Soluble recombinant pyruvate oxidase production in Escherichia coli can be enhanced and inclusion bodies minimized by avoiding pH stress

Abstract

BACKGROUND: Recombinant pyruvate oxidase (pod) is in high demand owing to its broad application in biochemical analysis and low yield from wild microbial strains. Recombinant Escherichia coli harboring pET28a-pod is an efficient producer of pod, facilitating its production in high yield. Optimizing cultivation using shake flasks is necessary for high-level soluble pod production. RESULTS:During recombinant E. coli cultivation, 96.9% of pod activity was lost, and 3.1% of residual pod activity remained in a 25 mL working volume in 250 mL flasks owing to accumulation of pod in insoluble inclusion bodies. pH stress was the factor responsible for this phenomenon based on investigating dissolved oxygen and carbon source addition. Residual pod activity was increased to 43.3, 88.0 and 61.5% by maintaining the pH at 5.0, 6.0 and 7.0 respectively during the induction phase, which decreased inclusion body formation and increased production of soluble pod. Glycerol addition increased the cell density and volumetric pod activity to 21 243.3 U L −1 at pH 6.0 after 64 h induction. CONCLUSION: Soluble pod is converted to inclusion bodies under pH stress conditions, and this can be avoided by pH maintenance.

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

“…Microbial transglutaminase (MTGase) monomers from Streptoverticillium mobaraensis , for example, were shown to have a high tendency to cross-link and oligomerize during intracellular expression [ 25 ]. MTGase was also observed to be expressed at low abundance in S. mobaraensis —at concentrations inadequate for subsequent experimental work—which is an issue frequently observed in similar studies [ 45 – 49 ]. Additionally, enzyme purification methods from native host sources often require a combination of methods such as salt precipitation and/or a series of chromatographic separations to deliver a product with sufficient purity.…”

“…Furthermore, inferences from this type of analysis can guide the choice of media. It was observed that factors including maintaining a pH 6 medium [ 45 ], the addition of betaine as an osmolyte [ 106 ], and the addition l -arginine [ 173 ] could improve the solubility of the final product in three studies, however ad hoc methods led to this discovery.…”

Lacto-N-biose synthesis via a modular enzymatic cascade with ATP regeneration

“…Based on the BRENDA and NCBI databases, we selected the enzymes GalK and AcK from Escherichia coli ( Yang et al., 2003 ; Yao et al., 2019 ), LnbP from Bifidobacterium bifidum ( Wada et al., 2008 ), and PyoD from Aerococcus viridans ATCC 10400 ( Zhang et al., 2019 ) for constructing pathway II. All enzymes were expressed functionally in E .…”