DK3137483T3 - Fremgangsmåde til reducering af DNA-indholdet i en fermenteringsbouillon - Google Patents

Fremgangsmåde til reducering af DNA-indholdet i en fermenteringsbouillon Download PDF

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DK3137483T3
DK3137483T3 DK15722474.2T DK15722474T DK3137483T3 DK 3137483 T3 DK3137483 T3 DK 3137483T3 DK 15722474 T DK15722474 T DK 15722474T DK 3137483 T3 DK3137483 T3 DK 3137483T3
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bacillus
fermentation broth
minutes
dna
protein
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DK15722474.2T
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Cedric Hobel
Heidi Winterberg Andersen
Peter Frode Pind
Cecilia Jansson Kepka
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Novozymes As
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/08—Reducing the nucleic acid content
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • 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)
    • C12N9/82—Asparaginase (3.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • 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)
    • C12Y305/01001—Asparaginase (3.5.1.1)

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  • Wood Science & Technology (AREA)
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  • Microbiology (AREA)
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  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
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Claims (21)

1. Fremgangsmåde til at fjerne DNA fra en fermenteringsbouillon, der indeholder et protein af interesse og en mikroorganisme, der producerer proteinet af interesse, hvilken fremgangsmåde omfatter: a) opvarmning af fermenteringsbouillonen til mindst 70°C; b) tilsætning af polyaluminiumklorid til fermenteringsbouillonen, og c) separation af den flokkulerede mikroorganisme fra fermenteringsbouillonen.
2. Fremgangsmåde ifølge krav 1, hvor proteinet af interesse er et peptid, som f.eks. et antimikrobielt peptid, et lipopeptid eller brazzein; eller et polypeptid som f.eks. et enzym.
3. Fremgangsmåde ifølge krav 2, hvor proteinet af interesse er et enzym valgt blandt: hydrolaser, lyaser, proteaser, amylaser glucoamylaser, pectinaser, pectatlyaser, cellulaser, xylanaser, arabinaser, arbinofuranosidaser, mannanaser, carrageenanaser, xanthanaser, endoglucanaser, chitinaser, asparaginaser, lipaser, phospholipaser, cutinaser, lysozymer, phytaser, peroxidaser, lactase, glucoseisomeraser, xyloseisomeraser, esteraser og phosphodiesteraser.
4. Fremgangsmåde ifølge krav 3, hvor enzymet er et termostabilt enzym.
5. Fremgangsmåde ifølge krav 4, hvor det termostabile enzym har en termostabilitet, som ved 70°C og pH 7.0 viser en halveringstid på mindst 30 minutter, fortrinsvist mindst 40 minutter, fortrinsvist mindst 50 minutter; fortrinsvist mindst 60 minutter, fortrinsvist mindst 70 minutter, fortrinsvist mindst 80 minutter, fortrinsvist mindst 90 minutter, fortrinsvist mindst 100 minutter.
6. Fremgangsmåde ifølge krav 4 eller 5, hvor det termostabile enzym er en amylase eller en asparaginase.
7. Fremgangsmåde ifølge ethvert af de foregående krav, hvor mikroorganismen er en bakterie eller en svamp.
8. Fremgangsmåde ifølge krav 7, hvor mikroorganismen er en svamp valgt fra gruppen bestående af Trichoderma- og Aspergillus-værtsceWer, fortrinsvist fra Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride\, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus n/ger eller Aspergillus oryzae.
9. Fremgangsmåde ifølge krav 7, hvor mikroorganismen er en bakterie fra gruppen, der omfatter grampositive bakterier, som f.eks. en Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, eller Streptomyces, eller en gramnegativ bakterie, som f.eks. en Campylobacter, Escherichia, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, eller Ureaplasma, som f.eks. Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearotermophilus, Bacillus subtilis eller Bacillus thuringiensis.
10. Fremgangsmåde ifølge ethvert af de foregående krav, hvor fermenteringsbouillonens temperatur hæves til mindst 75°C, fortrinsvist mindst 80°C.
11. Fremgangsmåde ifølge ethvert af kravene 1 til 10, hvor fermenteringsbouillonens temperatur hæves til en temperature på mellem 70°C og 110°C.
12. Fremgangsmåde ifølge ethvert af kravene 1 til 11, hvor temperaturen holdes på en temperatur over 70°C i en periode på mindst 10 min., fortrinsvist mindst 20 min., fortrinsvist mindst 30 min., fortrinsvist mindst 40 min., fortrinsvist mindst 50 min., fortrinsvist mindst 60 min., fortrinsvist mindst 70 min., fortrinsvist mindst 80 min.
13. Fremgangsmåde ifølge ethvert af de foregående krav, hvor polyaluminumkloridet tilsættes i en mængde på 0,1-10 % (w/w) beregnet per kg fermenteringsbouillon.
14. Fremgangsmåde ifølge ethvert af de foregående krav, hvor et eller flere flokkuleringsmidler, fortrinsvist valgt fra gruppen bestående af salte og polymerer; tilsættes udover polyaluminumkloridet.
15. Fremgangsmåde ifølge krav 14, hvor polymeren er en anionisk eller en kationisk polymer.
16. Fremgangsmåde ifølge ethvert af de foregående krav, hvor separationen i trin c) udføres ved centrifugering eller filtrering.
17. Fremgangsmåde ifølge krav 1, hvor pH justeres til en pH i området pH 2 til pH 11 efter tilsætning af polyaluniniumkloridet.
18. Fremgangsmåde ifølge ethvert af de foregående krav, hvor DNA-niveauet reduceres til et niveau under 1 pg/ml, fortrinsvist under 500 ng/ml, fortrinsvist under 200 ng/ml, fortrinsvist under 100 ng/ml, fortrinsvist under 50 ng/ml, fortrinsvist under 20 ng/ml, fortrinsvist under 10 ng/ml, fortrinsvist under 5 ng/ml, fortrinsvist under 2 ng/ml, fortrinsvist under 1 ng/ml, fortrinsvist under 500 pg/ml.
19. Fremgangsmåde ifølge krav 18, hvor DNAet er under detektionsgrænsen.
20. Fremgangsmåde ifølge krav 19, hvor detektionsgrænsen bestemmes af en PCR-amplifikation af et hvilket som helst segment af genomisk DNA, der er tilstede i en enkelt kopi i et haploidt genom efterfulgt af gel-elektroforese og ethidiumbromid-farvning, hvor farvningen ikke viser nogen synlige bånd.
21. Fremgangsmåde ifølge ethvert af de foregående krav, hvor fermenteringsbouillonen kan være fortyndet med vand op til 2000% (w/w).
DK15722474.2T 2014-04-30 2015-04-30 Fremgangsmåde til reducering af DNA-indholdet i en fermenteringsbouillon DK3137483T3 (da)

