WO2009013611A2 - Estérase modifiée et ses applications - Google Patents

Estérase modifiée et ses applications Download PDF

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Publication number
WO2009013611A2
WO2009013611A2 PCT/IB2008/001942 IB2008001942W WO2009013611A2 WO 2009013611 A2 WO2009013611 A2 WO 2009013611A2 IB 2008001942 W IB2008001942 W IB 2008001942W WO 2009013611 A2 WO2009013611 A2 WO 2009013611A2
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WO
WIPO (PCT)
Prior art keywords
esterase
enzyme
modified
amino acid
mtcc
Prior art date
Application number
PCT/IB2008/001942
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English (en)
Other versions
WO2009013611A3 (fr
Inventor
Micheal Durairaaj
Vasu Vinayagam
Twinkle Jasmine Masilamani
Ramanan Thirumoorthy
Cavery Manian Krishnan
Ravi Kanth Harit
Original Assignee
Orchid Chemicals & Pharmaceuticals Limited
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Publication date
Application filed by Orchid Chemicals & Pharmaceuticals Limited filed Critical Orchid Chemicals & Pharmaceuticals Limited
Publication of WO2009013611A2 publication Critical patent/WO2009013611A2/fr
Publication of WO2009013611A3 publication Critical patent/WO2009013611A3/fr

