Production Of γ-Cyclodextrin Glycosyltransferase By Bacillus Macorous WSH02-06

Cyclodextrin glycosyltransferase or cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) is one unique member of α-amylase family. CGTase has the capacity ofconverting starch and related substrates into cyclodextrins (CDs) via a transglycosylationreaction. CDs are homogeneous cyclic non-reducing oligosaccharides constituted by avariable number of units of glucose. As one kind of molecular capsule, CDs can easily forminclusion complexes with organic substances and have been applied in large number ofindustrial fields, e.g., in analytical chemistry, food processing, agricultural products,medicine and cosmetics industry. The most common form of CDs are α-, β-, and γ-CD.Until now, Only α-and β-CGTases are widely investigated and commercially produced.Compared to α-, β-CDs, γ-CD presents advantages of having a larger diameter nonpolarcavity and higher solubility, which facilitate the preparation of highly concentratedsolutions of active large molecules. However, there are few reports associated withγ-CGTase production.Bacillus macorous WSH 02-06, selected and conserved in our lab, has the ability toproduce high γ-CGTase. In this study, γ-CGTase production by this strain wassystematically investigated for the purpose of high enzymatic yield and productivity. Thebiochemical characteristics and dynamic properties of fermentation were explored.1. Corn starch was selected as the best carbon source for the γ-CGTase production by B.macorous WSH02-06. Peptone, corn steep liquor and ammonium sulfate were alsoselected as the suitable nitrogen sources. Cell growth and γ-CGTase yield weresignificantly promoted by the addition of cations such as magnesium, manganese and zinc.By the implement of an orthogonal array experiment design, the importance order offactors on enzyme production was obtained as such, corn starch > manganese sulfate >peptone > zinc sulfate. The optimal initial pH, temperature and medium volume in 500 mlshaking flasks for enzyme fermentation were 7.0, 37 ℃ and 80 ml. The appropriateinoculum size was 10 %. Compared to the result obtained before optimization, themaximal yield of γ-CGTase reached 375.3 U·ml-1, which was increased by 7-folds. Theinvestigation of carbon and nitrogen sources feeding in shaking flasks demonstrated thatthe nitrogen source feeding at 6 h and carbon source feeding at 12 h facilitated cell growthand enzyme production.2. The effects of individual and combined addition of magnesium, manganese and zinc onthe γ-CGTase production were tested in shaking flasks experiment. The combinedaddition of 4.88 mmol·L-1 magnesium ion, 2.96 mmol·L-1 manganese ion and 0.174mmol·L-1 zinc ion resulted in 392.5 U·ml-1 of γ-CGTase yield, which was three timeshigher than that obtained in the case without metal ions addition. Cell growth also reachedthe highest in this case, was 5.2 g·L-1.3. The addition of metal ions enhanced greatly the concentration of ATP and ATP/ADP ratioin cells and thus changed the energy status of the cells. ATP/ADP ratio increased from1.86 in the case without cations addition to 2.72 in the case with the addition of thesecations. It strengthened the energy reservation in cells and favored cell growth andγ-CGTase synthesis. It was also found that the increases of cell growth and γ-CGTaseyield were in linear relationship to the consumption of magnesium. The promoting effectsof manganese and zinc were also undeniable.4. The influences of various agitation speeds and dissolved oxygen (DO) levels on γ-CGTaseproduction by B. macorous WSH 02-06 were investigated in a 5 L fermentor. When theagitation speed was controlled at 300 rpm or DO was controlled at 40 %, γ-CGTase yieldreached the maximal value. The comparison of dynamic pattern of specific γ-CGTaseproduction rate revealed that the higher specific γ-CGTase production rate could beobtained with agitation speed of 300 rpm or 40 % of DO in the earlier stage offermentation, and the higher specific γ-CGTase production rate could be gained withagitation speed of 200 rpm or 20 % of DO in the later stage of fermentation. Hence, atwo-stage agitation speed control strategy and a two-stage DO