Vol.4 No.4 2012

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Research paper : Efficient production of active form of vitamin D3 by microbial conversion (Y. Yasutake et al.)−232−Synthesiology - English edition Vol.4 No.4 (2012) and the VD3 is thought to enter the cell diffusely after it breaks off from CD. However, the actual mechanism of the intake of VD3 into the cell is completely unknown (Fig. 5). Therefore, considering the possibility that the VD3 may be transported by some kind of membrane protein (transporter), we attempted the identification of a gene that might increase the VD3 conversion activity, by conducting the random gene destruction experiment using transposon and by using the R. erythropolis genome information. However, we have not been able to find such genes.Therefore, we changed our way of thinking and investigated whether the CD-VD3 complex could be delivered directly to the intracellular enzyme by physically making a hole in the cell. We focused on the antibacterial substance called nisin[11]. Nisin is an antibacterial peptide composed of 34 amino acids derived from Lactococcus lactis, and is approved as food additive. The action mechanism of nisin has been studied thoroughly, and it shows antibacterial activity by creating pores with diameters of about 2-2.5 nm in the membrane of the gram-positive bacteria, and the intracellular low molecular substances leak outside of the cell through the pores[12]. Although excessive addition of nisin will cause bacteriolysis, the R. erythropolis cell is characterized by bacteriolysis resistance compared to other bacteria. Therefore, by adjusting the amount of nisin added, it is possible to create a unique situation where pores will be formed while the cell structure is maintained without bacteriolysis. In theory, proteins such as ferredoxin and P450 cannot leave the cell, so such a cell can be used as a reaction vessel packed with high concentration of enzymes. To experimentally investigate whether CD and VD3 could freely enter and leave the cell through the pores, green chemiluminescence CD was added to the nisin-treated cell, to observe the intracellular intake of the substances. As a result, it was confirmed that the luminescence level from the cell increased depending on the nisin concentration and treatment time, and the pores could be used as the passage of CD.Next, the nisin-treated cell was used to conduct the VD3 hydroxylation experiment under various conditions, and we found that the hydroxylation capacity increased depending on the amount of enzyme present in the nisin-treated cell, unlike the untreated cells. Moreover, it was found that it was important to have a NADH regeneration system in the Fig. 6 Conceptual diagram of the hydroxylated VD3 production using the nisin-treated R. erythropolis cellFig. 5 Conceptual diagram of the permeability of cell membraneIn the cell membrane in an ordinary state (A), only VD3 may permeate the membrane by natural diffusion. In the cell membrane with pores created by nisin-treatment (B), the low molecular weight substance including cyclodextrin can move through freely. + + + Cell membrane Inside cell Outside cell Cell membrane Inside cell Outside cell A Cyclodextrin VD3VD3/cyclodextrin (clathrate) B Intracellular low molecular weight substances leak but conversion will continue without bacteriolysis.CyclodextrinPores made by nisin treatment (Nisin-Lipid II pore)25(OH)VD3VD3Rhodococcus erythropolisElectron(e-)Electron(e-)Glucose dehydrogenase (GDH)GluconolactoneGlucoseNAD+NADHNAD+NADHOHP450(Vdh)Ferredoxin (AciB)Ferredoxin reductase (AciC) +Nisin-NisinTotal amount of converted 25(OH)VD3 (µg)040080012001600 cell

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