Enzymatic Synthesis Of Glucose-6-phosphate By Glucokinase Coupled With ATP Regeneration By Acetate Kinase

The gene ack of Acetate kinase (Ackase), the gene glk of Glucose kinase (Glk) from Escherichia coli (E.coli) and the gene ack, glk from Geobacillus thermoglucosidasius (G.thermo) was cloned respectively. Four recombinant strains, pACKE, pACKG pGLKE and pGLKG were constructed. These four recombinant strains were cultured by IPTG and target enzyme proteins were analysised by SDS-PAGE. A large amount of soluble target proteins could be expressed in these four recombinant strains. The activity of Ackase in pACK.E was7.57U/mg and5.12U/mg in pACKG. The activity of Glkase in pGLKE was10.12U/mg and7.32U/mg in pGLK.G. All were much higher than that in control.Among them, the activity of Ackase in pACKE which was pretreatmented in37℃was the highest but its thermal stability was poor. The thermal stability of the Ackase in Lactobacillus bulgaricus or pACKG which was pretreatmented in42℃was good. The Ackase in pACK which was pretreatmented in60"C had good thermal stability.Glucose-6-phosphate was enzymatic synthesed by Glkase and Ackase expressed in above constructed recombinant strains. The conversion yield was higher than97%when the reaction solution containing lOmM glucose,20mM acetyl phosphate sodium,0.5mM ATP,5mM phosphate buffer (pH7),4.856U Glkase and3.632U Ackase from E.coli was put into37℃water bath for1h. The conversion yield was79%when4.856U Glkase and3.632U Ackase from G. therm which was pretreatmented in60℃were used as biocatalysts under same reaction conditions..