Effects of water activity and polyethylene glycol on enzymatic synthesis of cephalexin

Abstract

The effects of the controlled water activity (Aw) of the reaction medium and the addition of polyethylene glycol(PEG) on enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-$\alpha$ -phenylglycine methylester (PGM) using Xanthomonas citri enzyme were investigated. When the Aw of the reaction medium was depressed, the synthetic activity and conversion yield were markedly enhanced. Aw depressors used to control the Aw of reaction medium were sorbitol, sucrose and glycerol, and the enhancing effects on conversion yield were up to 270\%, 230\% and 170\%, respectively. Aw depressors could also increase the enzyme stability to some extent, but this stabilizing effect was not related proportionally to the enhancing effects on conversion yield. These enhancements of conversion yield were probably resulted from the change of thermodynamic equilibrium and maximized at a range of Aw from 0.96 to 0.97. Based on the experimental results that show the decreasing of D-$\alpha$ -phenylglycine (PG, the hydrolysate of PGM and an inhibitor on CEX synthesis) accumulation rate in depressed-Aw system, it was noted that the conversion yield of CEX was enhanced due to the more sufficient PGM amount as an available substrate resulted from the suppression of the PGM hydrolytic reaction. With enzyme concentration of 0.5 unit/ml, it was possible to achieve sixfold reduction in necessary PGM amount to obtain the same yield as control by Aw depression of the reaction medium. And the addition of polyethylene glycol(PEG) to the reaction mixture also increased synthetic yield markedly, but did not affect PGM hydrolytic and CEX hydrolytic reaction rates. The concentrations of PEG used in these experiments could not depress water activity so significantly that water activity might affect the reaction system. PEG could also improve the stability of the enzyme, but this was not related proportionally to the enhancement of CEX synthetic yield as the c...