Syntheses of Umami Peptides with Immobilized Papain in Water-Immiscible Organic Solvent Systems

L-谷氨酸二聚体和L-谷氨酸二聚体是一种具有肉汤味和苦味掩蔽作用的物质。在本研究中，我们尝试用固定化木瓜酶在有机溶剂体系中合成GluGlu、GluAsp、AspGlu和AspAsp四种多肽的前体。底物的酸组分为N-(苄氧羰基)-L-谷氨酸和N-(苄氧甲酰基)-L-天冬氨酸，胺组分为L-谷氨酸二乙酯和L-天冬氨酸二乙酯。本论文主要研究了N-(苄氧甲酰基)-L-谷氨酰基-L-谷氨酸二乙酯(Z-GluGlu(OEt)_2)在30ﾟC下的合成。结果表明：1)合成反应不在缓冲溶液中进行；2)在以乙酸乙酯为有机溶剂的水-有机两相反应体系中，Z-Glu(OEt)_2的最高产率可达70%左右。3)将该酶固定化在不同的多孔载体…上。更多的运动。该酶首先被吸附在载体上，然后用戊二醛进行交联。当酶被吸附在多孔陶瓷载体(SM10-C1，Nippon Gaishi K.)上时，酶的活性最高。4)固定化酶的活性强烈地依赖于外有机溶剂中的水含量。在水分含量为2.5%时，固定化的酶活力最高，底物中的胺组分被非酶催化转化为L-焦谷氨酸二乙酯。为了降低非酶反应的影响，提高固定化酶的浓度是最有效的。用100 mM的Z-Glu和200 mM的Glu(OEt)_2，固定化酶浓度为8%，产率可达90%以上。6)在相同的反应条件下，其他三种二肽前体也得到了较高的产率。7)产物经核磁共振、质谱分析和红外光谱鉴定。较少

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 … More 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