Study On The Production Of Fructose Using The Immobilized Glucose Isomerase

Glucose isomerase,also known as xylose isomerase,is one of the most pivotal enzymes in industrial production of high fructose corn syrup.It is capable to catalyze D-glucose to D-fructose as well as the preparation of xylulose from D-xylose,which has been applied widely in food and pharmaceutical industries.High fructose corn syrup?HFCS?is the mixture of the unreacted glucose and the produced fructose.According to the content of fructose,it is classified into three categories:HFCS-90,HFCS-42 and HFCS-55.HFCS-55 has been applied in beverage industry.At present,the market of glucose isomerase is monopolized by the foreign companies.HFCS-42 obtained under continuous catalytic reaction at 60 ^oC.To achieve HFCS-55,pure fructose obtained by chromatographic separation is added to HFCS-42,which contributes to high production cost.The recombinant strain E.coli BL21?DE3?/pET28b-TEGI-W139F/V186T,which can reach 53%-55%yield of fructose,is acquired in our lab,but the catalytic activity and stability of the strain still need to be improved.Therefore this paper first focuses on the study on the growth and conditions of enzyme production of recombinant bacteria.The experimental results shows that the optimum culture condition is to add citric acid to LB fermentation medium as supplementary carbon source,with the initial fermentation pH being 7.0;the concentrations of Mg²⁺and Co²⁺are 10 mM and 0.2 mM,with 0.1 mM IPTG as the inductive agent.The induction occasion is 2 hours and the induction time is 10 hours at 28 ^oC.The optimized process acquires 4.1 g?WCW?/L biomass weight and 509 U/g?WCW?enzyme activity,which is 1.25 times higher than before.After applying various approaches to immobilize glucose isomerase,glutaraldehyde cross-linking method is chosen.The further research on immobilization condition indicates that under the condition of 4%?v/v?polyethyleneimine,1 hour flocculation time,0.5%?v/v?glutaraldehyde,10mM Mg²⁺,0.3%?w/v?diatomite and 0.3%?w/v?bentonite,the activity recovery reaches 72%and dry-granulation can be carried out at 25 ^oC.Finally,the research on the catalytic properties of high fructose corn syrup prepared by immobilized enzyme demonstrates that the optimum pH and temperature for immobilized and free enzyme are identical?7.0 and 90 ^oC?.When studying on stability,it is noticed that immobilized enzyme shows better stability in phosphate buffer solution?pH 7.0?;the thermostability is better at85 ^oC than it is at 90 ^oC.The pH and the temperature stability of immobilized enzyme are better than free enzyme.20 mM Mg²⁺and 1 mM Co²⁺are also added to the reaction system.The kinetic constant of immobilized enzyme is168.45 mM,compared with free enzyme is increased and the affinity of substrate is decreased.After immobilized enzyme catalyzed 10 batches under the optimal conditions,yield of fructose was still reserved 43%.