Dipartimento di Scienze del Farmaco and Italian Biocatalysis Center, Via Taramelli 12, Università degli Studi di Pavia, 27100 Pavia, Italy.
Enzyme Microb Technol. 2011 Jun 10;49(1):52-8. doi: 10.1016/j.enzmictec.2011.03.011. Epub 2011 Apr 7.
Homodimeric thymidine phosphorylase from Escherichia coli (TP, E.C. 2.4.2.4) was immobilized on solid support with the aim to have a stable and recyclable biocatalyst for nucleoside synthesis. Immobilization by ionic adsorption on amine-functionalized agarose and Sepabeads(®) resulted in a very high activity recovery (>85%). To prevent undesirable leakage of immobilized enzyme away from the support, the ionic preparations were cross-linked with aldehyde dextran (MW 20 kDa) and the influence of the dextran oxidation degree on the resulting biocatalyst activity was evaluated. Although in all cases the percentage of expressed activity after immobilization drastically decreased (≤ 25%), this procedure allowed to obtain an active catalyst which resulted up to 6-fold and 3-fold more stable than the soluble (non immobilized) enzyme and the just adsorbed (non cross-linked) counterpart, respectively, at pH 10 and 37°C. No release of the enzyme from the support could be observed. Covalent immobilization on aldehyde or epoxy supports was generally detrimental for enzyme activity. Optimal TP preparation, achieved by immobilization onto Sepabeads(®) coated with polyethyleneimine and cross-linked, was successfully used for the one-pot synthesis of 5-fluoro-2'-deoxyuridine starting from 2'-deoxyuridine or thymidine (20mM) and 5-fluorouracil (10mM). In both cases, the reaction proceeded at the same rate (3 μmol min(-1)) affording 62% conversion in 1h.
来自大肠杆菌（TP，E.C. 2.4.2.4）的二聚体胸苷磷酸化酶通过离子吸附固定在胺功能化琼脂糖和 Sepabeads（®）上，实现了核苷合成的稳定可回收生物催化剂。固定化酶的酶活回收率非常高（>85%）。为了防止固定化酶从载体上不可避免地泄漏，离子制备物用醛化葡聚糖（MW 20 kDa）交联，并评估了葡聚糖氧化度对所得生物催化剂活性的影响。尽管在所有情况下，固定化后表达的活性百分比都急剧下降（≤25%），但该方法仍能获得活性催化剂，与可溶性（非固定化）酶相比，其在 pH 10 和 37°C 下分别稳定 6 倍和 3 倍；与仅吸附（非交联）的对应物相比，分别稳定 6 倍和 3 倍。未观察到酶从载体上释放。醛或环氧载体的共价固定化通常对酶活性有害。通过固定在涂有聚乙烯亚胺的 Sepabeads（®）上并交联，可获得最佳的 TP 制剂，该制剂成功地用于从 2'-脱氧尿苷或胸苷（20mM）和 5-氟尿嘧啶（10mM）一锅法合成 5-氟-2'-脱氧尿苷。在这两种情况下，反应速率相同（3 μmol min（-1）），1 小时转化率为 62%。
