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P. Xue et al. / Chinese Chemical Letters 21 (2010) 765–768
bisacrylamide (MBAA) in the presence of Fe3+ and Fe2+ dispersed in formamide, was used for ampicillin synthesis for
the first time, and a high yield and the S/H ratio were successfully achieved in ethylene glycol at 20 8C. The
immobilized PGA can be separated magnetically and recycled effectively.
1. Experimental
Preparation of magnetic hydrophilic polymer microspheres: 0.67 g of FeCl2ꢁ4H2O and 1.8 g of FeCl3ꢁ6H2O were
dissolved in 25 mL formamide, and then concentrated aqueous ammonia was slowly added to adjust the pH to 9. After
heating to 90 8C and stirring for 2 h, followed by another 4 h at 60 8C, 2.6 g of GMA and 1.9 g of MBAAwere added to
form a polymerizing mixture. The mixture was dispersed in the suspension which consisted of 109 mL of n-heptane,
35 mL of tetrachloroethylene and 0.1 g of span 60 as well as 5.0 g of calcium stearate. The polymerization was
initiated with 2,2-azobis(2-methylpropionitrile) under nitrogen at 60 8C for 5 h. The resultant microspheres were
separated magnetically, washed with acetone, dried under vacuum for 24 h. The sample was named MHPM.
Immobilization of PGA on MHPM: 0.06 g of MHPM support was immersed in 2 mL of solution prepared by
diluting ten times original enzyme solution with pH 7.0 phosphate buffer (PB). The mixture above was stirred gently
for 48 h at 30 8C. The immobilized PGA on MHPM was separated by a magnet and washed with phosphate buffer
eight times. The sample was named PGA/MHPM.
Typical synthesis of ampicillin: 50 mmol/L of 6-APA, 100 mmol/L of D-PGME and 2 mL of solvent were mixed in
a reactor. The wet PGA/MHPM was added and shaken at certain temperature. The PGA/MHPM was magnetically
separated at the end of reaction and kept at 4 8C for further use. The mixed solution was analyzed by HPLC (FL2200)
equipped with a UV detector and Venusil XBP reversed-phase C18 column (4.6 mm ꢂ 200 mm).
2. Results and discussion
The designed and prepared MHPM support for immobilizing PGA possesses the multiple functional actions.
Fig. 1a shows that the MHPM has perfect spherical shape and the average size of 100–200 mm. A typical
magnetization curve for the MHPM is observed in Fig. 1b in which there is no hysteresis in the magnetization with
both remanence and coercivity being zero as well as 6.4 emu/g of saturation magnetization. Therefore, the MHPM
displays superparamagnetic behavior, and their separation in liquid media can be controlled by magnetic fields.
Furthermore, there is an abundance of the active oxirane groups on the surface of MHPM that can couple covalently
the enzyme under mild conditions. The MHPM has high specific surface area and large average pore diameter
(128.2 m2/g and 17.1 nm, respectively), which is the advantage for the immobilization of PGA molecular with bulk
volume.
The apparent activity and enzyme coupled to the PGA/MHPM was 569 IU/g and 34.6 mg/g according to literature
[3,4], respectively. The results of the PGA/MHPM catalyzed 6-APA and D-PGME to synthesize ampicillin in some
conventional solvents are listed in Table 1. The ampicillin yield and S/H ratio reach 96.7% and 1.60 using PGA/
MHPM in pure ethylene glycol. While using free PGA, the values were 82.1% and 1.40, respectively. This result shows
that the synthesis activity of PGA can be effectively improved by the immobilization on the MHPM support, especially
in the S/H ratio. Furthermore, the reaction medium of the ampicillin synthesis by PGA/MHPM is a significant factor.
Fig. 1. (a) SEM photograph and (b) magnetization curves of MHPM.
Xue, Ping