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    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the present invention relates a modified esterase from Bacillus subtilis MTCC 121, having increased deacetylation activity when expressed in Escherichia coli towards substrates like cephalosporin C and 7-aminocephalosporanic acid (7-ACA) or its acyl amino derivatives.
  • the modified esterase from Bacillus subtilis is found useful in the bioprocess for the preparation of 7- amino-3-hydroxymethyl cephalosporanic acid (HACA) or its acyl amino derivative and 3-deacetyl cephalosporin C.
  • 7-ACA serves as a starting material for a large number of semi-synthetic cephalosporins like Cefalotin, Cefazolin, Cefotaxime, Cefcapene pivoxil, Cefuroxime etc., by modification of C7 and C3 positions (as shown in the scheme- 1). Deacetylation of C3 position is required for the attachment of appropriate side chain.
  • Chemical hydrolysis of acetate group at C3 position from cephalosporins under acidic conditions lead to poor yield (Kukolja, S., J. Med, Chem. 11 : 1067-1069, 1968), while hydrolysis at alkaline pH imposes significant strain on utilities and reduce productivity.
  • enzymatic hydrolysis at neutral pH offprs a cost-effective route for the manufacture of deacetyl derivatives of cephalosporins such as 7-ACA in good quality.
  • R is hydrogen or acyl group; Rl is hydrogen or alkoxy; R' is standard cephalosporin substituent like carbamoyl group.
  • CAH has been cloned, sequenced and expressed, like from, Rhodosporidium toruloids (US 5,869,309), Aureobasidium (US , 4,517,299), Bacillus subtilis (US 6,465,233, WO 99/55881), etc.
  • Rhodosporidium toruloids US 5,869,309
  • Aureobasidium US , 4,517,299
  • Bacillus subtilis US 6,465,233, WO 99/55881
  • Several processes have been described for enzymatic cephalosporin deacetylation (US 5,869,309, US 4,517,299, US 6,465,233, WO 99/55881, US 5,338,676, EP 0454478) using esterase from Bacillus subtilis, expressed in Escherichia coli with varying level of hydrolysis.
  • Bacillus subtilis has been sequenced and is known to encode esterase of 318 amino acids. Comparison of nucleotide and amino acid sequences has revealed significant sequence homology among esterase produced by several species of Bacillus subtilis such as ATCC 6633 , SHS 103 and DS 1152 (KCCM- 10143).
  • an object of the current invention is to provide a modified CAH from B. subtilis having increased activity when expressed in E. co.li with substrates such as 7-ACA, cephalosporin C when compared with the wild-type esterase.
  • Figure 1 Wild-type nucleotide and amino acid sequence of esterase from B. subtilis MTCC 121 is given as SEQ ID No: 1.
  • An object of this invention is to provide a modified esterase from B. subtilis MTCC 121 with improved capability for deacetylation than that occurs in the wild type esterase.
  • Another object of this invention is to provide a modified esterase having increased deacetylation activity on substrates like 7-ACA or its acyl amino derivative and cephalosporin C. Summary of the invention:
  • the present invention provides a mutated CAH from B. subtilis
  • MTCC 121 which consists of amino acid substitution at one or more amino acid residues corresponding to the wild type esterase for the following group of residues consisting of Aspartic acid at position 43, Methionine at position 138, Tyrosine at position 222 and Arginine at position 231.
  • the invention provides esterase mutants with amino acid substitutions at one or more positions of amino acid residues of Ml 38V, R231G and D43G; Y222H, wherein the residue positions of the amino acid substitutions corresponds to those of a wild-type esterase.
  • the process of invention is to provide a modified CAH gene encoding the mutated cephalosporin esterase.
  • Another embodiment of the invention is to provide an esterase protein with modified deacetylation activity for substrates such as cephalosporin C and 7-ACA.
  • the invention provides a recombinant vector specifically an expression vector, which comprises the modified esterase gene.
  • the present invention further relates to a host strain that contains the expression vector with the modified CAH gene.
  • Another embodiment of the invention is to provide a method of expression of esterase in a host strain that contains the expression vector with the modified CAH gene;
  • Another embodiment of the invention is to provide a method of isolation of the expressed enzyme.
  • Another embodiment of the current invention is for a method of deacetylation of cephalosporins using such isolated esterase either as soluble or immobilised form.
  • the primary embodiment of the present invention is to provide a mutant esterase having increased specificity (deacetylation activity at the 3 rd position) for substrates such as 7-ACA, (7-aminocephalosporanic acid) or (7-acyl aminocephalosporanic acid) and cephalosporin C than the wild type esterase, such that the mutant esterase is from the bacteria B. subtilis MTCC 121 , expressed in E. coli.
  • the wild type gene (CAH gene) has been cloned and characterized (Kunst, F., et al, Nature, 390, 249-256, 1997).
  • the wild-type nucleotide and amino acid sequence of esterase from B. subtilis is given in SEQ ID No: 1 ( Figure 1).
  • a mutated esterase having enhanced deacetylation activity with amino acid substitutions at one or more amino acid residues corresponding to the wild type esterase for the group of residues consisting of Aspartic acid at position 43, Methionine at position 138, Tyrosine at position 222 and Arginine at position 231.
  • the invention provides mutants with amino acid substitutions at one or more amino acid residues of Ml 38V, R231G and D43G; Y222A, wherein the residue positions of the amino acid substitutions corresponds to those of a wild type esterase from B. subtilis MTCC 121.
  • the following are the variations in the amino acid residues from the sequence in Seq. ID. NO. 1 :
  • Another embodiment of the invention is to provide an isolated nucleic acid molecule those codes for the mutated esterase.
  • this isolated nucleic acid molecule is obtained by mutating the wild type esterase.
  • the mutagenesis technique could be by chemical, error-prone PCR or site-directed approach.
  • the suitable mutagenesis technique can be selected and used for introducing mutations.
  • the mutated nucleic acid molecule can be cloned, expressed and the property of the polypeptide can be studied.
  • the mutated nucleic acid molecule may be incorporated into a recombinant vector, which is capable of expression or replication when transferred into a host cell.
  • Expression of the polypeptide can be controlled by a regulatory sequence probably a promoter.
  • the recombinant vector can be introduced into a host strain to produce the mutated esterase.
