Cloning and expression of a novel trans-anethole oxygenase gene from Paraburkholderia sp. MR185

• Published in: Journal of general and applied microbiology Vol. 68; no. 3; pp. 163 - 167
• Main Authors: Lin, Qian, Li, Jieni, Ling, Xinru, Zhang, Xinmei
• Format: Journal Article
• Language: English
• Published: Tokyo Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 01-01-2022; Japan Science and Technology Agency
• Subjects: Affinity chromatography; Anethole; Carbon sources; Cellulose; Cloning; E coli; Enzymatic activity; Enzyme activity; Enzymes; Gel electrophoresis; High performance liquid chromatography; Liquid chromatography; Molecular weight; Oxidation; Oxygenase; p-anisaldehyde; Paraburkholderia; Proteins; Pseudomonas putida; Sodium lauryl sulfate; trans-anethole; trans-anethole oxygenase
• ISSN: 0022-1260; 1349-8037
• DOI: 10.2323/jgam.2021.10.001

trans-Anethole oxygenase (TAO) is the key enzyme responsible for the oxidation of trans-anethole to p-anisaldehyde. A strain, Paraburkholderia sp. MR185, was isolated from soil in Yulin star anise-planting regions using trans-anethole as a sole carbon source and a gene which encodes a protein with high similarities to a hypothetical protein of Paraburkholderia sp. MM5384-R2 which shows 61.27% identies with TAO from Pseudomonas putida JYR-1 was cloned and sequenced. The gene, tao, was expressed in E. coli cells and its protein product was purified by affinity chromatography through regenerated amorphous cellulose (RAC). SDS-PAGE analysis indicated a clear band of recombinant protein TAO, and its molecular weight, 38.3 kDa, was consistent with the theoretical value. Its enzyme activity of producing p-anisaldehyde from trans-anethole was detected by DNPH (2,4-dinitrophenylhydrazine) chromogenic reaction and HPLC, and the specific activity of TAO reached 3.93 U/mg protein. Immobilized TAO on RAC was used to catalyze the production of p-anisaldehyde from trans-anethole, and the enzyme retained more than 60% of its initial activity after 10 uses. This is the first report on Paraburkholderia TAO.