DD266710A3 - A process for the biotechnical preparation van alkaline phosphatase - Google Patents

A process for the biotechnical preparation van alkaline phosphatase Download PDF

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Publication number
DD266710A3
DD266710A3 DD25178483A DD25178483A DD266710A3 DD 266710 A3 DD266710 A3 DD 266710A3 DD 25178483 A DD25178483 A DD 25178483A DD 25178483 A DD25178483 A DD 25178483A DD 266710 A3 DD266710 A3 DD 266710A3
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alkaline phosphatase
process
invention
culture
alkaline
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DD25178483A
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German (de)
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Ulrich Dipl Biol Korn
Brigitte Dipl Biol Lebentrau
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Ve Forschungszentrum Biotechnologie
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Abstract

Die Erfindung betrifft ein Verfahren zur biotechnischen Herstellung von alkalischer Phosphatase mittels Bacillus licheniformis. The invention relates to a process for the biotechnological production of alkaline phosphatase by Bacillus licheniformis. Ziel der Erfindung ist die Entwicklung eines technischen Verfahrens zur Herstellung von alkalischer Phosphatase. The aim of the invention is to develop an industrial process for the production of alkaline phosphatase. Das Ziel der Erfindung wird durch die Verwendung des Stammes Bacillus licheniformis 41p - ZIMET 10 911 - erreicht. The object of the invention is the use of the strain Bacillus licheniformis 41P - reached - ZIMET 10911. Die C-, N- und P-Quellen des Naehrmediums werden so kombiniert, dass durch die metabolische Wirkung der Kultur in der Phase der intensiven alkalische Phosphatase-Akkumulation der Glucosegehalt des Naehrmediums 0%, der Phosphatgehalt 7 mM und die NH4-Konzentration nicht mehr als 20 bis 30 mg/ml betraegt. The C, N and P sources of the nutrient medium are combined so that, by the metabolic action of the culture in the phase of intensive alkaline phosphatase accumulation the glucose content of the nutrient medium 0%, the phosphate content of 7 mM and the NH4-concentration is not more AMOUNT as 20 to 30 mg / ml. Optimierungen des Verfahrens koennen durch Glucosefeeding in der Uebergangsphase der Kultur, durch intensive Durchmischung des Fermentationsmediums und Einhaltung einer bestimmten Fermentationstemperatur erzielt werden. Optimization of the process can by Glucosefeeding in the transition phase of the culture, are achieved by intensive mixing of the fermentation medium and maintaining a certain fermentation temperature.

Description

-7- -7-

Titel der Erfindung Title of the invention

Verfahren zur bioteohnisohen Herstellung von alkalischer Phosphat ase A process for the preparation of alkaline phosphate bioteohnisohen ase

Anwendungsgebiet der Erfindung Field of the Invention

Die Erfindung betrifft ein Verführen zur bioteohniaohen Herstellung von alkalischer Phosphatasθ mit einem Bacillus lichen if or mis-Stamm. The invention relates to a seduction for bioteohniaohen preparation of alkaline Bacillus Phosphatasθ with a union or if mis strain. Das Enzym kenn in der klinischen Diagnostik sowie in der bioohemisohen und molokularbiologisohen Forschung eingesetzt werden. The enzyme label can be used in clinical diagnostics and in the bioohemisohen and molokularbiologisohen research.

Charakterisierung der bekannten technischen Lösungen Characterization of the known technical solutions

