DD266710A3 - Process for the biotechnical production of alkaline phosphatase - Google Patents
Process for the biotechnical production of alkaline phosphatase Download PDFInfo
- 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
- Authority
- DD
- German Democratic Republic
- Prior art keywords
- alkaline phosphatase
- production
- culture
- alkaline
- fermentation
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 102000002260 Alkaline Phosphatase Human genes 0.000 title abstract description 21
- 108020004774 Alkaline Phosphatase Proteins 0.000 title abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 0.000 claims abstract description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 10
- 239000008103 glucose Substances 0.000 claims abstract description 10
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 8
- 239000010452 phosphate Substances 0.000 claims abstract description 8
- 238000009825 accumulation Methods 0.000 claims abstract description 7
- 230000007704 transition Effects 0.000 claims abstract description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 3
- 230000002503 metabolic effect Effects 0.000 abstract description 2
- 238000002156 mixing Methods 0.000 abstract description 2
- 241000194108 Bacillus licheniformis Species 0.000 abstract 2
- 238000005457 optimization Methods 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000005018 casein Substances 0.000 description 5
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 5
- 235000021240 caseins Nutrition 0.000 description 5
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 4
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000006916 nutrient agar Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- -1 gluoose Chemical compound 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000013587 production medium Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-SOOFDHNKSA-N D-ribopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@@H]1O SRBFZHDQGSBBOR-SOOFDHNKSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
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- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- GZCGUPFRVQAUEE-KCDKBNATSA-N aldehydo-D-galactose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 description 1
- GZCGUPFRVQAUEE-KVTDHHQDSA-N aldehydo-D-mannose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-KVTDHHQDSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
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- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000013882 gravy Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
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- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Die Erfindung betrifft ein Verfahren zur biotechnischen Herstellung von alkalischer Phosphatase mittels Bacillus licheniformis. Ziel der Erfindung ist die Entwicklung eines technischen Verfahrens zur Herstellung von alkalischer Phosphatase. Das Ziel der Erfindung wird durch die Verwendung des Stammes Bacillus licheniformis 41p - ZIMET 10 911 - erreicht. 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. Optimierungen des Verfahrens koennen durch Glucosefeeding in der Uebergangsphase der Kultur, durch intensive Durchmischung des Fermentationsmediums und Einhaltung einer bestimmten Fermentationstemperatur erzielt werden.The invention relates to a method for the biotechnical production of alkaline phosphatase by means of Bacillus licheniformis. The aim of the invention is the development of a technical process for the production of alkaline phosphatase. The aim of the invention is achieved by the use of the strain Bacillus licheniformis 41p - ZIMET 10 911. The C, N and P sources of the Nährmediums are combined so that the metabolic activity of the culture in the phase of intensive alkaline phosphatase accumulation of the glucose content of the Nährmediums 0%, the phosphate content 7 mM and the NH4 concentration is no longer than 20 to 30 mg / ml. Optimizations of the method can be achieved by glucose feeding in the transition phase of the culture, by intensive mixing of the fermentation medium and maintaining a certain fermentation temperature.
Description
-7--7-
Titel der ErfindungTitle of the invention
Verfahren zur bioteohnisohen Herstellung von alkalischer Phosphat aseProcess for the bioteouric production of alkaline phosphate ase
Anwendungsgebiet der ErfindungField of application of the invention
Die Erfindung betrifft ein Verführen zur bioteohniaohen Herstellung von alkalischer Phosphatasθ mit einem Bacillus lichen if or mis-Stamm. Das Enzym kenn in der klinischen Diagnostik sowie in der bioohemisohen und molokularbiologisohen Forschung eingesetzt werden.The invention relates to a tempting for bioteohniaohen production of alkaline Phosphatasθ with a Bacillus lichen if or mis strain. The enzyme can be used in clinical diagnostics as well as in bioohemiotic and molecular biology research.
Charakterisierung der bekannten technischen LösungenCharacterization of the known technical solutions
Alkalische Fhosphatase wird von einer Vielzahl von Mikroorganismen synthetisiert. Unter den Bakterien eind zwar mehrere Spezies des Genus Baoillus dazu befähigt, so z. B. Bacillus subtilis, Bacillus oereus und Baoillus licheniformis (J.G. HANSA et al. 1981, Bioohim. Biophys. Aota £5£, 390 bis 401; O. HYDRKAN et al. 1977, J-. Biol. Chem. 2£2>, 6806; W.B. CHBSBHO & J.O. LAMPEN 1968, Baoteriol. 2£, 428 bis 437), aber die biotechnische Herstellung von alkalisoher Phosphatase mit Baoillen ist bisher nooh nicht bekannt.Alkaline phosphatase is synthesized by a variety of microorganisms. Among the bacteria, although several species of the genus Baoillus enabled, so z. B. Bacillus subtilis, Bacillus oereus and Baoillus licheniformis (JG HANSA et al., 1981, Bioohim., Biophys. Aota £ 5l, 390 to 401; O. HYDRKAN et al., 1977, J. Biol. Chem., 2 £ 2) 6806, WB CHBSBHO & JO LAMPS 1968, Baoteriol, £ 2, 428-437), but the biotechnical production of alkaline phosphatase with baoils has hitherto not been known.
