嗜热芽孢杆菌CHB1环糊精酶基因优化及其在毕赤酵母中的表达

为实现新型CGTase在毕赤酵母中的高效表达，提取嗜热芽孢杆菌CHB1基因组DNA为模块，PCR扩增野生型基因CGT1，编码680个氨基酸。将该基因进行密码子优化CGT2，与野生型基因CGT1分别插入分泌型载体pPICZαA转化毕赤酵母GS115，获得两株酵母工程菌GS115/pPICZαA-CGT1和GS115/pPICZαA-CGT2；经甲醇诱导表达120 h后，CGT2活力达0.62 U/mL，是优化前CGT1（0.37 U/mL）活性的1.7倍；进一步对GS115/pPICZαA-CGT2进行摇瓶发酵条件优化，确定其最优发酵条件为：pH 6.5、28 ℃、200 r/min、每24小时补加体积分数1.5%的甲醇诱导，120 h后其胞外酶活力达到1.26 U/mL，是优化前的两倍。
To achieve high-level expression of cyclodextrin glycosyltransferase from Gebacillius sp.CHB1 in methylotrophic yeast Pichia pastoris GS115，the DNA sequence（CGT1） encoding 680 aminno acids was amplified. The CGT2 was synthesized based on the codon preference of Pichia pastoris. Then CGT1 and CGT2 were respectively fused to the pPICZαA and expressed in Pichia pastoris. Two strains （GS115/pPICZαA-CGT1and GS115/pPICZαA-CGT2）were abtained. After methanol induction for 120 h in a shake flask ，the enzyme activity of CGT2 reached 0.62 U/mL，1.7 times higher than CGT1（0.37 U/mL）. Shake flask experiments were conducted to optimize the fermentation conditions. Highest enzyme activity achieved to 1.26 U/mL by induction for 120 h under the optimal conditions.（pH 6.5，28℃，200 r/min，inoculation amount of 1.5% methanol），two times higher than before