Alkalische Fhosphatase wird von einer Vielzahl von Mikroorganismen synthetisiert. Alkaline Fhosphatase is synthesized by a variety of microorganisms. Unter den Bakterien eind zwar mehrere Spezies des Genus Baoillus dazu befähigt, so z. Among the bacteria Although several species of the genus Baoillus eind capable as such. B. Bacillus subtilis, Bacillus oereus und Baoillus licheniformis (JG HANSA et al. 1981, Bioohim. Biophys. Aota £5£, 390 bis 401; O. HYDRKAN et al. 1977, J-. Biol. Chem. 2£2>, 6806; WB CHBSBHO & JO LAMPEN 1968, Baoteriol. 2£, 428 bis 437), aber die biotechnische Herstellung von alkalisoher Phosphatase mit Baoillen ist bisher nooh nicht bekannt. B. Bacillus subtilis, Bacillus oereus and Aota £ £ 5 Baoillus licheniformis (JG HANSA et al 1981 Bioohim Biophys, 390 to 401;.... HYDRKAN O. et al 1977 J Biol Chem 2 £ 2>... , 6806;. & JO WB CHBSBHO LAMPS 1968 Baoteriol 2 £, 428-437), but the biotechnological production of alkalisoher phosphatase with Baoillen is previously Nooh not known.

Ss existieren Verfahren zur Herstellung von alkalischer Phosphatase mit Hilfe von Mikroorganismen anderer Gattungen, so ua Aotinomyoes ooelioolor A-66 (SU-UR 943 279)» Actinomyces streptomycin (SU-UR 178 337) sowie mit E. coli und Serratia maroesoens, die neukombinierte Plasmide mit Genen für alkalische Phosphatase enthalten (DB-OS 2 931 999). Ss methods exist for the production of alkaline phosphatase by means of microorganisms of other genera, so among others Aotinomyoes ooelioolor A-66 (SU-UR 943 279) "Actinomyces streptomycin (SU-UR 178 337) as well as E. coli and Serratia maroesoens, the new combined plasmids containing genes for alkaline phosphatase include (DB-OS 2,931,999).

*** C-, mm *** C, mm

Die bekannten Verfahren - bis auf die, bei denen klonierte Mikroorganismen verwendet werden - bringen nur geringe Enzymausbeuten, so daß zur Zeit noch vorrangig die alkalische Phosphatase aus Kalb er darm gewonnen wird. The known methods - up to the are where cloned microorganisms used - bringing only low enzyme yields, so that it is obtained primarily bowel at the time nor the alkaline phosphatase from calf.

Ziel der Erfindung OBJECT OF THE INVENTION

Zif>l der Erfindung ist es, ein Verfahren zur teohnisohen Gewinnung von alkalisoher Phosphatase mittels Baoillus lioheniformis zu entwickeln. Zif> l of the invention is to develop a process for recovery of teohnisohen alkalisoher phosphatase means Baoillus lioheniformis.

Darstellung des Wesens der Erfindung Representation of the nature of the invention

Der Erfindung liegt die Aufgabe zugrunde, mit einem besonderen Bacillus lioheniformis-Stamm eine alkalische Phoaphatase in hoher Ausbeute zu produzieren. The invention has for its object to produce a specific Bacillus strain lioheniformis an alkaline Phoaphatase in high yield.

Erfindungsgemäß wird für die Herstellung der alkalischen Phosphatase Baoillus lioheniformis 41p - ZJMET 10 911 - verwendet, der sich vom Typstamm Bacillus lioheniformis ATCC 14 580 in mehreren Positionen unterscheidet (vgl. dazu nachfolgende Charakteristik mit BERGET's Manual of Determinative Bacteriology, 8 th ed. (1974)). ZJMET 10 911 - - According to the invention for the production of alkaline phosphatase Baoillus lioheniformis 41p used which differs from the type strain of Bacillus lioheniformis ATCC 14580 in a plurality of positions (see to subsequent characteristic with BERGET's Manual of Determinative Bacteriology, 8 th ed (1974.. )).

