Heterologous Expression Of Glucose Isomerase And Its Application In Production Of High Fructose Corn Syrup

Glucose isomerase(GI)is an important enzyme in the industry.GI could isomerize D-glucose and D-xylose into D-fructose and D-xylulose,respectively.Therefore,it has been applied in the industrial production of high fructose corn syrup(HFCS).At present,the companies of HFCS manufacturing in China still relied on the commercial GI from abroad.However,the optimum reaction temperature of commercial GI was about 60℃,at which only 42-45%of HFCS can be obtained.In order to get 55%HFCS,the separation process with chromatographic system had to be employed.Consquently,the production cost was elevated.In this work,genome mining was used to screen the genes of GI.The amino acid sequences of GI from Thermotoga neapolitana DSM 5068 and Streptomyces murinus were chosen as templates to blast in NCBI.At last the gene sequences of GI from Thermotoga petrophila RKU-1(TPGI)and Thermoanaerobacter ethanolicus CCSD1(TEGI)were obtained.The gene codon of tegi and tpgi were optimized and then synthesized,respectively.The length of genes of tpgi and tegi is 1332 bp and 1314 bp,respectively.The genes of tpgi and tegi were inserted into E.coli expression plasmids pET-28b,respectively.The recombinant expression plasmids pET-28b-TPGI and pET-28b-TEGI were successfully constructed and then transformed into E.coli BL21(DE3)competent cells,yielding two recombinant strains E.coli BL21(DE3)/pET-28b-TPGI and BL21(DE3)/pET-28b-TEGI.Through fermentation cultivation and IPTG induction,TPGI and TEGI were successfully heterologously expressed.The SDS-PAGE results showed that The molecular weights of TPGI and TEGI were about 50 kDa.The acitvity of TPGI was181 U/g(WCW)and that of TEGI was 195 U/g(WCW).Affinity chromatography with Ni-NAT column was used for separation and purification of TPGI and TEGI.The two enzymes were purified to electrophoretic homogeneity.With D-glucose as the substrate enzymes properties of purified TPGI and TEGI were investigated.The results showed that,the optimum temperature of TPGI and TEGI were 85℃ and 90℃,respectively.The optimum pH of TPGI and TEGI were 7.0 and 6.5.Both of them had a satisfactory thermostability.TEGI still retained more than 60%activity after heating treatment for 9 h at 90℃.TPGI still retained more than 80%activity after heating treatment for 1 h at 85℃.The activities of TPGI and TEGI both need metal ions.Co2+played an important role in enzyme activity and thermostability.After adding 1 mM Co2+and 15 mM Mg2+,enzyme activities could reach the maximum levels.However,the enzyme activities could also be inhibitied by Ni2+,Zn2+,Cu2+,Fe2+and Ca2+.There was 50%loss of enzyme activity when the concentration of Ca2+reached about 18 mM.The degree of inhibition of Ca2+to TEGI was relatively lower compared that in the case of TPGI.The Km and Vmax values of TPGI and TEGI for D-glucose were 373 mM,13.4 U/mg and 421 mM,27 U/mg,respectively.The Km and Vmax values of TPGI and TEGI for D-fructose were 897 mM,38.1 U/mg and 487 mM,15.6 U/mg,respectivity.The conversion rate for both GIs reached 53%with 10%glucose(m/v).When the concentration of glucose was 20%and 30%(m/v),the conversion rate for TEGI was 42%and 38%,respectively.But it was only 30%and 21%for TPGI,respectively.After comprehensive comparison,E.coli BL21(DE3)/pET28b-TEGI was selected as the GI producing strain for further optimization of enzyme production conditions.The results showed that,the optimum fermentation conditions were as follows:seed cultivation time was 10 h.The fermentation medium was glycerol medium.The initial fermentation pH was 7.0.The iron metal was 0.4 mM Co2+.The concentration of IPTG was 0.15 mM.The induction initial time and end time was 3-4 h after fermentation cultivation and 12 h after induction,respectively.The temperature in the induction phase was 25℃.After the optimized conditions,the biomass(WCW)reached 11.05 g/L which was 2-fold compared with the control.The enzyme activity was 228 U/g(WCW),12%more that of the control.