Study On Fermentation Of Glucose Dehydogenase Producing Engineering Strain And Properties Of Recombinant Glucose Dehydrogenase

Glucose dehydrogenase (GDH, EC 1.1.1.47) is a kind of NAD(P) dependent oxidoreductase, which can catalyze the conversation of beta-D-glucose into D-glucono-δ-lactone with the concomitant reduction of NAD(P) into NAD(P)H. It is not only widely applied in biocatalysis to regenerate NAD(P)H, but also used in biofuel cells.The enzyme is not rich in nature because it is only exisited in animal liver and Bacillus. In this paper, on the basis of preliminary optimized culture condition of shaking flask, the high density cultivation of E. coli BL21(DE3)/ pETl la (+)-GDH was investigated. Futhermore, the properties of GDH were studied. The main research results of this paper were as follows:(1)The fermentation condition in shaking flask was optimized by comparing the specific enzyme activity of GDH in different culture condition.The optimized culture medium was as follows:glucose 1%, peptone 2%, yeast powder 0.5%, pH 7.5, trace element addition volume 2 mL/L. The optimized culture conditions:medium volume 50 mL in 250 mL shaking flask, inoculum volume 6% of medium volume. The optimized induced condition was that GDH biosynthesis was induced by adding IPTG (final concentration was 1 mM) at the early stage of logarithmic growth of the recombinant bacteria, and that the induction was conducted for 6h at 32 ℃. The specific enzyme activity reached 17.44U/mg under the optimized condition.(2) Batch fermentation of E. coli BL21(DE3)/pET11a (+)-GDH was carried out in a 15L automatic fermentor and the corrsepongding fermentation kenetic equation was set up. On the basis of batch fermentation, the fed-batch fermentation based on pH-stat condition was investigated. The higher cell density and enzyme activity were obtainted with 18 g/L (DCW) and 130 U/mL, which were 2.25 times and 3.8 times of those under batch fermentation.(3) Two cell cracking methods of high pressure and ultrasonic were optimized and compared. Using the high pressure craking method under its optimized condition of the craking time 7 min and the cell concentration 0.083 g/mL, the enzyme activity and its cell disruption rate were 141.8U/mL and 98% respectively. While using ultrasonic cell craking method under its optimized condition of the power 900W, the craking time 9 min, and the cell concentration 0.083g/mL, the enzyme activity and cell disruption rate were only 99.6 U/mL and 87% respectively, which were just 70% and 88% of those through the high pressure method.(4) The enzymatic properties of the recombinat GDH was studied after purification by salting out with (NH4)SO4. SDS-PAGE analysis showed that the molecular mass of the recombinant GDH was around 32 kDa. The optimal reaction temperature and pH value were 40 ℃ and 8 for the recombinant GDH. The activation energy of de-active reaction was found to be 98.137KJ-moL-1 indicating the GDH thermal stability was sensitive to temperature. The GDH was stable during pH 6～8. The substrate specificity study showed that the enzyme almost had no catalytic effect on other substrate except glucose. The recombinant GDH could be activated by K+, Fe3+, Ca2+, Na+ and Mg2+ and inhibited by Cu2+ and Mn2+. The recombiant GDH was very unstable in some organic solvents, such as n-propanol, acetonitrile and tetrahydrofuran.(5) The GDH catalytic reaction mechanism was ordered BiBi mechanism. The apparent Km of NADP was 0.154 mM, the apparent Km of glucose was 8.85 mM, and the Vmax was 211.42 μmoL·L-1·min-1.