Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Patent
1996-08-16
1999-10-05
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
435174, 435176, 435175, 435177, 435178, 435179, 435180, 435188, 435195, 435280, C12P 1304, C12N 1100
Patent
active
059622792
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a composite immobilized enzyme preparation and a process for producing D-amino acids using the preparation. In particular, the composite immobilized enzyme preparation of the present invention is useful for the production of D-.alpha.-amino acids which are intermediate compounds for the production of antibiotics, such as D-(p-hydroxyphenyl)glycine to be used for the production of the antibiotic, amoxycillin and the like.
BACKGROUND OF THE INVENTION
Optically-active D-amino acids are important compounds as intermediate compounds for drugs and it has been known that they can be produced efficiently by combining an asymmetric hydrolysis reaction of 5-substituted hydantoins into the corresponding D-N-carbamyl-.alpha.-amino acids with the enzymes, hydantoinases (hereinafter sometimes abbreviated as "Hase") (JP-B 62-30785), and an conversion reaction of the resultant D-N-carbamyl-a-amino acids into the corresponding D-.alpha.-amino acids with the enzymes, D-N-carbamyl-.alpha.-amino acid .alpha.-midohydrolases (hereinafter sometimes abbreviated as "decarbamylase" or "DCase") (PCT/JP91/01696: WO 92/10579).
In addition, JP-A 63-185382, WO 92/00739 and the like disclose that the respective reactions are carried out more efficiently by using these enzymes in the form of so- called immobilized enzymes wherein they are immobilized on supports such as ion exchange resins and the like.
However, a two-step reaction has been employed for carrying out these reactions because the optimal and stable pH's of both enzymes are considerably different from each other. Therefore, both immobilized enzymes should be prepared separately and complicated reaction operations are required.
OBJECTS OF THE INVENTION
The present invention relates to a technique for producing D-.alpha.-amino acids efficiently by using an immobilized enzyme resin obtained by immobilizing both hydantoinase and decarbamylase on one immobilizing resin support in a coexisting state of the enzymes (hereinafter referred to as a "composite enzyme"), simultaneously.
In these two enzymatic reactions for converting 5-substituted hydantoins into D-.alpha.-amino acids, in general, the optimal pH of the hydantoinase reaction is pH 8 to 9 and the solubility of the substrate is increased as increase in pH. In addition, the racemic reaction of the hydantoin ring is promoted in an alkaline range. Therefore, it is desired to carry out the hydantoinase reaction in the pH ranging from 7 to 10, preferably in an alkaline range. On the other hand, in general, the optimal pH of the decarbamylase reaction is pH 6.5 to 9.0 but the hindrance of the reaction by ammonia formed is remarkably increased as increase in pH. Therefore, it is desired to carry out the decarbamylase reaction at pH about neutrality.
If so-called one-step reaction can be employed for carrying out these reactions, i.e., these two enzymatic reactions can be carried out in one reaction vessel simultaneously, in comparison with so-called two-step reaction wherein two different enzymes react with the substrates separately, reaction operations become simple and the overall reaction time can be shortened. In addition, by combining the hydantoinase reaction which is a reversible reaction and the decarbamylase reaction which is a irreversible reaction, the conversion yield of hydantoin can be improved as well as the subsequent purification of D-amino acids can be simplified. Thus, it is expected to significantly reduce the production cost. However, when the respective enzymes are immobilized on different immobilizing supports and they are mixed upon using them, the reactivities become inferior because of the difference in optimal pH and there is a problem in the stability of the immobilized enzymes.
The present inventors have intensively studied to produce a composite immobilized enzyme preparation wherein both enzymes are immobilized on one immobilizing support simultaneously. If these two enzymes are immobilized simultaneously, two enzymatic reactions occur suc
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patent: 5283182 (1994-02-01), Powell et al.
Lee et al. "Isolation of thermostable D-hydantoinase-producing . . . " Biotech. Lett. 16(5), 461-466, May 1994.
Fersht, A. "Enzyme Structure and Mechanism" second edition, W. H. Freeman and Com., Chapter 5, pp. 155-168, 1977.
Ikenaka Yasuhiro
Nanba Hirokazu
Ohashi Takehisa
Takahashi Satomi
Takano Masayuki
Kanegafuchi Kagaku Kogyo & Kabushiki Kaisha
Nashed Nashaat T.
Wax Robert A.
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