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Kuniyo Inouye - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis of N-carbobenzoxy-l-aspartyl-l-Phenylalanine Methyl Ester catalyzed by thermolysin variants with improved activity
    Enzyme and Microbial Technology, 2009
    Co-Authors: Masayuki Kusano, Kiyoshi Yasukawa, Kuniyo Inouye
    Abstract:

    Abstract Thermolysin is industrially used for the synthesis of N -carbobenzoxy- l -aspartyl- l -Phenylalanine Methyl Ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N -carbobenzoxy- l -aspartic acid (ZD) and l -Phenylalanine Methyl Ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, I168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009;145:103–13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 °C, the molecular activity k cat values of the variants were 1.6–3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant K m values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 μM, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis.

  • Effects of Salts on Thermolysin: Activation of Hydrolysis and Synthesis of N-Carbobenzoxy-L-Aspartyl-L-Phenylalanine Methyl Ester, and a Unique Change in the Absorption Spectrum of Thermolysin.
    Journal of biochemistry, 1992
    Co-Authors: Kuniyo Inouye
    Abstract:

    It has been reported that neutral salts such as NaCl activate the thermolysin-catalyzed hydrolysis of substrates containing glycine at the P1 position (carboxylic side of the cleavage bond) [Holmquist, B. & Vallee, B.L. (1976) Biochemistry 15, 101-107]. In this paper, we demonstrate that high concentrations (1-4 M) of neutral salts greatly enhance the thermolysin activity in both hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-Phenylalanine Methyl Ester (ZAPM), a precursor of a peptide sweetener, aspartame, in which the L-aspartyl residue is the P1 residue. The enzyme activity is enhanced with an increase in salt concentration in a pseudo-exponential fashion. The degree of activation by salts was in the order LiCl > NaCl > KCl. The rate of ZAPM hydrolysis in the presence of 3.8 M NaCl was 6-7 times higher than that in its absence, and 50 times or more activation is expected in saturated NaCl solution. The activation is brought about solely through an increase in the catalytic constant (kcat), and the Michaelis constant (Km) is not affected at all by the presence of NaCl. On mixing thermolysin with NaCl, a unique absorption difference spectrum suggesting a conformational change of the enzyme was observed. The intensity increased in a pseudo-exponential fashion with increase of NaCl concentration up to 3 M, and this dependence is similar to that of the enzyme activity.

P. Madhusudanan Nair - One of the best experts on this subject based on the ideXlab platform.

  • Stabilization of Phenylalanine ammonia lyase containing Rhodotorula glutinis cells for the continuous synthesis of l-Phenylalanine Methyl Ester/96/
    Enzyme and Microbial Technology, 1996
    Co-Authors: Godwin B. D'cunha, Vaduvatha Satyanarayan, P. Madhusudanan Nair
    Abstract:

    Abstract A procedure for the direct one-step synthesis of l -Phenylalanine Methyl Ester, a precursor of the artificial sweetener, aspartame, by using Phenylalanine ammonia lyase (PAL)-containing Rhodotorula glutinis cells in a 2:1 heptane: 0.1 m Tris-sulfate buffer pH 9.0 biphasic system was reported earlier; however, the yeast cells lost PAL activity rapidly during the reaction and were rendered unsuitable for repeated use. Stabilization of the enzyme during the biotransformation for the continuous use is described. Immobilization of PAL containing Rhodotorula glutinis cells by different conventional methods could not prevent the inactivation of the enzyme. The addition of a low concentration of Mg2+ to the incubation mixtures resulted in significant stabilization of PAL which was further enhanced by the presence of glycerol. At 4 m m MgSO4 and 10% glycerol concentrations, the system could produce l -Phenylalanine Methyl Ester for nine cycles of repeated use while the controls lost activity by the fourth cycle. The total yield of l -Phenylalanine Methyl Ester in nine cycles was 92 g l−1.

