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WO2021140526A1 - Acylases mutantes de pénicilline g d'achromobacter ccm4824 - Google Patents

Acylases mutantes de pénicilline g d'achromobacter ccm4824 Download PDF

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Publication number
WO2021140526A1
WO2021140526A1 PCT/IN2021/050022 IN2021050022W WO2021140526A1 WO 2021140526 A1 WO2021140526 A1 WO 2021140526A1 IN 2021050022 W IN2021050022 W IN 2021050022W WO 2021140526 A1 WO2021140526 A1 WO 2021140526A1
Authority
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WIPO (PCT)
Prior art keywords
amino acid
acylase
penicillin
replacement
mutation
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.)
Ceased
Application number
PCT/IN2021/050022
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English (en)
Inventor
Anupama Datla DESAI
Prashant NAGRE
Jagdish TAMORE
Muralidharan KRISHNA
Trupti ASHAR
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.)
Fermenta Biotech Ltd
Original Assignee
Fermenta Biotech 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
Application filed by Fermenta Biotech Ltd filed Critical Fermenta Biotech Ltd
Priority to KR1020227017795A priority Critical patent/KR20220125219A/ko
Publication of WO2021140526A1 publication Critical patent/WO2021140526A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase

Definitions

  • Penicillin G acylase has been widely reported for application, possibly because of the presence of this enzyme in variety of micro- organisms and its characterization for application in hydrolysis and synthesis reaction. Penicillin G acylase from different microorganisms has been widely studied for its substrate specificity and selectivity.
  • Input of substrate is one of the key factors which influence process economics.
  • the kinetics of reaction ratio demands that nucleus to acyl donor ratio used in the process be optimum to drive the synthetic reaction, usually the acyl donor is in excess.
  • ratio should be minimum which is influenced by the type of enzyme used. Enzyme input w.r.t. substrate is critical from the point of achieving maximum conversion in suitable time period.
  • the present invention provides unique ter-polymers which are custom designed for binding of Penicillin acylase enzymes to form immobilized biocatalyst.
  • Each of the disclosed polymers from the range of polymers used for immobilized biocatalyst are uniquely suitable for different antibiotic synthesis and recyclability by virtue of pore size, porosity, specific surface area, epoxy and cross-linking density. Amination of the said polymer and cross-linking thereafter creates additional spacer arm and further improves enzyme binding making the biocatalyst more stable for recyclability and reduces enzyme leaching from the polymer on use.
  • the present invention provides a process for enzymatic synthesis of semi-synthetic ⁇ -lactam antibiotic using the said mutant penicillin acylase enzyme and single pot enzymatic synthesis using dual enzyme system of parent and mutant penicillin acylase enzymes and its commercial viability for both the processes and distinct advantage over the native Penicillin G acylase (pNPGA) from Achromobacter sp CCM 4824 disclosed in US 8,039,604.
  • pNPGA Penicillin G acylase
  • the series of semi-synthetic ⁇ -lactam antibiotics which can be synthesized by enzymatic acylation of the said enzyme include amoxicillin, ampicillin, cephalexin, cefadroxil, cefaclor, cefprozil and cefradine.
  • Figure 2 depicts the antibiotic synthesis activity comparison of mNPGA mutants
  • the underlined region of the sequence is the alpha portion of the penicillin G acylase, the bold underline region is the beta potion of the penicillin G acylase.
  • the signal peptide corresponds to 40 amino acids - in italics, the a-subunit (209 amino acids-single underlined), the linker peptide (57 amino acids) and the ⁇ -subunit (557 amino acid-underlined in dark).
  • the a-subunit amino acids are numbered A1-A209 and the ⁇ -subunit amino acids are numbered B1 to B557.
  • the signal peptide as well as the connecting peptide are removed during post-translational maturation thus yielding an enzymatically active penicillin acylase composed of a and ⁇ subunit.
  • Position A27 in the penicillin G acylase derived from Achromobacter sp. CCM4824, harbours an Isoleucine amino acid residue.
  • a preferred mutation at position A27 comprises the replacement of isoleucine (I) by asparagine (N) or proline (P).
  • a highly preferred mutation would be Asparagine (N).
  • the SEQ ID No. 2 of the present invention has 54% homology as compared to wild type E. coli Penicillin acylase.
  • the SEQ ID No: 2 depicted herein above corresponds to the amino acid sequence of Penicillin G acylase produced by Achromobacter sp.CCM4824.
  • a naturally occurring Penicillin G acylase is selected from microorganisms which have at homology in the range of 40-100% to SEQ ID No.2. More preferred penicillin acylases which are 50% to 100% homologous to SEQ ID No.2.
  • an embodiment describes epoxy ter-polymer combination obtained by suspension polymerization of acrylic monomers as beads of specific surface area and porosity and particle size, modification process to include an additional spacer arm in epoxy beads by amination with various aliphatic amines to enable maximum expression of enzyme bound on these support with improved mechanical stability, filtration ability and recyclability on use of immobilized enzyme in suspension reaction for antibiotic synthesis.
  • each monomers and the quantity used on polymerization with itself and other monomers impart specific characteristic to the beads resulting in variable epoxy content, cross-linking density, porosity, hydrophilicity and rigidity.
  • the molar ratios of monomers like Ethylene glycol dimethacrylate, divinyl benzene to other monomers result in variable cross-linking density in polymer beads.
  • Epoxy content varies with quantity of glycidyl methacrylate or allyl glycidyl ether.
  • the acrylonitrile content imparts rigid backbone to the polymer beads making them mechanical stronger than ones without the component.
  • Each of the mutant clones were obtained in the above examples were grown in 10 mL Luria Bertani (LB) medium with kanamycin concentration of 30-50 ⁇ g / mL at 28°C for 18 h at 150 rpm. The culture growth was checked for pH, optical density at 600 nm and centrifuged to obtained cell pellet which was washed and resuspended to dry weight of 3-4%.
  • LB Luria Bertani
  • Examples 4-8 poly(GMA-ter-EGDM -ter-AN) ter-polymers also referred to as
  • Example 18 ter-polymer system prepared by the procedure as per Example-8, replacing cyclohexanol with mixture of isoamyl alcohol and cyclohexanol in ratio of 10:90 by weight.
  • Immobilized biocatalysts prepared by immobilizing enzyme isolated by process described in Indian Patent No. 253959 from mNPGA mutants of the present invention cultures as described in Example 3.
  • the following determination method of immobilization on polymer beads is familiar per se to the person skilled in the art of covalent immobilization and is detailed only for the sake of completeness.
  • reaction is carried out at 20-25 °C, pH 6.3-6.8 for 2 hrs in a stirred tank reactor.
  • Reaction mixture consists of 100 mM 6APA and 105-110 mM PGME hydrochloride form with 150-170 U of immobilized enzyme catalyst per gram of 6APA in 10 mL of water.
  • Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
  • reaction mixture consists of 100 mM 7ADCA and 130 mM HPGME form with 250 U of immobilized enzyme catalyst per gram of 7ADCA in 10 mL of water.
  • Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Cephalosporin Compounds (AREA)

