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-EJB 97 0741/4 D-Threonine aldolase is an enzyme that catalyzes the cleavage of D-threonine into glycine and acetaldehyde. Its activity was found in several genera of bacteria such as Arthrobacter, Alcaligenes, Xanthomonus, and Pseudonzonas, but not in yeasts or fungi. The enzyme was purified to homogeneity from one strain, Arthrobacter sp. DK-38. The enzyme appeared to consist of a single polypeptide chain with an apparent molecular mass of 51 kDa. This enzyme, as we11 as L-threonine aldolase, requires pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme. Unlike other pyridoxal-P enzymes, D-threonine aldolase also requires a divalent cation such as Co2+, Ni2+, Mnz+, or Mgz+ for its catalytic activity. The enzyme completely lost its activity in the absence of either pyridoxal-P or a divalent cation. A divalent cation was also essential for the thermal stability of the enzyme. The metal-free enzyme tends to become thermally unstable, resulting in the irreversible loss of its catalytic activity. The enzyme is strictly D-specific for the a-position, whereas it cannot distinguish between threo and erythro forms at the /$position. Thus, D-threonine and D-allothreonine act as substrates of the enzyme, but their kinetic parameters are different : the K,,, and V,,;,, values are 3.81 mM and 38.8 pmol . min-l . mg-' toward D-threonine, and 14.0 mM and 102 pmol . min-' . mg-l toward D-allothreonine, respectively. The aldolase reaction is reversible, and the enzyme is therefore able to produce nearly equimolar amounts of D-threonine and D-allothreonine through C-C bond formation between glycine and acetaldehyde. The enzyme also acts, in the same manner, on several other D-P-hydroxy-a-amino acids, including D-P-phenylserine, D-p-hydroxy-a-aminovaleric acid, ~-p-3,4-dihydroxyphenylserine, and D-~-3,4-methylenedioxyphenylserine.Keywords: D-threonine aldolase; threonine; Arthrohacter; pyridoxal %phosphate; divalent cation.Several enzymes are involved in L-threonine metabolism. Some are enzymes concerned in the biosynthesis of L-threonine from aspartic acid via homoserine 11, 21. Others are enzymes responsible for L-threonine degradation, such as L-threonine deaminase, which converts L-threonine into n-ketobutyrate, an intermediate in ~-isoleucine biosynthesis [3-51.L-Threonine aldolase, one of the L-threonine-degrading enzymes, cleaves L-threonine into glycine and acetaldehyde [6-91. Its enzyme activity has been found in several microorganisms, in such genera as Pseudomonas [lo], Clostridium 18,11, 121, Bacillus [13], and Candida [14, 151, as well as in mammalian organs [16, 171. L-Threonine aldolase has been extensively investigated because of its possible role in amino acid metabolism [lo, 111. Studies on the mechanism underlying its catalytic action revealed that L-threonine aldolase belongs to a group of enzymes that require pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme L-Threonine aldolase can produce the L-forms of threonine stereoisomers via C-C bond formation between glycine and acetaldehyde, though it is highly sele...

The concept of a charged membrane bioreactor (CMBR) has been proposed for continuous reactions of enzymatic reduction dependent upon the nicotinamide coenzyme NADP(H). It was found that a composite membrane with a negative charge, NTR 7410, could retain NADP(H) selectively without any chemical modification. Several permeation experiments have revealed that the retainment of a coenzyme is based on electrostatic repulsion of negative charges between the membrane and the phosphate moiety of NADP(H). The retainment ratio was reduced by the addition of inorganic salt, although it could be restored to 0.8 in the presence of albumin. A reactor equipped with a charged membrane as the coenzyme separator module was constructed and used in the continuous production of sorbitol. NADPH-dependent aldose reductase isolated from Candida tropicalis IAM 12202 was used for the production of sorbitol from glucose. The coenzyme oxidized in this reaction was enzymatically regenerated by conjugation with glucose dehydrogenase, together with the coproduction of gluconic acid from glucose. With a substrate conversion of 85%, 100 g/L sorbitol was produced and equimolar gluconic acid was coproduced for more than 800 h, indicating that the reaction was efficiently coupled to the enzymatic regeneration. The initial high retainment ratio of the membrane was almost maintained throughout the entire reaction. Consequently, the turnover number of the coenzyme reached 106,000.

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