双孢蘑菇酪氨酸酶基因在酿酒酵母中的异源表达及其酶学特性

Expression and characterization of tyrosinase from Agaricus bisporus in Saccharomyces cerevisiae

【目的】在酿酒酵母中异源表达双孢蘑菇来源的酪氨酸酶基因PPO2,并研究酪氨酸酶在酿酒酵母胞内及胞外的酶学特性。【方法】提取双孢蘑菇总RNA,通过RT-PCR克隆酪氨酸酶基因PPO2,构建表达载体pSP-G1-PPO2,并转化至酿酒酵母进行表达,采用镍亲和层析纯化蛋白并研究其酶学性质。【结果】在酿酒酵母中正确表达了大小为65 kDa的酪氨酸酶蛋白。重组酶能催化底物酪氨酸产生黑色素。体外活性测定表明,酪氨酸酶催化最适温度为45°C,以酪氨酸和多巴为底物时最适pH分别为7.0和8.0。在酿酒酵母中测得底物酪氨酸浓度低于2.5 mg/mL时,黑色素的产量与底物浓度呈现正相关性。【结论】来源于双孢蘑菇的酪氨酸酶基因PPO2在酿酒酵母中成功表达,重组酶具有良好的酶学特性。利用酪氨酸酶产物黑色素的产量与底物浓度呈现正相关性这一特性,可将其作为细胞酪氨酸产量的传感器,为高通量筛选酪氨酸高产菌株提供了思路。

[Objective] The aim of this research was to express the tyrosinase-coding PPO2 gene from Agaricus bisporus in Saccharomyces cerevisiae and to investigate the characteristics of tyrosinase in vitro and in vivo. [Methods] We cloned PPO2 gene by RT-PCR using the total RNA extracted from Agaricus bisporus. The expression vector pSP-G1-PPO2 was constructed and transformed into yeast. The recombinant protein was purified with Ni-NTA and the tyrosinase enzyme properties were evaluated. [Results] The optimum temperature of tyrosinase in vitro was 45 ℃. And the optimum pH was 7.0 and 8.0 using tyrosine and L-3,4-dihydroxyphenylalanine （L-DOPA） as substrate, respectively. In Saccharomyces cerevisiae, the yield of melanin increased with the rise in substrate concentration within 2.5 mg/mL. [Conclusion] We achieved heterologous expression of tyrosinase-coding gene PPO2 from Agaricus bisporus in Saccharomyces cerevisiae and characterized the enzyme properties. The melanin production positively correlates with the concentration of substrate tyrosine which indicates that tyrosinase could be a biosensor to report the content of tyrosine in yeast and could be used for the high throughput screening of high-yield tyrosine strains.