Charakteristik des Baoillus lioheniformis 41p - ZIMET 10 Characteristic Baoillus lioheniformis 41p - ZIMET 10

Morphologie morphology

Form der Spore: oval The form of the spore: oval

Lage der Spore: zentral Location of the Spore: central

Sporulationsverhalten: 95 % (Gerstenschrotaßav 10 d/ Sporulationsverhalten: 95% (Gerstenschrotaßav 10 d /

37 0 C) 37 0 C)

Beweglichkeit: beweglioh Agility: beweglioh

Gr amv er halten: grampositive Kurz stäbe heu Gr he AMV hold: gram positive short rods hay

Koloniemorphologie auf unterschiedlichen Nährböden Colony morphology on different culture media

Bluplatte: Kolonie hellbeige» milchig, matb- Bluplatte: Colony light beige "milky, matb-

nach 48 h/37 0 C after 48 h / 37 C 0

Nähragar (NA): Nutrient Agar (NA):

NA + Glue + Cas.-Ao id: NA + Glue + Cas.-Ao id:

Biochemische Merkmale Säurebildung aus: Biochemical characteristics of acid from:

Kolonie hellbeige, milohig, mattglänzend, gelappt Kolonie maude/roeö, mattglän· aend, Färbstoffbildung Colony light beige, milohig, matte, lobed colony maude / roeö, mattglän · aend, Färbstoffbildung

-Xylose, Gluoose, d-Mannose, d-Fruotose, Maltose, Trehalose, Saccharose, Raffinose, Rhamnose, D-Mannit» d-Sorbit, D-Ribose, Aesoulin -Xylose, Gluoose, d-mannose, d-Fruotose, maltose, trehalose, sucrose, raffinose, rhamnose, D-mannitol "d-sorbitol, D-ribose, Aesoulin

L-Arabinose naoh 2^ h, d-Galactose L-arabinose NaOH 2 ^ h, d-galactose

keine Säurebildung aus: no acid formation from:

Indolnaohweiss Indolnaohweiss

VPRs VPRS

C it r atv er w er imng: C r it atv he w he imng:

Ureasenaohweis.t Ureasenaohweis.t

Arginind.xhydr olase t Arginind.xhydr olase t

Waohstumsverhalten und bioohemisohe Merkmale Waohstumsverhalten and bioohemisohe Features

Wachstum in Nährbouillon: Growth in nutrient broth:

Waohstum auf Gluoose-Nitrat-Agar: Waohstum on Gluoose nitrate agar:

Waohstum auf Gluoose-Asp ar ag in-Ag art Waohstum on Gluoose-Asp ar ag-Ag art

Waohstum in NaCl-Bouillons Waohstum in NaCl bouillons

Gasbildung aus Nitrat unter anaeroben Bedingungen: Nitratreduktion: Katalase: Stärkehydrolyse: Caseinhydrolyse: Gelatinohydrolyse: Lezithinase: Gas formation from nitrate under anaerobic conditions: Nitrate reduction: catalase: starch hydrolysis: casein hydrolysis: Gelatinohydrolyse: Lezithinase:

Hautbildung, Trübung, Bodensatz starkes Waohstum Skinning, turbidity, sediment strong Waohstum

sohlechtes Waohstum sohlechtes Waohstum

3 - 7 % NaCl gutes Wachstum 10 % NaCl schwaches Wachstum 3-7% NaCl good growth of 10% NaCl weak growth

HZ HZ 2 2 mm mm
HZ HZ 1 1 mm mm
HZ HZ mm mm

HZ ss Hydrolysezone HZ ss hydrolysis

Die Herstellung des Enzyms kann im Submersv erfahren in zwangsbelüfteten, sterilisierbaren Fermentationstanks unter üblichen bioteohnologisohen Bedingungen erfolgen. The production of the enzyme can learn in Submersv made in forced air, sterilizable fermentation tanks under customary bioteohnologisohen conditions. Es wurde Jedoch gefunden, daß den Eigenschaften des Stammes angepaßte Verfahrenobedingungen zu höheren Enzymausbeuten führen. It was found, however, that lead to the properties of the strain adapted Verfahrenobedingungen to higher enzyme yields.

