• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.704-706
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• 1992

A model peptide, N-carbobenzoxy-L-phenylalanyl-L-phenylalanine methyl ester, was synthersized with free and immobilized thermolysin in a microaqueous system. The model peptide was formed mostly during the initial phase of the reaction. The yields of the compound with free and immobilized thermolysin after 4hr of reaction were 77.8 and 71.2, respectively.

• Korean Journal of Food Science and Technology
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• v.20 no.2
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• pp.245-251
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• 1988

Thermolysin was immobilized on Dowex MWA-1 with 10% glutaraldehyde and incorpo rated into a fluidized-bed continuous coagulation scheme to make Cheddar type cheese. The activity yield of thermolysin was 25%. The immobillized thermolysin was stable at $60^{\circ}C$ in the presence of 1/200M calcium ions and the half-life value is 16 days at the temperature. Raw milk alkalified to pH 7.0 was passed through a column of thermolysin beads at $55^{\circ}C$, cultivated with Streptococcus cremoris and allowed to coagulate. A typical milk curd was formed to make Cheddar type cheese, avoiding troublesome microbial contamination successfully during continuous hydrolysis process. During ripening of this cheese for 6 months at $10^{\circ}C$, its ripening ratio and taste were similar to those of cheese prepared by the traditional method.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.204-209
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• 1994

The synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methylester (Z-APM), a precursor of aspartame, from N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methylester hydrochlolide($L-PM\cdot HCI$) was investigated in a saturated-ethylacetate single phase system using immobilized thermolysin. Among the various supports tested, glyceryl-CPG was found to be most efficient for retaining enzyme activity. The enzyme immobilized onto glyceryl-CPG also showed the highest activity for Z-APM synthesis in saturated ethyl acetate. Z-APM conversion yield in saturated ethylacetate was half of that obtained in an ethyl acetate-buffer two-phase system under the same reaction conditions. However, as the mole ratio of $L-PM \cdot HCI$ to Z-Asp was increased to 4.0, the conversion yield reached 95 %. When continuous synthesis of Z-APM was canied out in a plug flow reactor (PFR) with 80 mM of L-PMㆍHCI and 20 mM of Z-Asp in saturated ethylacetate (pH 5.5), more than 95 % of Z-Asp was converted to Z-APM with a space velocity of 1.16 $hr^{-1} at 40^{\circ}C$. Although the operational stability in PFR was reduced rapidly, more than 80% of initial activity was maintained in CSTR even after a week of operation.

• Korean Journal of Food Science and Technology
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• v.27 no.4
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• pp.564-570
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• 1995

N-Benzoyl-L-aspartyl-L-phenylalanine methyl ester(BzAPM), a novel aspartame precursor, was investigated for its enzymatic synthesis by immobilized thermolysin using a water-miscible organic solvent system. The substrate used were N-benzoyl-L-aspartic acid(BzAsp) and L-phenylalanine methyl ester (PheOMe). Synthetic conditions such as substrates concentration, temperature, pH, and some metallic ions were varied to study their effects on BzAPM synthesis. The synthetic reaction rate increased linearly as the PheOMe concentration increased at a constant concentration of BzAsp(100 mM), and the maximum reaction rate was obtained at BzAsp concentration of 200 mM when 300 mM PheOMe was used. The optimum pH and temperature were found to be 6.1 and $40^{\circ}C$, respectively. The metallic ions such as $Zn^{2+},\;Mg^{2+},\;Mn^{2+},\;Fe^{2+},\;Pb^{2+}\;and\;Cu^{2+}$ at 5 mM level showed inhibitory effect on BzAPM synthesis, while $Co^{2+}$ and $Ca^{2+}$ ion increased synthesis. $Co^{2+}$ ion was also found to have synergistic effect with $Ca^{2+}$ ion. Benzoic acid, L-phenylalanin and NaCl showed inhibitory effect.

• Korean Journal of Food Science and Technology
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• v.24 no.5
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• pp.504-510
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• 1992

Several single or mixed water-miscible organic solvent systems were investigated to develop the most effective solvent system for enzymatic synthesis of N-benzoylaspartame(BzAPM). The BzAPM was prepared by immobilized thermolysin with using N-benzoyl-L-aspartic acid(Bz-Asp) and L-phenylalanine methyl ester(PheOMe). The solubilities of BzAPM and L-phenylalanine were highest in 4.5% methanol(1.89 and 1.79%, respectively) among the solvents system investigated while a mixed solvent system of 25% dimethyl sulfoxide(DMSO) and 20% polyethylene glycol(PEG) 200 showed relatively high values. The synthetic activity of BzAPM as well as initial reaction rate were found to be high in 45% methanol, 45% DMSO and a mixed solvent of 25% DMSO and 20% PEM 200. The imobilized thermolysin was most stable in 25% DMSO and 20% PEG 200 during storage at $40^{\circ}C$ for 42 days. PheOMe in the same solvent system was also found fairly stable against non-enzymatic decomposition at $40^{\circ}C$. Based on the synthetic efficiency and stability, the solvent system containing 25% DMSO and 20% PEG 200 was selected to be appropriate for the enzymatic synthesis of BzAPM.