Continuous regeneration of NAD(H) covalently bound to a cysteine genetically engineered into glucose dehydrogenase.
We introduced a cysteine residue on the surface of glucose dehydrogenase from Bacillus subtilis using site-directed mutagenesis. To this mutant, an NAD-analogue was covalently attached by a disulphide bridge so that it was active intramolecularly. The glucose dehydrogenase-cys44-NAD complex, which contained one reactive NAD molecule per subunit of glucose dehydrogenase, was operated together with lactate dehydrogenase in a coupled enzymatic regeneration of NAD(H) in a hollow fiber reactor. L-lactate and gluconic acid were continuously produced from pyruvate and D-glucose, respectively, with a turnover number of 45 cycles per minute for each NAD molecule. The total turnover per coenzyme was 135,000 for the first 2.5 days.
Pure and Applied Biochemistry, Chemical Center, University of Lund, Sweden.
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M
Persson
M O
MO
Månsson
L
L
Bülow
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K
Mosbach
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides; D013438:Sulfhydryl Compounds / Q000138:chemical synthesis / Q000378:metabolism
D009841:Oligonucleotides; D013438:Sulfhydryl Compounds; D009243:NAD; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D003545:Cysteine