生物技术通报2024，Vol.40Issue(3)：322-332,11.DOI:10.13560/j.cnki.biotech.bull.1985.2023-0996
地衣素合成酶关键模块LchAD蛋白的性质和功能研究
Study on the Properties and Functions of LchAD Protein,a Key Module of Lichenysin Synthase
摘要
Abstract
[Objective]Lichenysin,as a kind of lipopeptide substance,can promote the formation of Baijiu flavor.To study the properties and functions of thioesterase module LchAD protein in lichenysin synthase,Bacillus strains were isolated from Daqu using traditional separation and purification methods.[Method]The fermentation products were detected by primary and secondary mass spectrometry,and the species relationship of strains was analyzed by morphological,physiological and biochemical experiments and phylogenetic tree construction.LchAD gene was cloned using genomic DNA of a selected strain as template,and LchAD protein was heterologously expressed in Escherichia coli and purified by nickel column.Bioinformatics analysis was performed with online software to explore the properties and functions of LchAD protein.[Result]The strain YC7 isolated from Daqu was Bacillus licheniformis,and its fermentation products contained the homologue of lichenysin A(D or G),indicating that strain YC7 was a lichenysin producing bacterium.The study on the soluble expression of LchAD protein showed that LchAD protein was induced to form soluble protein at the temperature of 20℃and the final concentration of IPTG was 100 μg/mL,with a molecular weight of 27.62 kD.The results of nickel column purification showed that the relatively pure LchAD protein was obtained when the imidazole concentration was 250 mmol/L.The results of bioinformatics analysis showed that LchAD protein was a stable hydrophilic protein without signal peptide,with a theoretical isoelectric point(pI)of 7.23 and a conserved GrsT domain.The secondary structure of LchAD protein consisted of 43.5%α-helix,13.82%β-folding and 5.69%β-turn,and its tertiary structure had a high similarity with the thioesterase module of surfactant synthetase.[Conclusion]The isolated B.licheniformis strain YC7 can produce lichenysin,and the protein encoded by the LchAD gene of this strain is a hydrophilic protein of 27.62 kD.Soluble LchAD protein can be obtained by heterologous expression,which may be functionally similar to the thioesterase module of surfactant synthetase.关键词
LchAD蛋白/地衣素/地衣芽孢杆菌/质谱检测/可溶性表达/生物信息学Key words
LchAD protein/lichenysin/Bacillus licheniformis/mass spectrometry detection/soluble expression/bioinformatics引用本文复制引用
杨伟杰,杨周林,朱浩东,魏煜,刘君,刘训..地衣素合成酶关键模块LchAD蛋白的性质和功能研究[J].生物技术通报,2024,40(3):322-332,11.基金项目
五粮液集团公司产学研合作项目(CXY2020ZR002),四川省科技厅自然科学基金青年项目(2022NSFSC1782),四川轻化工大学研究生创新基金资助项目(Y2022091) （CXY2020ZR002）
