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Gluconobacter oxydans 2-ketoreductase enzyme and applications thereof

a technology of gluconobacter oxydans and ketoneductase, applied in the field of 2ketoneductase, can solve the problem that the large-scale synthesis of s-(+)-2-pentanol requires a large cell mass

Inactive Publication Date: 2006-12-28
NANDURI VENKATA +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a novel 2-ketoreductase isolated from the bacterium, Gluconobacter oxydans, and variants, modifications, and fragments thereof. The invention also provides isolated polynucleotides, nucleic acid probes and primers, vectors, host cells, and recombinant enzyme. The invention also provides methods of using the enzyme in enzymatic reactions requiring the synthesis of chiral alcohols, as well as methods of purifying the enzyme. The technical effects of the invention include improved methods for producing and purifying a novel 2-ketoreductase with improved activity and selectivity.

Problems solved by technology

However, large-scale synthesis of S-(+)-2-pentanol requires a large cell mass, i.e., a ratio of 2-pentanone to cell mass of 1 kg:50 kg.

Method used

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  • Gluconobacter oxydans 2-ketoreductase enzyme and applications thereof
  • Gluconobacter oxydans 2-ketoreductase enzyme and applications thereof
  • Gluconobacter oxydans 2-ketoreductase enzyme and applications thereof

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embodiments

[0121] This invention encompasses, but is not limited to, the following embodiments: [0122] An isolated nucleic acid comprising a nucleotide sequence encoding amino acid sequence SEQ ID NO:2. [0123] An isolated nucleic acid comprising a nucleotide sequence encoding at least 12 contiguous residues of amino acid sequence SEQ ID NO:2. [0124] An isolated nucleic acid comprising a nucleotide sequence encoding at least 12 contiguous residues of the short chain dehydrogenase domain of amino acid sequence SEQ ID NO:2. [0125] An isolated nucleic acid comprising nucleotide sequence SEQ ID NO:1. [0126] An isolated nucleic acid comprising at least 21 contiguous nucleotides of nucleotide sequence SEQ ID NO:1. [0127] An isolated nucleic acid comprising a nucleotide sequence which is at least 54% identical to nucleotide sequence SEQ ID NO:1. [0128] An isolated nucleic acid comprising a nucleotide sequence which is complementary to a nucleotide sequence of the invention (above). [0129] A vector com...

example 1

Purification of G. Oxydans 2-Ketoreductase

[0183] Fermentation: Gluconobacter oxydans (SC13851) was grown on a glycerol-containing medium as follows. Cultures were grown in 500 ml Erlenmeyer flasks for 24 hr in 100 ml medium A (5% glycerol, 0.5% yeast extract, 0.05% ammonium sulfate, 0.3% peptone, 0.05% K2HPO4, 0.02% MgSO4.7H2O, 0.001% NaCl, 0.001% FeSO4.7H2O, and 0.001% MnSO4.7H2O). After 24 hr, the flask cultures were used to inoculate (1% v / v inoculum) a 15 L fermentor containing medium A. The fermentation was carried out at 28° C. for 24 hr. A 4000 L fermentor (Expend Industries, Inc., Brooklyn, N.Y.) was inoculated with 10 L inoculum from the 15 L fermentor. The 4000 L fermentor contained medium A with 0.05% antifoam SAG 5693. The fermentor was operated at 28° C., 100 LPM airflow, 690 mbar pressure, and 620 rpm agitation for 48 hr.

[0184] Cell recovery: The fermentor broth was cooled to 8° C. at the harvest. The tank was pressurized to 15 psig and broth was diverted to a Sharpl...

example 2

Analysis of Purified G. Oxydans 2-Ketoreductase

[0189] Protein assay: The Bio-Rad protein assay was used to determine protein concentration. The assay was performed according to the manufacturer's protocol (Bio-Rad). Samples containing 1-10 μl of enzyme fraction were brought to a volume of 0.8 ml with water. Next, 0.2 ml of the Bio-Rad reagent was added to the 0.8 ml sample solution. This was mixed thoroughly. The absorbance of the solution was measured at 595 nm. The protein concentration (mg / ml) was calculated from the standard curve using bovine serum albumin as standard protein.

[0190] Enzyme activity units: One unit (U) of enzyme activity was defined as one micromole of S-2-pentanol formed in 1 hr under the conditions described above. Results from the protein analysis of G. oxydans 2-ketoreductase are summarized below.

TABLE 1Enzyme ActivitySp. ActivityS-2-PentanolStepsVolume (mL)(Units)Protein (mg)(Units / mg)(e e)Purification FoldCell extract300390.00729.000.501.00DEAE Cellulo...

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Abstract

This invention relates to an novel Gluconobacter oxydans 2-ketoreductase useful for the synthesis of chiral alcohols. Also related are isolated nucleic acids encoding G. oxydans 2-ketoreductase enzymes, and enzyme fragments and variants thereof, as well as vectors and host cells comprising these nucleic acids. Further related are isolated G. oxydans 2-ketoreductase polypeptides, and fragments and variants thereof, and antibodies that specifically bind to G. oxydans 2-ketoreductase polypeptides, fragments, or variants. The invention also relates to methods of obtaining isolated G. oxydans 2-ketoreductase nucleic acids, polypeptides, and antibodies, and methods of using G. oxydans 2-ketoreductase in various reactions for industrial or pharmaceutical applications.

Description

[0001] This invention claims priority from provisional U.S. application Ser. No. 60 / 341,933 filed Dec. 19, 2001, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] This invention relates to a novel 2-ketoreductase isolated from Gluconobacter oxydans which catalyzes the reduction of 2-pentanone to S-(+)-2-pentanol. The invention also relates to isolated nucleic acids comprising nucleotide sequences that encode G. oxydans 2-ketoreductase polypeptides. Also related are vectors and host cells comprising these nucleic acids, isolated G. oxydans 2-ketoreductase polypeptides (e.g., recombinant polypeptides), and antibodies that specifically bind to G. oxydans 2-ketoreductase polypeptides. The invention further relates to methods of obtaining isolated G. oxydans 2-ketoreductase nucleic acids, polypeptides, and antibodies, and methods of using G. oxydans 2-ketoreductase in reactions required for the synthesis of industrial or pharmaceutical compounds. BAC...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P7/02C12P21/06C07H21/04C12N1/21C12N15/74C12N9/02C12Q1/26C12Q1/32
CPCC12N9/0004G01N2333/04C12Q1/32C12Q1/26
Inventor NANDURI, VENKATAJOHNSTON, ROBERTGOLDBERG, STEVENCINO, PAULPATEL, RAMESH
Owner NANDURI VENKATA
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