  • the mutated esterase when expressed in the host strain is capable of converting the substrate 7-ACA or its acyl amino derivative to HACA (7- amino-3-hydroxymethyl cephalosporanic acid) and cephalosporin C to deacetyl-cephalosporin C by deacetylation. ' ⁇ ⁇ . ⁇
  • the modified peptide has amino acid sequence different from that of SEQ ID NO: 1.
  • This polypeptide is one, which has deacetylation activity i.e. catalyze the deacetylation of C3-acetyl side chain of 7-ACA or its acyl amino derivative to HACA or its acyl amino derivative.
  • the deacetylation activity of the polypeptide is modified or increased.
  • the invention provides a modified CAH protein, having an enhanced catalytic activity or increased specificity for substrates such as 7-ACA when compared with the wild-type esterase.
  • polypeptides thus produced from the mutated nucleotide sequence can be used to produce chimeras from portions of other esterase polypeptides.
  • Polypeptides from the present invention can be purified with varying level of homogeneity and can be used for other purposes.
  • the invention can be used for the manufacture of modified cephalosporins either as enzymatic or in vivo fermentation based technologies.
  • the detailed procedures, such as transformation and fermentation of such cells, purification and isolation can be found in the literature.
  • MTCC Microbial Type Culture Collection
  • Oligonucleotides were synthesized and supplied by Microsynth Gmbh, Switzerland or Sigma, India. Restriction enzymes, pUC19 vector for cloning were obtained from New England Biolabs Inc, USA. dNTPs were purchased from ABgene, England. pET24a vector, Escherichia coli BL21 (DE3) strain and Bugbuster reagent were purchased from Novagen, USA. Bacillus subtilis MTCC 121 was obtained from the Microbial Type Culture Collection, Chandigarh, India (MTCC). Bradford reagent was purchased from Biorad, USA. Cis columns (150 x 4.6mm, 5 ⁇ , ODS3) were obtained from GL science, Japan. GeneElute Bacterial genomic DNA isolation Kit Mini was supplied by Sigma, India and growth media components were obtained from Becton Dickinson, USA.
  • the B. subtilis strain (MTCC No.121) was grown on LB media containing Tryptone 10 gm, Yeast extract 5 gm and sodium chloride 10 gm made up to 1 liter with distilled water at pH 7.0. The culture was incubated at 30 0 C and 220 rpm for 24 hours. Cells were harvested from 1.5 ml culture and used for'genomic DNA isolation.
  • cephalosporin acetylesterase accesion number: Z99105
  • the gene coding for cephalosporin acetylesterase was amplified using 20 pmole of primers 5' CATATGCAACTATTCGATCTGCCGCTCGAC 3' and 5' TCAGCCTTTAAGATGCTGCTTAAAGAAAGC 3', 200 ⁇ M dNTPs, deep vent DNA polymerase buffer, 2.5U deep vent DNA polymerase enzyme, and water in a final reaction volume of 100 ⁇ l.
  • PCR condition consists of an initial denaturation for 5 min at 95 0 C followed by 30 cycles consisting of denaturation at 95 0 C for 40 sec, annealing at 50 0 C for 30 sec, extension at 72 0 C for 2 min with a final extension at 72 0 C for 10 min.
  • An amplified product of length of about l kb of the CAH gene fragment was verified by agarose gel electrophoresis.
  • the putative esterase gene fragment observed after amplification was purified by
  • the pOBTES vector served as template for error-prone PCR mutagenesis.
  • the amplification x was carried out with 20 pmole of
  • pOCPLES templete was used for mutagenesis using hydroxylamine.
  • E.coli BL21 (DE3) strain was inoculated in 96-well plate containing LB medium with Kanamycin (75 ⁇ g/ml) for overnight growth at 37 °C at 220 rpm. Overnight culture was subcultured again in 96-well deep well plates and grown till OD 6 oo reached 0.6 and induced with 0.1 mM isopropyl- ⁇ -D-thioga ⁇ actopyranoside (IPTG). After induction, the culture was allowed to grow for 3 hours at 25 0 C and pellets were harvested by centrifugation in a micro plate centrifuge at 4,000 rpm for 10 min at 4 0 C. The pellets were resuspended in a buffer containing 50 mM Tris. HCl (pH 7.5), 0.1 mM DTT, 0.01 mM EDTA, 10% Glycerol, 50 mM Glucose and stored at -8O 0 C. "
  • E. coli BL21 harbouring pOCPLES or modified esterase gene was cultivated in a medium containing 2 g of (NH 4 ) 2 HPO 4 , 6.75 g Of KH 2 PO 4 , 0.85 g of citric acid, 0.7 g of MgSO 4 .7H 2 O, and 5 ml of a trace metal solution that contains (1O g of FeSO 4 JH 2 O, 2.25 g of ZnSO 4 .7H 2 O, 1 g of CuSO 4 .5H 2 O, 0.5 g of MnSO 4 .5H 2 O, 0.23 g Of Na 2 B 4 O 7 .10H 2 O, 2 g of CaCl 2 .2H 2 O, and 0.1 g Of (NH 4 ) 6 MO 7 O 24 ) per litre) and glucose (20 gm/litre) at 37 0 C at 220 rpm in a shake flask (KI J.
  • 0.5mM EDTA pH 8.0, 500 ⁇ l of IM DTT, 200 ⁇ l of I M PMSF and 200 ⁇ l of I M benzamidin HCl was added.
  • the resulting cell suspension was separated into 250ml aliquots and sonicated for 8 minutes using 50% amplitude, 0.5 cycle time, probe dia 10mm, approx. 80mm long (Cat. -No. 8535671 ) in LABSONIC M sonicator. Subsequently, 125 ml of lOOmg/ml lysozyme was added to the suspension and sonication was repeated twice under same conditions with an interval of 5 minutes.
  • the suspensions were pooled together and 5 ml of 200mg/ml streptomycin sulphate was added and left in ice for 30 minutes.
  • the samples were centrifuged for 30 minutes at 12500 rpm and 4 0 C in a Beckman coulter centrifuge and the supernatant was stored at 4C until further use.
  • Protein concentration was estimated using the BIORAD dye concentrate as recommended by the supplier. Larger pellets were disrupted using dynomill (Multilab, Willy A. Bachofen AG, Switzerland) as recommended by the supplier.
  • 250 ml of enzyme solution containing 4.8 mg/ml of protein was made up to 300 ml with an addition of 50 mL of 4M potassium phosphate buffer (pH 7.5) and added to 50gm of Eupergit CM matrix for immobilization and left at 25 0 C. After 36 hours, the suspension was filtered through cloth and stored at 4 0 C after washing thoroughly with
  • Amino cephalosporanic acid was dissolved in water using bases such as sodium hydroxide, sodium bicarbonate, ammonia or sodium carbonate.
  • bases such as sodium hydroxide, sodium bicarbonate, ammonia or sodium carbonate.
  • esterase described herein was added and the pH was maintained 7.2- 7.8 till completion of reaction, after completion of reaction the reaction mass optionally diluted with oraganic solvent like acetone, THF, iso-propyl alcohol and pH was adjusted using acid like formic acid, acetic acid, dil HCl and the like to yield deacetylated 7-ACA (HACA).
  • the pellets were thawed and enzyme from expressed clones was released using bugbuster reagent.
  • the deacetylation reaction was assayed with 40 ⁇ l of 10% 7-ACA solubilized using aqueous ammonia, in 80 ⁇ l of 100 mM k-PO 4 buffer at pH 8.0.
  • the progression of reaction was analyzed colorimetrically and short-listed isolates were reconfirmed using HPLC.
  • pH indicator dye neutral red 2 ⁇ l was added to the assay reaction and the progression of reaction was monitored by change in color at 560 nm.