In Nährmedien mit für bakterielle Verfahren häufig verwendeten C-Quellen, wie Glucose, Maltose, Mais- bzw. Kartoffelstärke und Getreideschroten, und übHohen N-Quellen, wie Sojaprotein, Casein, Mager milchpulver, Erbsen- und Bohnenmehl sowie Gelatine, bildet der erfindungsgemäße Stamm in ökonomisch interessanten Mengen a^alisohe Phosphatase. In culture media with commonly used for bacterial Method C-sources, such as glucose, maltose, corn or potato starch and grain crushing and übHohen N sources such as soy protein, casein, skim milk powder, pea and bean flour and gelatin, the strain according to the invention alisohe in economically interesting amounts of a ^ phosphatase.

Bei einer bevorzugten Ausführungsform des Verfahrens zur bloteohnisohen Herstellung von alkalischer Phosphatase mit Bacillus lioheniformis 41p - ZIMET 10 911 - werden die C- und N-Quellen des Nährmediums so kombiniert, daß durch die cietabolisohe Wirkung der Kultur in der Phase der Intensiven alkalische Phosphatase-Akkumulation dor Glucosegehalt des Nährmediums O %, der Phosphatgehalt < 7 mM und die NH^-Konzentration nicht mehr als 20 bis 3OyUgZmI beträgt. In a preferred embodiment of the method for bloteohnisohen production of alkaline phosphatase by Bacillus lioheniformis 41p - ZIMET 10 911 - the C and N sources of the nutrient medium are combined so that by cietabolisohe effect of culture in the phase of intensive alkaline phosphatase accumulation dor glucose content of the nutrient medium O%, the phosphate content of not more than 20 to 3OyUgZmI is <7 mM and NH ^ concentration. Die Akkumulation der alkalischen Phosphatase wird durch ein GIuoosefeeding in der Übergangsphase und durch intensives Durchmischen des Fermentationsmediums gesteigert. The accumulation of alkaline phosphatase is increased by a GIuoosefeeding in the transition phase, and by intensive mixing of the fermentation medium. Die Permentationstemperatur beträgt 34 bis 40 0 C, insbesondere 37 0 C The Permentationstemperatur is 34 to 40 0 C, in particular 37 0 C

Bacillus licheriformis 41p - ZIMET 10 911 - synthetisiert alkalische Phosphatase, die zu über 90 % an Partikel bzw. Membranbruchstücke assoziiert ist. Bacillus licheriformis 41p - ZIMET 10 911 - synthesized alkaline phosphatase, which is associated to more than 90% of particulate or membrane fragments. Das Enzym kann in bekannter Welse, z. The enzyme can be in a known catfish, z. B. duroh MgCIg, von den Membranbruchstüoken gelöst und anschließend gereinigt werden (FM* HULETT-COWLING 1971, Blechern, $&> 1364 bis 1371; G. SCHAFF 1 SL et al· 1978, Biochim. Biophys· Acta 52£, 457 bis 467). B. duroh MgCIg be solved by the Membranbruchstüoken and then cleaned (FM * HULETT-COWLING 1971, Tinny, $ &> 1364 to 1371; G. SCHAFF 1 SL et al · 1978, Biochim Biophys Acta 52 · £, 457 to. 467).

Prozeßanalysen gaben wichtige wissenschaftliche Grundlagen für die oben angegebene Verfahrensführung: Process analysis provided important scientific basis for the above mentioned process management:

- Die intensive Akkumulation an alkalischer Phosphatase er- - ER The intense accumulation of alkaline phosphatase

f-ro f ro

folgt in der stationären Waohstumsphese der Kultur, und zwar dann, worin das Mbrmedium total an Glucose verarmt und dor Phosphatgehalt unter 7 mM abgesunken ist. follows in the stationary Waohstumsphese the culture, and that then, wherein the Mbrmedium totally depleted of glucose and dor phosphate content has fallen below 7 mM. Der Phosphatgehalt ist etwa 50fach höher als bei in dor Literatur (V. JEANNODA & G. BALASSA (1978) Moleo. gen. Genet. 163 . 65 bis 73) erwähnten alkalische Phosphatase-Synthesen mit Baoillen. The phosphate content is about 50-fold higher than in dor literature (V. G. & JEANNODA BALASSA (1978) Moleo. Gen. Genet. 163rd 65 to 73) mentioned alkaline phosphatase syntheses Baoillen.

