Bernardino Susana M S A, Fernandes Pedro, Fonseca Luís P
ESTM - Escola Superior de Turismo e Tecnologia do Mar, Instituto Politécnico de Leiria, Peniche, Portugal.
Biotechnol J. 2009 May;4(5):695-702. doi: 10.1002/biot.200800287.
The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 microm, as determined by scanning electron microscopy. An immobilization yield of 95-100% and a recovered activity of 50-65% at 37 degrees C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g(dry weight)) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14 degrees C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.
本工作聚焦于一种新型磁性生物催化剂的开发与基本特性研究，该生物催化剂即青霉素G酰化酶（PGA），其固定于具有磁性的溶胶 - 凝胶基质中，最终旨在应用于头孢氨苄（CEX）的合成。基于从四甲氧基硅烷（TMOS）起始制备的多孔干凝胶二氧化硅基质，制备了一种机械稳定的载体，通过扫描电子显微镜测定，得到了中等粒径为30微米的微载体。通过青霉素G（PG）水解（pH STAT法）测定，在37℃下观察到固定化产率为95 - 100%，回收活性为50 - 65%。这些结果明显超过了先前关于PGA固定于溶胶 - 凝胶的工作中所报道的结果，在该工作中仅回收了10%的活性。活性值在6个月内保持恒定。固定化PGA（682 U/g（干重））在连续十次PG水解运行中均保持高比活性，表明生物催化剂具有足够的稳定性。CEX的合成在14℃下进行，使用游离和固定化的PGA在水性介质中。苯甘氨酸甲酯用作酰基供体，浓度为90 mM，7 - 氨基去乙酰氧基头孢烷酸为限量底物，浓度为30 mM。1小时反应后CEX的化学计量产率分别接近68%（23 mM CEX/h）和65%（19 mM CEX/h）。
