase can hydrolyze glucosinolates to produce isothiocyanates. In this study, BoTGG1 from broccoli and BMYR from cabbage aphid were expressed in Pichia pastoris for the first time, respectively. The recombinant BMYR showed higher activity. And Pichia pastoris X33 combining with vector pPICZα were more conducive for the expression of myrosinase than GS115 and pPIC9K. High-cell density fermentation of the recombinant strain X33/pPICZα-BMYR was performed in a 3 L fermenter, and the activity of myrosinase reached 0.64 U/mL, which was 8-fold higher than that in shaking flask. Conversion of 3 g/L glucoraphanin using myrosi- nase at pH 4.5 and 45 ℃, 1.21 g/L of sulforaphane was obtained within 60 min, with the molar conversion rate of 99.6%, which is the highest level for the preparation of sulforaphane at present. This study provides a strategy for the production of myrosinase and preparation of sulforaphane.
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