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| Proteins, Expression, Isolation and Analysis | Production of xylitol dehydrogenase was performed as reported earlier [22]. The enzyme was purified from cell lysate by Ni-NTA affinity chromatography. A buffer containing 25?mM NaH2PO4, 150?mM NaCl, and 200?mM imidazole (pH 8.0) was used for proteins elution, After Ni-NTA purification, xylitol dehydrogenase was further purified by gel filtration on Sephadex G-25 with 50?mM Tris-Cl, 200?mM NaCl, and pH 8.0-buffer solution as a mobile phase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a 12% (w/v) acrylamide gel at a constant current of 200?mA. Low molecular weight protein standard was used as a size marker (GenScript, NJ, USA). After electrophoresis, gels were colored using Coomassie brilliant blue G-250 via standard procedures. Phoretics 1D Non-linear Dynamics (Newcastle, UK) software was used for densitometry analysis of SDS-PAGE gel. | Get A Quote |
D-threitol (hereinafter as threitol) is a diastereoisomer of erythritol with wider applications in green chemistry, food, pharmaceutical, and medicine. Herein, xylitol dehydrogenase from?Scheffersomyces stipitis?CBS 6054 was demonstrated able to catalyze highly efficient threitol formation from erythritol with erythrulose as an intermediate. When the corresponding gene?Ss-XDH?was overexpressed in the erythritol-producing?Yarrowia lipolytica?strain CGMCC7326, threitol was produced with a titer of 112?g·L?1?and yield of 0.37 from glucose. Unexpectedly, gene encoding mannitol dehydrogenase was upregulated in the threitol producing stain, leading to mannitol accumulation in the culture broth. In order t... More
