Triple fusion of d-amino acid oxidase from Trigonopsis variabilis with polyhistidine and Vitreoscilla hemoglobin

Abstract

Fusion proteins of D-amino acid oxidase from Trigonopsis variabilis (TvDAAO) with Vitreoscilla Hemoglobin (VHb) and (His) 6 -tag were constructed and expressed in recombinant Escherichia coli. A fusing-position effect was revealed that (His) 6 -tag's N-terminal fusion with TvDAAO (HDAAO) reduced the specific activity by *29%, while the C-terminal fusion (DAAOH) remained unreduced. The N-terminal fusion of VHb with TvDAAO and DAAOH significantly improved their activity. As in a 5 l fermentor, the activity of the triple fusion VHb-TvDAAO-(His) 6 (VDAAOH) reached 2.53 U/mg dry cell at 9 h, *58% increase than that of DAAOH together with *40% biomass increase, confirming the positive effect of VHb expression on cell level. After purification, the UV-visible and fluorescence spectrum of DAAOH and VDAAOH were characterized. Enzyme kinetics studies further indicated that VDAAOH behaved a higher K cat , but a weaker substrate affinity of K m relative to DAAOH, revealing two distinct impacts of VHb-coupling with TvDAAO on protein level.

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli

“…According to this method ( Tang et al, 2020a ), the recombinant strains were cultured, induced, collected, and lysed by ultrasonication. The lysate supernatant was purified to electrophoretic purity by Ni-chelation affinity chromatography ( Ma et al, 2009 ; Tang et al, 2018a , b ) and centrifuged at 4°C and 8,000 rpm for 5 min to collect the induced bacterial cells. The collected cells were resuspended in 5 ml of ice-bath binding solution, then 25 μl of PMSF (10 mg ml –1 ) solution was added, followed by ultrasonication of lyse cells on ice.…”

Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel d-Mandelate Dehydrogenase with High Catalytic Activity

“…The induced cells were harvested by centrifugation and lysed by ultrasonication, and the cell lysate was centrifuged at 10000 × g and 4 °C for 30 min. The supernatant was then purified to homogeneity by Ni-chelating affinity chromatography …”

Exploitation of cold‐active cephalosporin C acylase by computer‐aided directed evolution and its potential application in low‐temperature biosynthesis of 7‐aminocephalosporanic acid

“…After cultivation at 37 °C for 2.5 h, IPTG was added to a final concentration of 0.1 mM, and induced for 12 h at 26 °C. Then, the cells were harvested by centrifugation and lysed by ultrasonication, and the cell lysate was centrifuged at 13684 g and 4 °C for 30 min, before the supernatant was purified to homogeneity by Ni‐chelating affinity chromatography . The specific activity of the purified mutant at 13 °C was respectively determined to rescreen these mutants …”