Optimization Of The Fermentation Conditions Of Engineering Strain Producing Glucose Oxidase

Glucose oxidase is widely existed in microbial,it is a very important enzyme for enzyme-using technology. It has been widely applied in field of food、medicine and biology etc,so it has a broad development prospect.The optimal of fermentation conditions is an important process to improve the volume of enzyme for foreign gene expression system. Fermentation medium of Pichia Yeast is expensive,we had constructed a genetically engineered strain in the early . We have studied the growth and inductive medium of the glucose oxidase engineering bacterium,and the fermentation conditions are optimized. Finally,a cheap high yield production process was established. Based on the shake flask conditions we constructed the 5 L fermenter amplification test.The fermentation conditions of pichia pastoris HL-69,including carbon source,nitrogen source,inorganic salt,metal iron and so on,were investigated in shake flask level. It was found that the concentrations of glucose,Soybean powder,KH2PO4 and MgSO4 had more obvious influences. The fermentation medium were studied via single factor test and orthogonal design of the four factors and three levels.The optimal growth medium was determined as follows:glucose 1.5%,Soybean powder 3.0%,KH2PO4 0.2%,MgSO4·7H2O 0.03%;The optimal fermentation medium was determined as follows:methanol 1.5%,NH3·H2O 3.5%,KH2PO4 0.5%,MgSO4·7H2O 0.03%.On the process of optimizing fermentation condition,impact of inoculation amount, methanol supply,growth initial pH,induce initial pH, medium volume on fermentation have been studied in shake flask level. And the best fermentation conditions were:inoculating time 18 h,inoculating amount 2%,medium volume 50 mL in 300 mL flask,growth initial pH 5.0,induce initial pH 6.0.Orthogonal test of the four factors three levels was designed to study the impact of enzyme production of the methanol supply,induce initial pH,growth initial pH,flask liquid volume. The results showed that R flask liquid volume > R methanol supply > R induce initial pH> R growth initial pH,the best combination of the four factors is: methanol supply 1.5%,medium volume 50 mL in 300 mL flask,initial pH 5.5,induce pH 5.5.Under such conditions,the activity of glucose oxidase enzyme was 6.516U/mL,which was 7.50 times of the original strain.Based on the results at shake flask level,scale up studies were carried out in 5 L fermentor. The metabolizing parameters including dissolution oxygen,pH,total sugar,deoxidize sugar,protein and glucose enzyme activity were tested.It laid a foundation for large scale fermentation.