Optimization Of Conditions Of Recombinant Cyclodextrin Glycosyltransferase And Its Immobilization

Cyclodextrin glucosyltransferase catalyzes the cyclization of glucose units of from starch,producing cyclodextrin（CD）.As cyclodextrin has great application in the fields of chemical industry,medicine and food,etc,it is necessary to provide high amount of CGTase.Currently,there are two issues accompanying the biocatalytic process of CD production.The first one is the low yield of CGTase.CGTase from wild microbial strains is low.And genetic engineering and fermentation is necessary to enhance CGTase production.The second one is poor production specificity of CGTase forα,β,γ-CD production specificity.Therefore,this subject optimized the fermentation conditions of recombinant Komagataella phaffii strain to increase the yield of CGTase firstly,and then improved the specificity of CGTase by immobilization.The main results were stated as following:（1）The basic inorganic salt medium was optimized for expression of recombinant CGTase at medium composition,induction conditions and additions.First,phosphate acid and ammonium hydrooxide of the original medium were replaced with phosphate buffer and ammonium sulfate,and theβ-CGTase andγ-CGTase activity of CGTase increased from 0 to 49.06 U/mL and 3.84 U/m L,respectively.Then,the optimum fermentation condition was obtained as:100 mmol/L phosphate buffer,1%glycerol,1%ammonium sulfate,induction temperature of 30°C,induction pH of 6.5,8%inoculum,1.5%methanol,and the addition of 10 mmol/L of Tween 80.Finally,theβ-CGTase andγ-CGTase activities improved up to 122.03 U/mL and 8.84 U/m L,respectively.（2）The high specific conformation of CGTase was formed at high temperature and retained in the form of cross-linked aggregates by cross-linking action of glutaraldehyde,and then used for high-purityβ-CD production.The optimal conditions of CLEA-CGTase preparation was the cross-linking at 85°C,0.1%glutaraldehyde concentration,75 U/m L CGTase loaded,the cross-linking at pH 9.0 for 10 minutes.Theβ-CGTase of CLEA-CGTase was more stable thanγ-CGTase.After storaged at 50°C for 8 h and storaged at4°C for 42 d,β-CGTase remained 47.9%and 86.8%,respectively.The kinetic parameters determined that the substrate affinity,maximum reaction rate（V_m）and catalytic efficiency(K_cat/K_m)ofβ-CGTase were 4.0 fold,4.7 fold and 18.7 fold ofγ-CGTase,respectively.In addition,the potato starch conversion by the CLEA-CGTase also showed that the CLEA-CGTase had high product specificity,and the ratio ofβ-CD reached above 87%.At the same time,CLEA-CGTase also could be used repeatedly to reduce the cost of CD production.（3）For recycling of CGTase,CGTase was immobilized on magnetic nanoparticles using dopamine in an alkaline solution.Then the magnetic nanoparticles coated with polydopamine were modified by loading abundant active groups of polyethyleneimine on the surface to improve the profermence of immobilized CGTase.Firstly,the optimal conditions for the preparation of the magnetic nanoparticles were determined to be 2.0mg/mL dopamine,polymerization for 12 h.And then four organic reagents such as dimethyl sulfoxide,N,N-dimethylformamide,acetylacetone and polyethyleneimine（PEI）were added to increase the specificity of immobilized CGTase.It showed that 0.5%PEI improved specificity and activity recovery of CGTase.The immobilization conditions were optimizedas 300 U/g of CGTase,immobilizing at pH 6 for 18 h.The optimum reaction temperature ofβ-CGTase activity of immobilized enzyme（CGTase/PEI@PDA-Fe₃O₄）had increased from 50°C to 55°C,and the optimum reaction temperature ofγ-CGTase activity did not change.This result benefied theβ-cyclization of CGTase at high temperature.Compared with the free CGTase,the efficiency ofβ-cyclization andγ-cyclization was increased by 3.1 times and 2.4 times,respectively,after CGTase immobilization.The stability of CGTase/PEI@PDA-Fe₃O₄ was also improved.After storaged at 55°C for 8 h and storaged at 42°C for 42 d,β-CGTase remained 38.29%and83.72%,respectively.In addition,the result of continuous batch conversion showed that CGTase/PEI@PDA-Fe₃O₄ had a good recycling ability.After 10 batches of transformation,γ-CGTase activity was completely lost,onlyβ-CGTase activity remained as 19.05%.Therefore,the research of CGTase/PEI@PDA-Fe₃O₄ was very important for saving the production cost of cyclodextrin and the purification cost in the later stage.This research increased the yield of CGTase by optimizing the fermentation of engineering bacteria,and successfully improved the recycling efficiency by using immobilized enzyme technology,and improved the catalytic specificity of CGTase,which has a good reference value for industrial production CD.