Study Of Trans10, Cis12-Conjugated Linoleic Acid By Biocatalysis

trans10, cis12-Conjugated linoleic acid(t10, c12-CLA) has many biological and physiological functions, such as reducing the uptake of lipid, inhibiting the carcinogenesis and tumorigenesis, enhancing the immune, so it could be used in the development of weight loss and pharmaceutical products and possess high application values in foods, health care products, and drugs industry. Biosynthesis is the alternative approach to produce safe and highly specific t10, c12-CLA. However, the production of t10, c12-CLA using biosynthesis is too low to meet the industrial purpose.. Linoleic acids isomerases(PAI) from Propionibacterium acnes could catalyze the conversion of c9, c12-LA into single t10, c12-CLA. The recombinant Yarrowia lipolytica that PAI has been successfully heterologously expressed was constructed in our laboratory, which provided a basic for the biosynthesis of t10, c12-CLA.This paper focused on the purification of PAI from recombinant Yarrowia lipolytica, characterization and catalytic reaction kinetics of PAI, optimization of conditions and systems for production of t10, c12-CLA by whole cell catalysis.Firstly, the crude enzyme of the PAI was purified by successive chromatography on Hitrap DEAE FF, Hitrap Butyl HP and Superdux 200 10/300 GL. The results indicated PAI was successfully purified to homogeneity level, the molecular weight was about 48 kDa, yield of enzyme was about 28.35% and purification fold was about 9.78 times compared to original enzyme.Secondly, enzymatic properties and catalytic reaction kinetics of PAI were investigated. The optimum conditions for temperature and p H were 37℃ and 7.5, The stability of PAI behaved was significantly difference different when kept PAI was incubated at different temperature. Al3+ could improve enzyme activity about 8%, Mg2+ had no influence but the other metal ions displayed inhibiting effects. 0.1 M Tween-80 and CTAB(low concentration) increased PAI activity, and high concentration surfactant inhibited enzyme activity. Other fatty acids restrained catalysis reaction process of PAI. The data indicated that PAI was not subject to substrate inhibition when c9, c12-LA was used as substrate. The Michaelis constant Km of PAI was 84.68 μmol/L and Vmax was 2098.79 nmol/min/mg. Besides, product inhibition of PAI was observed and showed to be reversible and competitive.Thirdly, the preparation for whole-cell catalyst was studied. Adding 5 g/L oleic acid to the culture of Yarrowia lipolytica increased the periplasmic space which was observed by transmission electron microscopy(TEM). The findings might result in improving transport speed of substrates and products between cells and environments, so as to decrease conversion time by 20 h. Compared to the results of non-permeabilized cells, the treatment with freeze/thaw increased the t10, c12-CLA production with 13.81% and the cell viability remained 97.77%.Finally, the optimal conditions and systems of whole cell catalysis was achieved: 28℃, pH 7, 200 rpm, aerobic, 0.1 M phosphate buffer, 1.5 g/L sodium acetate, 25 g/L LA and reaction time 40 h. Furthermore, under current conditions, whole-cell catalyst can be reused for three cycles, with the t10, c12-CLA extracellular production remained above 10 g/L. The conversion process was carried out for 120 h with 75 g/L LA reusing whole-cell catalyst 3 cycles, the total production of extracellular t10, c12-CLA was 31.11 g/L and the conversion yield was 41.18%, which was the highest production of single t10, c12-CLA reported.This work was investigated on catalysis using enzyme and cell respectively, which provided a solid foundation for industrialized biosynthesis of t10, c12-CLA.