氨基树脂固定化L-苏氨酸醛缩酶及其应用*

L-苏氨酸醛缩酶(L-Threonine aldolase,L-TA)可以催化甘氨酸和醛合成β-羟基-α-氨基酸。β-羟基-α-氨基酸具有两个手性中心,是多种手性药物的中间体。但是,游离的L-TA难以重复利用,分离纯化困难,严重阻碍了工业化应用。固定化技术可以有效解决这些问题。利用氨基树脂NAA固定化来源于Bacillus nealsonii的L-苏氨酸醛缩酶,采用戊二醛作为交联剂,经过条件优化确定最佳固定化条件为:加酶量13 U、载体量0.6 g、0.4%(V/V)戊二醛、活化时间2 h、pH 8.5、35℃、固定化5 h。在此条件下,固定化酶酶活回收率为85.7%。在30℃下半衰期可达59天,为游离酶的6.5倍。将其应用于合成L-syn-对甲砜基苯丝氨酸,使用460 h后,残余酶活为79.4%。进一步开发了载体再利用策略,将失活固定化酶表面的氨基用戊二醛活化后,再与新的游离酶进行固定化,实现载体的再利用。利用该方法载体可重复利用两次,制备的固定化酶仍能使用460 h。该方法大大降低了固定化成本,为固定化L-TA的工业化应用打下坚实的基础。

Immobilized L-threonine Aldolase by Amino Resin and Its Application

L-threonine aldolase (L-TA) synthesizes β-hydroxy-α-amino from glycine and aldehyde. β-hydroxy-α-amino acids with two chiral centers is an important pharmaceutical intermediate for the synthesis of many drugs. However, free threonine aldolase is difficult to be reused and separated, which seriously hinders industrial application. Immobilization technology can effectively solve these problems. L-threonine aldolase was immobilized by amino carrier NAA and glutaraldehyde was used as the crosslinking reagent. After optimization of conditions, the optimal immobilization conditions were determined as: enzyme loading 13 U, 0.6 g carrier, glutaraldehyde concentration of 0.4 %(V/V), activation time of 2 h, pH of 8.5 and temperature of 35℃, and immobilization time of 5 h. Under these conditions, the activity recovery of immobilized enzyme was 85.7%. The half-life at 30℃ can reach 59 days, which was 6.5 times that of the free enzyme. The immobilized enzyme was applied in the synthesis of L-syn-3-4-(methylsulfonyl)phenylserine] (L-syn-MTPS) for 460 h still remained activity of 79.4%. Furthermore, the method to reuse carrier was developed. In this method, the residual amino groups on the surface of the inactivated immobilized enzyme were first activated by glutaraldehyde, and then further combined with the fresh enzyme to realize the reuse of the resin. Using this method, the carrier can be reused twice and the prepared immobilized enzyme can still be used for 460 h. This method greatly reduces the cost of immobilization and lays a solid foundation for the industrial application of immobilized L-TA.