- Eine um 50 % höhere Akkumulation an alle al lecher Phosphatase wird duroh ein Gluoosef eeding in der Ube.?gangsphaee der Kultur erreioht. - A 50% increase in accumulation of all al Lecher phosphatase is a duroh Gluoosef eeding erreioht in the Ube.?gangsphaee the culture. Die Teobnologie des Gluoosefeedings erfolgt in der Weise, daß die Konzentration an freier Glucose in der KuItürlösung etwa 2 Stunden nach Beendigung der Zufütterung O % beträgt. The Teobnologie of Gluoosefeedings carried out in such a manner that the concentration of free glucose in the KuItürlösung about 2 hours after completion of the feeding O%.

- Die Syntheeerate der alkalischen Phosphatase von Bacillus lioheniformis wird ganz entsoheidend duroh die Intensität der Durohmisohung der Kulturlösung beschleunigt. - The Syntheeerate of alkaline phosphatase by Bacillus lioheniformis is quite entsoheidend duroh the intensity of the culture solution Durohmisohung accelerated. So wird allein duroh die Erhöhung der Hührgesohwindigkeit cieo Laboriermenters von 600 auf 800 rpm bei gleichbleibendem Lufteintrag zum gleichen Fermentationszeitpunkt eine um das lOfaohe höhere alkalische Phosphatsso-Aktivität gemessen * Thus, the increase in the Hührgesohwindigkeit cieo Laboriermenters from 600 to 800 rpm with a constant air input to the same fermentation time is measured alone duroh a higher around the lOfaohe alkaline Phosphatsso activity *

- Die optimale Fermentationstemperatur liegt bei 37 0 C Eine Abweichung um t 3 0 C bewirkt bereits eine merkliohe Senkung der Enzymausbeute. - The optimum fermentation temperature is 37 0 C. A deviation of t 3 0 C already causes a merkliohe reduction of enzyme yield.

- Um eine ungestörte alkalische Phosphatase-Akkumulation zu erreichen, ist <?τ.β Nährmedium aus den aufgeführten C- und N-Quellen so zusammenzusetzen, daß duroh die metabolische Wirkung der Kultur in der Phase der intensiven Produktbildung die Konzentration des Phosphates < 7 mM ist und nioht mehr als 20 bis 30yug/ml Ammonium freigesetzt werden. - To achieve an undisturbed alkaline phosphatase accumulation is <τ.β broth from the listed C and N sources to assemble so that duroh the metabolic effect of the culture in the phase of intensive product training, the concentration of the phosphate <7 mM? and nioht more than 20 to 30yug / ml ammonium are released.

Mit dem erfindungsgemäßea Verfahren können Enzymaktivitäten erreioht »ardan, die die Höhe der aus Kälberdarm gewinnbaren With the method erfindungsgemäßea enzyme activities may erreioht "ardan that the amount recoverable of calf intestine

Aktivitäten errelohen, so daß eine technische Produktion bakterieller alkalischer Phosphataso ökonomisch sinnvoll ist. Activities errelohen so that industrial production bacterial alkaline Phosphataso makes economic sense.

Gegenüber der bisher durchgeführten klolntechnlechon Produktion von alkalischer Phosphatase mit E. coli hat Bacillus lioheniformis 41p - ZIMBT 10 911 - den Vorteil, toxikologisch und pathologisch unbedenklich zu Bein. Compared to the previously conducted klolntechnlechon production of alkaline phosphatase with E. coli Bacillus lioheniformis 41p has - ZIMBT 10,911 - the advantage of toxicological and pathological safe to leg.

