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

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.