Cyclodextrin production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R.

Cyclodextrins (CD) are cyclic oligosaccharides with multiple applications in the food, pharmaceutical, cosmetic, agricultural and chemical industries. In this work, the conditions used to produce CD with cyclodextrin glycosyltransferase from Bacillus circulans DF 9R were optimized using experimental designs. The developed method allowed the partial purification and concentration of the enzyme from the cultural broth and, subsequently, the CD production, using the same cassava starch as enzyme adsorbent and as substrate. Heat-treatment of raw starch at 70 degrees C for 15 min in the presence of adsorbed cyclodextrin glycosyltransferase allowed the starch liquefaction without enzyme inactivation. The optimum conditions for CD production were: 5% (w/v) cassava starch, 15 U of enzyme per gram of substrate, reaction temperature of 56 degrees C and pH 6.4. After 4h, the proportion of starch converted to CD reached 66% (w/w) and the weight ratio of alpha-CD:beta-CD:gamma-CD was 1.00:0.70:0.16.

Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R.

BACKGROUND: The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related alpha-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans. RESULTS: CGTase activity produced from Bacillus circulans DF 9R was optimised in shake flasks using a combination of conventional sequential techniques and statistical experimental design. Effects of nutrients, including several carbon, nitrogen and mineral sources, were assayed. The selected minimal medium consisted of 1.5 % cassava starch, 0.4 % ammonium sulphate, 0.1 M phosphate buffer, 0.002 % MgSO4 and 0.002 % FeSO4. The optimal concentrations of carbon and nitrogen sources were determined using a central composite design. Maximum CGTase activity obtained in supernatants was 5.8 U/mL in 48 h of incubation. Optimal conditions for enzyme production also included an initial pH of 8.3 and 37 degrees C as the incubation temperature.Cell growth and CGTase production profile were not linked to each other, suggesting that enzyme production/secretion is not growth-associated but mainly a late-log phase event. CONCLUSION: We have screened conditions for optimal CGTase production. The selected minimal medium contained starch, ammonium, Mg2+ and Fe2+ as essential nutrients. As an additional advantage, this medium does not require complex nitrogen sources with varying and unknown composition.