Selection Of Novel Thermostable Glucose Isomerase And Its Application Research

Glucose isomerase（GI）is able to catalyze D-glucose to D-fructose.The obtained high fructose corn syrup（HFCS）serving as sweetener,is widely used in various food fields.Based on the concentration of D-fructose,HFCS has been traditionally classified as HFCS-42,HFCS-55 and HFCS-90.Among them,HFCS-55 with higher concentration of D-fructose is more favorable for industrial application.Currently,HFCS-42 is the main industrial product via bioconversion approach using GIs.55% HFCS must be derived from chromatographic enrichment of 42% grade to 90% and blending the both.In order to realize one-step production of HFCS-55,the major researches were summarized as follows.Genome mining for novel thermophilic GIs.Novel thermostable GIs from Thermoanaerobacterium xylanolyticum（Tx GI）,Thermus oshimai（ToGI）,Geobacillus thermocatenulatus（GtGI）and Thermoanaerobacter siderophilus（Ts GI）were mined from the National Center for Biotechnology Information（NCBI）database using Thermotoga maritima（TmGI）as template.The genes were synthesized and inserted into pET28b（+）and heterologously expressed in E.coli BL21（DE3）.The activity of ToGI and TsGI were 2.42 and 2.54 U/（mg·total protein）,respectively,which were higher than that of GtGI and TxGI.Finally,ToGI and TsGI with higher activity were finally chosen as biocatalysts for the following research.Purification of recombinant ToGI and TsGI and enzymatic characteristics analysis.First,the crude enzyme solution was heated at high temperature and the cell suspension was removed by centrifugation,then,the supernatant was injected to the Bio-Rad purifier through nickel-NTA superflow column.As a result,the best enzymatic activator for ToGI and TsGI are Mn²⁺ and Mg²⁺,respectively.The purified ToGI had an optimum temperature of 95 oC,higher than that of TsGI.When ToGI was incubated with 20 mM Mn²⁺ at 85 oC,it could remarkably retain more than 80% of initial activity after incubation for 48 h.This result suggested that holo-ToGI had higher thermostability at 85 oC than that of other representative thermostable GIs.The optimum pH of TsGI and ToGI were around p H 6.5 and pH 8.0,respectively.According to the Lineweaver-Burk plot,the K_m value of To GI and TsGI were estimated to be 81.46 mM and 199.03 mM,respectively.Therefore,the superiority of substrate’s affinity of ToGI could be illustrated owing to its lower K_m value toward D-glucose.In addition,the k_cat/K_m value of ToGI was 21.77 min^-1·mM^-1,higher than 14.54 min^-1·mM^-1 that of TsGI,indicating that To GI had the advantage of higher catalytic efficiency toward D-glucose over TsGI.ToGI with excellent high thermostability was finally chosen as biocatalyst for the following research.Optimization of fermentation and conversion conditions of E.coli BL21（DE3）/pET28b/To GI.The results showed that the optimum enzymatic production condition were at 28 oC by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside（IPTG）and 5 mM Mn²⁺.Then,the scale up reaction was performed with 20 g/L wet cell,10 mM Mn²⁺ and 200 g/L D-glucose.After incubation of 4 h at pH 7.5 and 95 oC,the yield of D-fructose reached 57.34%.According to the homology modeling and molecular docking studies of ToGI,site-directed mutagenesis was performed on several speculative important residues（Trp136Phe,Glu216 Asp,Thr352Asp,Leu342 Ile,Val228Cys）to improve the activity of ToGI towards D-glucose.Finally,two mutants Val228 Cys and Thr352 Asp were obtained,which exhibited 1.07-fold and 1.04-fold higher activity towards D-glucose than the wild-type To GI.In summary,a novel superiorly thermostable To GI was screened,overexpressed and purified.Followed research revealed the better thermostability of ToGI than other reported thermophilic glucose isomerase,which renders this enzyme to be a suitable catalyst to achieve the one-step production of HFCS-55,that was beneficial to the simplification of the enrichment and separation steps,conducive to energy conservation.Some key residues closely related to the enzyme activity had been identified based on molecule docking,providing basis for the subsequent molecular modification of ToGI.