《植物生理学报》 2011, 47(10): 1017-1023

盐生植物盐地碱蓬酪氨酸酶的酶学特性

陈天舒, 修妍伟, 王琼, 王宝山*

山东师范大学生命科学学院逆境植物重点实验室, 济南250014

通信作者：王宝山；E-mail: bswang@sdnu.edu.cn；Tel: 0531-86180197

摘　要：

酪氨酸酶是植物甜菜素生物合成的限速酶, 但是, 其酶学特性尚不了解。以黑暗培养3 d的盐地碱蓬幼苗为材料, 采用NaF抽提、饱和硫酸铵沉淀法提取盐地碱蓬中的酪氨酸酶, 以研究其酶学特性。结果表明, 酪氨酸酶氧化活性的最适温度为35 ℃, 最适pH值为6.6, 最适条件下Km=1.09 mmol·L-1, Vmax=71.43 μmol·g-1 (FW)·min-1; 酪氨酸酶羟化活性的最适温度为40 ℃, 最适pH值为6.6, 最适条件下Km=3.16 mmol·L-1, Vmax=0.645 μmol·g-1 (FW)·min-1。Na2S2O3是盐地碱蓬酪氨酸酶的强效抑制剂, 0.05 mol·L-1 Na2S2O3几乎完全抑制酪氨酸酶氧化及羟化活性。而0.01 mmol·L-1的Cu2+可以显著激活酪氨酸酶的氧化及羟化活性, 分别为对照的126%和128.2%。这些结果表明盐地碱蓬中酪氨酸酶的羟化活性是影响甜菜红素合成速率的关键, 也为深入研究盐地碱蓬酪氨酸酶在甜菜素合成中的作用及其与环境之间的关系奠定了基础。

关键词：盐地碱蓬; 酪氨酸酶; 氧化活性; 羟化活性; 影响因子

收稿：2011-08-04   修定：2011-09-19

资助：国家自然科学基金(30870138和31070158)和国家支撑计划项目(2009BADA7B05)。

Enzymatic Characteristics of Tyrosinase in Euhalophyte Suaeda salsa

CHEN Tian-Shu, XIU Yan-Wei, WANG Qiong, WANG Bao-Shan*

Key Lab of Plant Stress, College of Life Sciences, Shandong Normal university, Jinan 250014, China

Corresponding author: WANG Bao-Shan; E-mail: bswang@sdnu.edu.cn; Tel: 0531-86180197

Abstract:

Tyrosinase is the first key enzyme in betalains biosynthesis of plants while its enzymatic characteristics remain unclear. In order to investigate the properties of tyrosinase, 3-day-old Suaeda salsa seedlings (cultured in dark) were used and the tyrosinase was partially purified by ammonium sulphate [(NH₄)₂SO₄] precipitation after being extracted by sodium fluoride (NaF). The results indicated that, in terms of O-diphenol oxidation activity, the optimum temperature and pH values were 35 ℃ and 6.6, the Km=1.09 mmol·L^-1 and V_max=71.43 µmol·g^-1 (FW)·min^-1; and as for the activity of monophenol hydroxylation, the optimum temperature and pH value were 40 ℃ and 6.6, the Km=3.16 mmol·L^-1 and V_max=0.645 µmol·g^-1 (FW)·min^-1. Na₂S₂O₃ was the potent inhibitor of tyrosinase, and 0.05 mol·L^-1 Na₂S₂O₃ almost completely inhibited the activity of O-diphenol oxidation and monophenol hydroxylation. Moreover, the activity of O-diphenol oxidation and monophenol hydroxylation was significantly activated by 0.01 mmol·L^-1 Cu²⁺, 126%、128.2% of control respectively. These results suggested that tyrosinase hydroxylation activity of S. salsa played a key role in betalains biosynthesis and laid the foundation of deep investigating the role of tyrosinase in betalains biosynthesis in plants and its interaction with environment factors.

Key words: Suaeda salsa L.; tyrosinase; oxidation activity; hydroxylation activity; impact factor