Expression and characterization of fusion protein of vitreoscilla hemoglobin with spinach glycolate oxidase

Abstract

A glycolate oxidase (GO) gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from the spinach leaves, and the recombinant E. coli BL21(DE3)/pET-GO was constructed. To know whether bacterial hemoglobin can be used as an oxygen donor to oxygen-dependent GO, Vitreoscilla hemoglobin (VHb) gene was fused with GO gene by overlap-PCR and the fusion protein VGO was expressed in E. coli BL21(DE3). The recombinant protein GO and VGO were purified by one-step Ni-NTA chelating chromatography and then applied for further characterization. The specific activity of GO was 2.97U/mg, while VGO was 4.13U/mg, 39% higher than that of GO, suggesting that fusion of VHb could enhance specific activity of oxygen-consuming GO. Under the conditions of high phosphate buffer concentration, pH and temperature, the activity of VGO was more stable than that of GO. With glycolate as substate, K m values of GO and VGO were found to be 0.22mM and 0.16mM, respectively, indicating the substrate affinity of VGO is higher. It was also observed that growth and enzyme activity of BL21(DE3)/pET-VGO were less affected by aeration condition than those of BL21(DE3)/pET-GO. The higher cell density of BL21(DE3)/pET-VGO at the same culture condition suggested that the hemoglobin in fusion protein also benefit the growth of recombinant strain.