Optimum Extracellular Production Of Recombinant Cyclodextrin Glucanotransferase From Anaerobranca Gottschalkii And Its Cyclodextrin Products

Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme that catalyzes starchbioconversion into cyclodextrins through its action of intramolecular transglycosylation.Cyclodextrins are non-reducing cyclic oligosaccharides consisting of6(α-cyclodextrin),7(β–cyclodextrin) and8(γ–cyclodextrin) glucopyranose units linked by α-(1,4) bonds. Due totheir special truncated structure, cyclodextrins form inclusion complexes with many guestmolecules which enabled their use of in various crucial applications such as agriculture,biotechnology, pharmacy, food and cosmetics, analytical chemistry, environment, etc. Here inthis thesis paper we have successfully transformed the target gene of CGTase fromAnaerobranca gottschalkii into expression host, E. coli BL21(DE3) and investigated somecrucial approaches for enhancing its extracellular secretion. Use of molecular and chemicalapproaches has been implemented successfully to enhance the extracellular secretion of therecombinant CGTase and its products.In this study, the enzymatic preparation parameters of recombinant CGTase and itscyclodextrin products in general and α-cyclodextrin (α-CD) in was optimized. Theoptimization was done both in shaking flask and bioreactor level(1) The recombinant CGTase production was optimized in shaking flask level and a maximalextracellular activity of10.00U/mL was achieved in the presence of medium additive,Tween-80.(2) An orthogonal analysis of the shaking flak optimization factors such as carbon source,nitrogen source, and medium additives was also done and was found out that a maximalactivity of12U/mL could be achieved in the presence of a particular concentrationcombination of the crucial factors.(3) The crude extracellular recombinant CGTase was characterized in which a maximaltemperature of65°C and pH of8.0were found out to be suitable. Additionally, theenzyme stability at storage (4°C) was investigated and was found out that it retained66.7%of its original activity in storage time of about96h.(4) Bioconversion optimization including reaction time, starch source (potato, cassava, andsoluble starch), the enzyme dosage, temperature, pH, polar organic solvents in theirindividual and/or coupled effects (ethanol, cyclohexane, butanol, and decanol), and starchconcentration was investigated. The condition was optimized at a concentration of:10%soluble starch,5U of CGTase per gram of starch, pH8.0,5%of organic solvents24h atan optimal temperature of65°C. A In this optimized condition, a total CD yield of45% was achieved in the presence of ethanol and a91%specific conversion of starch to α-CDwas obtained in the presence of decanol. Additionally the coupled effect of organicsolvents on the yield and specificity was investigated and was found out that the coupledeffect of decanol and ethanol resulted in the highest conversion yield of77%α-CD.(5) Although the condition at higher temperature of65°C favoured the enzyme activity, areduced temperature of40°C was suitable for bioconversion in the presence of organicsolvents.(6) In fed-batch fermentation an overall enzyme activity of36U/mL was achieved out ofwhich78%was extracellular. This yield of enzyme activity from large scale fermentationwas2.4times higher than the total enzyme activity from shaking flasks fermentation(15U/mL).