Continuous Synthesis of a Tripeptide by Successive Condensation and Transesterification Catalyzed by Two Immobilized Proteinases in Organic Solvent

Abstract

The tripeptide Z-GlyPheLeuNH2 was continuously synthesized in a high yield from three amino acid derivatives, Z-Gly, PheOMe, and LeuNH2, by immobilized thermolysin (IMT) and immobilized alpha-chymotrypsin (IMC) in an organic solvent, ethyl acetate. The optimal conditions for the synthesis of Z-GlyPheOMe were established theoretically. The yield of Z-GlyPheOMe with IMT in ethyl acetate saturated with buffer was more than 88% after continuous synthesis for 116 hr. The optimal conditions for the synthesis of Z-GlyPheLeuNH2 from Z-GlyPheOMe and LeuNH2 by IMC through transesterification was established in batch reaction experiments. When the concentration of water in the reaction solution was 17-20 microliters/ml, the activity of IMC was highest. The equilibrium between the water concentration in the reaction solution and that in the resin used for enzyme immobilization depended on the resin and was not affected by the presence of the enzyme immobilized. Z-GlyPheLeuNH2 was synthesized from Z-GlyPheOMe and LeuNH2 with a yield of 100%, by continuous reaction for 160 hr. The reactor for synthesis of this tripeptide was efficient and stable because of the use of transesterification and the choice of an appropriate organic solvent. The series plug-flow reactor was successfully operated for 220 hr with a yield of more than 80%. The residual activity of IMT was 94% and that of IMC was 100%.

Synthesis of Peptides Consisting of Essential Amino acids by a Reactor System Using Three Proteinases and an Organic Solvent

“…In a biphasic system at pH 7 when a-chymotrypsin or IMC was used, synthesis did not proceed, contrary to our previous finding 8 > The partition coefficient (P value) of nondissociated Z-TW-OEt was 2000, higher than that of Z-GF-OMe (600), so the concentration of Z-TW-OEt in the aqueous phase was low. a-Chymotrypsin has lower esterase activity at pH 7 than at pH 9-10 12 > The low concentration of Z-TW -OEt and the low activity of esterase probably caused the poor yield at pH 7.…”

Enzyme Immobilization Using the Cellulose-Binding Domain of a Cellulomonas Fimi Exoglucanase

“…Since the 1970s, immobilization technology has advanced beyond the simple one-enzyme type of hydrolytic/ isomeric reactions to include many novel immobilization processes utilizing a great variety of natural and chemically-modified supports. Recent examples include the use of immobilized cells/proteins for multi-step enzymatic reactions (Karkare, 1991;Kimura etal., 1990a); for biochemical synthesis (Jayakumari & Pillai, 1990;Yoshida et al, 1990;Kimura et al, 1990b); for clinical diagnosis (Roda et al, 1991;Chien et al, 1991) and analytical biosensors (Guilbault, 1984;Lowe, 1984;Guilbault, 1988;Guilbault etal, 1991;Huang etal, 1991;; for detoxification of pesticides (Caldwell & Raushel, 1991); and for purification of target proteins (Baneyx & Georgiou, 1989;Greenwood et al, 1989;Chen et al, 1991). Several excellent reviews and monographs deal with the various aspects of immobilization technology (Chibata, 1978;Buchholz, 1979;Bernath & Venkatasubramanian, 1987;Hartmeier, 1988;.…”

Continuous Production ofN-(Benzyloxycarbonyl)-L-Glycyl-L-Phenylalanine Methyl Ester Utilizing Extractive Reaction in Aqueous/Organic Biphasic Medium

“…Z-GlyPhe and its derivatives were synthesized as intermediate products in the enzymatic synthesis of a tripeptide 3 and Leu-enkephalin. 4 To date, however, no effective method for synthesizing these derivatives from the viewpoint of its industrial-scale production has been proposed.…”