DE4238842A1 - Enzymes prodn. by fermentation of Ciliates - in high cell density fermentation using medium contg. particulate nutrients - Google Patents

Enzymes prodn. by fermentation of Ciliates - in high cell density fermentation using medium contg. particulate nutrients

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Publication number
DE4238842A1
DE4238842A1 DE19924238842 DE4238842A DE4238842A1 DE 4238842 A1 DE4238842 A1 DE 4238842A1 DE 19924238842 DE19924238842 DE 19924238842 DE 4238842 A DE4238842 A DE 4238842A DE 4238842 A1 DE4238842 A1 DE 4238842A1
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prodn
ciliates
medium
cells
useful
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DE19924238842
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German (de)
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Arno Prof Dr Tiedtke
Thomas Dipl Biol Kiy
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • 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
    • C12N1/10—Protozoa; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine

Abstract

Process for high cell density fermentation of ciliates (species : Tetrahymena, Colpidium) in axenic medium, esp. for recovery of secreted natural substances, whereby the Ciliates are supplied with a cheap nutrient medium that contains the nutrient substances in predominantly particulate form, while the spent nutrient medium, enriched with useful natural substances, is recovered via a polypropylene membrane system with simultaneous retention of the cells in the fermentation vessel. USE/ADVANTAGE - The process is useful for technical scale prodn. of enzymes (useful in diagnostic procedures, food technology, analysis and molecular biology), melanin (which due to its UV absorbing characteristics is useful in the prodn. of sun protection creams) and extrusions. The process gives rapid cell multiplication to cell densities of over 2 x 107 cells/ml (corresponding to a dry mass of 50 g/l) to give high rates of prodn. of useful natural prods., and the same cells can be used over long periods of continuous prodn. without use of complex methods for separating the cells from the medium.

Description

Es ist bekannt, daß einige Ciliatenspezies, wie etwa Tetrahymena, bestimmte Enzyme in das umgebende Medium sezernieren (Müller, 1972). Die sezernierten Enzyme wurden bisher nur im Labormaßstab gewonnen, wobei einfache batch-Fermentationen durchgeführt wurden. Dabei werden z. B. 500-ml-Erlenmeyerkolben, die 130 ml frisches Medium enthalten, mit Zellen angeimpft (Blum, 1975). Nach dem Erreichen der Stationärphase werden die Zellen durch Zentrifugationsschritte von dem enzymhaltigen Medium abgetrennt. Bisher wurden Zuchtmedien verwendet, die sich z. B. aus Proteose Pepton und Hefe Extrakt oder Leber Extrakt zusammensetzen und die Nährstoffe in überwiegend gelöster Form enthalten. Bei dem beschriebenem Verfahren liegt die maximale Zellkonzentration unter 10⁶ Zellen/ml (Rasmussen & Modeweg-Hansen, 1973) und die Generationszeit bei über 2 h, wenn etwa Tetrahymena thermophila fermentiert wird. Die maximalen Enzymausbeuten betragen z. B. 100 mU β- Hexosaminidase und saure Phosphatase pro ml Medium. Obwohl man weiß, daß Ciliaten wie z. B. Tefrahymena ein besseres Wachstum aufweisen, wenn die Nährstoffe in partikulärer statt gelöster Form vorliegen (Rasmussen & Modeweg-Hansen, 1973), wurde solch ein axenisches Zuchtmedium bisher nicht beschrieben.It is known that some ciliate species, such as Tetrahymena, have certain enzymes in them secrete the surrounding medium (Müller, 1972). The secreted enzymes were previously only obtained on a laboratory scale, with simple batch fermentations were carried out. Here, for. B. 500 ml Erlenmeyer flask containing 130 ml fresh Contain medium, inoculated with cells (Blum, 1975). After reaching the The cells become stationary phase by centrifugation steps from the enzyme-containing Medium separated. So far, cultivation media have been used which, for. B. from proteose Peptone and yeast extract or liver extract and put the nutrients in contain mostly dissolved form. In the described method lies the maximum cell concentration below 10⁶ cells / ml (Rasmussen & Modeweg-Hansen, 1973) and the generation time at over 2 h, if about Tetrahymena thermophila is fermented. The maximum enzyme yields are e.g. B. 100 mU β- Hexosaminidase and acid phosphatase per ml of medium. Although you know that Ciliates such as B. Tefrahymena have better growth if the nutrients are in particulate rather than dissolved form (Rasmussen & Modeweg-Hansen, 1973), such an axenic breeding medium has not previously been described.

