Production And Purification Of A Thermostable Cyclodextrin Glycosyltransferase With A High Starch Hydrolytic Activity From Bacillus Licheniformis Sk13.002 And Its Application For β-cyclodextrin Production

Cyclodextrin glycosyltransferases (CGTases; EC 2.4.1.19) are industrially important enzymes that produce cyclicα-(1,4)-linked oligosaccharides called cyclodextrins (CDs) from starch as a major carbon source and can also be used to produce oligosaccharides with novel properties. A thermostableβ-CGTase was produced from an alkalitolerant Bacillus licheniformis SK 13.002 strain using different carbon, nitrogen and metal ion sources. The CGTase was produced from all tested starches and the highest activity was achieved from soluble starch, maltodextrin and dextrin from maize starch. There was no significant difference between them for CGTase production. Glucose and maltose showed catabolite repressive effect on CGTase production. Presence of soy peptone and yeast extract as organic nitrogen sources in the fermentation medium was very essential for CGTase production. Inorganic nitrogen sources were not suitable for CGTase production. MgS04 and FeCl2 were found to be the best metal ion sources for enzyme production. The effect of FeCl2 on the production of CGTase in fermentation has not been reported before except for stability tests in the presence of this metal. Addition of iron containing metal ions to the fermentation media promoted the biosynthesis of CGTase enzyme. ZnSO4,ZnCl2 and CuSO4 completely inhibited CGTase production.B. licheniformis SK 13.002 CGTase could significantly hydrolyze starch into short linear saccharides, in addition to production of CDs. Hydrolysis activity was four times more than the cyclization activity. While enhancement of the hydrolytic side reaction of CGTase may have useful applications in the bread baking industry, it has a detrimental effect on the overall production of cyclodextrins since it produces short saccharides that are responsible for accelerating the breakdown of the cyclodextrin ring during the coupling reaction, thus limiting the final product yields. B. licheniformis SK 13.002 CGTase hydrolytic activity is thought to be due to partial retention of ancestral enzyme function from evolution over time, hence this CGTase is another example of an enzyme at an intermediary stage in between "true" a-amylases and "true" CGTases.The CGTase optimum cyclization and hydrolysis activities were at pHs 7.0 and 6.0, respectively. Optimal temperature for both reactions was at 65℃. The CGTase also displayed high temperature activities for both reactions, indicating a potential application in processes that use high temperature. It exhibited 75% residual cyclization activity after pretreatment at 65℃for 30 min and 65% residual activity after 1 h pretreatment respectively, whereas at 70℃the CGTase was inactivated. A good CGTase thermal stability was obtained without detailed studies including the presence of different concentrations of Ca2+, substrate or other reagents. The thermostability of B. licheniformis SK 13.002 CGTase gives it a potential for use in industrial CD production as the starch solution would have to be cooled down to a lesser extent after the jet-cooking step for CGTase action, and this would make the process cheaper. There will also be no need to pre-treat starch with thermostableα-amylase enzyme. Thermostability property also offers protein engineers a genetic template from which to create a highly stable enzyme with the desired properties.The B. licheniformis SK 13.002β-CGTase produced predominantlyβ-CD andγ-CD as a minor secondary product from 5% hydrolyzed starch without any accumulation ofα-CD. A 50% maximal conversion of starch to CDs was obtained by the crude CGTase without addition of any additives or optimization of the bioconversion conditions. Theβ-CD produced was at 83% andγ-CD was at 17% of the total CDs yield and noα-CD was formed after 24 h of reaction. The producedβ-andγ-CDs corresponded to 20.9 g/l and 4.2 g/l, respectively. Production of CDs with economical conversion rate without any toxic complexing agents is commercially important. The established thermostability and production of onlyβ-andγ-CDs are interesting features of this enzyme for cheap, easy purification of these CDs based on their solubility differences in water and also gives a potential forβ-CD industrial production.The B. licheniformis SK 13.002 CGTase was eventually purified to homogeneity using both anion exchange chromatography and gel filtration and displayed two CGTase isozymes on SDS PAGE which were found to have molecular weights of 67.6 kDa and 47.3 kDa respectively. The purification protocol resulted in increased CGTase specific activity from 0.49 U/mg for crude enzyme to 9.46 U/mg and 8.83 U/mg for the individual isozymes, respectively, with a purification fold of 37.3 and 19.1% yield recovery. Therefore Bacillus licheniformis SK 13.002 strain is another kind of Bacilli capable of producing CGTase isozymes.