Studies On The Fermentation Of Cycoldextrin Glycosy Transferase Strain And The Immobilization Of The Enzyme

In this study, a higher Cycoldextrin Glycosy Transferase (CGTase) production strain HAUV-4 was obtained, and an emphasis was placed on optimization of fermentation conditions for production of the CGTase by the strain HAUV-4 by flask culture and in 5 L-fermentor, the condition of CGTase immobility and some enzymatic characteristics between immobilized enzyme and free enzyme were studied. The results are summarized as follows:1. At first, the original strain Bacillus alcalophilus IS whose enzyme productivity is 1764.7U/mL. Then it was mutated by HNO₂, acridine yellow, UV and UV+LiCl. The optimal mutation effect were obtained by HNO₂ 0.25mol/L, acridine yellow 20μg/mL and 50s UV+1% LiCl; We use a rapid method for choosing the higher production strain, also the stability of the mutants were studied, finally the mutant HAUV-4 was obtained and its enzyme productivity was 5389.7U/mL, and it is 3.05 higher than that of original strain.2. The optimal fermentation conditions for CGTase production were determined by orthogonal experiment. The compositions of fermentation medium were maize powder 2%, corn steep liquor 6%, K₂HPO₄ 0.15%, MgSO₄·7H₂O 0.02%; The incubation temperature is 30℃; amount of inoculum is 10%; pH 9.8. The load of 250mL-flask was 40 mL, and the shaking incubation took 36 h at 180 r/min and the enzyme activity could reach as high as 5770.4U/mL.3. In 5L-fermentor,the discipline was studied of HAUV-4, at the same time the fermentation conditions were optimized. By increasing the dissolution oxygen of medium at the exponential phase, the time to reach enzyme activity peak was shortened. In 5L-fermentor, the strain could produce as much as 6054.4U/mL. Also the change of total sugar was studied in 5L-fermentor.4. The optimal condition of immobilized CGTase by sodium alginate is sodium alginate 7% (the final concentration is 2.3%) , CaCl₂ 3% ,the ratio of sodium alginate and enzyme 1:2,2.5h immobilization time.5. The prime temperature of immobilized enzyme and free enzyme are 60℃, the prime pH between them are 5.0 and 9.0.But the immobilized enzyme is higher than free enzyme in temperature stability, also the pH stability of immobilized enzyme is more abroad.