EP1121446A1 - Aminoacylase et son utilisation pour la preparation de d-acides amines - Google Patents

Aminoacylase et son utilisation pour la preparation de d-acides amines

Info

Publication number
EP1121446A1
EP1121446A1 EP99949259A EP99949259A EP1121446A1 EP 1121446 A1 EP1121446 A1 EP 1121446A1 EP 99949259 A EP99949259 A EP 99949259A EP 99949259 A EP99949259 A EP 99949259A EP 1121446 A1 EP1121446 A1 EP 1121446A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
acetyl
substrate
concentration
process according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99949259A
Other languages
German (de)
English (en)
Inventor
Stephen John Clifford Taylor
Robert Christopher Brown
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chirotech Technology Ltd
Original Assignee
Chirotech Technology Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9822947.9A external-priority patent/GB9822947D0/en
Priority claimed from GBGB9907739.8A external-priority patent/GB9907739D0/en
Application filed by Chirotech Technology Ltd filed Critical Chirotech Technology Ltd
Publication of EP1121446A1 publication Critical patent/EP1121446A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C12P41/007—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving acyl derivatives of racemic amines

Definitions

  • This invention relates to an enzyme having D-aminoacylase activity and to its use in the production of D-aminoacids, by resolving a racemic mixture of N-acyl aminoacids and deprotecting optically-enriched N-acyl aminoacids.
  • D-Aminoacids are commercially important intermediates in the production of various pesticides, antibiotics and other pharmaceuticals.
  • phenylglycine and p-hydroxyphenylglycine are used in the synthesis of semi-synthetic penicillins and cephalosporins.
  • D-aminoacids There is also much demand for novel D-aminoacids as building blocks for new drug substances.
  • D-Aminoacids may be accessed by physical separation, for example by crystallisation of salts, or by asymmetric chemocatalysis by way of hydrogenation of an enamide precursor.
  • Chemocatalysis provides a general method ofbroad applicability, e.g. for unnatural aminoacids, but requires subsequent N-deacylation for which conventional chemical hydrolysis often results in partial racemisation of the product.
  • biocatalytic methods also, for example by the hydrolysis of hydantoins using a D-specific hydantoinase.
  • the resulting D-carbamoyl aminoacid still requires enzymic or chemical deprotection to the aminoacid.
  • L-aminoacids by means of an L-specific aminoacylase-catalysed hydrolysis of the racemic N-acetylaminoacid is a technology that is well established. This uses the enzyme from Aspergillus oryzae and has been operated on a commercial basis at very large scale, to produce L-methionine, L-valine and L-phenylalanine. Such a large- scale technology does not exist for production ofD-aminoacids, although D-aminoacylase activity has been identified in several microbial strains of Pseudomonas, Streptomyces and Alcaligenes. See Sugie and Suzuki, Agric. Biol. Chem.
  • This enzyme obtained from Alcaligenes xylosoxydans subsp. xylosoxydans ( Alcaligenes A-6 M ), NCUvffi 10771, does not hydrolyse N-acetyl-D-tryptophan. It is reported that the activity of D-aminoacylase is inhibited by 37% and 40% by D- phenylalanine and N-acetyl-D-alloisoleucine at a very low concentration of 2mM. This suggests that the enzyme is susceptible to severe product and substrate inhibition.
  • US-A-5206162 discloses a D-aminoacylase obtained from Alcaligenes faecalis, CCRC 14817.
  • EP-A-0896057 discloses a D-aminoacylase obtained from Amycolatopsis orientalis, IFO 12806. Summary of the Invention
  • the present invention was made following a screen for D-aminoacylase activity performed on a collection of bacteria, and from this screen several were identified as having a D-aminoacylase. Five of these strains were used for genomic DNA preparation. It was then possible, by examining a known literature sequence, to design oligonucleotide primers, and use these in PCR experiments to generate a 1.4kb fragment possessing D- aminoacylase activity. The recombinant fragment was sub-cloned into pTrc99C expression vector. The recombinant plasmid carrying the D-amino acylase fragment was then transformed in to E. coli DH5 for over-expression.
  • the volume efficiency is low, which increases the cost of recovering the product and reduces the economic viability of the process.
  • the enzyme is effective at lOOg/l of substrate; even at 200g/l good activity was demonstrated. It is useful at high volume efficiency, of about lOOg/l, for the deprotection of several (D)-N- acetylaminoacids. This allows an economical process to be developed.
  • an isolated enzyme according to the present invention is capable of hydrolysing N-acetyl-D-tryptophan at a substrate concentration of 10 g 1.
  • it is capable of the desired activity at the given concentration, and also at higher concentrations.
  • it unlike the enzymes disclosed in US- A-5206162 and in EP-A- 0896057, it exhibits the ability to convert (R)-N-acetyl-2-thienylalanine, and also to convert it faster than (R)-N-acetyl-4-chlorophenylalanine.
  • the substrate used in the invention may be part of a mixture of the (L)- and (D)-N-acylaminoacids.
  • the (D)-N-acylaminoacid may be enantiomerically enriched, e.g. essentially optically pure.
  • the novel enzyme may be used to produce natural and unnatural aminoacids.
  • One class of the latter is aryl/heteroaryl-substituted aminoacids.
  • the enzyme may suffer from substrate inhibition.
  • a high substrate concentration may merely lead to a low conversion to product, so that a volume efficient reaction is not possible.
  • this effect can be overcome by the simple expedient of adding the substrate in several batches over the course of thebiotransformation, and, if kept at low concentration, a high product accumulation is possible.
  • substrate hydrolysis is poor.
  • the enzyme will hydrolyse 15 g/1 efficiently and, by making several additions of the substrate, it is possible to accumulate about 75 g/1 of D-2-naphthylalanine.
  • the enzyme may be used in whole cell or isolated form. It may be immobilised, if desired, by methods known to those of ordinary skill in the art.
  • the enzyme may be produced from the deposited organism (details given below). Alternatively, it may be produced by recombinant technology.
