双交联剂体系氨基酰化酶交联聚体的制备

Preparation of Cross-Linked Aggregates of Aminoacylase by Using Dual Cross-Linkers

采用酶交联聚集体技术(CLEAs)制备氨基酰化酶(EC 3.5.1.14)交联聚集体,优化了制备条件。90%(体积比)乙醇为沉淀剂,戊二醛和乙二醇二缩水甘油醚(EGDE)作为混合交联剂,以酶、戊二醛和EGDE的质量比为1.00∶0.75∶2.00,在30℃下交联12 h。制备所得氨基酰化酶CLEA酶活保留率为63.42%。氨基酰化酶经固定化后热稳定性得到增强,热失活过程由自由酶的一阶指数失活模型变为二阶指数失活模型,分子间亚基的结合力得到显著加强。自由酶在37℃下保存24 h后几乎损失了所有活力,而氨基酰化酶CLEA则保留了43%的酶活力,在57℃下保存24 h后仍有32%的酶活力保留。此外,氨基酰化酶CLEA重复使用性能得到明显提升,在重复水解底物10次后仍保留了72%的酶活力。

The cross-linked enzyme aggregate（CLEA） of aminoacylase（EC 3.5.1.14） from aspergillus melleus was prepared by precipitation with ethanol and cross-linking with glutaraidehyde and ethylene glycol diglycidyl ether（EGDE） as cross-linkers.The various cross-linked parameters were optimized.Almost 63.42% activity of CLEA of aminoacylase remained by using 90%（V/V） ethanol,followed by cross-linking 12 h with glutaraidehyde and EGDE at 30 ℃,the mass ratio was 1.00∶0.75∶2.00.The stability of aminoacylase was enhanced after immobilization.The thermal deactivation model was changed from one-exponential model（free enzyme） to two-exponential model.Free enzyme lost almost all of activity,while aminoacylase CLEA 43% of initial activity.Even at 57 ℃ after 24 h,32% of initial activity was retained.Additionally,the reusability of the CLEA was evaluated.Aminoacylase CLEA showed 72% residual activity after 10 cycles of repeated use.