茶黄素(theaflavins,TFs)是影响红茶品质的关键成分,在医药、保健、食品、美容等领域有着广泛的应用前景,但红茶中TFs含量极低且提取困难,因此添加外源多酚氧化酶(polyphenol oxidase,PPO)酶促合成TFs成为工业化生产的关键途径。以马铃薯为外源PPO供体,经过磷酸缓冲液浸提、硫酸铵沉淀和DEAE-Sepharose Fast Flow阴离子交换层析分离纯化出马铃薯PPO,并进一步研究其酶学性质和酶促合成TFs的性能。试验结果表明:马铃薯PPO的比活力为21.79 U/mg,相对分子质量约为42 kDa,最适pH和温度分别为6.0和30 ℃,添加金属离子Al3+、Mg2+和Zn2+对其酶活力有激活作用,Cu2+和Mn2+有抑制作用,Na+、K+和Ca2+无明显作用;马铃薯PPO粗酶和纯酶均可酶促合成TFs,其最大生成量分别是(0.096 1±0.000 5)和(0.237 8±0.013 2)g/L,转化率分别为(9.24±0.05)%和(22.86±1.27)%。综上,马铃薯PPO可以作为外源PPO酶促合成TFs,且PPO纯酶比粗酶更具催化效果。
Theaflavin (TF) is a key component affecting the quality of black tea, and it is widely used in various fields, including medicine, health care, food, beauty etc. The content of TFs in black tea is extremely low and it is difficult to extract. Therefore, adding exogenous polyphenol oxidase (PPO) to promote the synthesis of TFs has become the key path of industrial production. Potato PPO was separated and purified by extraction, precipitation and anion exchange chromatography. Then, the PPO was used to analysis its enzymatic properties and ability to enzymatic synthesis of theaflavins. The results showed that the specific activity of PPO was 21.79 U/mg and the relative molecular weight was about 42 kDa. The optimum reaction pH and temperature were 6.0 and 30℃. Metal ions Al3+, Mg2+ and Zn2+ could activate the enzyme activity of PPO, whereas Cu2+ and Mn2+ inhibit it. Na+, K+ and Ca2+ showed no significant effects on its activity. Both crude enzyme and purified enzyme of PPO could enzymatically synthesize TFs, the maximum production amounts were (0.096 1±0.000 5) and (0.237 8±0.013 2) g/L, respectively. And the conversion rates were (9.24±0.05)% and (22.86±1.27)%, respectively. In conclusion, potato PPO can synthesize TFs enzymatically. And the catalytic effect could be promoted by purified PPO.
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