  • DNA and amino-acid sequence provided herein, a person skilled in the art can readily construct fragments or mutations of the genes and enzymes disclosed herein. These fragments and mutations, which retain the activity of the exemplified enzyme, are within the scope of the present invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino-acid sequences disclosed herein. It is well within the skill of one of ordinary skill in the art to create these alternative DNA sequences encoding the same, or similar, enzymes. These DNA sequences are within the scope of the present invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino-acid substitutions, deletions, additions or insertions which do not materially affect activity. Fragments retaining activity are also included in this definition.
  • genes of this invention can be isolated by known procedures and can be introduced into a wide variety of microbial hosts. Expression of the gene results, directly or indirectly, in the intracellular production and maintenance of the enzyme.
  • the gene may be introduced via a suitable vector into a microbial host.
  • a DNA construct may include the transcriptional and translational regulatory signals for expression of the gene, the gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.
  • the construct can involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3* of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region.
  • This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker.
  • the gene can be introduced between the transcriptional/translational initiation and termination regions, so as to be under the regulatory control of the initiation region.
  • This construct can be included in a plasmid, which could include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host.
  • one or more markers may be present, as described above.
  • the plasmid will desirably include a sequence homologous with the host genome.
  • the transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for activity.
  • Suitable host cells include prokaryotes and eukaryotes.
  • An example is E. coli.
  • Genomic DNA was prepared from 5 Alcaligenes strains held in the Chirotech culture collection; CMC3352, 3353, 2916, 3378, 3823. From these genomic preparations PCR was carried out to amplify the D-aminoacylase reported by Wakayama et al (1995), supra. Primers were synthesised according to the published sequence of the dan gene from Alcaligenes A-6. The 5' PCR primer in SEQ ID NO. 1 ; the 3' PCR primer is SEQ ID NO. 2.
  • a 1.4 kb PCR fragment was amplified from strains CMC 3352 and 3353. These fragments were then cloned into the PCR cloning vector from Stratagene, pCR-script and transformed into E. coli. Resultant clones were analysed by restriction mapping to ascertain the presence of a 1.4kb acylase fragment. Clones harbouring this fragment were sequenced to verify that the putative acylase showed homology to the reported sequence. DNA sequence analysis show the majority of cloned fragments to include SEQ ID NO. 3. The deduced aminoacid sequence is given below as SEQ ID NO. 4.
  • residues of the recombinant D-acylase differ from the published sequence as follows; Ser 2 to Ala; Gin 3 to Glu; Ala 14 to Val; ; Gly 126 to Arg; Gly 240 to Arg; Glu 242 to Lys.
  • the recombinant fragment was sub-cloned into pTrc99C expression vector via the 5' Nc ⁇ l and 3' BamY ⁇ . engineered restriction sites.
  • the recombinant plasmid carrying the D-amino acylase fragment was transformed into E. coli DH5 for over-expression.
  • the recombinant cells E. coli strain CMC 4406, have been deposited at NCIMB, 23 St. Machar Drive, Aberdeen AB243RY, Scotland.
  • the accession number is NCIMB 40965.
  • the recombinant cells were grown by fermentation in a medium containing glucose, peptides and salts. The seed culture was inoculated from plates, and incubated overnight in TSB medium containing 0.1 g/1 ampicillin at 37 °C.
  • the inoculum (5ml, OD 5.0) was grown in 1.51 of the following medium which contained (amounts in g.l "1 unless otherwise indicated): KHTO 8
  • the reaction was monitored by chiral GC as follows: 0.5 ml of the reaction mixture was taken and acidified to pH 2.0 with cone. HC1. The aqueous was extracted with EtOAc which was dried (MgSO 4 ) and filtered and treated with OJml of TMS-diazomethane. The derivatised product was assayed by chiral GC (Chrompack Chirasil L-Nal, 25m, 20psi He, 60°C for 10 mins, 5°C/min to 200°C, holding for 10 minutes, FID detection). After 1 hour the ee of the substrate had decreased to 68%, after 2 hours it was 24% and after 22 hours was 7%.
  • Table 1 reports D-acylase reactions using a range of unnatural (R)-N-Ac- phenylalanine and (R)-N-Ac-alanine derivatives, and (R)-N-Ac-4-fluorophenylglycine.
  • coli CMC4406 containing recombinant D-acylase were immobilised on a reactive soluble polymer (RSP).
  • the RSP was prepared by reaction of polyethyleneimine (0.8g) with aqueous 25% w/v glutaraldehyde (1.6ml), to a total volume of 20 ml H 2 O.
  • the RSP was then mixed with 1 Og of cells resuspended in 20ml H 2 O. This was stirred vigorously for 30 minutes, after which the immobilised cells, having the consistency of foam rubber, were recovered by filtration.
  • the final product (20g) had a specific activity of 20.55 U/g and the recovery of activity was 43% of the whole cells used in the immobilisation.
  • 1 Unit of activity is defined as the hydrolysis of 1 ⁇ mol/min N-Ac- D-tryptophan to D-tryptophan measured at a substrate concentration of lOmM at 25 °C, pH7.5.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Enzyme isolé capable d'hydrolyser N-acétyl-D-tryptophane à une concentration de substrat de 10 g/l et exerçant une conversion plus rapide de (R)-N-acétyl-2-thiènylalanine que de (R)-N-acétyl-4-chlorophénylalanine. Cet enzyme est utile pour préparer des D-acides aminés.
EP99949259A 1998-10-20 1999-10-20 Aminoacylase et son utilisation pour la preparation de d-acides amines Withdrawn EP1121446A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9822947 1998-10-20
GBGB9822947.9A GB9822947D0 (en) 1998-10-20 1998-10-20 Aminoacylase and its use in the production of d-aminoacids
GB9907739 1999-04-01
GBGB9907739.8A GB9907739D0 (en) 1999-04-01 1999-04-01 Aminoacylase and its use in the production of D-aminoacids
PCT/GB1999/003458 WO2000023598A1 (fr) 1998-10-20 1999-10-20 Aminoacylase et son utilisation pour la preparation de d-acides amines