Ss existieren Verfahren zur Herstellung von alkalischer Phosphatase mit Hilfe von Mikroorganismen anderer Gattungen, so u. a. 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).There are processes for the production of alkaline phosphatase by means of microorganisms of other genera, so u. a. Aotinomyoes ooelioolor A-66 (SU-UR 943 279) "Actinomyces streptomycin (SU-UR 178 337) as well as E. coli and Serratia maroesoens containing recombined plasmids with genes for alkaline phosphatase (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 - except for those in which cloned microorganisms are used - bring only low enzyme yields, so that at present still primarily the alkaline phosphatase of calf intestine he is gained.
Ziel der ErfindungObject 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 method for teohnisohen extraction of alkalisoher phosphatase by Baoillus lioheniformis.
Darstellung des Wesens der ErfindungPresentation of the essence 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 an alkaline Phoaphatase in high yield with a special Bacillus lioheniformis strain.
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, 8th 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 10Characteristic of Baoillus lioheniformis 41p - ZIMET 10
Morphologiemorphology
Form der Spore: ovalShape of the spore: oval
Lage der Spore: zentralLocation of the spore: central
Sporulationsverhalten: 95 % (Gerstenschrotaßav 10 d/Sporulation Behavior: 95 % (Barley Gravy 10 d /
37 0C)37 0 C)
Beweglichkeit: bewegliohAgility: agile
Gr amv er halten: grampositive Kurz stäbe heuGr amv keep it: Grampositive short rods hay
Koloniemorphologie auf unterschiedlichen NährbödenColony morphology on different nutrient media
Bluplatte: Kolonie hellbeige» milchig, matb-Blaupatte: colony light beige »milky, matb-
nach 48 h/37 0Cafter 48 h / 37 ° C.
Nähragar (NA):Nutrient Agar (NA):
NA + Glue + Cas.-Ao id:NA + Glue + Cas. Ao id:
Biochemische Merkmale Säurebildung aus:Biochemical features Acid formation from:
Kolonie hellbeige, milohig, mattglänzend, gelappt Kolonie maude/roeö, mattglän· aend, FärbstoffbildungColony light beige, milky, matte, lobed Colony maude / roeö, frosted, staining
-Xylose, Gluoose, d-Mannose, d-Fruotose, Maltose, Trehalose, Saccharose, Raffinose, Rhamnose, D-Mannit» d-Sorbit, D-Ribose, AesoulinXylose, gluoose, d-mannose, d-frutose, maltose, trehalose, sucrose, raffinose, rhamnose, D-mannitol d-sorbitol, D-ribose, aesouline
L-Arabinose naoh 2^ h, d-GalactoseL-arabinose, 2h, d-galactose
keine Säurebildung aus:no acid formation from:
IndolnaohweissIndolnaohweiss
VPRsVPRS
C it r atv er w er imng:C it r atv er w im imng:
Ureasenaohweis.tUreasenaohweis.t
Arginind.xhydr olase tArginine oxydrolase t
Waohstumsverhalten und bioohemisohe MerkmaleWaohstumsverhalten and bioohemisohe features
Wachstum in Nährbouillon:Growth in nutrient broth:
Waohstum auf Gluoose-Nitrat-Agar:Growth on Glucose-Nitrate-Agar:
Waohstum auf Gluoose-Asp ar ag in-Ag artGrowth on Gluoose-Asp ar ag in-Ag art
Waohstum in NaCl-BouillonsGrowth in NaCl brothels
Gasbildung aus Nitrat unter anaeroben Bedingungen: Nitratreduktion: Katalase: Stärkehydrolyse: Caseinhydrolyse: Gelatinohydrolyse: Lezithinase:Nitrate gas formation under anaerobic conditions: nitrate reduction: catalase: starch hydrolysis: casein hydrolysis: gelatinohydrolysis: lecithinase:
Hautbildung, Trübung, Bodensatz starkes WaohstumSkin formation, turbidity, sediment strong growth
sohlechtes Waohstumso-good Waohstum
3 - 7 % NaCl gutes Wachstum 10 % NaCl schwaches Wachstum3 - 7 % NaCl good growth 10 % NaCl weak growth
HZ ss HydrolysezoneHZ ss hydrolysis zone