Der im Patentanspruch 1 angegebenen Erfindung liegt das Problem zugrunde, daß die Ciliaten sich bei den bisher verwendeten Zuchtverfahren und -medien relativ schlecht vermehren ließen, woraus niedrige Ausbeuten bezüglich der Biomasse und wertvoller sezernierter Naturstoffe wie Enzyme, Melanine und Extrusomen resultierten. Weiterhin mußten die Zellen bisher in arbeitsaufwendigen Zentrifugations- bzw. Filtrationsschritten vom enzymhaltigen Kulturmedium abgetrennt werden.The invention specified in claim 1 is based on the problem that the Ciliates are relatively poor in the breeding methods and media used to date increased, resulting in low yields in terms of biomass and more valuable secreted natural products such as enzymes, melanins and extrusomes resulted. Farther Up to now, the cells had to be laboriously centrifuged or Filtration steps are separated from the enzyme-containing culture medium.

Dieses Problem wird durch die im Patentanspruch 1 aufgeführten Merkmale gelöst.This problem is solved by the features listed in claim 1.

Die mit der Erfindung erzielten Vorteile bestehen insbesondere darin, daß Ciliaten der Gattungen Tetrahymena und Colpidium sich sehr schnell vermehren (Generationszeit: 1,4 h) und Zelldichten von über 2×107 Zellen/ml (entspricht einer Trockenmasse von etwa 50 g/l) erreichen, wodurch eine enorm hohe Produktivität bezüglich der sezernierten Substanzen gegeben ist. Der kontinuierliche Austausch des Kulturmediums über ein Membransystem, das die Zellrückhaltung im Fermenter gewährleistet, ermöglicht es kontinuierlich über Monate große Mengen an Enzymen, Melaninen und Extrusomen zu gewinnen. Weitere Vorteile sind, daß dieselben Zellen über einen langen Zeitraum zur Produktion genutzt werden können, und daß aufwendige Arbeitsschritte zur Abtrennung des Mediums von den Zellen entfallen. Das gute Wachstum (und somit die hohen Ausbeuten) wird durch den Einsatz eines Kulturmediums, das im Gegensatz zu den bisher üblichen axenischen Medien die Nährsubstanzen überwiegend in partikulärer Form enthält, ermöglicht. Dies wird erreicht durch die Verwendung von Magermilchpulver als Nährsubstrat. Durch Autoklavieren des Mediums wird bewirkt, daß das Milcheiweiß koaguliert und aus der löslichen in die partikuläre Form übergeht. Dieses Kulturmedium enthält im wesentlichen Magermilchpulver und kostet somit nur Bruchteile des bislang verwendeten PPYS Mediums, was als Vorteil für eine kommerzielle Nutzung des Systems gesehen werden muß.The advantages achieved by the invention are in particular that ciliates of the genera Tetrahymena and Colpidium multiply very quickly (generation time: 1.4 h) and cell densities of over 2 × 10 7 cells / ml (corresponds to a dry matter of about 50 g / l ) achieve, which gives an enormously high productivity regarding the secreted substances. The continuous exchange of the culture medium via a membrane system, which ensures cell retention in the fermenter, enables large quantities of enzymes, melanins and extrusomes to be obtained continuously for months. Further advantages are that the same cells can be used for production over a long period of time and that complex work steps for separating the medium from the cells are eliminated. The good growth (and thus the high yields) is made possible by the use of a culture medium which, in contrast to the axenic media previously used, mainly contains the nutrient substances in particulate form. This is achieved through the use of skimmed milk powder as a nutrient substrate. Autoclaving the medium causes the milk protein to coagulate and change from the soluble to the particulate form. This culture medium essentially contains skimmed milk powder and thus only costs a fraction of the previously used PPYS medium, which must be seen as an advantage for commercial use of the system.

Ein Ausführungsbeispiel der Erfindung ist dem beigelegten Manuskript einer Publikation, die am 19.11.92 erscheinen soll, zu entnehmen.An embodiment of the invention is the enclosed manuscript Publication to be published on November 19, 1992.