Publications (1)

Publication Number Publication Date
EP1121446A1 true EP1121446A1 (fr) 2001-08-08

Family

ID=26314541

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99949259A Withdrawn EP1121446A1 (fr) 1998-10-20 1999-10-20 Aminoacylase et son utilisation pour la preparation de d-acides amines

Country Status (6)

Country Link
EP (1) EP1121446A1 (fr)
JP (1) JP2002527110A (fr)
KR (1) KR20010075649A (fr)
AU (1) AU6222799A (fr)
CA (1) CA2347079A1 (fr)
WO (1) WO2000023598A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4526157B2 (ja) 2000-01-27 2010-08-18 ダイセル化学工業株式会社 Ｄ−アミノアシラーゼ、およびそれをコードする遺伝子
KR20020087948A (ko) * 2001-02-01 2002-11-23 미쯔이가가꾸가부시끼가이샤 신규 ｄ-아미노아실라제를 코딩하는 ｄｎａ 및 그것을사용한 ｄ-아미노산의 제조 방법
EP1435388B1 (fr) 2002-12-24 2012-12-12 Daicel Corporation Mutants de la D-aminoacylase d'Alcaligenes denitrificans pour une meilleure producion d'aminoacides-D
CN108624577B (zh) * 2017-03-22 2021-07-27 中国科学院天津工业生物技术研究所 用于催化n-乙酰-d-色氨酸水解生成d-色氨酸的新酶
KR20220096237A (ko) 2020-12-30 2022-07-07 이종학 수산물 유통 서비스 시스템

Family Cites Families (8)

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Publication number Priority date Publication date Assignee Title
JPS5037277B2 (fr) * 1972-08-31 1975-12-01
JPS5233194B2 (fr) * 1973-04-24 1977-08-26
JPS62126976A (ja) * 1985-11-26 1987-06-09 Agency Of Ind Science & Technol Ｄ−アミノアシラ−ゼの製造法
JPH0783711B2 (ja) * 1987-06-29 1995-09-13 ダイセル化学工業株式会社 新規なｄ―アミノアシラーゼの製造法
US4981799A (en) * 1987-08-21 1991-01-01 Takeda Chemical Industries, Ltd. Acylamino acid racemase, production and use thereof
JP2869793B2 (ja) * 1989-03-07 1999-03-10 第一化学薬品株式会社 酸性ｄ―アミノ酸に作用するｄ―アミノアシラーゼ及びその製造法
US5206162A (en) * 1991-10-17 1993-04-27 National Science Council Of Republic Of China Process for making D-aminoacylase
JP4063400B2 (ja) * 1997-07-31 2008-03-19 ダイセル化学工業株式会社 Ｄ−アミノアシラーゼ

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0023598A1 *

Also Published As

Publication number Publication date
JP2002527110A (ja) 2002-08-27
WO2000023598A1 (fr) 2000-04-27
KR20010075649A (ko) 2001-08-09
AU6222799A (en) 2000-05-08
CA2347079A1 (fr) 2000-04-27

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