黑曲霉单宁酶基因在毕赤酵母中的表达

Expression of recombination Aspergillus niger tannase in Pichia pastoris

单宁酶(EC3.1.1.20)在食品、化工及医药等领域应用广泛,期望通过单宁酶在毕赤酵母中的表达来提高单宁酶的产量。以经室温常压等离子体诱变技术诱变得到的较高产单宁酶的黑曲霉B1401的总DNA为模板,PCR扩增获得单宁酶基因(tan)后构建重组表达载体pPIC9K-tan,通过电转化至毕赤酵母GS115,利用甲醇诱导培养重组菌获得单宁酶。单宁酶的最适反应温度为30℃,最适反应pH为5.0,最高酶活力可达到34.25 U/g,相较于黑曲霉B1401产单宁酶活力提高了1.38倍,Ca^(2+)、Fe^(3+)、Mn^(2+)等金属离子对重组单宁酶酶活力影响不大。实验结果表明实现了单宁酶在毕赤酵母中的高效表达。

Tannase（EC3.1.1.20） is widely used in food, chemical and pharmaceutical fields. This study hoped to increase tannase yield through the expression of tannase in Pichia pastoris. Based on the research model of（DNA） Aspergillus niger（B1401） which obtained by room temperature and atmospheric pressure plasma mutagenesis, we amplified the tannase gene by PCR and then cloned the gene into the expression vector pPIC9K. pPIC9K-tan has been transformed into the P. pastoris GS115 by electroporation method. Methanol was added to induce recombinant bacteria to produce tannase. The optimal pH and temperature of recombinant tannase were 5.0 and 30 ℃. The highest enzyme activity reached 34.25 U/g. Ca^2＋, Fe^3＋, Mn^2＋ and other metal ions have little effect on enzyme activity. In conclusion, the tannase gene from Aspergillus niger was successfully expressed by recombinant P. pastoris.