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Continuous enzymatic production of peptide precursor in aqueous/organic biphasic medium
Author(s) -
Murakami Yoshihiko,
Yoshida Tatsunori,
Hayashi Shigemitsu,
Hirata Akira
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000705)69:1<57::aid-bit7>3.0.co;2-j
Subject(s) - aspartame , chemistry , aqueous solution , yield (engineering) , aqueous two phase system , substrate (aquarium) , extraction (chemistry) , peptide synthesis , enzyme , peptide , protecting group , aspartic acid , amino acid , chromatography , organic chemistry , biochemistry , materials science , alkyl , metallurgy , oceanography , geology
N ‐(benzyloxycarbonyl)‐ L ‐aspartic acid (Z‐ L ‐Asp) has generally been used as a carboxyl substrate for the enzymatic synthesis of a precursor of aspartame (synthetic sweetener); however, alternative inexpensive protection groups have been in demand for lowering the total cost of its industrial‐scale production. A formyl group (F‐) was found to be a more desirable protecting group for the N ‐terminus of amino acid derivatives due to its low cost of preparation, introduction, and removal. The yield of F‐AspPheOMe ( N ‐formyl‐ L ‐aspartyl‐ L ‐phe‐ nylalanine methylester), however, was found to be <10% in a conventional aqueous medium. We found that F‐ L ‐Asp and L ‐PheOMe were partitioned mainly to the aqueous phase in an aqueous/organic biphasic medium, whereas F‐AspPheOMe partitioned to the organic phase, especially when some extracting agents were added. In this study, simultaneous operation of an enzymatic reaction and a product separation by liquid–liquid extraction was thus applied to the F‐AspPheOMe synthesis. We succeeded in synthesizing F‐AspPheOMe continuously in an aqueous/tributylphosphate (TBP) biphasic medium with >95% yield, which was about tenfold higher than that in an aqueous monophasic medium. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 57–65, 2000.