Die Herstellung des Enzyms kann im Submersv erfahren in zwangsbelüfteten, sterilisierbaren Fermentationstanks unter üblichen bioteohnologisohen Bedingungen erfolgen. Es wurde Jedoch gefunden, daß den Eigenschaften des Stammes angepaßte Verfahrenobedingungen zu höheren Enzymausbeuten führen.The production of the enzyme can be carried out in submersed in forced-ventilation, sterilizable fermentation tanks under normal bioteohnologisohen conditions. However, it has been found that process conditions adapted to the properties of the strain result in 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 nutrient media with C sources commonly used for bacterial processes, such as glucose, maltose, corn or potato starch and cereal grains, and H from high N sources, such as soy protein, casein, skimmed milk powder, pea and bean flour and gelatin forms the strain of the invention in economically interesting amounts a ^ alisohe 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. Die Akkumulation der alkalischen Phosphatase wird durch ein GIuoosefeeding in der Übergangsphase und durch intensives Durchmischen des Fermentationsmediums gesteigert. Die Permentationstemperatur beträgt 34 bis 40 0C, insbesondere 37 0CIn a preferred embodiment of the process for the bloteless production of alkaline phosphatase with Bacillus lioheniformis 41p - ZIMET 10 911 - the C and N sources of the nutrient medium are combined so that by the cietabolisohe effect of the culture in the phase of intensive alkaline phosphatase accumulation dor glucose content of the nutrient medium O %, the phosphate content <7 mM and the NH ^ concentration is not more than 20 to 3OyUgZmI. The accumulation of alkaline phosphatase is increased by a Giuoosefeeding in the transition phase and by intensive mixing of the fermentation medium. The permentation temperature 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. Das Enzym kann in bekannter Welse, z. B. duroh MgCIg, von den Membranbruchstüoken gelöst und anschließend gereinigt werden (F.M* HULETT-COWLING 1971, Blechern, $&> 1364 bis 1371; G. SCHAFF1SL et al· 1978, Biochim. Biophys· Acta 52£, 457 bis 467).Bacillus licheriformis 41p - ZIMET 10 911 - synthesizes alkaline phosphatase, which is more than 90% associated with particles or membrane fragments. The enzyme may be in known catfish, z. MgCIg, are dissolved by the membrane breakage agents and then purified (FM * HULETT-COWLING 1971, Blechern, & # 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 analyzes provided important scientific foundations for the above-mentioned procedure:
- Die intensive Akkumulation an alkalischer Phosphatase er-- The intensive accumulation of alkaline phosphatase
f-rof 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. 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.follows in the stationary Waohstumsphese the culture, namely, in which the Mbrmedium totally depleted of glucose and the phosphate content has dropped below 7 mM. The phosphate content is about 50 times higher than in alkaline literature with the Bao alkaline phosphatase syntheses (V.JEANNODA & G. BALASSA (1978) Mol. Gen. Genet., 163, 65 to 73).
- Eine um 50 % höhere Akkumulation an alle al lecher Phosphatase wird duroh ein Gluoosef eeding in der Ube.?gangsphaee der Kultur erreioht. 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.- A 50 % higher accumulation of all alike phosphatase is achieved by glucose secretion in the primary phase of the culture. The Teobnologie of Gluoosefeedings takes place in such a way that the concentration of free glucose in the KuItürlösung is 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. 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 *The rate of synthesis of the alkaline phosphatase of Bacillus lioheniformis is accelerated very significantly by the intensity of the dehydration of the culture solution. Thus, solely by increasing the stirring speed of the cieo laboratory fermenter from 600 to 800 rpm while maintaining the air intake at the same fermentation time, the alkaline phosphatoso activity, which is higher by 10%, is measured *
- Die optimale Fermentationstemperatur liegt bei 37 0C Eine Abweichung um t 3 0C bewirkt bereits eine merkliohe Senkung der Enzymausbeute.- The optimum fermentation temperature is 37 0 C A deviation by t 3 0 C already causes a merkliohe lowering of the 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, <. Τ.β nutrient medium from the listed C and N sources should be composed so that duroh the metabolic effect of the culture in the phase of intensive product formation, the concentration of phosphate <7 mM and not more than 20 to 30yug / ml of ammonium are released.
