Separation, Purification And Structure Analysis Of Mycolic Acids In Corynebacterium Glutamicum

Corynebacterium glutamicum,Brevibacterium flavum,Brevibacterium lactofermentum are widely used for producing amino acids. These bacteria contain mycolic acids in their cell walls. Mycolic acids greatly reduce the permeability of cell wall and are closely related to the secretion of amino acids. Therefore, it is important to study the structure of mycolic acids in detail.In this paper, three wild strains (C. glutamicum ATCC 13032, B. lactofermentum ATCC 14047, B. flavum ATCC 14067) and two amino acid producing strains (IWJ-1 and VWJ-1) were used to study the separation, purification and structural analysis of mycolic acids. The results are listed below:1). The methods for separation and purification of mycolic acids were optimized; the method for detection of mycolic acids on thin-layer chromatography (TLC) were fetermined. Mycolic acids from five different strains were purified and analyzed by using TLC.2). The structure of mycolic acids was analyzed by ESI/MS. More than twenty molecular species of mycolic acids, differ in the length (20-40 carbons) and the saturation (0–3 double bonds), were observed in all five strains. The composition of mycolic acids in the five strains was different. ESI/MS/MS analysis of the three dominant species of mycolic acids (m/z 495.4, 521.4, 547.5) showed that the two hydrocarbon chains were similar in length (14-18 carbons), and the meromycolate chain usually contained double bonds.3). The influence of temperature on the structure of mycolic acids was investigated. As the growth temperature of cells increased from 30℃to 34℃, the proportion of mycolic acid species containing shorter and unsaturated hydrocarbon chains increased.4). The influence of incubation time on the composition of mycolic acids was investigated. The relative intensity of shorter and saturated mycolic acids (m/z 355.2, 411.2 and 439.3) firstly decreased then increased with the incubation time, but the saturated mycolic acids (m/z 495.3) showed the opposite pattern. Some unsaturated mycolic acids (m/z 437.3 and 547.3) were only observed in the stationary phase.5). The influence of isoniazid on the structure of mycolic acids was investigated. The cell growth rate was increased when isoniazid was added. ESI/MS analysis of mycolic acids from cells grown with 6mg/L isoniazidshowed that the relative intensity of shorter mycolic acids (m/z 315.2, 383.3, 411.3, 439.4 and 467.4) and the saturated mycolic acids (m/z 383.3, 411.3, 439.4, 467.4 and 537.4) decreased, while the relative intensity of unsaturated myclic acids (m/z 521.4 and 547.4) increased.