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

La présente invention concerne une estérase modifiée ayant une activité de désacétylation améliorée pour 7-ACA ou son dérivé acyle amino et la céphalosporine C pour la fabrication respectivement de HACA et de 3-désacétyl céphalosporine C. Ladite estérase modifiée porte une ou plusieurs modifications d'acide aminé à des positions de résidu par comparaison avec l'estérase de type sauvage (MTCC 121) parmi le groupe suivant de résidus, l'acide aspartique en position 43, la méthionine en position 138, la tyrosine en position 222 et l'arginine en position 231.
PCT/IB2008/001942 2007-07-26 2008-07-25 Estérase modifiée et ses applications WO2009013611A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019211969A1 (fr) * 2018-05-02 2019-11-07 天野エンザイム株式会社 Estérase modifiée et son application
CN113637653A (zh) * 2021-08-05 2021-11-12 云南师范大学 一种活性提高的酯酶突变体Est8-XL及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0454478A1 (fr) * 1990-04-27 1991-10-30 SHIONOGI SEIYAKU KABUSHIKI KAISHA trading under the name of SHIONOGI & CO. LTD. Gène codant pour une acétylhydrolase de la céphalosporine et protéine codée par celui-ci
WO1999055881A1 (fr) * 1998-04-24 1999-11-04 Daesang Corporation Cephalosporine desacetylase, gene codant pour celle-ci, et preparation de composes de cephalosporine desacetylee au moyen de celle-ci.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0454478A1 (fr) * 1990-04-27 1991-10-30 SHIONOGI SEIYAKU KABUSHIKI KAISHA trading under the name of SHIONOGI & CO. LTD. Gène codant pour une acétylhydrolase de la céphalosporine et protéine codée par celui-ci
WO1999055881A1 (fr) * 1998-04-24 1999-11-04 Daesang Corporation Cephalosporine desacetylase, gene codant pour celle-ci, et preparation de composes de cephalosporine desacetylee au moyen de celle-ci.

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [Online] 02 January 2009 'false' Database accession no. NLM9384377 & KUNST F. ET AL: 'The complete genome sequence of the gram-positive bacterium Bacillus subtilis' NATURE vol. 390, no. 6657, 1997, *
MITSUSHIMA, K. ET AL.: 'Gene cloning, nucleotide sequence, and expression of a cephalosporin-C deacetylase from Bacillus subtilis.' APPLIED AND ENVIRONMENTAL MICROBIOLOGY vol. 61, no. 6, 1995, pages 2224 - 2229 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019211969A1 (fr) * 2018-05-02 2019-11-07 天野エンザイム株式会社 Estérase modifiée et son application
CN113637653A (zh) * 2021-08-05 2021-11-12 云南师范大学 一种活性提高的酯酶突变体Est8-XL及其应用
CN113637653B (zh) * 2021-08-05 2023-05-23 云南师范大学 一种活性提高的酯酶突变体Est8-XL及其应用

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