To promote the biocatalytic production of fructose-6-phosphate by recombinant of fructokinases, novel fructokinases from Sorangium cellulosum were isolated and well analyzed.The fructokinase genes SoCeFruK1852 and SoCeFruK7579 were cloned by designing primers from the genomic DNA of S.cellulosum.The recombinant plasmids of pET30a-SoCeFruK1852 and pET30a-SoCeFruK7579 were constructed and transformed into Escherichia coli (DE3) for overexpression.The recombinant fructokinases were purified.The activity and biochemical characterization of fructokinases were determined.The molecular weights of recombinant fructokinase SoCeFruK1852 and SoCeFruK7579 were about 31.2 kDa and 32.8 kDa.The protein concentrations were 2.04 and 1.8 mg/mL.The specific activities were 35.8 and 18.3 U/mg, respectively.The optimal temperature and pH of recombinant SoCeFruK1852 were 37 ℃ and 4.5.The activity of SoCeFruK1852 was inhibited by Co2+, Fe3+, Zn2+, SDS, EDTA and Tween 20.However, Mg2+and Mn2+ could enhance its activity.The Km and Vmax values of SoCeFruK1852 were 0.54 mmol/L and 1.28 μmol/s when fructose was used as the substrate.SoCeFruK1852 could specifically catalyze fructose, whereas it also showed slight activity to D-tagatose.The recombinant fructokinases SoCeFruK1852 and SoCeFruK7579 isolated from bacteria S.cellulosum were successfully obtained.SoCeFruK1852 showed good enzymatic activity, characterization and highly substrate specificity, laying a foundation for the research related to recombinant fructokinases.
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