2,5-Diketo-gluconic acid reductase from Corynebacterium glutamicum: Characterization of stability, catalytic properties and inhibition mechanism for use in vitamin C synthesis

Evaluation of the food grade expression systems NICE and pSIP for the production of 2,5-diketo-D-gluconic acid reductase from Corynebacterium glutamicum

“…In this and previous studies (Kaswurm et al 2012 , Pacher 2006 ) the dkr gene was cloned and expressed such that the third in-frame ATG codon of the complete open reading frame (ORF) (GenBank accession JQ407590.1) was used as translation start (Figure 1). This is a consequence of previous experiments conducted in our laboratory that demonstrated the presence of two protein bands with distinct electrophoretic mobilities (both identified as dkr gene products by MALDI-TOF analysis) when the complete dkr ORF (His 6 -tagged) was expressed with an E. coli expression system ( Pacher 2006 ).…”

“…Interestingly, the corresponding production yields were in the same range as those previously obtained by dkr expression with E. coli /pET21d (approx. 200 U L -1 fermentation broth) (Kaswurm et al 2012 ). Additionally, this is the highest expression level so far reported for this enzyme and shows that LAB systems are suitable for dkr expression as well.…”

“…2,5-DKG, the substrate for 2,5-DKG reductase assays, was produced by fermentation of glucose with Pectobacter cypripedii (Pacher et al 2008 ) and further purified as previously described (Kaswurm et al 2012 ). All oligonucleotide primers used in this study are displayed in Table 1 and were synthesized by VBC-Biotech (Vienna, Austria).…”

“…2,5-DKG reductase activity assay was performed spectrophotometrically as previously described (Kaswurm et al 2012 ). One unit of 2,5-DKG reductase activity is defined as the enzyme quantity required to reduce 1 μmol of 2,5-DKG per min under assay conditions, which is equivalent to the production of 1 μmol of NADP + per min (Kaswurm et al 2012 ).…”

“…One unit of 2,5-DKG reductase activity is defined as the enzyme quantity required to reduce 1 μmol of 2,5-DKG per min under assay conditions, which is equivalent to the production of 1 μmol of NADP + per min (Kaswurm et al 2012 ). Protein concentrations were determined by the dye binding method of Bradford (Bradford, 1976 ) using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories Inc.).…”