牛肝β-糖苷酶是一种能催化核苷类化合物区域选择性糖基化反应合成二糖核苷的生物催化剂。在已有的研究报道中，采用的生物催化剂都是游离酶，不利于其大规模应用。交联酶聚合物(CLEAs)作为一种新型的无载体固定化酶，具有制备简便、成本低、体积酶活高等优点。本文研究牛肝β-糖苷酶CLEAs的制备，并对其酶学性质和结构进行了研究，探讨了其在二糖核苷合成中的应用。最适的沉降剂、交联剂及交联时间分别为70%饱和度的(NH4)2SO4、50% (V/V)葡聚糖多醛及8 h。在最适条件下，CLEAs的活性回收率为58%。该CLEAs的最适pH、温度、Vmax及Km分别为8.5、60 ℃、0.13 μmol/(min mg)及1.43 mM。并且该CLEAs具有较好的操作稳定性，重复利用6批次后，其催化合成5’-O-β-葡萄糖基-2’-脱氧尿苷的相对产率仅下降20%。牛肝β-糖苷酶CLEAs呈无定形态。

β-glycosidase from bovine liver is a biocatalyst that can catalyze the synthesis of disaccharide nucleosides via regioselective glycosylation of nucleosides. In previous reports, the used biocatalysts were free enzymes, which limit the large-scale applications. As novel carrier-free immobilized enzymes, cross-linked enzyme aggregates (CLEAs) have many advantages, such as easy preparation, low cost, and high enzyme volumetric activities. CLEAs of β-glycosidase from bovine liver were prepared, their enzymatic properties and structures were studied, and their application for the synthesis of disaccharide nucleoside was explored. The optimal precipitant, cross-linking agent and cross-linking time were 70% saturated ammonium sulfate, 50% (V/V) dextran polyaldehyde, and 8 h, respectively. Under optimal conditions, the activity recovery of CLEAs reached 58%. The optimal pH, temperature, Vmax and Km of the CLEAs were 8.5, 60 ℃, 0.13 μmol/(min?mg), and 1.43 mM, respectively. Additionally, the CLEAs showed good operational stability. After they were reused for six batches, the relative yield in the CLEA-catalyzed synthesis of 5′-O-β-glucosyl-2′-deoxyuridine had decreased only by 20%. The CLEAs had an amorphous structure.

珠江科技新星专项基金（2012J2200011）；中央高校基本科研业务费项目(2014ZG0045)