Abstract

La présente invention concerne des antibiotiques bêta-lactame semi-synthétiques mutants synthétisant des enzymes de pénicilline G acylase d'Achromobacter sp. CCM4824 généré par des mutations dirigées sur site avec substitution d'une ou de plusieurs positions d'acides aminés. La séquence génique codant pour la pénicilline acylase mutante portée sur le plasmide pKARA9 est exprimée dans E. coli BL21. Les enzymes ainsi obtenues à partir D'E. coli BL21 recombiné ont montré une activité de pénicilline acylase, un rapport S/H, un rapport d'entrée inférieur de donneur d'acyle et des rendements améliorés par rapport à l'enzyme parente acylase de pénicilline d'Achromobacter sp. CCM 4824.
PCT/IN2021/050022 2020-01-08 2021-01-08 Acylases mutantes de pénicilline g d'achromobacter ccm4824 Ceased WO2021140526A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020227017795A KR20220125219A (ko) 2020-01-08 2021-01-08 아크로모박터 ccm4824의 돌연변이 페니실린 g 아실라아제

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202021000782 2020-01-08
IN202021000782 2020-01-08

Publications (1)

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WO2021140526A1 true WO2021140526A1 (fr) 2021-07-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113584008A (zh) * 2021-07-30 2021-11-02 湖南福来格生物技术有限公司 一种青霉素g酰化酶突变体及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003055998A2 (fr) * 2001-12-27 2003-07-10 Dsm Ip Assets B.V. PROCEDE DE PREPARATION D'UNE ss-LACTAMINE
WO2010072765A2 (fr) * 2008-12-23 2010-07-01 Dsm Ip Assets B.V. Acylases de pénicilline g mutantes
IN2012MU03159A (fr) * 2012-10-31 2014-05-02
WO2017143944A1 (fr) * 2016-02-23 2017-08-31 上海星维生物技术有限公司 Mutant de la pénicilline g acylase
IN201824049419A (fr) * 2018-12-27 2019-09-13

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003055998A2 (fr) * 2001-12-27 2003-07-10 Dsm Ip Assets B.V. PROCEDE DE PREPARATION D'UNE ss-LACTAMINE
WO2010072765A2 (fr) * 2008-12-23 2010-07-01 Dsm Ip Assets B.V. Acylases de pénicilline g mutantes
IN2012MU03159A (fr) * 2012-10-31 2014-05-02
WO2017143944A1 (fr) * 2016-02-23 2017-08-31 上海星维生物技术有限公司 Mutant de la pénicilline g acylase
IN201824049419A (fr) * 2018-12-27 2019-09-13

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113584008A (zh) * 2021-07-30 2021-11-02 湖南福来格生物技术有限公司 一种青霉素g酰化酶突变体及其应用

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