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EP14166721 2014-04-30
PCT/EP2015/059497 WO2015166037A1 (en) 2014-04-30 2015-04-30 Method for reducing the dna content of a fermentation broth

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DK3137483T3 true DK3137483T3 (da) 2019-04-23

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US (1) US10259841B2 (da)
EP (1) EP3137483B1 (da)
CN (1) CN106255698B (da)
CA (1) CA2941536C (da)
DK (1) DK3137483T3 (da)
MX (1) MX2016012822A (da)
RU (1) RU2687153C2 (da)
WO (1) WO2015166037A1 (da)

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Publication number Priority date Publication date Assignee Title
CN109251872A (zh) * 2018-07-31 2019-01-22 北京林业大学 废弃物堆肥用复合菌剂、其制备方法及废弃物堆肥方法
CN111040966B (zh) * 2019-12-23 2022-06-24 河北科技大学 一种地衣芽孢杆菌KD-1、其生产的β-甘露聚糖酶及其应用
EP3926039A1 (en) 2020-06-17 2021-12-22 AB Enzymes GmbH Use of a nuclease for reducing the viscosity and/or preventing an increase in viscosity of a fermentation broth

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Publication number Priority date Publication date Assignee Title
DE60027406T2 (de) * 1999-01-25 2006-11-23 Novozymes A/S Rückgewinnung eines proteins bei hohem ph-wert
EP1567657A1 (en) * 2002-11-26 2005-08-31 Novo Nordisk A/S Process for purifying a fermentation-derived product
NZ540895A (en) 2003-01-09 2007-03-30 Genentech Inc Purification of polypeptides with ethacridine lactate
EP1685256A1 (en) * 2003-10-31 2006-08-02 Novozymes A/S Method for flocculation of a fermentation broth comprising a fungus
ATE462714T1 (de) * 2004-02-27 2010-04-15 Dow Global Technologies Inc Verfahren zur extraktion eines intrazellulären proteins aus einer fermentationsbrühe
EP2089422A1 (en) * 2006-11-27 2009-08-19 Natimmune A/S Methods of separating nucleic acids and protein
JP6105590B2 (ja) * 2011-09-22 2017-03-29 ダニスコ・ユーエス・インク Ｄｎａ含有量を減少させる内因性ｄｎアーゼ活性
US9249432B2 (en) * 2012-07-13 2016-02-02 Alliance For Sustainable Energy, Llc Enzymes for improved biomass conversion

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US10259841B2 (en) 2019-04-16
RU2016138692A3 (da) 2018-10-11
RU2016138692A (ru) 2018-05-30
MX2016012822A (es) 2016-12-09
US20170044209A1 (en) 2017-02-16
WO2015166037A1 (en) 2015-11-05
CA2941536A1 (en) 2015-11-05
CN106255698B (zh) 2021-08-03
RU2687153C2 (ru) 2019-05-07
CN106255698A (zh) 2016-12-21
CA2941536C (en) 2023-01-17
EP3137483B1 (en) 2019-01-16
EP3137483A1 (en) 2017-03-08

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