重组黄杆菌肝素酶Ⅲ的纯化、表征及培养条件对酶生产的影响

肝素酶Ⅲ是一种特异性地裂解乙酰肝素的酶,在大肠杆菌中表达时容易形成包涵体.为实现肝素酶Ⅲ的可溶性表达,利用谷胱甘肽-S-转移酶(GST)与肝素酶Ⅲ融合性能,通过构建相应的表达质粒pGEX-heparinaseⅢ,在大肠杆菌中实现了肝素酶Ⅲ的可溶性表达.粗酶通过一步亲和纯化其纯度可达95%以上.通过对LB培养基摇瓶培养Escherichia coli BL21的诱导时机,诱导剂用量、诱导时间等培养条件的优化,确定了该可溶性肝素酶Ⅲ融合蛋白的最适生产条件.通过对纯酶的最适反应温度、pH、Ca~(2+)浓度等一系列性质研究,确定了该酶的最适反应条件.

Purification and properties of recombinant GST-haparinase Ⅲ and optimizationm of cultivation conditions

Heparinase Ⅲ is an enzyme that specifically cleaves certain sequences of heparan sulfate.Previous reports showed that this enzyme expressed in Escherichia coil was highly prone to aggregation in inclusion bodies and lacks detectable biological activity.In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase Ⅲ gene and expressed the fusion protein in Escherichia cell to develop an expression system of soluble heparinase Ⅲ.As a result, approximately 80% of the fusion protein was soluble.The protein was then purified to near homogeneity via one-step affinity chromatography.A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein.This represented 32.3% recovery of the total activity of recombinant GST-heparinase Ⅲ.The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-β-D-thiogalactoside at 15℃ for 12 h.The enzyme showed maximum activity at 30℃ and pH 7.5.And the enzyme activity was stimulated by 1 mmol/L Ca~(2+) and 150 mmol/L NaCl.