运动发酵单胞菌共表达α-淀粉酶和葡萄糖淀粉酶发酵甘薯生产乙醇

Co-expression of α-amylase and Glucoamylase in Zymomonas mobilis and Direct Ethanol Production from Sweet Potato

运动发酵单胞菌是乙醇发酵的极佳菌种,但其所能利用的发酵底物范围狭窄,不能利用淀粉作为发酵底物.为增加其利用底物的范围使其能够水解淀粉,本研究构建了3种表达淀粉酶的运动发酵单胞菌菌株:1)Zymomonas mobilis(pAmyE)表达α-淀粉酶;2)Z.mobilis(pGA)表达葡萄糖淀粉酶;3)Z.mobilis(pAmyGA)共同表达α-淀粉酶和葡萄糖淀粉酶.DNS法测定淀粉酶活显示,每种转化菌株的胞外淀粉酶活性均高于胞内,且两种淀粉酶共表达的酶活高于这两种淀粉酶单独表达的酶活之和,说明这两种淀粉酶能够协同作用降解淀粉.对于重组菌株Z.mobilis(pAmyGA),约59.3%的淀粉酶活性都在胞外检测到.用淀粉含量高且耐贮存的徐薯18匀浆加少量葡萄糖作为培养基直接用上述3个菌株发酵生产乙醇.结果显示,共表达α-淀粉酶和葡萄糖淀粉酶的重组菌株Z.mobilis(pAmyGA)的乙醇产量为54.7 g/L,达到了理论值的83.2%,表明本研究得到了能够直接高效利用淀粉生产乙醇的运动发酵单胞菌的菌株.

The gram-negative bacterium Zymomonas mobilis is one of the best ethanol producers found in nature,however,this ethanologenic bacterium cannot hydrolyze starch abundantly existed in nature because of its narrow spectrum of fermentable carbohydrates.In this research,three genetic recombined strains were constructed to express amylolytic enzymes：（1） α-amylase expressing strain Z.mobilis（pAmyE）,（2） glucoamylase expressing strain Z.mobilis（pGA） and（3） α-amylase and glucoamylase co-expressing strain Z.mobilis（pAmyGA）.Most of the enzyme activity was detected in the medium,and the enzyme activity of amylolytic enzyme in co-expression strain was higher than the sum of each enzyme expressed individually,suggesting there was combined effect between α-amylase and glucoamylase in the hydrolysis of starch.For recombinant strain Z.mobilis（pAmyGA）,59.5% of the enzyme activity was detected in the extracellular fraction.Using the raw material of sweet potato xushu 18 with high starch content and good storage character and adding 5 g/L of glucose as medium,the direct ethanol production was determined via fermentation of the three recombined Z.mobilis strains.The results revealed that the recombinant Z.mobilis（pAmyGA） exhibited the highest level of ethanol production,54.7 g/L ethanol in 3.5 days,which was 83.2% of the theoretical yield.