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.
Persson
M
M
Pure and Applied Biochemistry, Chemical Center, University of Lund, Sweden.
Månsson
M O
MO
Bülow
L
L
Mosbach
K
K
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