Study On Immobilization Of Marine Cyclodextrin Glucosyltransferase And Its Properties

Cyclodextrin glycosyltransferase?CGTase,EC 2.4.1.19?,who is an important member of the?-amylase No.13 family,can be used to catalyze hydrolysis reactions,cyclization reactions,coupling reactions and disproportionation reactions.Meanwhile,CGTase is widely used to produce cyclodextrin?CD?and 2-O-?-D-glucosyl-L-ascorbic acid?AA-2G?.However,some problems such as poor reusability,poor stability,and high reaction costs are found during the enzymatic reaction that catalyzed by free CGTase enzyme,which limit the use of CGTase;VC is an essential vitamin for human physiological activities,But VC has an unstable molecular structure and is easy to oxidize,which has caused difficulties in its industrial application.Meanwhile,AA-2G is considered to be a derivative with the closest physiological activity to VC,and has broad application prospects;Amino resin,a kind of artificially synthesized mesoporous polymer,is widely used as a carrier for immobilized enzymes at present,which can improve the enzyme activity,increase the thermal stability,and improve the efficiency of immobilization.In this paper,magnetic nanoparticles and amino-functional resins are used to immobilize CGTase produced by Bacillus marine,and the immobilized CGTase are applied to the catalytic reaction of AA-2G that synthesized by L-ascorbic acid?VC?and?-cyclodextrin.The main contents are as follows.?1?Preparation and Characterization of Magnetic Fe₃O₄,Fe₃O₄@SiO₂ and Fe₃O₄@SiO₂-NH₂nanoparticles:In this part,Silicon-coated magnetic materials are obtained by coating Fe₃O₄ particles with tetraethyl orthosilicate?TEOS?as silicon source.Then,the Aminosilanized magnetic nanoparticles were synthesized via bonding 3-aminopropyltriethoxysilane?APTES?to the silicon-coated magnetic materials through chemical bonds.Finally,it's confirmed that the Fe₃O₄@SiO₂-NH₂ nanoparticles have a particle size of approximately 22 nm,which is successfully modified by amino.Meanwhile,the nanoparticles exhibit a variety of characteristics,including high crystallinity,good stability,and good superparamagnetism.?2?Selection of optimal reaction conditions of marine CGTase immobilized by Fe₃O₄@SiO₂-NH₂magnetic molecules:in this part,an optimal reaction conditions of enzyme protein concentration:0.1 mg/m L,immobilization time:6 h,immobilization temperature:20?,rotation speed:200 rpm,p H:7,under which conditions the enzyme recovery rate is 62.7%,protein loading rate is 81%.At the same time.compared with the free CGTase,the CGTase immobilized on magnetic carrier shows a significant enhancement in thermal and p H stability.Besides,after 30 days of storage,the CGTase immobilized on magnetic carrier retains 61%of initial enzyme activity while the free CGTase retains only 50%.What's more,after 10 epochs of catalytic reaction,the enzyme activity of immobilized CGTase is still 66.3%remaining.?3?Selection and evaluation of aminated resin with optimal fixation effect:in this part,the fixation effect of 7 types of aminated resin,MC-300EP?MA-WP8?MI-BSI?MA-P9?MI-BN4?LX-1000EP?LX-1000HA,are analyzed and evaluated.Which confirmed that the MI-BSI shows the best fixation effect.With the MI-BSI being the matrix of immobilized CGTase,the enzyme recovery is 46.83%and the portion loading rate is 70.03%,which is significantly higher than the other 6 resins.Then,the reaction conditions,enzyme protein concentration,immobilization time,immobilization temperature,speed and p H,are analyzed and optimized as 0.067 mg/m L,8 h,35?,200rpm,8,the enzyme recovery and portion loading rate are raised to 64.9%and 88.4%under the optimized reaction conditions.Meanwhile,compared with the free CGTase,the CGTase immobilized on aminated resin shows a significant enhancement in thermal and p H stability,stronger resistance to metal ions and organic reagents,good storage stability and operational stability.Besides,after 30 days of storage,the CGTase immobilized on magnetic carrier retains 58.6%of initial enzyme activity while the free CGTase retains only 50%.What's more,after 10 epochs of catalytic reaction,the enzyme activity of immobilized CGTase is still 68.2%remaining.?4?Analysis of AA-2G synthesis catalyzed by free and amino resin immobilized CGTase:in this part,catalyzed,by free and immobilized CGTase,AA-2G is synthesized using VC and?-cyclodextrin as substrates.The optimal single factor reaction conditions of Free-CGTase-catalyzed reaction are temperature:35?,p H:5,substrate concentration:40 g/L,reaction time:24 h,and amount of enzyme:5 U.Meanwhile,he optimal single factor reaction conditions of immobilized-CGTase-catalyzed reaction are temperature:35?,p H:5,substrate concentration:50 g/L,reaction time:24 h,and amount of enzyme:5 U.Under optimal conditions,the yield of AA-2G produced by conversion of free enzyme to VC was 6.8 g/L,while the yield of AA-2G produced by immobilized enzyme was 7.29 g/L,so the yield of AA-2G increased by 6.7%.