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Preparation and characterization of a laccase-like enzyme from Thermomicrobium roseum

文献类型： 外文期刊

作者： Hao, Mengyao 1 ; Huang, Aimin 1 ; Li, Bingjie 1 ; Xin, Yu 1 ; Zhang, Liang 1 ; Gu, ZhengHua 1 ; Sun, Haiyan 3 ; Li, Youran 1 ; Shi, Guiyang 1 ;

作者机构： 1.Jiangnan Univ, Engn Res Ctr Cereal Fermentat & Food Biomfg, Wuxi 214122, Peoples R China

2.Jiangnan Univ, Jiangsu Prov Engn Res Ctr Bioact Prod Proc, Wuxi 214122, Peoples R China

3.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Hainan Key Lab Trop Microbe Resources, Haikou 571101, Peoples R China

4.Jiangnan Univ, 1800 Lihu Rd, Wuxi 214122, Jiangsu, Peoples R China

关键词： Thermomicrobium roseum Lac -like; Bacillus subtilis; Characterization; Inosine

期刊名称：INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES （ 影响因子：8.2； 五年影响因子：7.8 ）

ISSN： 0141-8130

年卷期： 2023 年 242 卷

页码：

收录情况： SCI

摘要： In this study, a laccase-like gene from Thermomicrobium roseum DSM 5159 (TrLac-like) (NCBI: WP_012642205.1) was recombinantly expressed in Bacillus subtilis WB600. The optimum temperature and pH for TrLac-like were 50 degrees C and 6.0, respectively. TrLac-like showed high tolerance to mixed systems of water and organic solvents, indicating its potential for large-scale application in various industries. It showed 36.81 % similarity with YlmD from Geobacillus stearothermophilus (PDB:6T1B) in sequence alignment; therefore, 6T1B was employed as the template for homology modeling. To improve catalytic efficiency, amino acid substitutions within 5 angstrom of the inosine ligand were simulated to reduce the binding energy and promote substrate affinity. Single and double substitutions (44 and 18, respectively) were prepared, and the catalytic efficiency of the mutant A248D was increased to approximately 110-fold that of the wild type, while the thermal stability was maintained. Bioin-formatics analysis revealed that the significant improvement in catalytic efficiency could be attributed to the formation of new hydrogen bonds between the enzyme and substrate. With a further decrease in the binding energy, the catalytic efficiency of the multiple mutant H129N/A248D was approximately 14-fold higher than that of the wild type but lower than that of the single mutant A248D. This is possibly because kcat also decreased with the decrease of Km; consequently, the substrate could not be released in time owing to the enzyme with the combination mutation not being able to release the substrate at a high rate.

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