Oxygen Vector Enhanced Oxygen Transfer To Promote The Synthesis Of AD By Mycobacterium

Androst-4-ene-3,17-dione(AD)is an important intermedium of sterol hormones,which can be obtained via the side chain cleavage of phytosterol(PS)by Mycobacterium.PS transformation by Mycobacterium is strongly dependent on dissolved oxygen concentration.The dissolved oxygen has important influence on the AD accumulation.Two-phase fermentation system with the addition of oxygen vectors become a positive trend in this field.In this paper,the biotransformation of PS to AD by Mycobacterium neoaurum TCCC 11028 M3 AksdD(MNR M3△ksdD)was selected as a model system for evaluating different oxygen vectors on AD yield.The mechanism underlying the effects of oxygen vector on the sidechain degradation of PS were also analyzed.Firstly,a reliable sampling method was established,secondly,several oxygen vectors(n-hexadecane,perfluorohexane,soybean oil,polydimethyl siloxane,polymethylphenylsiloxane)were selected to investigate their transformability for PS.The rusults showed that 16%(v/v)soybean oil was proved to be the most suitable for AD production,the largest molar yield of AD was 52.68%at the fifth day.Optimization results of soybean oil indicated that the addition of 16%(v/v)soybean oil to the liquid medium at the time of inoculation was found to be optimal,the peak molar yield of AD(57.50%)was achieved at the third day,increased by 6-fold higher than the control group.The effects of soybean oil on dissolved oxygen were investigated.The results showed that soybean oil could improved the oxygen volumetric mass transfer rate(kLa),when the concentration of soybean oil>2.5%,kLa increased almost linearly with the increase of soybean oil.The intracellular nucleotide levels and the enzyme activities involved in TCA cycle were monitored.Higher ATP level and a lower NADH/NAD+ratio were obtained with 16%soybean oil in broth,both of the activities of isocitrate dehydrogenase(ICDHc)and a-ketoglutarate dehydrogenase(a-KGDH)were obviously up-regulated.Analysis of metabolic pathways showed that the electron transfer chain(ETC),oxidative phosphorylation and tricarboxylic acid(TCA)cycle of MNR were stimulated with soybean oil,which resulted in enhanced AD yield.Based on this,we deduced that the main cofactor metabolism of PS side chain cleavage in MNR.The effects of soybean oil on the cell dehydrogenase activity and permeability were studied.After pretreatment by 16%soybean oil,the dehydrogenase activity remained at 85%above,the alteration of cell permeability was observed but cells did lose neither catalytic activity nor cell integrity,which also possibly favorable influenced sterol uptake.This may be another reason that soybean oil raised the PS conversion.The synergistic effects of oxygen vector and different surfactants for AD biosynthesis were studied.Results showed both of lecithin and Tween 80/Span 80 exhibited promoting effect on AD biosynthesis.The best effection is lecithin,the percentage yield of AD reach 68.09%with 0.6%lecithin in soybean oil.