Literaturliterature

Blum, J. J. (1975) Effects of Metabolites Present During Growth of Tetrahymena pyriformis on the Subsequent Secretion of Lysosomal Hydrolases. J. Cell. Physiol. 86: 131-142.Blum, J.J. (1975) Effects of Metabolites Present During Growth of Tetrahymena pyriformis on the Subsequent Secretion of Lysosomal Hydrolases. J. Cell. Physiol. 86: 131-142.

Müller, M. (1972) Secretion of Acid Hydrolases and its Intracellular Source in Tetrahymena pyriformis. J. Cell Biol. 52 : 478-487.Müller, M. (1972) Secretion of Acid Hydrolases and its Intracellular Source in Tetrahymena pyriformis. J. Cell Biol. 52: 478-487.

Rasmussen, L. and Modeweg-Hansen, L. (1973) Cell multiplication in Tetrahymena cultures after addition of particulate material. J. Cell Sci. 12 : 275-286.Rasmussen, L. and Modeweg-Hansen, L. (1973) Cell multiplication in Tetrahymena cultures after addition of particulate material. J. Cell Sci. 12: 275-286.

AusbeutenExploit

1 ml zellfreier Überstand aus unserem Verfahren enthält ca. 25000 mU saure Phosphatase und 14000 mU β-Hexosaminidase, während 1 ml zellfreier Überstand aus herkömmlichen batch-Verfahren nur etwa 100 mU beider Enzyme enthält. Ähnliche Verhältnisse findet man bei allen weiteren Enzymen. Für 50000 mU β-Hexosaminidase (aus Rinderniere) der Fa. Boehringer Mannheim zahlt man 274,- DM und für 60000 mU saure Phosphatase 70,- DM. Weiterhin lassen sich mit unserem System große Mengen PDE I, die heute noch aus Schlangengiften gewonnen wird, PDE II, Protease, alpha-Mannosidase, alpha-Glucosidase, β-Glucosidase, Phospholipase C, Phospholipase A1 u. a. Enzyme produzieren (siehe auch Abb. 1). Durch den Einsatz geeigneter Mutanten konnten außerdem große Mengen Melanin gewonnen werden.1 ml of cell-free supernatant from our process contains approx. 25000 mU of acid phosphatase and 14000 mU of β-hexosaminidase, while 1 ml of cell-free supernatant from conventional batch processes only contains around 100 mU of both enzymes. Similar relationships can be found with all other enzymes. You pay 274 DM for 50,000 mU β-hexosaminidase (from beef kidney) from Boehringer Mannheim and 70 DM for 60,000 mU acidic phosphatase , PDE II, protease, alpha-mannosidase, alpha-glucosidase, β-glucosidase, phospholipase C, phospholipase A 1 and others produce enzymes (see also Fig. 1). Large amounts of melanin could also be obtained by using suitable mutants.

AnwendungsbeispieleExamples of use

Die genannten Enzyme sind von kommerzieller Bedeutung und finden Verwendung in Bereichen der Diagnostik, Lebensmittelchemie, Analytik und Molekularbiologie. Da geeignetere Quellen bisher nicht zur Verfügung standen, wird die PDE I z. B. noch aus Schlangengift, die PDE II aus Kalbsmilz, die β-Hexosaminidase aus Rinderniere und die β-Glucosidase aus Süßmandeln gewonnen. Melanine werden aufgrund ihrer Eigenschaft, UV Strahlen zu absorbieren, zur Herstellung von Sonnenschutzcremes eingesetzt.The enzymes mentioned are of commercial importance and are used in Areas of diagnostics, food chemistry, analytics and molecular biology. There More suitable sources have not yet been available, the PDE I z. B. still out Snake venom, the PDE II from calf spleen, the β-hexosaminidase from bovine kidney and the β-glucosidase obtained from sweet almonds. Melanins are made because of their Property to absorb UV rays for the production of sun protection creams used.

