Pawlik Anna, Drozd Radosław, Janusz Grzegorz
Department of Biochemistry and Biotechnology, Institute of Biological Sciences, Maria Curie-Sklodowska University, Akademicka 19 St., 20-033 Lublin, Poland.
Department of Microbiology and Biotechnology, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology in Szczecin, 45 Piastow Avenue, 71-311 Szczecin, Poland.
Biomolecules. 2025 Apr 4;15(4):531. doi: 10.3390/biom15040531.
Due to their catalytic performance, laccases constitute one of the most promising groups of enzymes for potential applications in modern biotechnology. In this study, we aimed to chemically modify () and laccase and comparatively characterize the structures of both enzymes. The most characteristic feature was the spatial localization of lysine residues, predominantly positioned distal to the active site region for both compared enzymes. The solvent-accessible surface area (SASA) analysis showed that bacterial laccase was characterized by a larger hydrophobic SASA than the fungal enzyme. The pK prediction identified only one Lys in the laccase structure susceptible to modification. Modifications were achieved by using mono- and bifunctional crosslinking agents, and glycosylations were also performed. The degree of protein modification ranged from 0% for glucose- and galactose-modified laccase and citraconic anhydride-modified (CA) laccase to 62.94% for the palmitic acid N-hydroxysuccinimide ester-modified enzyme. The stability of covalently modified laccases over a wide pH and temperature ranges and in the presence of inhibitors was investigated. Protein modifications with polymeric sucrose (PS) and ethylene glycol bis-(succinimidyl succinate) (EGNHS) significantly increased the activity of the bacterial and fungal laccases by 15 and 19%, respectively. Although pH optima remained relatively unchanged by modifications, certain variants, especially CA-modified bacterial protein and EGNHS-modified enzyme, exhibited improved stability at near-neutral pH (6-7). Modification of the bacterial enzyme with glutaraldehyde-carbodiimide (GA-CDI-ver) and of the fungal enzyme with CA was the most effective in improving its thermal stability. Chemical modifications using GA, CDI, GA-CDI, and PS allowed L 3.8 laccase to retain full activity in the presence of 5 mM NaI, whereas CA-, PS-, and EGNHS-modified variants retained their activity even at elevated NaCl concentrations. The results clearly demonstrate that the outcome of chemical modifications is closely linked to enzyme-specific structural features and that selecting an appropriate modification strategy is critical to achieving the desired effect.
由于其催化性能，漆酶是现代生物技术中最具应用潜力的酶类之一。在本研究中，我们旨在对（）和漆酶进行化学修饰，并比较这两种酶的结构特征。最显著的特征是赖氨酸残基的空间定位，对于这两种被比较的酶而言，它们主要位于活性位点区域的远端。溶剂可及表面积（SASA）分析表明，细菌漆酶的疏水性SASA比真菌酶更大。pK预测表明，在漆酶结构中只有一个赖氨酸易于修饰。修饰是通过使用单功能和双功能交联剂实现的，并且还进行了糖基化。蛋白质修饰程度从葡萄糖和半乳糖修饰的漆酶以及柠康酸酐修饰的（CA）漆酶的0%到棕榈酸N-羟基琥珀酰亚胺酯修饰的酶的62.94%不等。研究了共价修饰漆酶在广泛的pH和温度范围内以及在抑制剂存在下的稳定性。用聚合蔗糖（PS）和乙二醇双（琥珀酰亚胺琥珀酸酯）（EGNHS）进行蛋白质修饰分别使细菌和真菌漆酶的活性显著提高了15%和19%。尽管修饰后最适pH相对保持不变，但某些变体，特别是CA修饰的细菌蛋白和EGNHS修饰的酶，在近中性pH（6 - 7）下表现出更高的稳定性。用戊二醛 - 碳二亚胺（GA - CDI - ver）修饰细菌酶以及用CA修饰真菌酶在提高其热稳定性方面最为有效。使用GA、CDI、GA - CDI和PS进行化学修饰可使L 3.8漆酶在5 mM NaI存在下保持完全活性，而CA、PS和EGNHS修饰的变体即使在较高NaCl浓度下仍保留其活性。结果清楚地表明，化学修饰的结果与酶的特定结构特征密切相关，选择合适的修饰策略对于实现预期效果至关重要。
