Molecular cloning of the gene and the crystal structure of the prolyl aminopeptidase [EC 3.4.11.5] from Serratia marcescens have been studied by us [J. Biochem. 122, 601-605 (1997); ibid. 126, 559-565 (1999)]. Through these studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned with substrate recognition. To elucidate the details of the mechanism for the substrate specificity, the site-directed mutagenesis method was applied. The F139A mutant showed an 80-fold decrease in catalytic efficiency (k(cat)/K(m)), but the Y149A mutant did not show a significant change in catalytic efficiency. The catalytic efficiency of the E204Q mutant was about 4% of that of the wild type. The peptidase activity of the mutant (R136A) was markedly decreased, however, arylamidase activity with Pyr-bNA was retained as in the wild-enzyme. From these results, it was clarified that the pyrrolidine ring and the amino group of proline at the S1 site were recognized by Phe139 and Glu204, respectively. P1' of a substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was resistant to it.

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Glycyl aminopeptidase was purified 600-fold from a cell extract of Actinomucor elegans by ammonium sulfate fractionation and sequential chromatography on DEAE-Toyopearl, Toyopearl HW65C, and FPLC-Superdex 200 HR, with recovery of 3.3% of the activity. The enzyme highly specifically hydrolyzed Gly-X (amino acid, peptide, or arylamide) bonds. The enzyme hydrolyzed other amino acid residues but at a rate of less than one fifth that with Gly. The order was Gly >> Ala >> Met > Arg > Ser > Leu. The Km value for glycyl-2-naphthylamide was 0.24 mM. The enzyme was most active at pH 8.0 with glycyl-2-naphthylamide as the substrate and its optimal temperature was 40 degrees C. The enzyme was inhibited by iodoacetic acid, and p-chloromercuribenzoate but not done by diisopropylfluorophosphate, o-phenanthroline, or EDTA. Magnesium and calcium had no effect on enzymic activity, but the activity was suppressed by cadmium, zinc, and copper ions. The molecular mass was estimated to be 320 kDa by gel filtration on FPLC-Superdex 200 HR and 56.5 kDa by SDS-PAGE, so the enzyme probably was a hexamer.

The potential of producing L-isoleucine crystals with the aid of electric potential and its effect on the nucleation kinetics of L-isoleucine were probed using polythermal and isothermal crystallisation techniques, assisted with 5 V, 9 V, and 20 V electric potentials. The polythermal experiments were conducted with cooling rates of 0.1 °C/min–0.7 °C/min, whilst isothermal crystallisation was conducted with a supersaturation of 1.30–1.70, and both were carried out in a 200 mL temperature-controlled jacketed reactor. Prediction of the nucleation rate and its associated parameters for isothermal crystallisation was carried out using a molecular dynamics simulation. In both crystallisation techniques, electric potentials increased the nucleation rate, but the intensity of the electric potential had less impact on the measured parameters. Nucleation rates for 5 V isothermal crystallisation were in the order of 1010 higher than for polythermal crystallisation. Electric potential doubled the nucleation rates for polythermal crystallisation and increased the nucleation rates 12-fold in isothermal crystallisation. The isothermal technique produced the form B polymorph, but mixtures of forms A and B were produced in polythermal crystallisation. The predicted critical number of molecules, N*, and the critical radius, r*, were in good agreement with the experimental data, with a higher predicted nucleation rate in the order of 102.