Continuous synthesis of a tripeptide by successive condensation and transesterification catalyzed by two immobilized proteinases in organic solvent.

Kimura, Yukitaka; Yoshida, Tomoko; Muraya, Koji; Nakanishi, Koji; Matsuno, Ryuichi

doi:10.1271/bbb1961.54.1433

Cited by 18 publications

(1 citation statement)

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“…Some studies on long-term stability have been carried out in organic media under continuous operation using R-chymotrypsin (5,6) and thermolysin (7)(8)(9)(10)(11)(12)(13). And there are a few studies on operational stability in repeated batch operation using thermolysin CLECs (14) and immobilized R-chymotrypsin (15)(16)(17) in organic solvents.…”

Section: Introductionmentioning

confidence: 99%

Operational Stability of High Initial Activity Protease Catalysts in Organic Solvents

Fernandes

Halling

2002

Biotechnology Progress

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The first studies on the operational stability of cross-linked enzyme crystals (CLECs) in organic media are described. Although these catalysts display high initial specific activity, they inactivate rapidly, losing more than 50% of the initial activity within the first 4 h under continuous flow. Furthermore, the inactivation is not reversible when returned to an aqueous medium. The same rapid inactivation occurs with adsorbed protease preparations that show similar high initial specific activity (propanol-rinsed enzyme preparations (PREPs) of subtilisin and alpha-chymotrypsin).

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Section: Introductionmentioning

confidence: 99%

Operational Stability of High Initial Activity Protease Catalysts in Organic Solvents

Fernandes

Halling

2002

Biotechnology Progress

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Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

Nagayasu¹,

Miyanaga²,

Tanaka³

et al. 1994

Biotech & Bioengineering

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N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.

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Synthesis of aspartame precursor with an immobilized thermolysin in tert‐amyl alcohol

Nagayasu

Miyanaga

Tanaka

et al. 1994

Biotech & Bioengineering

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N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetner asparatame, was synthesized from N-(benzyloxycarbolyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) with an immobilized thermolysin in various organic solvents. We found that in tert-amyl alcohol containing a small amount of water the immobilized enzyme showed a high activity comparble to that in ethyl acetate with quite a high stability. The immobilized enzyme was fully stable up to 70 degrees C in tert-amyl alcohol in the absence of the subatrate, and up to 50 degrees C in the presence of the substrate. The high stability in the presence of the substrate was found due to the fact that the release of calcium ions, the stabilizing factor of thermolysin, is suppressed.The substrate concentration dependence of the initial synthetic rate with the immobilized enzyme was quite different from that with the free enzyme in the biphasic system, in contrast to that in ethyl acetate. Finally, Z-AspPheOMe was continuously synthesized in a column reactor using 200 mM PheOMe and 120 mM Z-Asp as the substrate for over 300 h at 45 degrees C and a space velocity of 1 h(-1) without any loss of acivity. (c) 1994 John Wiley & Sons, Inc.

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Continuous synthesis of a tripeptide by successive condensation and transesterification catalyzed by two immobilized proteinases in organic solvent.

Cited by 18 publications

References 1 publication

Operational Stability of High Initial Activity Protease Catalysts in Organic Solvents

Operational Stability of High Initial Activity Protease Catalysts in Organic Solvents

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

Synthesis of aspartame precursor with an immobilized thermolysin in tert‐amyl alcohol

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