Heterologous Expression,Crystallization And Product Specificity Of Marine Cyclodextrin Glycosyltransferase

Cyclodextrins?CDs?are a family of cyclic oligosaccharides with hydrophilic external and hydrophobic central cavities.CDs were widely used in cosmetics,pharmaceuticals,food,chemical industry,because they can embed hydrophobic molecules or groups to form water-soluble inclusion compounds.The cyclization reaction of cyclodextrin glucosyltransferase?CGTase?can produce CDs which is a mixture of?-,?-and?-CD from starch substrates.It greatly increases the cost of purification to obtain a single species of CDs.In recent years,improving product specificity of CGTase by molecular modification is a research hotspot.Based on the analysis of the simulated structure and amino acid sequence of marine CGTase,the amino acid position R81 which may have an effect on the specificity of the CD product was determined.The mutant R81T of which arginine at site 81 was mutated to threonine was constructed and expressed by site-directed mutagenesis,using the recombinant plasmid p ET24a-cgt as a template.In order to increase the protein expression of the mutant enzyme in E.coli BL21?DE3?,three single factors including the concentration of the IPTG,the induction time and the induction temperature were optimized.The results showed that the optimal IPTG induction concentration is 0.4 m M.,the induction is insufficient when the IPTG concentration is too low,leading to a low protein expression.It may have a toxic effect on the E.coli if the IPTG concentration is too high,or resulting in a fast expression which was too fast to generate active CGTase.The optimal induction temperature is20?.The growth of bacteria is slow when temperature was below 20?,the expression of protein was limited.However when the induction temperature was over 30?,the speed of protein folding is not as fast as translation and then it will easily aggregate to form inclusion bodies.The best induction time is between 15 h-18 h.The cell of E.coli will die and rupture when the culture time is too long,causing the loss of protein.The final induced condition was to inoculate the seed liquid into the LB liquid medium at 2%inoculation amount,culturing at 37°C,200r/min until the optical density at 600 nm(OD₆₀₀)reached about 0.6,then add IPTG at a final concentration of 0.4 m M,the induction was placed on a rotary shaker?200 rpm?at 25?for 15 h.4 L fermentation broth was fermented according to the optimized induction conditions above.400 m L of crude enzyme solution was obtained after ultrasonic disruption in ice and centrifugation.In this experiment,the crude enzyme solution was purified by two-step of chromatography.The first step was nickel column affinity chromatography,after that ultrafiltration was performed to remove salt and concentration.The specific activity of the CGTase was increased from 0.065 U/mg to0.26 U/mg,and the purification factor was 4.02.In order to remove other protein,the protein was further purified by gel filtration chromatography,and results SDS-PAGE proved that the CGTase was electrophoretic pure.The final concentration of the protein used as raw material for crystallization was 22 mg/m L after concentration by ultrafiltration.The protein crystallization experiment was performed by vapor diffusion method.The initial screening conditions was 0.2 M Ammonium acetate,0.1 M Tris p H8.5,45%V/V?+/-?-2-Methyl-2,4-pentanediol.The crystallization condition was optimized basing on the initial conditions.It was found that plate crystals which can be used as X-ray diffraction material were observed when the concentration of V/V?+/-?-2-Methyl-2,4-pentanediol was 45%or 60%and the concentration of Ammonium acetate was 0.2 M or 0.4 M.The appropriate amount of the mutant and wild CGTase were reacted with 1%starch solution.By monitoring the CD product generation using high-performance liquid analysis in real time,it was found that the yield of?-CD was decreased by 8%,the percentage of?-CD in cyclization product was increased from 64%to 71%,and the production of?-CD also increased significantly.The above results validate the speculation that the mutant R81T improves product specificity.The possible reasons is the side chain of threonine is shorter than arginine,which increases the space for substrate binding and is conducive to generating larger CD product.In addition,There was a change of the hydrogen bonding force.At the same time,the enzymatic properties of the mutant maintain the thermophily,thermostability,alkali resistance and p H stability of the wild-type.