高产高纯度果胶甲酯酶黑曲霉工程菌的构建

为获得一株高产高纯度的果胶甲酯酶的黑曲霉工程菌，提高果胶甲酯酶的产量，从果胶酶生产菌种中克隆了果胶甲酯酶基因pmeA，通过同源重组的原理，冻融法转化农杆菌、农杆菌介导法转化黑曲霉方法，成功构建了分泌表达果胶甲酯酶的纯合重组菌株TH-2（glaA::pmeA）。基本发酵培养基中发酵第9 d上清中最高酶活达到467.77 U/mL。进一步敲除重组菌株TH-2（glaA::pmeA）中背景蛋白酸稳定的α-淀粉酶的编码基因asaA，获得纯合重组菌株TH-2（glaA::pmeA?pyrG?asaA）。该菌株在添加1%的硫酸铵的发酵培养基中培养7 d后，发酵液上清中主要的背景蛋白均消失。但是与纯合重组菌株TH-2（glaA::pmeA）相比，果胶甲酯酶表达量有所下降，最高酶活为255.40 U/mL。重组果胶甲酯酶的最适作用温度为50 ℃，适合的温度范围是40~80 ℃，在80 ℃下仍能维持其酶活性的70%以上，适合的pH范围是3.0~5.0，最适pH为4.0。最终获得了一株温度和pH作用范围较宽的高产高纯度果胶甲酯酶的黑曲霉工程菌。

Construction of High-yield and High-purity Pectin Methylesterase Aspergillus niger Engineering Strain

In order to obtain an engineering strain of Aspergillus niger with high yield and purity of pectin methylesterase and improve the yield of pectin methylesterase, the pectin methylesterase gene pmeA was cloned from the pectinase-producing strain, and Aspergillus niger was transformed by agrobacterium-mediated method, the homozygous recombinant strain TH-2 (glaA::pmeA). The highest enzyme activity in the supernatant on the 9th day of fermentation in the basic fermentation medium reached 467.77 U/mL. At the same time, SDS-PAGE showed that the main background protein α-amylase disappeared, but the acid-stable α-amylase remained. Further the background protein acid-stable α-amylase encoding gene asaA in the recombinant strain TH-2 (glaA::pmeA) was knock out to obtain the homozygous recombinant strain TH-2 (glaA::pmeA?pyrG?asaA). After the strain was cultured in a fermentation medium supplemented with 1% ammonium sulfate for 7 days, the main background proteins in the supernatant of the fermentation broth disappeared. However, compared with the homozygous recombinant strain TH-2 (glaA::pmeA), the expression of pectin methylesterase decreased, and the highest enzyme activity was 255.40 U/mL. The optimum temperature of the recombinant pectin methylesteraseis 50 ℃, the suitable temperature range was 40~80 ℃, it could still maintain more than 70% of its enzyme activity at 80 ℃, the suitable pH range was 3.0~5.0, the optimum pH was 4.0. Finally, an engineering strain of Aspergillus niger with high yield and high purity pectin methylesterase was obtained.