Jin Ke-ming, Cao Xue-jun, Su Jin, Ma Li, Zhuang Ying-ping, Chu Ju, Zhang Si-liang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2008 Mar;28(3):360-2.
Immobilized penicillin acylase was used for bioconversion of penicillin PG into 6-APA in aqueous two-phase systems consisting of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB. Partition coefficients of 6-APA was found to be about 5.78 in the presence of 1% NaCl. Enzyme kinetics showed that the reaction reached equilibrium at roughly 7 h. The 6-APA mole yields were 85.3% (pH 7.8, 20 degrees C), with about 20% increment as compared with the reaction of single aqueous phase buffer. The partition coefficient of PG (Na) varied scarcely, while that of the product, 6-APA and phenylacetic acid (PA) significantly varied due to Donnan effect of the phase systems and hydrophobicity of the products. The variation of the partition coefficients of the products also affected the bioconversion yield of the products. In the aqueous two-phase systems, the substrate, PG, the products of 6-APA and PA were biased in the top phase, while immobilized penicillin acylase at completely partitioned at the bottom. The substrate and PG entered the bottom phase, where it was catalyzed into 6-APA and PA and entered the top phase. Inhibition of the substrate and products was removed to result in improvement of the product yield, and the immobilized enzyme showed higher efficiency than the immobilized cells and occupied smaller volume. Compared with the free enzyme, immobilized enzyme had greater stability, longer life-time, and was completely partitioned in the bottom phase and recycle. Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage. The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtered 450 nm light, while pH-sensitive polymer PADB could be recovered at the isoelectric point (pH 4.1). The recovery of the two copolymers was between 95% and 99%.
固定化青霉素酰化酶用于在由光敏聚合物PNBC和pH敏感聚合物PADB组成的双水相体系中将青霉素G（PG）生物转化为6-氨基青霉烷酸（6-APA）。在1% NaCl存在下，6-APA的分配系数约为5.78。酶动力学表明反应在大约7小时达到平衡。6-APA的摩尔产率为85.3%（pH 7.8，20℃），与单相水相缓冲液反应相比增加了约20%。PG（Na）的分配系数几乎没有变化，而产物6-APA和苯乙酸（PA）的分配系数由于相体系的唐南效应和产物的疏水性而有显著变化。产物分配系数的变化也影响了产物的生物转化产率。在双水相体系中，底物PG、产物6-APA和PA偏向于上相，而固定化青霉素酰化酶完全分配在下相。底物PG进入下相，在那里被催化成6-APA和PA并进入上相。底物和产物的抑制作用被消除，导致产物产率提高，并表明固定化酶比固定化细胞具有更高的效率且占用体积更小。与游离酶相比，固定化酶具有更高的稳定性、更长的使用寿命，并且完全分配在下相且可循环使用。使用固定化青霉素酰化酶在双相体系中的生物转化显示出突出优势。形成双水相体系的光敏共聚物可通过488 nm激光辐射或过滤450 nm光回收，而pH敏感聚合物PADB可在等电点（pH 4.1）回收。两种共聚物的回收率在95%至99%之间。
