US20120270285A1 - Stereospecific carbonyl reductases - Google Patents

Stereospecific carbonyl reductases Download PDF

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US20120270285A1
US20120270285A1 US13/379,942 US201013379942A US2012270285A1 US 20120270285 A1 US20120270285 A1 US 20120270285A1 US 201013379942 A US201013379942 A US 201013379942A US 2012270285 A1 US2012270285 A1 US 2012270285A1
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seq
amino acid
carbonyl
acid sequence
purified polypeptide
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Gaetano T. Montelione
Rong Xiao
Yao Nie
Yan Xu
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Jiangnan University
Rutgers State University of New Jersey
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Assigned to RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY reassignment RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MONTELIONE, GAETANO T., XIAO, RONG
Assigned to JIANGNAN UNIVERSITY reassignment JIANGNAN UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NIE, YAO, XU, YAN
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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

  • FIG. 1 Map of contig005802 of Candida parapsilosis genome including the four open reading frames, scr1, scr2, scr3, and cpadh.
  • FIG. 2 Amino acid sequence alignment of CPADH (GenBank accession number DQ675534; SEQ ID NO:1), SCR1 (GenBank accession number FJ939565; SEQ ID NO:4), SCR2 (GenBank accession number FJ939563; SEQ ID NO:3), and SCR3 (GenBank accession number FJ939564; SEQ ID NO:2) from C. parapsilosis . Gaps in the aligned sequences are indicated by dashes. Identical amino acid residues are enclosed in boxes.
  • FIG. 3 Analysis of the overexpression of SCR1, SCR2, and SCR3.
  • the proteins were separated on a 12% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue G-250. Lane 1, total protein for SCR1; Lane 2, soluble fraction for SCR1; Lane 3, total protein for SCR2; Lane 4, soluble fraction for SCR2; Lane 5, total protein for SCR3; Lane 6, soluble fraction for SCR3; Lane 7, molecular mass standard.
  • FIG. 7A-7D Substrate specificity of SCR1 and SCR3.
  • the enzyme activities of SCR1 (open bars) and SCR3 (shaded bars) ( 7 A) to various substrates ( 7 B- 7 D) were measured as described herein. Maximal enzyme activity observed was set as 100% relative activity for the enzymes to various substrates.
  • the amino acid sequence has at least 70% identity to SEQ ID NO:2.
  • the amino acid sequence has at least 95% identity to the Candida parapsilosis stereospecific carbonyl reductase (e.g., to at least one of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4).
  • the carbonyl reductase activity of the polypeptide is NADPH-dependent.
  • the sequence comprises SEQ ID NO:6 or of a degenerate variant of SEQ ID NO:6.
  • sequence encodes SEQ ID NO:4.
  • the reduction takes place in the presence of a coenzyme.
  • the coenzyme is NADPH.
  • the carbonyl substrate comprises an aryl ketone.
  • the aryl ketone is 2′-chloro-2-hydroxyacetophenone, 3′-chloro-2-hydroxyacetophenone, 4′-chloro-2-hydroxyacetophenone or 4′-methoxy-2-hydroxyacetophenone.
  • the carbonyl substrate is ethyl 4-chloro-3-oxobutyrate.
  • stereospecific oxidoreductases including alcohol dehydrogenases and carbonyl reductases
  • SDR short-chain dehydrogenase/reductase
  • AMR aldo-keto reductase
  • an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • comparison of nucleotide sequences for determination of percent sequence identity to another sequence may be made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the program.
  • substantially identical in the context of a peptide indicates that a peptide comprises an amino acid sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99%, sequence identity to a reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)).
  • ketoesters the enzymes both exhibited high activity to those with small groups, but compared with SCR3, SCR1 was more active to bulky ketoesters with phenyl ring and generally showed higher activities to ⁇ -ketoesters.
  • SCR1 preformed the highest activity for the reduction of ethyl 4-chloro-3-oxobutyrate, which affords ethyl 4-chloro-3-hydroxybutyrate, an important pharmaceutical intermediate (Thayer, Chem. Eng. News, 84, 26-27 (2006).
  • Enantiomers were eluted with hexane and 2-propanol (9:1) at a flow rate of 0.5 ml/min. The effluent was monitored at 215 nm, and the areas under each peak were integrated (Nie et al., Org. Process Res. Dev., 8, 246-251 (2004)).

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  • Chemical & Material Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
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  • General Chemical & Material Sciences (AREA)
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  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
US13/379,942 2009-06-23 2010-06-23 Stereospecific carbonyl reductases Abandoned US20120270285A1 (en)

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PCT/US2010/039666 WO2010151593A1 (en) 2009-06-23 2010-06-23 Sterospecific carbonyl reductases
US13/379,942 US20120270285A1 (en) 2009-06-23 2010-06-23 Stereospecific carbonyl reductases

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Cited By (1)

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US9422583B2 (en) 2009-06-23 2016-08-23 Rutgers, The State University Of New Jersey Stereospecific carbonyl reductases

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US9790524B2 (en) 2013-04-17 2017-10-17 Council Of Scientific & Industrial Research Designer cells for enantioselective reduction of ketones and use thereof in efficient production of enantioenriched alcohols
CN104372041B (zh) * 2013-08-12 2018-03-16 南京朗恩生物科技有限公司 一种全细胞催化制备(s)‑4‑氯‑3‑羟基丁酸乙酯的方法
CN105018439B (zh) * 2015-05-19 2019-01-15 上海弈柯莱生物医药科技有限公司 一种羰基还原酶及其在合成手性羟基化合物中的应用
CN106929491B (zh) * 2017-03-13 2019-09-17 江南大学 (s)-羰基还原酶异源聚合体及其在催化多苯环化合物的应用

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MX240992B (es) * 1999-07-09 2006-10-10 Degussa Secuencias de nucleotidos que codifican para el gen opca.
EP1811021A4 (en) * 2004-10-27 2008-05-28 Kaneka Corp NEW CARBONYL REDUCTASE, GENE FOR IT AND USE THEREOF
CN1884502A (zh) * 2006-06-20 2006-12-27 江南大学 近平滑假丝酵母cctcc m203011的(s)－羰基还原酶的基因序列和氨基酸组成
CN102482649B (zh) 2009-06-23 2014-09-03 江南大学 立体专一性羰基还原酶

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CN102482649A (zh) 2012-05-30
WO2010151593A8 (en) 2011-06-23
US9422583B2 (en) 2016-08-23
US20150167032A1 (en) 2015-06-18
CN102482649B (zh) 2014-09-03
WO2010151593A1 (en) 2010-12-29

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