Vol.4 No.4 2012

41/62

Research paper : Efficient production of active form of vitamin D3 by microbial conversion (Y. Yasutake et al.)−231−Synthesiology - English edition Vol.4 No.4 (2012) 3.4.2 Completely eliminating the enzymatic side reactionIn the active form of VD3 production by P. autotrophica, side reaction product, in which the carbon-26 is hydroxylated, was also produced at the rate of about 10 %. This was clearly an issue of the substrate recognition of the enzyme, and it was thought necessary to either fabricate the enzyme that tightly recognized the VD3, or to fine-adjust the bonding orientation of the substrate in the substrate binding pocket. Therefore, we attempted to determine the crystal structure of Vdh in complex with VD3, and based on the structural information, we introduced the mutations to the substrate-binding pocket. Vdh-WT had low substrate binding affinity, and substrate complex crystal could not be obtained. However, high activity mutant Vdh-K1 was successfully crystallized as the complex with VD3, and we were able to clarify how the enzyme recognized VD3 (Fig. 4)[8]. Of the amino acid residues that formed the substrate binding pocket, we focused on the amino acid residue positioned in proximity to the carbon-24 to -27 of VD3, and the variant (I88V) with lowered percentage of side reaction was obtained by saturated mutagenesis for those residues[9]. The quintuple variant of Vdh-K1+I88V decreased the side reaction rate to about 1 % in the bioconversion test by P. autotrophica, and the 26(OH)VD3 decreased to below detection limit in case of the single I88V variant. While this result was based on the structures, it was not entirely a rational design. It is extremely difficult to logically estimate which variation of what amino acid residue will reduce the side reaction. We succeeded in selecting the mutant that achieved the side reaction reduction by selecting the amino acid residues based on the 3D structure, and then by taking the strategy of saturated mutagenesis for the selected amino acid residues. Through this research result, we succeeded in increasing the production efficiency of the 25(OH)VD3, and the development for practical application is currently in progress. 3.5 Processing the cellIn producing the active form of VD3 by R. erythropolis with recombinant expression of Vdh, the final major issue was the cell membrane permeability. This is not a unique problem of R. erythropolis but a similar issue was observed also for P. autotrophica. In R. erythropolis, no correlation was recognized in the conversion rate of hydroxylated VD3 and the amount of intracellular enzyme. Even if the expression level of Vdh was changed, the conversion rate was fixed at a certain rate[10]. Even the Vdh-K1 (see section 3.4.1) in which dramatic high activity was confirmed using the in vitro reconstitution system did not show much significant difference from the wild type when the conversion was done in the P. autotrophica cell. This meant that the performance of the enzyme inside the cell was not utilized, and it was estimated that the cell membrane permeation of substrate VD3 acted as the rate limiting factor. The VD3 is a fat-soluble steroid and solubility in the water is extremely low. Therefore, in the current microbial conversion, the solubility is increased by adding CD to the culture to trap the VD3 in the cyclic structure of the CD. In fact, the VD3 hydroxylation of Vdh increases dramatically by the addition of CD to the solution both in vivo and in vitro experiments. However, the permeation of the high molecular weight CD-VD3 complex through the cell membrane is difficult, Fig. 3 Structural mechanism of activity enhancement of P450 VdhIn general, P450 is in equilibrium of open and closed structures, and the substrate is likely to bind with the closed structure. The equilibrium between these structures shift greatly according to the variation selected by evolutionary engineering. The population in closed form increased and the activity was enhanced. Fig. 4 Recognition mechanism of substrate in an anti-parallel orientation by P450 VdhThe VD3 (left) and 25(OH)VD3 (right) can bond to the enzyme in an anti-parallel orientation, and that gives them the capacity for two-step hydroxylation to 1,25(OH)2VD3. The amino acids that were candidates for variations to eliminate the side reaction were selected from the detailed structural information of the substrate binding site. M216L156R384R70E216V156E384T70Substrate (VD3)Evolutionary engineering (replacement of 4 amino acid residues)HemeHighly active Vdh variant (Vdh-K1)Wild type Vdh(Vdh-WT)Closed from(with high substrate affinity)Open form(with low substrate affinity)Substrate binding pocketAsn181 Asn181 Lys180 Lys180 Leu171 Leu171 Met86 Met86 Leu89 Leu89 Ile88 Ile88 Pro287 Pro287 Thr240 Thr240 Leu387 Leu387 Ala236 Ala236 Ile235 Ile235 Leu232 Leu232 Asn181 Asn181 Lys180 Lys180 Leu171 Leu171 Met86 Met86 Leu89 Leu89 Ile88 Ile88 Pro287 Pro287 Thr240 Thr240 Leu387 Leu387 Ala236 Ala236 Ile235 Ile235 Leu232 Leu232 VD3VD3 25(OH)VD325(OH)VD3

※このページを正しく表示するにはFlashPlayer9以上が必要です