Characterization of an efficient N-oxygenase from Saccharothrix sp. and its application in the synthesis of azomycin

被引:1
|
作者
Fan, Chuanle [1 ,2 ,3 ]
Zhou, Fang [1 ,2 ,3 ]
Huang, Wei [1 ,2 ,3 ]
Xue, Yi [1 ,2 ,3 ]
Xu, Chao [1 ,2 ,3 ]
Zhang, Rubing [1 ,2 ,3 ]
Xian, Mo [1 ,2 ,3 ]
Feng, Xinjun [1 ,2 ,3 ]
机构
[1] Chinese Acad Sci, Qingdao Inst Bioenergy & Bioproc Technol, CAS Key Lab Biobased Mat, Qingdao 266101, Peoples R China
[2] Shandong Energy Inst, Qingdao 266101, Peoples R China
[3] Qingdao New Energy Shandong Lab, Qingdao 266101, Peoples R China
来源
BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS | 2023年 / 16卷 / 01期
基金
中国国家自然科学基金;
关键词
Heme-oxygenase-like diiron oxygenase; Nitro compounds; Site-directed mutation; Whole-cell biocatalysis; Nitroimidazoles; DIIRON ENZYME; BIOSYNTHESIS; AURF; IDENTIFICATION;
D O I
10.1186/s13068-023-02446-5
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background The nitro group constitutes a significant functional moiety within numerous valuable substances, such as nitroimidazoles, a class of antimicrobial drugs exhibiting broad spectrum activity. Conventional chemical methods for synthesizing nitro compounds suffer from harsh conditions, multiple steps, and environmental issues. Biocatalysis has emerged as a promising alternative to overcome these drawbacks, with certain enzymes capable of catalyzing nitro group formation gradually being discovered in nature. Nevertheless, the practical application is hindered by the restricted diversity and low catalytic activity exhibited by the reported nitrifying enzymes.Results A novel N-oxygenase SaRohS harboring higher catalytic capability of transformation 2-aminoimidazole to azomycin was characterized from Saccharothrix sp. Phylogenetic tree analysis revealed that SaRohS belongs to the heme-oxygenase-like diiron oxygenase (HDOs) family. SaRohS exhibited optimal activity at pH 5.5 and 25 degrees C, respectively. The enzyme maintained relatively stable activity within the pH range of 4.5 to 6.5 and the temperature range of 20 degrees C to 35 degrees C. Following sequence alignment and structural analysis, several promising amino acid residues were meticulously chosen for catalytic performance evaluation. Site-directed mutations showed that threonine 75 was essential for the catalytic activity. The dual mutant enzyme G95A/K115T exhibited the highest catalytic efficiency, which was approximately 5.8-fold higher than that of the wild-type and 22.3-fold higher than that of the reported N-oxygenase KaRohS from Kitasatospora azatica. The underlying catalytic mechanism was investigated through molecular docking and molecular dynamics. Finally, whole-cell biocatalysis was performed and 2-aminoimidazole could be effectively converted into azomycin with a reaction conversion rate of 42% within 14 h.Conclusions An efficient N-oxygenase that catalyzes 2-aminoimidazole to azomycin was screened form Saccharothrix sp., its phylogenetics and enzymatic properties were analyzed. Through site-directed mutation, enhancements in catalytic competence were achieved, and the molecular basis underlying the enhanced enzymatic activity of the mutants was revealed via molecular docking and dynamic simulation. Furthermore, the application potential of this enzyme was assessed through whole cell biocatalysis, demonstrating it as a promising alternative method for azomycin production.
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页数:12
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