解脂耶氏酵母lip2脂肪酶水解谷维素产生阿魏酸的研究

Production of ferulic acid from oryzanol by Y. lipolytica lip2 enzyme-catalyzed

对解脂耶氏酵母(Yarrowia lipolytica)脂肪酶水解谷维素产生阿魏酸的酶反应体系进行了研究。实验发现解脂耶氏酵母全脂肪酶粉(105U/mg)在50mmol/L p H7.0 Tris-HCl(含7.5mmol/L黄胆酸钠),100mmol/L p H6.0磷酸钠缓冲液(含1000U脂肪酶)的体系中,水解产生阿魏酸的得率为2.94%。为了进一步提高脂肪酶水解效率,对解脂耶氏酵母脂肪酶中主要组分lip2脂肪酶基因进行了克隆,整合至毕赤酵母GS115基因组后发酵制取lip2脂肪酶粉(70.1U/mg),于上述酶解体系中进行水解谷维素实验。实验结果表明阿魏酸产率为2.87%。获得的lip2脂肪酶催化效率略低于全脂肪酶粉催化效率,但是获得了单一的脂肪酶基因,为进一步采取分子进化技术提高其催化能力奠定了基础。

An appropriate enzymatic system was preliminary explored for the Yarrowia lipolytica lipase. The study found that the optimal enzyme reaction system for the whole lipase powder was 50mmol/L p H7.0 Tris-HCl（containing 7.5mmol/L ammonium taurocholate）,100mmol/L p H6.0 PBS（containing 1000 U lipase powder）,and the ferulic acid yield was 2.94%. To further improve the efficiency of hydrolysis of lipase,lip2 lipase gene was cloned and integrated in Pichia pastoris GS115 genome,and the lip2 lipase powder was obtained by fermentation. Using the optimal enzyme reaction system to hydrolysis oryzanol,the results showed that the ferulic acid yield by lip2 lipase powder was 2.87%. This efficiency was slightly lower than the whole lipase powder hydrolysis efficiency. However,a single lipase gene was obtained,which would lay the foundation of improving the catalyze ability to hydrolysis oryzanol by molecular evolution technology.