重组真菌腈水解酶的发酵工艺条件及生物催化特性初探【需加急】

对产真菌腈水解酶的重组大肠杆菌的培养基种类、培养基成分、诱导剂种类和浓度、诱导条件、p H和温度进行了系统考察。摇瓶发酵优化结果显示:以甘油作为主要碳源,蛋白胨和酵母膏作为主要氮源,并添加微量元素的SOC培养基作为发酵培养基,最适接种量为0.5%;较优的诱导剂诱导条件为:采用0.5 mmol/L的IPTG诱导12 h,发酵p H=7.5,诱导温度25℃时产酶效果最佳。经过优化后,重组酶的酶活得到了显著提高,总酶活最高达到了3.84 U/m L,相比初始水平(0.84 U/m L)提高约4倍。5 L发酵罐的放大实验表明,产酶效果良好,总酶活和比酶活均与摇瓶水平基本持平。全细胞催化性质考察研究结果表明,该菌株所产腈水解酶催化反应的最适催化反应温度是45℃,最适反应p H约为7.2。

A Preliminary Study on Fermentation Optimization and Biocatalytic Properties of Fungal Nitrilase

The culture conditions of recombinant E. coli harboring fungal nitrilase were systematically investigated in the level of shake flask, including medium composition, inducers, induction condition, pH and temperature. The results showed that a modified SOC medium containing trace elements was used as the fermentation medium with glycerol as the carbon source and peptone plus yeast extract as the nitrogen sources. The preferred inoculation amount was 0.5% (v/v). The induction for nitrilase production was carried out at 25°C and pH 7.5 with 0.5 mmol/L IPTG as the inducer for 12 h. The enzyme activity of the nitrilase were substantially improved through optimization and the total activity reached up to 3.8 U/mL, which was 3-fold higher than that of the initial level (0.84 U/mL). Subsequently, the nitrilase production in a 5-L stirred tank fermentor was determined and proved to be almost the same as that of shake flask. Preliminary study on biocatalytic properties of resting cells showed that the optimum reaction temperature and pH of the fungal enzyme were 45°C and pH 7.2.