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Supplemental Information
2,5-diketo-gluconic acid reductase from Corynebacterium glutamicum: Characterization of stability, catalytic properties and inhibition mechanism for use in vitamin C synthesis
Vanja Kaswurm, Claudia Pacher, Klaus Dieter Kulbe and Roland Ludwig,*Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria
Supplementary Table S1. Media and growth conditions used in screening experiments.StrainMedium a)Growth Temperature a)(C)
Acetobacter hansenii36028
Acetobacter lovaniensis36028
Arthrobacter agilis5322
Arthrobacter roseus9222
Bacillus alvei130
Bacillus funiculus130
Brevibacterium imperiale5330
Brevibacterium testaceum5330
Corynebacterium barkeri5330
Corynebacterium glutamicum5330
Corynebacterium ilicis5330
Gluconobacter asaii62626
Kocuria kristinae5337
Kocuria varians5330
Micrococcus lylae5337
Pantoea stewarti subsp. stewartii126
Pectobacterium chrysanthemi126
Pimelobacter jenseii5330
Pseudomonas cichorii127
Pseudomonas syringae126
Staphylococcus muscae9237
Staphylococcus pulvereri9237
a) as recomended by DSMZSupplementary Table S2. Media components as listed by DSMZ.Medium IdentifierMedium NameFormula (L-1)
1Nutrient BrothPeptone 5.0 g
Meat extract 3.0 g
MnSO4 x H2O 10.0 mg
pH 7.0
53Corynebacterium BrothCasein peptone, tryptic digest 10.0 g
Yeast extract 5.0 g
Glucose 5.0 g
NaCl 5.0 g
pH 7.2 7.4
92Trypticase Soy-Yeast Extract MediumCasein, enzymatic digest of 17.0 g
Soybean meal, enzymatic eigest of 3.0 g
Yeast extract 3.0
Glucose 2.5 g
NaCl 5.0 g
K2HPO4 2.5 g
pH 7.2 7.4
360YPM (Yeast Extract Peptone Mannitol) MediumYeast extract 5.0 g
Peptone 3.0 g
Mannitol 25.0 g
pH not adjusted
6265% Sorbitol MediumD-Sorbitol 50.0 g
Yeast extract 10.0 g
Peptone 10.0 g
pH 6.0, adjusted with HCl
Supplementary Figure S1. Recombinant 2,5-DKG reductase production. Measured parameters: OD600 (squares), dry cell weight (crosses), glycerol consumption (triangles up), lactose concentration (triangles down) and volumetric activity of 2,5-DKG reductase (circles).
Supplementary Figure S2. SDS-PAGE (A) and isoelectric focusing (B) of purified 2,5-DKG reductase from C. glutamicum overexpressed in E. coli BL21 Star (DE3). (A) lane 1, purified recombinant 2,5-DKG reductase; lane 2, molecular mass marker (Bio-Rad). (B) lane 1, purified recombinant 2,5-DKG reductase; lane 2, isoelectric marker range 3.510.7 (Serva).
Supplementary Figure S3. Differential scanning calorimetry of 32 M 2,5-DKG reductase in the absence (A) and presence (B) of 200 M NADPH. The measurement was performed by a Microcal VP-Capillary DSC System (GE Healthcare) in 50 mM ammonium acetate buffer pH 6.4 using a temperature ramp of 1 K min-1. The buffer ramp was subtracted from the shown data. Buffer containing 200 M NADPH did not differ significantly from the pure buffer. Black lines indicate measured data, red lines fitted curves.