Secretion Expression In Escherichia Coli And Fermentation Optimization Of β-CGTase From Bacillus Xiaoxiensis STB08

Cyclodextrin glycosyltransferase（CGTase,EC 2.4.1.19）can utilize starch,malto-oligosaccharide and other glucose polymers to generate cyclodextrin through cyclization reaction.Among them,β-CGTase is the most widely used.The CGTase used in the industrial production of cyclodextrins are mainly from natural Bacillus,a small amount of which are genetically engineered bacteria,but most of the CGT enzymes have relatively low thermal stability,and half-life at 60°C is less than 10 min,while theβ-CGTase derived from Bacillus xiaoxiensis STB08 have a higher thermal stability（half-life at 60°C is 12.5 min）.The high thermal stability makes it suitable for industrial applications.But at present,due to the low level of fermentation enzymes produced by its natural strains,the enzyme activity is only about 10U/m L.So it is necessary to improve the expression ofβ-CGTase.Based on this,this study constructed an extracellular expression system ofβ-CGTase derived from B.xiaoxiensis STB08in Escherichia coli,and optimized the conditions for the production of the enzyme by shake flask fermentation.Analyses were performed and production scaled up in fermentors with a view to better directing the industrial production of the enzyme.The main research contents and results are as follows:（1）The cgt gene derived from B.xiaoxiensis STB08 was inserted into the plasmid p ET-20b（+）,and the E.coli expression system of the gene was constructed,and the extracellular expression ofβ-CGTase was successfully realized.The enzyme activity reached 11.46 U/m L after fermentation in the medium.Then,on the basis of TB medium,after optimization of temperature,fermentation time,initial p H value,carbon source and its concentration and compounding,nitrogen source and its concentration and compounding,etc.,corn steep liquor24 g/L,soy peptone 6 g/L,glucose 2 g/L,maltose 4 g/L,KH₂PO₄ 2.32 g/L,K₂HPO₄ 3H₂O16.43 g/L as optimal fermentation medium.The extracellular enzyme activity reached 34.66U/m L after the recombinant E.coli was fermented at 30°C and p H 7.0 for 96 h,which was138.1%higher than that before optimization（TB medium）.（2）When optimizing conditions such as metal ions and dissolved oxygen,it was found that changing dissolved oxygen and adding a certain final concentration of calcium ion had a significant impact on the growth,reproduction and enzyme production of recombinant E.coli.The enzyme activity reached 66.86 U/m L under the optimal dissolved oxygen（that is,the filling volume was 100 m L/250 m L）.And the addition of calcium ion with a final concentration of 25m M in 50 m L of fermentation broth could increase the enzyme activity to 83.15 U/m L.At the same time,it was found that dissolved oxygen and calcium ion have a synergistic effect on the growth and enzyme production of recombinant E.coli.When the dissolved oxygen is high（that is,the volume of the solution is 30 m L/250 m L）,the final concentration of calcium ion is 25m M,The enzyme activity finally reached 105.69 U/m L,which was 204.9%higher than that of the control group with no change in dissolved oxygen and no calcium ion added.（3）In order to explore the effect of dissolved oxygen and calcium ion on the growth,reproduction and enzyme production of recombinant E.coli.Subcellular localization analysis,flow cytometry analysis and cold field emission scanning electron microscopy were performed on the recombinant E.coli cells after fermentation.The effects of them on the permeability of the inner and outer membranes of the cells were determined during the fermentation process,respectively.The results show that lower dissolved oxygen can reduce the formation of recombinant E.coli inclusion bodies,and the cell activity is higher when the dissolved oxygen is low.Under the condition that the suitable growth concentration of the bacteria can be guaranteed,avoiding the rapid growth as much as possible is to improve the production.The key to the amount of enzyme.The addition of calcium ion can also reduce the formation of recombinant E.coli inclusion bodies,and calcium ion have a strong protective effect on cells,and the number of viable cells increases significantly after adding calcium ions.The morphology became longer,the surface area of the cells increased,and the permeability of the outer membrane of the cells increased significantly.Under the synergistic effect of dissolved oxygen and calcium ion,while ensuring a high growth concentration of bacteria,calcium ion increase the permeability of the outer cell membrane and protect cells,so that the extracellular enzyme activity reaches the highest level.（4）Finally,the industrial production of recombinantβ-CGTase was simulated at the fermenter level,and the fermentation conditions in the shake flask stage were verified.The results showed that the effects of dissolved oxygen and calcium ion on the fermentation of recombinantβ-CGTase were similar to shake flask stages are consistent.That is,under the condition that the normal growth concentration of recombinant E.coli can be guaranteed,avoiding its rapid growth as much as possible can improve the extracellular enzyme activity.When the DO value of dissolved oxygen is 30%,the enzyme activity is the highest,which is39.50 U/m L.Dissolved oxygen and calcium ion also have a synergistic effect on the growth of recombinant E.coli and extracellular enzyme production.Under higher dissolved oxygen DO（when the DO value is 50%）,adding calcium ion with a final concentration of 25 m M,extracellular.The enzyme activity reached the highest at 80.24 U/m L,which was 95.4%higher than that of the control group with DO of 30%and no calcium ion added.