该文从猕猴桃中克隆出果胶甲酯酶抑制剂（Pectin Methylesterase Inhibitor，PMEI）基因，经EcoRⅠ/XbaI双酶切后连接到表达载体pPICzαA上，电转化到毕赤酵母GS115筛选出阳性菌株GS115/pPICzαA-PMEI，并将重组酵母表达的PMEI浓缩纯化后应用于果酒发酵。研究结果表明：重组毕赤酵母表达菌株GS115/pPICzαA-PMEI成功构建，PMEI的发酵表达量为35.38 mg/L。将PMEI应用到果酒发酵中，桔子酒甲醇降低47.54%，含量由189.75 mg/L降低到99.55 mg/L；苹果酒甲醇降低21.52%，含量由118.15 mg/L降低到91.20 mg/L；葡萄酒甲醇变化不显著，含量均低于20.00 mg/L，表明PMEI对果胶丰富且内源性果胶酶强的桔子具有明显的降甲醇效果。桔子酒发酵工艺优化的PMEI最低抑制质量浓度8.84 mg/L，最适温度18 ℃，此时桔子酒中的甲醇降低66.40%到89.10 mg/L。这为低甲醇果酒的发酵提供了新的技术路线。

The pectin methylesterase inhibitor (PMEI) gene, cloned from the kiwi fruit, was linked to the expression vector pPICzαA through double digestion using EcoRI/XbaI. It was then transferred into Pichia pastoris GS115 by electro-transformation to obtain the positive strain GS115/pPICzαA-PMEI. After the PMEI expressed by GS115/pPICzαA-PMEI was concentrated and purified, it was applied in fruit wine fermentation. The results show that the recombinant P. pastoris expression strain GS115/pPICzαA-PMEI was successfully constructed, and the PMEI expression level was 35.38 mg/L. When PMEI was applied in fruit wine fermentation, the methanol in orange wine decreased by 47.54% from 189.75~99.55 mg/L. Similarly, the methanol content of apple wine dropped by 21.52% from 118.15~91.20 mg/L, whereas the methanol content of grape wine did not change significantly and remained below 20.00 mg/L. These results indicate that PMEI has a significant methanol reduction effect on oranges that are rich in pectin and endogenous pectinase. The minimum inhibitory concentration of PMEI for optimal orange wine fermentation was 8.84 mg/L and optimum temperature was 18 ℃. Under these conditions, the methanol content of orange wine reduced by 66.40%, to 89.10 mg/L. These findings provide a novel technique for producing low-methanol fruit wine by applying PMEI.

国家自然科学基金项目（51878291）