融合疏水蛋白在毕赤酵母中的表达及其性质研究
Study on expression of fusion hydrophobin in Pichia pastoris and its properties
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摘要: 运用基因改造方法，将不同来源的Ⅰ型疏水蛋白基因（平菇的Po.hyd1和香菇的Le.hyd）拼接后，构建融合表达载体pPIC9k-Po.hyd1-Le.hyd，并将其电转化到毕赤酵母GS115感受态细胞中，经PCR验证和遗传霉素G418的抗性筛选，得到3个高效表达的毕赤酵母转化子；运用三氟乙酸方法分离纯化发酵上清液中的融合疏水蛋白Po.hyd1-Le.hyd，经检测其单体分子量约为26 kD.运用单因素试验和响应面分析试验优化，得到毕赤酵母转化子的最佳诱导表达条件为碳源（甲醇）质量分数为0.62%，氮源（m（酵母提取物）：m（蛋白胨）=1：2）质量浓度为8.39 g/L，发酵培养基的初始pH值为6.05，YNB质量浓度为100 mg/L，装液量为50 mL，初始OD600值为1.2.此时融合疏水蛋白Po.hyd1-Le.hyd的产量最高可达30 mg/L.对融合疏水蛋白Po.hyd1-Le.hyd起泡性和乳化性的研究发现，其蛋白性质均优于从平菇菌丝中提取的疏水融合蛋白.Abstract: Two hydrophobin genes, Po.hyd1 of Pleurotus ostreatus and Le.hyd of Lentinula edodes were ligated to construct the expression vector pPIC9k-Po.hyd1-Le.hyd, and then it was transferred into the Pichia pastoris GS115 competent cells by electroporation. After PCR validation and geneticin G418 resistance screening, three highly expressed Pichia pastoris transformants were obtained. After separation and purification of fusion hydrophobin Po.hyd1-Le.hyd from fermentation supernatant by the trifluoroacetic acid method, it showed that its monomer was about 26 kD. The fermentation conditions of P. pastoris were optimized by single factor and response surface analysis and the best fermentation conditions were showed as followed:the mass fraction of carbon source (methanol) was 0.62%, the mass concentration of nitrogen source (m (yeast extract):m (peptone)=1:2) was 8.39 g/L, the initial pH value of the fermentation medium was 6.05, the mass concentration of YNB was 100 mg/L, the fluid amount was 50 mL and the initial OD600 value was 1.2.Under the above conditions, the maximum yield of the fusion hydrophobin Po.hyd1-Le.hyd could be up to 30 mg/L. The foaming and emulsifying properties of the fusion hydrophobin Po.hyd1-Le.hyd were investigated and they were all better than that of fusion hydrophobin extracted from the Pleurotus ostreatus mycelium.
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Key words:
- fusion hydrophobin /
- Pichia pastoris /
- transformant /
- response surface optimization /
- emulsibility /
- foamability
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