|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1990 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1989 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Dipeptides such as L-glutamic acid dimer and L-glutamic acid dimer are known as a substance posessing umami (brothy) taste and a bitter taste masking effect. In this study, we attempted to synthesize precursors of four peptides, i. e., GluGlu, GluAsp, AspGlu, and AspAsp with an immobilized papain in organic solvent systems. As an acid component of a substrate, N-(benzyloxycarbonyl)-L-glutamic acid and N-(benzyloxycarbonyl)-L-aspartic acid, and as an amine component L-glutanic acid diethyl ester and L-aspartic acid diethyl ester were used. In most cases, we focussed on the synthesis of N-(benzyloxycarbonyl)-L-glutamyl-L-glutamic acid diethyl ester (Z-GluGlu(OEt)_2) at 30 ^ﾟC.
The results obtained are Summarized as follows :
1) The synthetic reaction did not take place in buffer solution.
2) In the aqueous-organic biphasic reaction systems with ethyl acetate as an organic solvent, the maximum yield of Z-GluGlu (OEt) _2 reached about 70%.
3) The enzyme was immobilized onto various porous supp
orts. The enzyme was first adsorbed onto the support and then cross-linked with glutaraldehyde. The highest activity was obtained when the enzyme was adsorbed onto porous ceramic support (SM10-C1, Nippon Gaishi K. K.).
4) The activity of immobilized enzyme depended strongly on the water content in an outer organic solvent. The highest activity was obtained at a water content of 2.5% with the enzyme immobilized onto SM10-C1.
5) L-Glutamic acid diethyl ester, an amine component of the substrate was non-enzymatically coverted to L-pyroglutamic acid diethyl ester. To reduce the effect of non-enzymatic reaction, the increase of the concentration of the immobilized enzyme was found most effective. With 100 mM Z-Glu and 200 mM Glu (OEt) _2, and immobilized enzyme concentration of 8%, the yield became higher than 90%.
6) The other three dipeptides precursors were also obtained with quite a high yield under the same reaction conditions as shown in (5).
7) The products were identified by analyzing with nuclear magnetic resonance, mass spectrum, and infrared adsorption spectrum. Less