表达重组咖啡豆α-半乳糖苷酶的酵母工程菌的高密度发酵

High Cell-Density Fermentation of Pichia pastoris Producing Recombinant Coffee Bean α-Galactosidase

用5L发酵罐优化了重组咖啡豆α-半乳糖苷酶酵母工程菌pPIC9K-Gal/GS115(本室构建)的高密度发酵工艺。通过对发酵条件的优化,包括甘油补充量及补充时机、甲醇诱导量及诱导时机、溶氧控制、诱导时间等,重组咖啡豆α-半乳糖苷酶在毕赤酵母中得到了高效表达。利用所确定的最适条件进行发酵,菌体密度最终达到368g/L以上,每批发酵液离心后可获得3.5L的发酵上清,上清中的蛋白含量达到3g/L以上,目的蛋白占上清总蛋白的50%以上,含量约为1.5g/L,上清中α-半乳糖苷酶的活性维持在80U/ml左右。确立工艺后又进行了3次发酵试验,证明了工艺的可行性和稳定性。为重组咖啡豆α-半乳糖苷酶在B→O血型改造和酶解大豆低聚糖方面的应用奠定了基础。

Based on the previous study of construction of Piehia pastoris engineering Strain （ pPIC9K-Gal/GS115 ） expressing recombinant （ -galactosidase, this report described the optimal fermentation conditions of Pichia pastoris in a 5-liter-working-volume fermentor. 3 experiments were performed with various fermentation parameters, including the volume of inoculum seed, the volume and time of glycerol fed-batch, the volume and time of methanol fed-batch, dissolved oxygen, pH, agitation, temperature of glycerol batch phase and methanol fed-batch phase. For each experiment,0.4 liter pPIC9K-Gal/GS115 cells were inoculated into 4.5 liter basal salts medium. Then 3.5 liters of supernatant were harvested at the end of the fermentation. The results showed that the protein concentration in fermentation supernatant was 3 - 4.5 mg/ml, the activity of α-galaetosidase was 80 U/ml, and the enzyme activity ratio was 24 - 30 U/ mg. Based on the above experiments, 3 fermentations were carried out and confirmed the feasibility and stability of the technology. Therefore, this fermentation research laid a foundation for recombinant α-galactosidase purification to obtain sufficient amounts and highly purity of α-galactosidase.