Die Erfindung soll anhand von Aueführungsbeispielen näher . The invention is based on Aueführungsbeispielen closer. erläutert werden» explains "

Ausführungtibolspiole Ausführungtibolspiole

Beispiel 1 example 1

Der Bakterienrasen einer 48 h bei 37 0 O bebrüteten Sohräg» agaikultuB Baoillus lioheniformis 11p - ZBiEZP 10 911 - auf Caseinagar wird mit sterilem Leitungswasser abgeschwemmt und in 50 ml einesVorkulturmediums in 500 ml-Rundstehkolben übertragen. The bacterial lawn an incubated 48 h at 37 0 O Sohräg "agaikultuB Baoillus lioheniformis 11p - ZBiEZP 10,911 - on Casein Agar is washed away with sterile tap water and transferred into 50 ml einesVorkulturmediums in 500 ml round-bottomed flask. Naoh einer .Inkubation von 7 h bei 37 0 C auf einer Sohüttelmasohine mit 220 U/min werden ca. 4 bis 6 χ Naoh a .Inkubation of 7 h at 37 0 C on a Sohüttelmasohine with 220 U / min to about 4 to 6 χ

10 Zellen/ml erreioht. 10 cells / ml erreioht. Damit werden die Produktionsmedien so beimpft, daß etwa 3 x 1Cr Zellen/ml KuI tür lösung vorhanden sind. Thus, the production media are inoculated so that about 3 x 1Cr cells / ml Kui door solvent are present.

Der Fermentationsprozeß mit dem nachfolgend aufgeführten Produktionsmedium erfolgte in einem Laborfermonter mit 1,5 1 Nettovolumen. The fermentation process with the following production medium was performed in a Laborfermonter 1.5 1 Net volume. Die Fermentationstemperatur lag bei 37 0 C, der Lufteintrag betrug 1 vvm bei 800 U/min. The fermentation temperature was 37 0 C, the air entry was 1 vvm at 800 rpm / min. Der Anfang s-pH-Wert lag bei 6,5, zum Zeitpunkt des Abbaus lag er bei pH 7,0. The initial s pH was 6.5 at the time of the degradation he was at pH 7.0. Unter diesen Bedingungen bildet der erfindungsgemäße Bacillus Hoheniformis 11p durchschnittlich 2,2 IE alkalische Phosphatase pro ml Kulturζentr i fugat, das bei 6 000 U/min 10 Minuten zentrifugiert wurde. Under these conditions, the Bacillus Hoheniformis invention 11p forms an average of 2.2 IU alkaline phosphatase per ml Kulturζentr i fugat that was / centrifuged at 6000 rpm for 10 minutes.

Zusammensetzung der Nährböden für die Anzucht des Bakterienrasens und für die Vorkulturt Composition of the nutrient media for growing the bacterial lawn and the Vorkulturt

zu to

Casoinogar: Casoinogar:

Vor kulturmedium t P:jjoduktionsmedium: Before culture medium P t: jjoduktionsmedium:

— 7 — - 7 -

Gluoose Gluoose

Casein casein

Na 2 HPO^ . Na 2 HPO ^. 2 H 2 O 2 H 2 O

Agar-Agar Agar Agar

Gluoose Gluoose

Trookenhefe Trookenhefe

Weizenfeinsohrot H . Weizenfeinsohrot H. 2 H 2 O • / H 9 U 2 H 2 O • / H 9 U

Stärkehydjffolyseprodukt Stärkehydjffolyseprodukt

Sod&öohrot Casein Sod & öohrot casein

Na 2 HPO^ . Na 2 HPO ^. 2 H 2 O . 2 H 2 O. 7 H 0 O 7 H 0 O

0,5 0,5 0,6 0,1 2,5 0.5 0.5 0.6 0.1 2.5

2,0 0,5 0,5 2.0 0.5 0.5

0,1 0.1

1,6 0,8 1.6 0.8

Beispiel 2 example 2

Anzuoht und Fermentation erfolgen wie· im Beispiel 1, nur jiit dem Unterschied, daß die Rührung 700 U/min beträgt und von der 12. bis zur 20. Fermentatiousstunde insgesamt 9 8 Gluoose zugefüttert werden. Anzuoht and fermentation are carried out as in Example 1, ·, only jiit the difference that the agitation 700 rev / min and be fed from the 12th to the 20th Fermentatiousstunde total 9 8 Gluoose. Die Ausbeute konnte au£ 2,7 DS/ ml gesteigert werden. The yield was au £ 2.7 DS / ml can be increased.

