Mechanism On The Promoting 2-keto-L-gulonic Acid Production Of Ketogulonicigenium Vulgare By The Siderophores From Bacillus Pumilus In Vc Co-culture Fermentation

The mechanism of 2-keto-L-gulonic acid(2-KGA)production promoted by siderophores from Bacillus pumilus SH-B9 in vitamin C(Vc)co-culture fermentation composed with Ketogulonigenium vulgare 25B-1 and Bacillus pumilus SH-B9 were studied by using microbial physiology-biochemistry and molecular biology technology.The results were as followed:(1)We studied the different 2-KGA-productivities with different helper stains in the Vc co-culture fermentation system.We ascribed the different 2-KGA-productivities to the varied growth characteristics and siderophores production of the different helper strains.The slower growth with longer lag phase of the helper stain could prolong the fermentation time,in contrast,the rapid growth with shorter lag phase of the helper stain could lead to the lower 2-KGA production.The varied siderophores production of the different helper stains also could influence the 2-KGA-productivities.The higher siderophores production of the helper stain could lead to the higher 2-KGA production in the Vc co-culture fermentation system.Therefore,B.pumilus SH-B9 was selected as the research strain for the futher study.(2)The different concentration addition of Fe3+ could influence the production of siderophores and 2-KGA production in the mono-and co-culture fermentation system.With the increase of Fe3+ concentration,the production of siderophores in helper strain decreased gradually.In the co-culture fermentation system,the Fe3+ concentration increased,the 2-KGA production increased.In the mono-culture fermentation system,1 ?mol/L Fe3+ additional could increase the 2-KGA production,while further increasing the Fe3+ concentration could decrease the 2-KGA production,perhaps due to the iron metabolism balance was broken and effected the growth and 2-KGA production of K.vulgare 25B-1.(3)In order to identify the solubility of siderophores in organic solvents with different polarity,we got the ultrafiltration powder by vacuum freeze-drying technology after intercepting extracellular of B.pumilus SH-B9 with a molecular weight of not more than 5000 Da.Using chromeazurol S(CAS)to specific detect siderophores.It was found that a small part of the siderophores was soluble in chloroform,slight part was soluble in acetone,partial part was soluble in methanol.After,the insoluble part was filtered and dissolved in ultra pure water,the CAS test showed positive results.The siderophores produced by B.pumilus SH-B9 could be mixtures.The ultrafiltration powder was preliminarily separated and purified by Sephadex LH-20 eluted with 20% methanol water solution,and the CAS detection solution was used to detect the collected elution fractions liquid.The highest activity components were collected and analyzed by high performance liquid chromatography(HPLC).The results showed that Sephadex LH-20 had a higher separation effect and could get purer siderophores.(4)The purer siderophores could promote 2-KGA production in the mono-culture fermentation system.With increasing addition of the siderophores,the 2-KGA production was increased.With addition of 500 ?g/L the purer siderphores,the production of 2-KGA was increased by 71.45% in K.vulgare 25B-1 fermentatoin system.(5)Siderophores could improve the expression of important genes in the iron uptake and metabolism system of K.vulgare 25B-1.Siderophores could improve the iron-sulfur protein gene expression of respiratory chain and the key genes related to ATP synthesis expression in K.vulgare 25B-1.Siderophores also improved the gene expression of the antioxidant enzymes which could use iron as a cofactor in K.vulgare 25B-1 to resist its oxidative stress.Siderophores could improve the gene expression increase of L-sorbosone dehydrogenase and L-sorbose dehydrogenase to increase the 2-KGA production.