| 4"-O-isovalerylspiramycin is a new macrolide antibiotic produced by genetically engineered Streptomyces spiramyceticus transformed with the 4"-O-acyltransferase gene from S. mycarofaciens.4"-O-isovalerylspiramycin is a mixture of 4"-O-isovalerylspiramycin I, II, and III, which all possess similar molecular structures. During fermentation, 4"-O-isovalerylspiramycinⅢis the main and active product. However, separation of 4"-O-isovalerylspiramycinⅡandⅢis very difficult because they have very similar chemical structures and characteristics.In the present work, the separation process of 4"-O-isovalerylspiramycinⅡand III has been studied systematically. According to the difficulties of separation and purification, an innovative chromatographic technology routine has been presented, which can solve the difficulties in the separation of 4"-O-isovalerylspiramycin II and III.The main contents and conclusions are listed as follows:(1)With impure 4"-O-isovalerylspiramycin (4"-O-isovalerylspiramycinⅡ:Ⅲ=2:3, w/w) as raw material, the appropriate selective adsorption conditions for 4"-O-isovalerylspiramycinⅢ, which have been obtained by batch adsorption experiments, are as follows:HZ820 as adsorbent, ionic strength 0.5 mol/L, pH 6.5, KⅡⅢis 1.67, indicating that HZ820 has a good selectivity for 4"-O-isovalerylspiramycin III under these conditions. According to thermodynamics results, this adsorption is a spontaneous, entropy increase and endothermic process.(2)The competitive adsorption equilibrium data for the binary system of 4"-O-isovalerylspiramycinⅡandⅢonto a macroporous resin HZ820 were tested and modeled using the extended Langmuir, extended Langmuir-Freundlich and extended Jovanovic-Freundlich equation at different pH, ionic strengths and temperatures. For both 4"-O-isovalerylspiramycinⅡandⅢ, the adsorption was enhanced with the increase of pH, ionic strength and temperature. The extended Langmuir-Freundlich adsorption model gave the best competitive isotherm fits for both 4"-O-isovalerylspiramycin II and III. The relative errors of the extended Langmuir-Freundlich model were within 15%.(3)The effects of the operation parameters, which is mentioned above, on the adsorption kinetics of 4"-O-isovalerylspiramycin to HZ820 resin have been determined. Pseudo-first-order, Pseudo-second-order and intra-particle diffusion model each have been used to describe 4"-O-isovalerylspiramycin adsorption profile in thermostatic shaker. The results showed that Pseudo-second-order model successfully described the adsorption of 4"-O-isovalerylspiramycin on HZ820. The adsorption was favorable process.(4)The effect of flow velocity, feed concentration, and aspect ratio on the fixed-bed adsorption characteristics of 4"-O-isovalerylspiramycin was investigated by using a macroporous resin HZ820 as adsorbent. The film, porous and axial diffusion model taking the film mass transfer, pore diffusion and axial dispersion into account was adopted to describe the breakthrough curves. The mass transfer coefficients and the relative errors of the model were consequently determined. The results showed that the increase of flow velocity and feed concentration was not in favor of the fixed-bed adsorption performance, while the increase of aspect ratio could improve the mass transfer. It is clear that both liquid film mass transfer and pore diffusion play important roles in the fixed-bed adsorption of 4"-O-isovalerylspiramycin onto HZ820, whereas the axial dispersion could be negligible in the experimental range. The relative errors of the model were within 10%, indicating a satisfactory simulation.(5)On the basis of those above, the adsorption and desorption processes with impure 4"-O-isovalerylspiramycin as raw material have been studied by using the optimal conditions in fixed bed. It shows that a competitive adsorption between 4"-O-isovalerylspiramycin II andⅢoccurred; petroleum ether as eluent, flow velocity 1.20 BV/hr, the elution volume 6.00BV, KⅡⅢis 1.60, and thus 4"-O-isovalerylspiramycin III desorption rate can reach 94.2%.(6)A complete set of separation process for recovering 4"-O-isovalerylspiramycin III from fermentation broth was introduced, including membrane filtration, adsorption, impurity washing, desorption and crystallization in the eluate. The technological conditions and its practical effect of each unit operation were studied and optimized. The phosphate hydrate buffer solution (pH 8.0,1=0.2 mol·L-1)was chosen to wash impurities. Through the whole separation process, the content of 4"-O-isovalerylspiramycin III in product could meet the requirement.Aim at the difficulties of separation of 4"-O-isovalerylspiramycin II and III, an innovative technology routine for separation and purification of 4"-O-isovalerylspiramycin III has been presented in this paper. This research provides the experimental foundation for the development and design of large-scale industrial equipment. With the combination of membrane filtration and crystallization, an advanced 4"-O-isovalerylspiramycin production technology can be established, which can improve the purity and yield of 4"-O-isovalerylspiramycin III significantly and be of great prospect in practical application.
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