Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine, using artificial cells containing an immobilized multienzyme system and dextran-NAD+. 2. Yeast alcohol dehydrogenase for coenzyme recycling.
Semipermeable nylon-polyethylenimine artificial cells containing leucine dehydrogenase (EC 1.4.1.9), alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1.5), and dextran-NAD+ were prepared. Artificial cells could convert ammonia or urea into L-leucine, L-valine, and L-isoleucine. For batch conversion in 20.0 mM of ammonium acetate substrate solutions, in 2 h 0.2 ml of artificial cells could produce 4.48 mumol of L-leucine, 9.98 mumol of L-valine, or 5.96 mumol of L-isoleucine. The corresponding conversion ratios were 22.4, 49.9, and 29.8%. In 20.0 mM of urea substrate solutions, 13.71 mumol of L-leucine, 16.12 mumol of L-valine, or 13.44 mumol of L-isoleucine was produced and the conversion ratios were 68.6, 80.6, and 67.2%. The substrate specificity of leucine dehydrogenase for the reductive amination was determined. Of the three branched-chain amino acids produced, the production rates of L-valine were the highest. The apparent Km values were as follows: 0.32 mM for alpha-ketoisocaproate, 1.63 mM for alpha-ketoisovalerate, and 0.73 mM for Dl-alpha-keto-beta-methyl-n-valerate. The leucine dehydrogenase multienzyme system had a good storage stability. It retained 72.0% of the original activity with artificial cells were stored at 4 degrees C for 6 weeks. The optimum conversion pH and temperature were 8.5-9.0 and 35-40 degrees C. The effects of urea and ammonium salts on conversion rate were also studied. The relative activities in ammonium salts solutions were 45.1-75.9% of those in urea solutions.
Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
K F
KF
Gu
T M
TM
Chang
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000601:Amino Acids, Essential / Q000096:biosynthesis*; D000641:Ammonia / Q000378:metabolism; D001709:Biotechnology; D002214:Capsules; D003911:Dextrans; D004800:Enzymes, Immobilized; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000096:biosynthesis; D007930:Leucine / Q000096:biosynthesis; D009243:NAD; D013379:Substrate Specificity; D014508:Urea / Q000378:metabolism; D014633:Valine / Q000096:biosynthesis
D000601:Amino Acids, Essential; D002214:Capsules; D003911:Dextrans; D004800:Enzymes, Immobilized; D007532:Isoleucine; D009243:NAD; D000641:Ammonia; D014508:Urea; D000426:Alcohol Dehydrogenase; D007930:Leucine; D014633:Valine