Studies On The Screening And Fermentation Of Gyclodextrin Glucanotransferase Higher Production Strain MS-UD5 And Its Enzymatic Characteristics

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is a unique enzyme capable of converting starch and related substrates into cyclodextrins (CDs). Central gaps in CD molecules can be filled with chemical compounds, forming complex inclusions and leading to improvement of physical and chemical properties of the compounds. Therefore, CD products have been widely used in pharmaceutical, food, cosmetic, and plastic industries, which make it very important to study the production of CGTase. In this study, a higher CGTase production strain MG-UD5 was obtained, and an emphasis was placed on optimization of fermentation conditions for production of the CGTase by the strain MG-UD5 by flask culture and in 5 L-fermentor; also some enzymatic characteristics were studied. The results are summarized as follows:1. At first, The morphological physiological and biochemical characteristics of the original strain Bacillus alcalophilus IS was studied .Then it was mutated by UV, DES, He-Ne laser and UV+ DES. The optimal mutation effect were obtained by UV 50s , DES 40min, He-Ne laser 25min and 50s UV+30min DES; we use a rapid method for screening the higher production strain, also the stability of the mutants were studied, finally the mutant MS-UD5 was obtained and its enzyme productivity was 5278.8 U/mL, and it is 2.99 fold higher than that of original strain.2. The optimal fermentation conditions for CGTase production were determined by a single-factorial experiment and 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 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 5741.6 U/mL.3. In 5 L-fermentor ,it is seen that during the exponential phase the dissolution oxygen of medium was very low. It is also seen that the pH of medium dropped at the prophase and increased after the metaphase while the CGTase concentration increased continually. During the stationary phase, the dissolution oxygen, pH of medium returned to their original level, but the maximal yield of CGTase yield was obtained at the 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 5 L-fermentor, the strain could produce as much as 6032.5 U/mL.4. The optimal conditions for reaction of the CGTase produced by MS-UD5 was pH 6.0 at 60℃. The enzyme was stable in a range of pH 5.0-10.0 ,at the temperature below 70℃. The enzyme was inactivated at the presence of Cu²⁺,Ag⁺,Al³⁺,Fe²⁺ at the level of 1mmol/L, and 50% inactivated by Tween 80;whereas it was activated at the same level of Mn²⁺> Ca²⁺; However, Mg²⁺, K⁺,Zn²⁺,Cr³⁺,Tween20å’ŒTriton X-100 had insignificant effect on the activity of the CGTase concerned.