嗜热真菌单宁酶的克隆表达及在柿子汁中的应用

Cloning and Expression of Tannase Gene from Thermophilic fungi and Effect of Recombinant Tannase on Antioxidant Activity of Persimmon Juice

对嗜热烟曲霉中的单宁酶基因afTanA在毕赤酵母中进行异源表达,分析重组酶的酶学性质及其对柿子汁抗氧化性的影响。研究表明,afTanA基因由1 767 bp碱基组成,编码588个氨基酸和一个终止密码子,存在1个Kex2酶切位点(Lys315-Arg316),其催化活性位点为Ser202、Asp455和His501。重组菌株经48 h诱导,重组AfTanA酶活力达到678U/mL。AfTanA的最适反应温度为40℃,最适pH5.0。不同金属离子浓度对酶活力的影响存在差异,在低浓度(1mmol/L)条件下,Zn2+使AfTanA酶活力提高49.65%,而Cu2+和Fe3+使该酶活力分别降低约78%和98%;在高浓度(5mmol/L)条件下,Zn2+使AfTanA酶活力降低47.12%,Cu2+和Fe3+能够完全抑制AfTanA活性。柿子汁经重组单宁酶AfTanA处理后,其抗氧化性能力显著提高,DPPH、ABTS自由基和·OH清除率分别提高15.79%,12.78%,3.4%。单宁酶AfTanA在毕赤酵母中实现了高效表达,并在果汁加工工业中具有良好的应用潜力。

The heterologous expression of the tannase gene afTanA in Pichia pastoris and the recombinant enzyme were analyzed. The effects of recombinant tannase on the antioxidant activity of persimmon juice were also discussed. It was found that the afTanA gene consisted of 1 767 bp base, encoding 588 amino acids and one stop codon, and there was one Kex 2 restriction site(Lys315-Arg316), and the catalytic sites were Ser202, Asp455 and His501. The recombinant strain was induced by 48 h, and the activity of AfTanA was 678 U/mL. The optimum reaction temperature for AfTanA is 40 ℃ and the optimum pH is 5.0. Zn2+ increased the activity of AfTanA by 49.65% under low concentration(1 mmol/L), while Cu2+ and Fe3+ decreased the activity of the enzyme by about 78% and 98%, respectively. At high concentration(5 mmol/L), Zn2+ reduced the activity of AfTanA by 47.12%, The activity of AfTanA was inhibited completely by Cu2+ and Fe3+. The antioxidant ability of persimmon juice increased significantly, and after treated with recombinant tannase AfTanA the DPPH, ABTS free radical and ·OH scavenging rate increased by 15.79%, 12.78% and 3.4%, respectively. In summary, tannase AfTanA has been highly expressed in Pichia pastoris and has good application potential in the juice processing industry.