Heterologous Expression of Pectin Methylesterase Inhibitor and its Application in Reducing Methanol in Fruit Wine
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.
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