细菌酪氨酸酶高效异源表达及其在生物染发和丝素多肽多巴修饰中的应用

Heterologous expression of bacterial tyrosinase and its applications in biological hair dyeing and DOPA modification of hydrolyzed silk fibroin

摘要:

酪氨酸酶是一种含铜的多酚氧化酶，广泛应用于食品、日化、医药等领域。目前商品化酪氨酸酶主要依赖于真菌提取，存在价格高、纯度低、比酶活低和稳定性差等问题。本研究旨在获得高效表达并具有工业化应用前景的细菌酪氨酸酶。通过在大肠杆菌(Escherichia coli) BL21(DE3)中异源表达5种不同来源的细菌酪氨酸酶，筛选获得了巨大芽孢杆菌(Bacillus megaterium)和多刺疣微菌(Verrucomicrobium spinosum)来源的酪氨酸酶TyrBm和TyrVs，酶活分别为(16.1±0.2) U/mL和(48.6±0.9) U/mL。纯化后通过比较TyrBm与TyrVs的酶学性质，证明了TyrVs具有更高的热稳定性和底物专一性。在表征TyrVs高催化性能的基础上，建立了基于TyrVs催化的酶法生物染发体系，实现了原位催化染发，水洗色牢度实验测得模拟14 d清洁后的色差值△E低于7.38±0.64。为便于催化产物与酶快速分离，成功构建了依赖于自组装标签CipA的固定化酶TyrVs-CipA催化体系并应用于水解丝素多肽(hydrolysed silk fibroin, HSF)的多巴修饰(DOPA modification)，酶连续催化达7次以上，单次多巴修饰度超过70.00%。进一步研究表明，多巴修饰使HSF对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-trinitrophenylhydrazine, DPPH)自由基与O₂^-自由基清除活性分别提高了507.80%与78.23%。本研究为基于酪氨酸酶的绿色生物染发剂及组织工程生物材料开发提供了技术基础。

关键词:细菌酪氨酸酶;固定化酶;生物染发剂;水解丝素;多巴修饰

Abstract:

Tyrosinase is a copper-containing polyphenol oxidase widely applied in the food, cosmetics, pharmaceutical, and other industries. Currently, the production of commercial tyrosinase primarily relies on extraction from fungi, which has high costs, low purity, low specific activity, and poor stability. The objective of this study is to obtain highly expressed bacterial tyrosinase with potential for industrial applications. The bacterial tyrosinases from five different sources were heterologously expressed in Escherichia coli BL21(DE3), and the tyrosinases TyrBm and TyrVs derived from Bacillus megaterium and Verrucomicrobium spinosum were obtained with the enzyme activities of (16.1±0.2) U/mL and (48.6±0.9) U/mL, respectively. After protein purification, we compared the enzymatic properties of TyrBm and TyrVs, which revealed that TyrVs exhibited better thermal stability and higher substrate specificity than TyrBm. On the basis of characterizing TyrVs with high catalytic performance, we established a biological hair dyeing system based on TyrVs catalysis to achieve in-situ catalytic hair dyeing. The color washing fastness test measured the △E value less than 7.38±0.64 after simulated 14-day cleaning. To facilitate the rapid separation of catalytic products and enzymes, we successfully constructed an immobilized enzyme TyrVs-CipA dependent on self-assembly label CipA and applied this enzyme in the DOPA modification of hydrolyzed silk fibroin (HSF). The immobilized enzyme continuously catalyzed HSF for more than seven cycles, resulting in a single DOPA modification degree exceeding 70.00%. Further investigations demonstrated that DOPA modification enhances the scavenging activity of HSF towards DPPH and O2- radicals by 507.80% and 78.23%, respectively. This study provides a technical foundation for the development of environmentally friendly biological hair dye based on tyrosinase and biomaterials for tissue engineering.

Key words:bacterial tyrosinase;immobilized enzyme;biological hair dye;hydrolyzed silk fibroin;DOPA modification