Abstract：
Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.
摘要：
酪氨酸酶能够氧化蛋白质中的酪氨酸残基，导致分子间蛋白质交联，可以改变食物的蛋白质网络，改善食物的质地。用微生物细胞工厂获得重组酪氨酸酶，而不是从双孢蘑菇中分离酪氨酸酶，本研究构建了一个TYR表达盒。将表达盒电穿孔到里氏木霉Rut-C30中，并整合到其基因组中，产生重组菌株C30-TYR。用微晶纤维素诱导7天后，C30-TYR可以成功表达和分泌重组酪氨酸酶，相当于摇瓶培养物中约2.16g/L酪氨酸酶。通过硫酸铵沉淀和凝胶过滤纯化重组TYR，纯化的TYR的生物活性为45.6U/mL。纯化的TYR可以催化大豆球蛋白的交联，TYR处理的大豆球蛋白乳液的乳液稳定性指数比未处理的大豆球蛋白乳液提高了30.6％。与通过天然大豆球蛋白稳定的乳液相比，通过TYR交联大豆大豆球蛋白导致水包油乳液的性质改变。因此,用这种重组酪氨酸酶交联是改善蛋白质稳定的乳液和凝胶性能的可行方法。
