Molecular Modification Of Glucose Isomerase And Its Application In Biosynthesis Of High Frucose Corn Syrup

High fructose corn syrup?HFCS?is an alternative of sweetener to sucrose that is isomerized by commercial glucose isomerase?GI?.Currently,only 42-45%of HFCS could be obtained in domestic factories because of the poor thermostability and low activity of foreign commercial GI under the temperature about 60 ^oC.A costly purification procedure by chromatography was still required to obtain the great demand product of 55%HFCS.In this paper,the recombinant E.coli BL21?DE3?/pET28b-TEGI was set as parental strain to construct a robust recombinant GI with conversion more than 55%.At first,we searched a protein crystal structure of GI in the PDB database with high homology to TEGI.Then molecular docking with D-glucose as substrate in Autodock was performed.From the results analysis,on the basis of improving the enzyme activity,we chose some feasible mutational sites and designed the primers,then used the site-saturation mutagenesis and site-directed mutagenesis to perform directed evolution of glucose isomerase.Finally,we got a mutant strain E.coli BL21?DE3?/pET28b-TEGI-W139F/V186T with the best performance.TEGI-W139F/V186T with a six His-tag was purified by a Ni-NTA column to gain the pure enzyme.Then the enzymetic properties of pure enzyme was investigated in detail,including the optimum temperature,thermal stability,optimum pH,pH stability,the influence of metal ions on the enzyme activity,and reaction kinetic parameters and so on.Final results showed that the optimum temperature of the enzyme was 90 ^oC,the residual activity of mutant TEGI-W139F/V186T could retain 68.4%after incubation at 90 ^oC for 24 h,which was 1.2 fold than parental TEGI.The optimum pH was 6.5,the residual activity of mutant TEGI-W139F/V186T could retain 82.4%after incubation at pH 6.5 for24 h,a little more stable than TEGI.Co²⁺had an activation effect on enzyme activity,Mg²⁺,Mn²⁺,Ba²⁺were also had positive impact.The tolerance of mutant TEGI-W139F/V186T to Ca²⁺had been improved compared with wild type TEGI.The K_m value of TEGI-W139F/V186T was lower than TEGI indicating higher affinity of D-glusoce with the mutant.The catalytic efficiency k_cat/K_m was 1.9 fold than TEGI.The biotransformation conditions of D-glucose to HFCS using whole cells of mutant TEGI-W139F/V186T as biocatalysts was investigated.The optimal reaction conditions were as follows:50mM phosphates buffer,pH 6.5,90 ^oC,15 mM Mg²⁺and 2 mM Co²⁺.Wet cells of mutant TEGI-W139F/V186T?3%w/v?was used to catalyze 200 mg/mL D-glucose under the optimal reaction condition,the isomerization gradually reached equilibrium within 1.5 h and the product D-fructose yield maintained above 53%,finally reached a maximum yield of 55.4%.This study paved foundation for the production of 55%HFCS using the mutant thermostable TEGI.