Die quantitative Bestimmung der alkalisohen Phosphatase erfolgte untttP optimalen Bedingungen bei 37 0 O in 1 M !Oiäthanolamin-HCl, ρΊ 9,5 (modifiziert naoh K. YAMANB und 5. MABKO (1978) J, Beoteriol. Ij^, 100). Quantitative determination of alkalisohen phosphatase was carried untttP optimal conditions at 37 0 O in 1 M! Oiäthanolamin-HCl, ρΊ 9.5 (modified naoh K. YAMANB and 5th MABKO (1978) J, Beoteriol. IM, 100). Als Substrat diente 5,5 mM p-Nitrophc>ny !phosphat. As the substrate, 5.5 mM p-served Nitrophc> ny! Phosphate.

Eine IE (internationale Einheit) ist definitionsgemäß die Menge alkalische Phosphatase, die die Freisetzung von 1 y uMol p-Nitrophenol Je Minute unter den Bedingungen der Prüfmethode bewirkt. One IU (International Unit) is defined as the amount of alkaline phosphatase, which causes the release of y 1 micromole of p-nitrophenol per minute under the conditions of the test method.

Claims (5)

- 8 - ZC(, Ϊ10 Erfindungsanspruch - 8 - ZC (, Ϊ10 invention claim
1. Verfahren zur biotechnischen Herstellung von alkalischer Phosphataseι dadurch gekennzeichnet, daß der Stamm Bacillus lichenif ormie 41p - ZIMBT 10 911 - verwendet wird. 1. A process for the biotechnical preparation of alkaline Phosphataseι characterized in that the strain Bacillus lichenif ormie 41p - ZIMBT 10 911 - is used.
2» Verfahren nach Punkt 1, dadurch gekennzeichnet, daß die C-, N- und P~Quellen des Nährmediums so kombiniert werden, daß durch die metabolisohe Wirkung der Kultur in der Phase der intensiven alkalische Pho ep hat aoe-Akkumulation der Gluoosegehalt des Nährmediums O %, der Phosphatgehalt C7 mM und die NH^-Konzentration nicht mehr als 20 bis 30/Ug/ml beträgt. 2 »The method of item 1, characterized in that the C, N and P ~ sources of the nutrient medium are combined so that by metabolisohe effect of culture in the phase of intensive alkaline Pho ep has aoe accumulation of Gluoosegehalt of the nutrient medium O%, the phosphate content and the C 7 mM NH ^ concentration is not more than 20 to 30 / ug / ml.
3. Verfahren nach den Punkten 1 und 2, dadurch gekennzeichnet, daß in der Übergangsphase ein Gluoosefeeding erfolgt. 3. The method according to items 1 and 2, characterized in that takes place in the transition phase, a Gluoosefeeding.
4. Verfahren naoh den Punkten 1 bis 3, dadurch gekennzeichnet, daß das FermentationsDiedium intensiv durchmischt wird. 4. The method NaOH points 1 to 3, characterized in that the FermentationsDiedium is intensively mixed.
5. Verfahren nach den Punkten 1 bis 4, dadurchgekennzeiohnet, daß die Fermentation bei einer Temperatur von 34 bis 40 0 C, insbesondere bei 37 0 C, erfolgt. 5. The method according to items 1 to 4, dadurchgekennzeiohnet that the fermentation is carried out at a temperature from 34 to 40 0 C, especially at 37 0 C.
DD25178483A 1983-06-06 1983-06-06 A process for the biotechnical preparation van alkaline phosphatase DD266710A3 (en)

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