Abstract：Mycoplasma bovis (Mb) is one of the most important pathogens causing diseases in beef cattle (Bos taurus) and cow, which cause great economic loss to cattle industry.Enolase (eno) is the key rate-limiting enzyme in glycolysis pathway and catalyzes the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP).In order to study the enzymatic activity of Mb eno, the specific primers were designed according to the eno gene sequence of Mb PG45 strain in GenBank (NC_014760.1) and the eno gene of Mb Wuwei strain was amplified by PCR.Based on the sequence analysis and the gene optimization, the prokaryotic expression vector pET-eno was constructed and transformed into Escherichia coli Transetta (DE3).Then the recombinant protein was expressed with induction by isopropyl β-D-1-thiogalactopyr-Anoside (IPTG).Subsequently, the expression product was purified and the assay of its enzymatic activity was completed.The results showed that the optimized eno gene of Mb Wuwei strain was successfully expressed in DE3 in soluble form and the relative molecular weight of the recombinant protein His-eno was approximately 53 kD.The results of enzymatic activity analysis indicated that the catalytic activity of His-eno was slightly higher than that of rabbit muscle enolase, the optimum temperature was 42 ℃ and the optimal pH was 8.0.The michaelis constant (Km) and maximum reaction velocity (Vmax) were 4.1×10-3 mol/L and 3.4 μmol/(L·min), respectively.The results of this study could provide basic data for further study on the biological function of Mb eno.
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