N-乙酰-D-葡萄糖胺-2-差向异构酶基因的克隆及表达

Cloning，Expression and Enzymatic Properties of N-Acetyl-D-Glucosamine-2-Epimerase in Escherichia coli BL21（DE3）

摘要:

N-乙酰-D-葡萄糖胺-2-差向异构酶（AGE）是合成N-乙酰-D-神经氨酸（Neu5Ac）的关键酶。该酶的高效表达是提高全细胞细胞催化法合成Neu5Ac的有效途径。根据集胞藻（Synechocystis sp. PCC 6803）AGE编码基因slr1975序列信息和大肠杆菌密码子偏好性，优化密码子并人工合成目的基因序列slr975-co。将该基因克隆到表达载体pET28a中，构建了重组大肠杆菌BL21（DE3）/pET28-slr并实现了目的基因的诱导表达。SDS-PAGE分析表明，重组蛋白质的相对分子质量在43 000左右，与预期大小一致，纯化产物蛋白质质量浓度约为1.7 g/L。重组AGE的最适反应温度为42 ℃，最适反应pH为6.0。AGE对GlcNAc、ManNAc的Km值分别为39.0、46.5 mmol/L。以分别表达AGE和N-乙酰神经氨酸裂合酶（NanA）的重组大肠杆菌作为催化剂，以N-乙酰-D-葡萄糖胺为底物，实现了N-乙酰-D-神经氨酸的全细胞催化合成。结果表明，对AGE密码子进行优化实现了该基因在大肠杆菌中的高效表达，为全细胞催化法高效合成N-乙酰-D-神经氨酸奠定了坚实的基础。

Abstract:

Overexpression of N-acetyl-D-glucosamine 2-epimerase（AGE），as a critical enzyme for N-acetyl-D-neuraminic acid（Neu5Ac） synthesis ，is an efficient strategy for improvement of Neu5Ac production. Based on the information about the slr1975 gene encoding Synechocystis sp. PCC6803 AGE and the codon bias of Escherichia coli，slr1975-co was synthesized and cloned into the pET28a expression vector. The recombinant plasmid pET28-slr was purified，and then transformed into E. coli BL21（DE3） strain. The recombinant enzyme was overexpressed by induction of IPTG and SDS-PAGE showed that the molecular mass of the recombinant AGE protein is about 43 kDa，which was in agreement with their theoretical value. The recombinant enzyme was characterized and the reuslts showed that the optimal temperature is 42 ℃ and the optimal reaction pH was 6.0. Kinetic studies indicated that the Km for N-acetyl-D-glucosamine（GlcNAc） and N-acetyl-D-mannosamine（ManNAc） was 39.0 mmol/L and 46.5 mmol/L，respectively. Moreover，Neu5Ac production was evaluated by the recombinant enzmye AGE and the results demonstrated that recombinant enzmye AGE coupled with Neu5Ac aldolase（NanA） could catalyze N-acetyl-D-glucosamine to produce Neu5Ac effectively. In summary，codon-optimization and overexpression of AGE provide paved a way for efficient Neu5Ac production by whole-cell catalysis.