Claims (1)

Verfahren zur Hochzelldichte-Fermentation von Ciliaten (Gattungen: Tetrahymena, Colpidium) auf axenischem Medium insbesondere zur Gewinnung von sezernierten Naturstoffen dadurch gekennzeichnet, daß die Ciliaten kontinuierlich mit einem billigen Nährmedium versorgt werden, das die Nährstoffe in überwiegend partikulärer Form enthält, während das mit wertvollen Naturstoffen angereicherte, verbrauchte Kulturmedium, bei gleichzeitiger Rückhaltung der Zellen im Zuchtgefäß, über ein Polypropylenmembransystem geerntet wird.Process for high-cell density fermentation of ciliates (genera: Tetrahymena, Colpidium) on axenic medium, in particular for the production of secreted natural products, characterized in that the ciliates are continuously supplied with an inexpensive nutrient medium which contains the nutrients in a predominantly particulate form, while the valuable ones Consumed culture medium enriched with natural substances is harvested via a polypropylene membrane system while the cells are retained in the culture vessel.
DE19924238842 1992-11-17 1992-11-17 Enzymes prodn. by fermentation of Ciliates - in high cell density fermentation using medium contg. particulate nutrients Withdrawn DE4238842A1 (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0708176A2 (en) * 1994-10-21 1996-04-24 Hoechst Aktiengesellschaft Process for the cultivation of lipid-dependent tetrohymenids and a process for the production of biogenic products of lipid-dependent tetrahymenids
EP0725144A1 (en) * 1995-02-06 1996-08-07 Hoechst Aktiengesellschaft Process for the preparation of glycosides using glycosidases from ciliates
DE19626564A1 (en) * 1996-07-03 1998-01-08 Hoechst Ag Genetic transformation of ciliate cells by microcarrier bombardment with DNA-loaded gold particles
WO1999015634A1 (en) * 1997-09-19 1999-04-01 Aventis Research & Technologies Gmbh & Co. Kg Fermentation method with continuous mass cultivation of ciliates (protozoa) for producing biogenous valuable substances
EP1024199A2 (en) * 1999-01-27 2000-08-02 Axiva GmbH Recovery of gamma-linolenic acid from protozoa of the genus Colpidium
US6391351B2 (en) 1999-09-13 2002-05-21 German A. Valcarce Methods for treating foodstuffs using cell free extracts from ciliates
WO2005099748A1 (en) * 2004-04-13 2005-10-27 Cilian Ag Recombinant lysosomal enzymes comprising a glycosylation pattern typical of ciliates for treatment

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0708176A2 (en) * 1994-10-21 1996-04-24 Hoechst Aktiengesellschaft Process for the cultivation of lipid-dependent tetrohymenids and a process for the production of biogenic products of lipid-dependent tetrahymenids
EP0708176A3 (en) * 1994-10-21 1998-09-16 Hoechst Aktiengesellschaft Process for the cultivation of lipid-dependent tetrohymenids and a process for the production of biogenic products of lipid-dependent tetrahymenids
EP0725144A1 (en) * 1995-02-06 1996-08-07 Hoechst Aktiengesellschaft Process for the preparation of glycosides using glycosidases from ciliates
DE19626564A1 (en) * 1996-07-03 1998-01-08 Hoechst Ag Genetic transformation of ciliate cells by microcarrier bombardment with DNA-loaded gold particles
WO1999015634A1 (en) * 1997-09-19 1999-04-01 Aventis Research & Technologies Gmbh & Co. Kg Fermentation method with continuous mass cultivation of ciliates (protozoa) for producing biogenous valuable substances
US6716617B1 (en) * 1997-09-19 2004-04-06 Nutrinova Fermentation method with continuous mass cultivation of ciliates (protozoa) for producing biogenous valuable substances
EP1024199A2 (en) * 1999-01-27 2000-08-02 Axiva GmbH Recovery of gamma-linolenic acid from protozoa of the genus Colpidium
EP1024199A3 (en) * 1999-01-27 2002-03-06 Celanese Ventures GmbH Recovery of gamma-linolenic acid from protozoa of the genus Colpidium
US6391351B2 (en) 1999-09-13 2002-05-21 German A. Valcarce Methods for treating foodstuffs using cell free extracts from ciliates
WO2005099748A1 (en) * 2004-04-13 2005-10-27 Cilian Ag Recombinant lysosomal enzymes comprising a glycosylation pattern typical of ciliates for treatment

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