Studies On Fermentation Conditions Optimization And Stabilization Of Recombinant Lipoxygenase

Lipoxygenase(LOX,EC 1.13.11.12)was a kind of oxidoreductase that could catalyze polyunsaturated fatty acids with the specific structure forming hydrogen peroxides containing conjugated double bonds.This unstable compound could strengthen gluten of wheat flour and whiten it by oxidizing flour gluten proteins and carotenoids.Since having both effects on wheat flour and could be a potential substitute for chemical flour quality improvers,it had attracted wide attention of scholars at home and abroad.Currently,there had not yet appeared commercial grade LOX on the market.Soybean meal which was rich with LOX had many shortcomings,such as complex composition,ineffective function and so on.Nowadays,researchers began to focus on cloning and expression of heterologous LOX genes.However,LOX productivity was limited.In order to increase LOX production by recombinant Escherichia coli,this paper mainly screened fermentation medium and optimized culture conditions.On this basis,it studied on the batch fermentation conditions of recombinant E.coli for LOX production and its stabilization,which was intended to provide a foundation for industrial production and application of LOX.The main results were as follows:1.Optimization of fermentation conditions for LOX production by recombinant E.coli.Firstly,by screening of carbon sources,nitrogen sources and inorganic salts,the fermentation medium composed as follows:soy peptone 25 g/L,yeast extract 15 g/L,glycerol 6 g/L,KH2PO4 5 g/L,MgSO4·7H2O 2 g/L,MnCl2·4H2O 0.05 g/L.Secondly,we studied the effects of starting time of induction,inducer concentration,induction time,induction temperature,inoculum size,medium initial pH and medium volume on LOX production by single factor tests.Finally,three factors which had significant influences on fermentation were selected from these seven factors by using Plackett-Burman design.These were starting time of induction,induction time and medium initial pH.Simultaneously,the optimal LOX production conditions were determined by Box-Behnken design and response surface methodology,namely starting time of induction at OD600=1.6,inducing 34 h and medium initial pH at 7.0.The LOX activity was 21261.60±264.03 U/mL under these conditions,which was improved by 56.87%than before(13553.96±46.64 U/mL).2.Studies on batch fermentation conditions for LOX production by recombinant E.coli.Based on the optimal fermentation conditions in shake flask,batch fermentation curve of recombinant bacteria was measured to determine the cell density at about 3.0,5.0 and 7.0 to add inducer.By analyzing the curves of cell density,glycerol concentration,dissolved oxygen,pH and LOX activity with time,the optimal starting time of induction was at early logarithmic(OD600 at about 3.0),and LOX activity was 29498.40±244.38 U/mL.Controlling pH at 6.5,7.0 and 7.5 in the fermentation process showed that different constant pH had some influence on the enzyme production.When pH controlled at a relatively high level,cell growth and expression of recombinant LOX were promoted.The LOX activity was up to 31568.16±492.77 U/mL at pH7.0,which was improved by 48.47%than the optimization rseult.By controlling the constant oxygen levels in fermentation broth,cell growth was significantly accelerated and went to be stable after 24 h,and dissolved oxygen began to rebound.Although the activity was not improved much relatively,the fermentation period shortened from 64 h to 48 h approximately,increasing the intensity of LOX production.3.Studies on the stabilization of recombinant LOX.The stabilizers commonly used for liquid enzyme formulations in storage were screened by single factor tests,including metal salts,carbohydrates,polyols,macromolecular polymers,surfactants,amino acids,protease inhibitors and preservatives.It was given three stabilizers which had a significant protective effect on LOX enzyme activity,namely magnesium chloride,sucrose and Tween 80.Meanwhile,the optimum concentration range of these three additives were preliminarily determined.On this basis,the orthogonal experiment of three factors and three levels was designed,and through visual analysis and variance analysis,optimal composite stabilizer formulation was magnesium chloride 0.5%,sucrose 3.0%,and Tween 80 0.75%.Under this formulation,the enzyme activity retention rate was 80.14%and 74.16%when stored at 4? and 25? for thirty days respectively.