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

Abstract

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.

Efficient transesterification of sucrose catalysed by the metalloprotease thermolysin in dimethylsulfoxide

“…The comparable catalysis rate of aspartame and sucrose ester synthesis suggests that the latter reaction is enzymatic. In agreement with Nagasayu et al, aspartame synthesis could not be observed using DMSO as the solvent [16]. Polyols are known to stabilise proteins [17] and studies of thermolysin have shown that small carbohydrates contribute to the hydration and activation of the enzyme [18].…”

Kinetics of enzymatic solid-to-solid peptide synthesis: Synthesis ofZ-aspartame and control of acid-base conditions by using inorganic salts

“…These rates indicate that the specific activity of thermolysin in these reactions was around 12-fold higher than the industrial-grade Thermoase. These rates were also much higher than comparable thermolysin reactions in organic solvents, which were in the range of 2 to 10 nmol min −1 mg −1 (e.g., Nagayasu et al, 1993b). The yields of up to 80% product conversion were clearly higher than in comparable aqueous systems, which were usually well below 50% (e.g., Inouye, 1992;Murakami et al, 1996;Zhou and Huang, 1993).…”

“…Based on the available literature, the aspartame precursor Z-Asp-Phe-OMe is probably the most investigated model dipeptide. Enzymatic synthesis methods have used aqueous (e.g., Inouye, 1992;Murakami et al, 1996;Zhou and Huang, 1993), biphasic (e.g., Hirata et al, 1997;Murakami and Hirata, 1997;Nagayasu et al, 1993b), and organic-phase systems (e.g., Nagayasu et al, 1993a;Nakanishi et al, 1985) in both continuous (e.g., Hirata et al, 1997;Nakanishi et al, 1990) and batch mode (e.g., Inouye, 1992;Nagayasu et al, 1993b). Most of these methods have some drawbacks, usually either low yields or low reaction rates.…”

Operational Stability of High Initial Activity Protease Catalysts in Organic Solvents

“…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.…”