Mit dem erfindungsgemäßea Verfahren können Enzymaktivitäten erreioht »ardan, die die Höhe der aus Kälberdarm gewinnbarenWith the method according to the invention, enzyme activities can be achieved which increase the amount of calf intestine
Aktivitäten errelohen, so daß eine technische Produktion bakterieller alkalischer Phosphataso ökonomisch sinnvoll ist.Errelohen activities, so that a technical production of 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 with the previous production of alkaline phosphatase with E. coli, Bacillus lioheniformis 41p - ZIMBT 10 911 has the advantage of being toxicologically and pathologically harmless to the leg.
Die Erfindung soll anhand von Aueführungsbeispielen näher . erläutert werden»The invention is based on Aueführungsbeispielen closer. be explained »
AusführungtibolspioleAusführungtibolspiole
Der Bakterienrasen einer 48 h bei 37 0O 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. Naoh einer .Inkubation von 7 h bei 37 0C auf einer Sohüttelmasohine mit 220 U/min werden ca. 4 bis 6 χThe 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 a .Incubation of 7 h at 37 0 C on a Sohüttelmasohine with 220 rev / min are about 4 to 6 χ
10 Zellen/ml erreioht. Damit werden die Produktionsmedien so beimpft, daß etwa 3 x 1Cr Zellen/ml KuI tür lösung vorhanden sind.10 cells / ml achieved. This inoculates the production media so that there are about 3 x 1Cr cells / ml of glucose solution.
Der Fermentationsprozeß mit dem nachfolgend aufgeführten Produktionsmedium erfolgte in einem Laborfermonter mit 1,5 1 Nettovolumen. Die Fermentationstemperatur lag bei 37 0C, der Lufteintrag betrug 1 vvm bei 800 U/min. Der Anfang s-pH-Wert lag bei 6,5, zum Zeitpunkt des Abbaus lag er bei pH 7,0. Unter diesen Bedingungen bildet der erfindungsgemäße Bacillus Hoheniformis 11p durchschnittlich 2,2 IE alkalische Phosphatase pro ml Kulturζentrifugat, das bei 6 000 U/min 10 Minuten zentrifugiert wurde.The fermentation process with the production medium listed below was carried out in a laboratory fermenter with 1.5 1 net volume. The fermentation temperature was 37 0 C, the air intake was 1 vvm at 800 U / min. The initial s-pH was 6.5, at the time of degradation it was at pH 7.0. 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 VorkulturtComposition of the culture media for the cultivation of the bacterial lawn and for the Vorkulturt
zuto
Casoinogar:Casoinogar:
Vor kulturmedium t P:jjoduktionsmedium:In front of culture medium t P: Yioduktionsmedium:
— 7 —- 7 -
GluooseGluoose
Caseincasein
Na2HPO^ . 2 H2ONa 2 HPO ^. 2H 2 O
Agar-AgarAgar Agar
GluooseGluoose
TrookenhefeTrookenhefe
Weizenfeinsohrot H . 2 H2O • / H9UWheat fine red H. 2 H 2 O • / H 9 U
StärkehydjffolyseproduktStärkehydjffolyseprodukt
Sod&öohrot CaseinSod & oohrot casein
Na2HPO^ . 2 H2O . 7 H0ONa 2 HPO ^. 2H 2 O. 7 H 0 O
0,5 0,5 0,6 0,1 2,50.5 0.5 0.6 0.1 2.5
2,0 0,5 0,52.0 0.5 0.5
0,10.1
1,6 0,81.6 0.8
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. Die Ausbeute konnte au£ 2,7 DS/ ml gesteigert werden.Anzuoht and fermentation carried out as in Example 1, except that the difference is that the agitation is 700 rpm and from the 12th to the 20th fermentation hour a total of 9% glucose is fed. The yield could be increased to 2.7 DS / ml.
Die quantitative Bestimmung der alkalisohen Phosphatase erfolgte untttP optimalen Bedingungen bei 37 0O in 1 M !Oiäthanolamin-HCl, ρΊ 9,5 (modifiziert naoh K. YAMANB und 5. MABKO (1978) J, Beoteriol. Ij^, 100). Als Substrat diente 5,5 mM p-Nitrophc>ny !phosphat.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). The substrate used was 5.5 mM p-nitrophecyl phosphate.
Eine IE (internationale Einheit) ist definitionsgemäß die Menge alkalische Phosphatase, die die Freisetzung von 1yuMol 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)
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|---|---|---|---|
| DD25178483A DD266710A3 (en) | 1983-06-06 | 1983-06-06 | Process for the biotechnical production of alkaline phosphatase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DD25178483A DD266710A3 (en) | 1983-06-06 | 1983-06-06 | Process for the biotechnical production of alkaline phosphatase |
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| Publication Number | Publication Date |
|---|---|
| DD266710A3 true DD266710A3 (en) | 1989-04-12 |
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