The Effect Of Co²⁺/Mg²⁺ On Recombinant Thermobifida Fusca Glucose Isomerase And The Study Of Immobilized Cells

Glucose isomerase （EC5.3.1.5; xylose isomerase） can catalyze the reversibleisomerization of aldose （glucose, xylose or ribose） to ketose, and it is an important enzyme inthe food and beverage industry. Glucose isomerase has mainly been applied to isomerizeglucose syrup to an equilibrium mixture of glucose and fructose, widely known ashigh-fructose corn syrup （HFCS）.In our previous work, the gene of T. fusca glucose isomerase was cloned and expressedin E. coli BL21（DE3）. However, the enzyme activity is still low and the thermostability is notgood enough to make the enzyme operated at high temperature for long time. In order toincrease the catalytic efficiency and thermostability of recombinant T. fusca glucoseisomerase, Co²⁺/Mg²⁺were added into the culture medium and the recombinant cells wereimmobilized in the present study. The main results were listed as follows:（1） E. coli BL21（DE3）/pET-24a（+）-glu was constructed previously in our laboratory. Therecombinant cells were cultured in TB medium supplemented with5mmol L^-1Mg²⁺or1mmol L^-1Co²⁺at37°C. The maximum activity of the enzyme （GI） was20.6U mL^-1, andthere was no increase in enzyme activity from adding Mg²⁺into the culture medium （M-GI）.Whereas a significant increase from the supplementation of1mmol L^-1Co²⁺was obtained（C-GI）. The maximum activity of C-GI reached38.4U mL^-1,1.86-fold higher than that of GI.However, the maximum activity obtained in TB medium supplemented with both1mmol L^-1Co²⁺and5mmol L^-1Mg²⁺（MC-GI） was41.1U mL^-1. Comparing with C-GI, no furtherincrease in the enzyme activity of MC-GI was observed.（2） The recombinant enzymes were purified through a combination of heat treatment,ammonium sulfate fractionation, and chromatographic separations （DEAE-Sepharose anionexchange）. The purified C-GI exhibited a high specific activity of23.8U mg^-1, about1.97-fold higher than that of GI (12.1U mg^-1). At75°C, the half-life of C-GI was17.3h,nearly11.3-fold greater than that of GI （1.5h）. Furthermore, the thermostability of MC-GIwas better than that of C-GI, the half-life of MC-GI was24.0h, which was1.41-fold greaterthan that of C-GI. To the best of our knowledge, this is the first report to show that Co²⁺playsan important role in enhancing the catalytic efficiency and thermostability of glucoseisomerasefrom T. fusca. In addition, the metal-specificity of T. fusca glucose isomerase wasanalyzed preliminary through the crystal structure simulation and analysis circular dichroismof the enzyme.（3） The recombinant cells were immobilized by four different immobilized methods.Through comparing enzyme activity, activity recovery and hardness, the two-stepsimmobilization method （flocculation-crosslinking） was used in the following experiment. Theoptimal immobilization condition were0.015%（w/v） chitosan solution,0.05%（v/v）glutaraldehyde solution,1g L^-1diatomite and3h crosslinking reaction. The maximumactivity and activity recovery of the immobilized cells were2579U g^-1and70.5%,respectively. The hardness of immobilized cells was1797g.（4） The immobilized cells were also characterized. The optimal temperature and pH of the immobilized cells were85°C and8.0, which were the same with the free enzyme（MC-GI）. The half-life of the immobilized cells was29.1h at75°C,11.3-fold higher thanMC-GI （24h）. In the aspect of application, the conversion rate of the immobilized cells wasstill43.1%at the end of8cycles, meeting the conversion rate requirement. In addition, theimmobilized cells exhibited good column performance with converting1.9t glucose per kg ofimmobilized cells.