control strategy were putforward and applied in the γ-CGTase fermentation. These two two-stage oxygen supplystrategies were proved to be both effective for the improvement of γ-CGTase production.Compared to the case in constant agitation speed of 300 rpm, γ-CGTase yield andproductivity increased by 69.7 % and 50.9 %, respectively with two-stage DO controlstrategy.5. Analysis of the degradation of corn starch during fermentation by Gel chromatographyindicated that amylose and the linear chain part of amyl pectin were the carbon sourceused by B. macorous WSH 02-06 for cell growth and enzymatic production. At the sametime, the presentations of linear maltoligosacchrides (LM) in broth during fermentationwere detected by a HPLC method. It was testified that the addition of LM intofermentation media powerfully increased cell growth and decreased the γ-CGTase yield.The detection of metabolic intermediates of glycolysis in cell demonstrated that theconcentration of FDP and ATP increased abnormally when LM was added. So, the LMhad an important part in the carbon catabolite repression of γ-CGTase fermentation.6. The relation among cell growth, γ-CGTase yield and the concentration of LM werestudied at different agitation speeds, pH and temperature. The higher the specific growthrates were, the more LMs were accumulated, and the lower γ-CGTase yields were. Whenthe specific growth rate was higher than 0.2 h-1, the yield of γ-CGTase declined sharply.Thus, it was the best policy to control the specific growth rate to be no more than 0.2 h-1in the γ-CGTase fermentation.7. The exponential fed-batch fermentation with predetermined specific growth rate of 0.15,0.2 and 0.25 h-1 demonstrated that γ-CGTase yield reached the highest value, 2565 U·ml-1,at the predetermined specific rate of 0.2 h-1. The tests of the feeding time of carbon andnitrogen source displayed that the CGTase yield was higher in the case with a separatedcarbon and nitrogen sources feeding. On the base of the results of exponential fed-batchfermentation, genetic algorithm (GA) method was applied to optimize substrates feedingtrajectory to control cell growth and improve the cell growth, γ-CGTase yield andproductivity. After optimization, the γ-CGTase yield reached 3016 U·ml-1, biomass was8.9 g·L-1, the average specific production rate was 9.31 KU·g-1·h-1, productivity was 35.28KU·L-1·h-1. Compared to the results obtained in batch fermentation, the γ-CGTase yieldincreased 9.25 times, productivity 7.21 times, biomass 2.07 times at most in fed-batchfermentation.8. Different surface active substances were added and tested for their abilities to enhancethe γ-CGTase production in batch. At the addition concentration of 0.6 g·L-1, Tween-60increased the biomass, γ-CGTase yield, specific γ-CGTase production rate on DCW andon protein 45, 160, 11 and 9.2 %, respectively. At the addition concentration of 12.5ml·L-1, n-propyl alcohol elevated the biomass, γ-CGTase yield, specific γ-CGTaseproduction rate on DCW and on protein 8, 170, 62.6 and 61.7 %, respectively.9. The analysis of lipid fatty acid composition of cell membrane demonstrated, after additionof Tween-60, up to 49 % (molar percent) of unsaturated hexadecylic acids was presented,the molar percent of unsaturated eicosly acids increased and octadecyl and docosyl acidsdecreased. Finally, the ratio of saturated acids to unsaturated acids descended from 0.61 ofcontrol experiment to 0.21. In the case of n-propyl alcohol addition, the molar percent ofunsaturated eicosly acids increased, unsaturated octadecyl acids reduced. The ratio ofsaturated acid to unsaturated acids was almost kep the same.10. By continues supply of n-propyl alcohol in batch, the γ-CGTase yield obtained was 655.2U·ml-1, which was higher than that gained with a single supply of n-propyl alcohol. Theinvestigation of variation of fatty acids composition during fermentation indicated that themolar percent of unsaturated octadecyl acids increased and unsaturated eicosyl acidsdecreased when n-propyl alcohol was presented, the molar percent of saturated pentacosylacid, hexadecyl acid, heptadecyl acid, eicosyl acid and docosyl acid were not sensitive tothe addition of n-propyl alcohol.