Cloning And Expression Of Glycerol Dehydratase And Reactivation Factor From K.pneumoniae XJPD-Li And Their Reactivation Relation

To investigate the quantitative relation of glycerol dehydratase reactivation factor and other factors to inactived glycerol dehydratase in vitro, a new strain, XJPDLi-2 from Xinjiang was screened which could convert glycerol anaerobically to 1, 3-propanediol. The strain of XJPDLi-2 is a member of Klebsiella pneumoniae after identified, and named K. pneumoniae XJ-Li(XJPD-Li). The study of fermentation characteristic indicated that the optimal cultivation parameters for temperature and pH were determined as 40℃and 8.0, respectively; the optimized nitrogen source was 6.0g·L-1 of (NH4)2SO4, and the concentration of glycerol and glucose concentration were 20g·L-1 and 2.5g·L-1, respectively. Batch fermentation in 5L bioreactor was performed, and the results displayed that 20 g·L-1 of glycerol was consumed just in 8h with 12.2 g·L-1 of 1, 3-PD and the molar yield of 1,3-PD to glycerol 0.75 was achieved. Fed-batch fermentation also indicated a higher molar yield of 0.70, and 1,3-PD reached 38.1 g·L-1 by consumping 66.4 g·L-1 of glycerol after 48h. Thus, it was shown that this strain would be a excellent 1,3-PD producer.Research about the catalytic characteristic of glycerol dehydratase showed that the enzyme activity obviously goes simultaneously with the cell growth of XJPD-Li strain. The optimal reaction temperature and pH were 40℃and 8.0 respectively, and the enzyme was relatively stable under 40℃and pH 6.0~8.0. In all of the cation ions tested, Na+,Mg2+,Mn2+ and Fe2+ have activation effect, while Ca2+,Co2+,Fe3+,Zn2+ and Cu2+ have inhibition effect on the enzyme. The saturated concentration for catalyzing reaction of glycerol dehydratase was 0.15mol·L-1and 0.3mol·L-1, and Km was 5.57mmol·L–1 and 8.16 mmol·L–1 when glycerol and 1,2-propanediol were as substrate, respectively. The study also displayed that the activation energy of the catalyzing reaction of glycerol dehydratase was 18.904kJ·mol– 1.The genes of glycerol dehydratase(dhaBCE)and reactivation factor (dhaFG) were cloned by PCR from the genome DNA of XJPD-Li, respectively. The expression plasmids of PET-28a(+)-dhaBCE and PE-T28a(+)-dhaFG were conceived, and then were successfully transformed into E.coli BL21(DE3), respectively. The active proteins were induced after 5 hours by 0.4mMIPTG at 25℃while OD600 reached 0.6. The specific activity of glycerol dehydrates was 299 U·mg-1which was about 300 times compared with that of wild strain, and the reactivation factor could make the activity renew above 90% for glycerol and O2 -inactivated glycerol dehydratase.The studies of inactivation process of glycerol dehydratase displayed that the enzyme activity lossed 35% and 48% in 3 minutes, and only 2.7% and 1.5% after 1 hour when glycerol dehydratase reacted with glycerol and sparged with O2 at 40℃, respectively. The optimum reactivation conditions of inactivated glycerol dehydratase were ATP 50mmol·L-1,Mg2+10mmol·L-1,coenzyme B123μmol·L-1 and glycerol dehydratase/reactivation factor 4, and the activity of glycerol and O2 inactivated can be renewed 98.9% and 97.3% under these conditions, respectively. For the contributing extent to reactivate the inactivated glycerol dehydratase, glycerol dehydratase/reactivation factor and ATP are the most important to effective reactivation of inactivated glycerol dehydratase, but all factors are necessary to reactivation of inactivated glycerol dehydratase. The reactivation factor can reactivate effectively the glycerol and O2-inactivated glycerol dehydratase with ATP, Mg2 + and coenzyme B12. Compared with O2-inactivated glycerol dehydratase, glycerol -inactivated glycerol dehydratase can be reactivated more easily, and the concentration of propionaldelyde is 1.5 times after reactivation about 60 minutes.The inactivation and reactivation of glycerol dehydratase which was obtained by expressing in Ecoli BL21 were studied. This research will provide the guidance for the construction the newly catalyzing reaction and reactivation systems of glycerol dehydratase and also for the regulation of biosynthsis of 1,3-propanediol.