Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Republic of Korea.
Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Republic of Korea.
Biotechnol J. 2021 Dec;16(12):e2100216. doi: 10.1002/biot.202100216. Epub 2021 Sep 29.
Catechol-containing polymers such as mussel adhesive proteins (MAPs) are attractive as biocompatible adhesive biomaterials, and the catecholic amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) is considered a key molecule in underwater mussel adhesion. Tyrosinases can specifically convert tyrosine to DOPA without any cofactors. However, their catalytic properties still need to be adjusted to minimize unwanted DOPA oxidation via their diphenolase activity and catechol instability at neutral and basic pH values in the reaction products.
In this work, we constructed a novel functional tyrosinase, mTyr-CNK_CBM, by fusion of mTyr-CNK with a cellulose-binding motif (CBM) for oriented in situ immobilization on microcrystalline cellulose via the C-terminal CBM without any additional purification steps. mTyr-CNK_CBM showed optimal catalytic activity at pH 4.5-6.5 and room temperature and had a high monophenolase/diphenolase activity ratio (V mono/V di = 2.08 at pH 6 and 25°C). mTyr-CNK_CBM exhibited 2.17-fold higher (as a unimmobilized free enzyme) and similarly high (upon immobilization) in vitro DOPA modification of a bioengineered MAP compared to a commercially available mushroom tyrosinase. Moreover, the immobilized mTyr-CNK_CBM showed long-term storability and improved reusability.
These results clearly demonstrate a strong potential for practical use of immobilized mTyr-CNK_CBM as a monophenol monooxygenase in preparing biocompatible DOPA-tethered biomaterials and other catechol-containing polymers.
含儿茶酚的聚合物，如贻贝黏附蛋白（MAPs），作为生物相容的黏附生物材料具有吸引力，而儿茶酚氨基酸 3,4-二羟基苯丙氨酸（DOPA）被认为是水下贻贝黏附的关键分子。酪氨酸酶可以特异性地将酪氨酸转化为 DOPA，而无需任何辅因子。然而，它们的催化特性仍需要调整，以最小化通过其多酚氧化酶活性和在反应产物中中性和碱性 pH 值下的儿茶酚不稳定性而产生的不需要的 DOPA 氧化。
在这项工作中，我们通过融合 mTyr-CNK 与纤维素结合基序（CBM）构建了一种新型功能型酪氨酸酶 mTyr-CNK_CBM，通过 C 末端 CBM 可以在无需任何额外纯化步骤的情况下将其定向原位固定在微晶纤维素上。mTyr-CNK_CBM 在 pH4.5-6.5 和室温下表现出最佳的催化活性，并且具有高的单酚酶/二酚酶活性比（在 pH6 和 25°C 时 V mono/V di =2.08）。与市售的蘑菇酪氨酸酶相比，mTyr-CNK_CBM 表现出 2.17 倍更高的（作为未固定的游离酶）和类似的高（固定后）体外生物工程 MAP 的 DOPA 修饰。此外，固定化的 mTyr-CNK_CBM 具有长期储存能力和提高的可重复使用性。
这些结果清楚地表明，固定化的 mTyr-CNK_CBM 作为单酚单加氧酶在制备生物相容的 DOPA 键合生物材料和其他含儿茶酚的聚合物方面具有很强的实用潜力。
