Ispitivanje proizvodnje ciklodekstrina pomoću ciklodekstrin glukanotransferaze iz bakterije Bacillus megaterium

The process of producing cyclodextrins from starch with cyclodextrin glucanotransferase from Bacillus megaterium has been studied. The effect of starch and enzyme concentrations was evaluated using optimal composite designs and the response surface methodology. Mathematical models describing the process in two overlapping areas of variation of the independent variables were developed. The models were used to determine the optimal values of the variables. The increase in starch concentration resulted in an increase in the amount of β-cyclodextrin formed, but lowered the yield. The optimal starch concentration was considered to be 50.0 mg/mL. The critical cyclodextrin glucanotransferase concentration necessary for maximum β-cyclodextrin production was 2.0 U/g. Factors that limit the complete conversion of starch to cyclodextrins were established. The enzyme activity was strongly inhibited by the reaction products. The coupling activity of cyclodextrin glucanotransferase was proved. The enzyme was able to degrade high concentrations of β-cyclodextrin and to transform different types of cyclodextrins one into another. Cyclodextrin glucanotransferase formed α-, β- and γ-cyclodextrins in different ratios, depending on the duration of the process.Istražen je proces proizvodnje ciklodekstrina iz škroba pomoću ciklodekstrin glukanotransferaze iz bakterije Bacillus megaterium. Pomoću optimalno uređenog plana i metode odzivnih površina istražen je utjecaj koncentracije škroba i enzima na proizvodnju ciklodekstrina. Izrađeni su matematički modeli koji opisuju proces u dva područja varijacija nezavisnih varijabla koja se preklapaju. Modeli su upotrijebljeni za određivanje optimalnih vrijednosti varijabla. Povećanjem koncentracije škroba povećala se količina nastalog β-ciklodekstrina, a smanjio prinos. Optimalna koncentracija škroba bila je 50 mg/mL. Kritična je koncentracija ciklodekstrin glukanotransferaze, prijeko potrebna za maksimalnu proizvodnju β-ciklodekstrina, bila 2 U/g. Utvrđeni su faktori koji limitiraju potpunu konverziju škroba u ciklodekstrine i ustanovljeno je da produkti reakcije znatno inhibiraju aktivnost enzima. Dokazano je da ciklodekstrin glukanotransferaze može razgraditi velike koncentracije β-ciklodekstrina i transformirati ga u različite tipove ciklodekstrina. Djelovanjem enzima nastaju α-, β- i γ-ciklodekstrini u različitim omjerima, ovisno o dužini trajanja procesa

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering

Excretory over-expression of Bacillus sp. G1 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: Optimization of the cultivation conditions by response surface methodology

Co-expression of cyclodextrin glucanotransferase (CGTase) from Bacillus sp. G1 (cgt) with bacteriocin release protein (BRP) in Escherichia coli system resulted in the expression and excretion of the enzyme into the culture medium. The cultivation conditions were then optimized with the objective to enhance the production of extracellular recombinant CGTase using response surface methodology (RSM) that based on rotatable central composite design (CCD). The process consisted of a total of 50 experiments involving 10 star points and 8 replicates at the central points. The optimum predicted cultivation conditions for the maximum expression of extracellular recombinant CGTase were found to be comprised of: 20 °C post-induction temperature, induction-starting time when cell optical density is 0.3 at 600 nm, 1.0 mM xylose, 50 μM IPTG and 29 h post-induction time, with a predicted extracellular recombinant CGTase activity of 9144.28 U/ml. The experimental extracellular recombinant CGTase activity obtained was 9542.30 U/ml, which was very close to the predicted value. The expression of extracellular recombinant CGTase improved about 151-fold after the optimization was conducted