mmc1.doc

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.