  • Novel direct synthesis of l-Phenylalanine Methyl Ester by using Rhodotorula glutinis Phenylalanine ammonia lyase in an organic-aqueous biphasic system
    Enzyme and Microbial Technology, 1994
    Co-Authors: Godwin B. D'cunha, Vaduvatha Satyanarayan, P. Madhusudanan Nair
    Abstract:

    Abstract A procedure for the direct, one-step enzymatic conversion of trans-cinnamyl Methyl Ester to L-Phenylalanine Methyl Ester is described. The reverse reaction of Phenylalanine ammonia lyase from Rhodotorula glutinis was utilized for this conversion. Insolubility of substrate trans-cinnamyl Methyl Ester in aqueous buffer solution was overcome by employing an organic-aqueous biphasic system, heptane:0.1 m Tris-sulfate buffer, pH 9.0 (2:1). Different conditions were optimized for the maximal conversion such as time (16–18 h), temperature (30°C), pH (9.0), concentration of substrates, 0.1 m trans-cinnamyl Methyl Ester and 1 m (NH4)2 SO4, and nature of the organic solvent (heptane); about 70% conversion of substrate to product was obtained under these conditions. Formation of the product, l -Phenylalanine Methyl Ester, was identified by paper chromatography and was further confirmed by autoradiography and NMR spectral analyses.

Tsutomu Oishi - One of the best experts on this subject based on the ideXlab platform.

Godwin B. D'cunha - One of the best experts on this subject based on the ideXlab platform.

  • Stabilization of Phenylalanine ammonia lyase containing Rhodotorula glutinis cells for the continuous synthesis of l-Phenylalanine Methyl Ester/96/
    Enzyme and Microbial Technology, 1996
    Co-Authors: Godwin B. D'cunha, Vaduvatha Satyanarayan, P. Madhusudanan Nair
    Abstract:

    Abstract A procedure for the direct one-step synthesis of l -Phenylalanine Methyl Ester, a precursor of the artificial sweetener, aspartame, by using Phenylalanine ammonia lyase (PAL)-containing Rhodotorula glutinis cells in a 2:1 heptane: 0.1 m Tris-sulfate buffer pH 9.0 biphasic system was reported earlier; however, the yeast cells lost PAL activity rapidly during the reaction and were rendered unsuitable for repeated use. Stabilization of the enzyme during the biotransformation for the continuous use is described. Immobilization of PAL containing Rhodotorula glutinis cells by different conventional methods could not prevent the inactivation of the enzyme. The addition of a low concentration of Mg2+ to the incubation mixtures resulted in significant stabilization of PAL which was further enhanced by the presence of glycerol. At 4 m m MgSO4 and 10% glycerol concentrations, the system could produce l -Phenylalanine Methyl Ester for nine cycles of repeated use while the controls lost activity by the fourth cycle. The total yield of l -Phenylalanine Methyl Ester in nine cycles was 92 g l−1.

  • Novel direct synthesis of l-Phenylalanine Methyl Ester by using Rhodotorula glutinis Phenylalanine ammonia lyase in an organic-aqueous biphasic system
    Enzyme and Microbial Technology, 1994
    Co-Authors: Godwin B. D'cunha, Vaduvatha Satyanarayan, P. Madhusudanan Nair
    Abstract:

    Abstract A procedure for the direct, one-step enzymatic conversion of trans-cinnamyl Methyl Ester to L-Phenylalanine Methyl Ester is described. The reverse reaction of Phenylalanine ammonia lyase from Rhodotorula glutinis was utilized for this conversion. Insolubility of substrate trans-cinnamyl Methyl Ester in aqueous buffer solution was overcome by employing an organic-aqueous biphasic system, heptane:0.1 m Tris-sulfate buffer, pH 9.0 (2:1). Different conditions were optimized for the maximal conversion such as time (16–18 h), temperature (30°C), pH (9.0), concentration of substrates, 0.1 m trans-cinnamyl Methyl Ester and 1 m (NH4)2 SO4, and nature of the organic solvent (heptane); about 70% conversion of substrate to product was obtained under these conditions. Formation of the product, l -Phenylalanine Methyl Ester, was identified by paper chromatography and was further confirmed by autoradiography and NMR spectral analyses.

Yong-kyung Lee - One of the best experts on this subject based on the ideXlab platform.