| UDP-glucose4-epimerase, belonging to the enzyme superfamily of short-chain dehydrogenase/reductase, catalyzes the interconversion of UDP-glucose and UDP-galactose in galactose metabolism of bacterial and mammalian to realize the last step of the interconversion of UDP-glucose and UDP-galactose.Marinitoga piezophila KA3is a thermophilic, anaerobic, chemoorganotrophic, sulfur-reducing bacterium isolated in2002from enrichment cultures of deep-sea hydrothermal chimney samples at65℃under in situ hydrostatic pressure(30MPa), which is the first true thermopiezophilic bacterium. The species grows optimally at65℃and40MPa. Structurally,56%identical to the homologous Escherichia coli K12enzyme, each monomer of UDP-glucose4-epimerase from Marinitoga piezophila KA3consists of337amino acids with one molecule of NAD+tightly bound in the syn-conformation with its si-face oriented towards the sugar substate.In order to reduce biological barrier during the heterologous enzyme expression in Escherichia coli, according to the related gene sequence provided by NCBI, nucleotide sequence of UDP-glucose4-epimerase gene was optimized from the codon bias and mRNA secondary structure before the experiment, to ensure that Escherichia coli can express the target enzyme efficiently and stably.The target gene was amplified via designing specific primer using software Prime Primer5.0and polymerase chain reaction, and the molecular weight of specific band was identified about1kbp via1%agarose gel electrophoresis. The purified amplification product and vector pET-28a(+) were double digested by restriction enzyme Hind Ⅲ and Nde I, and then connected through the T4DNA ligase. The connecting product was washed, purified in the capacitance of25μF, voltage of2500V, resistance of500Ω and then transformed electrically into the clone strains Escherichia coli DH5α competent cells, followed by coated on the resistance plate, and then identified via polymerase chain reaction and double enzyme digestion. The results showed:3in10white colonies picked randomly were identified as positive clones, the others were identified as false positive clones. The recombinant plasmid extraction from the positive clone was transformed by CaCl2into expression strain Escherichia coli BL21(DE3) competent cell to obtain engineering strain Escherichia coli BL21(DE3)(pET-gale).The engineering strain Escherichia coli BL21(DE3)(pET-gale) was cultured of12-16h in the condition of37℃and250r/min, transferred1mL culture liquid to50mL LB liquid medium and induced by IPTG to produce target enzyme under certain conditions. When the final concentration of IPTG was0.1mM, the induction temperature was27℃, and the induction time was4h, target enzyme with activity was harvested most. As the adsorbent of Ni2+affinity column can binds specifically substance with6×His marker, the crude enzyme liquid through Ni2+affinity chromatography was identified by SDS-PAGE electrophoresis as specific protein having a molecular weight close to40kDa with a clear band and less excess protein. After purification, specific activity of target enzyme increased from0.517U/mg protein to9.440U/mg protein, the purification multiple and the recovery rate was18.26and57.08%, respectively.According to the following high performance liquid chromatographic conditions:column type was Athena NH2, the volume ratio of KH2PO4buffer solution (0.125M, pH3.6) and acetonitrile was40:60as mobile phase, the detection wavelength was254nm using ultraviolet absorption detector, the retention time of UDP-glucose standard and the UDP-galactose standard detected by high performance liquid chromatography were17.130min and15.748min, respectively. UDP-galactose appeared prior to UDP-glucose with sharp peaks and satisfying separation effect when mixed. Both UDP-glucose and UDP-galactose had good linearity with high sensitivity according to standard curves, of which correlation coefficients were0.9986and0.9988respectively and the detection limits were5.8ng and3.4ng respectively. Also the recovery rate of two kind of material mentioned above were95.57%and96.48%respectively and the relative standard deviation were6.2%and7.8%respectively, of which high precision meet the requirements. At35℃and pH8.0, target enzyme had high4-epimerization activity on the substrate UDP-glucose with the enzyme activity of19nmol/min and the conversion rate of77.71%.Using UDP-glucose as a substrate, the target enzyme showed high4-epimerization activity in a wide pH(7.0～9.0) and temperature(35℃～45℃), of which the optimum pH and the optimum temperature were8.0and40℃～45℃. At the optimum pH and the optimum temperature, the Michaelis constant and the maximum reaction rate of the target enzyme for UDP-glucose as a substrate were0.76mM and11.89nmol/min, respectively. In addition to the cofactor NAD+, the4-epimerization activity of the target enzyme was also affected by metal ions. Ca2+, Mn2+and Mg2+could activate the4-epimerization reaction, but Cu2+and Hg2+led to strong inhibitory effect on the enzyme reaction with the feature that low concentration of it could lead to the rapid decline of enzyme activity. Higher polarity of organic solvent had stronger inhibition on enzyme activity. SDS and PMSF could significantly inhibit the activity of enzyme, but Tween-80and EDTA had little inhibition on enzyme activity. |
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