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EP0236475A1 - Procede de preparation d'un systeme d'enzyme lytique de cellules de levure - Google Patents

Procede de preparation d'un systeme d'enzyme lytique de cellules de levure

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Publication number
EP0236475A1
EP0236475A1 EP86906046A EP86906046A EP0236475A1 EP 0236475 A1 EP0236475 A1 EP 0236475A1 EP 86906046 A EP86906046 A EP 86906046A EP 86906046 A EP86906046 A EP 86906046A EP 0236475 A1 EP0236475 A1 EP 0236475A1
Authority
EP
European Patent Office
Prior art keywords
activity
dilution rate
glucanase
yeast
beta
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP86906046A
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German (de)
English (en)
Other versions
EP0236475A4 (fr
Inventor
Juan A. Asenjo
Barbara A. Andrews
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Columbia University in the City of New York
Original Assignee
Columbia University in the City of New York
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Publication of EP0236475A1 publication Critical patent/EP0236475A1/fr
Publication of EP0236475A4 publication Critical patent/EP0236475A4/fr
Withdrawn legal-status Critical Current

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    • 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)
    • C12Y302/01039—Glucan endo-1,3-beta-D-glucosidase (3.2.1.39)
    • 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
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • 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)
    • C12Y302/01058—Glucan 1,3-beta-glucosidase (3.2.1.58)

Definitions

  • This invention is a method for preparing a yeast cell lytic enzyme system having desired beta (1 ⁇ 3) glucanase and protease activities.
  • the method provides an efficient and continuous way to synthesize a lytic enzyme system.
  • Yeast cell walls consist of two layers.
  • the outer layer is a mannan-protein complex and the inner layer is an alkali-insoluble glucan.
  • the inner layer is an alkali-insoluble glucan.
  • glucanase displays more profound lytic action than the lytic enzyme (glucanase and protease) alone after a brief treatment of the cells with lytic enzyme.
  • the protease appears to be important in the initial attack on the cell surface.
  • the opening of polypeptide chains by the protease makes the inner glucan layer of the wall accessible to the glucanase.
  • Yeast cell lytic enzyme systems may be produced by either batch or continuous fermentation.
  • the continuous process is superior to the batch process with regard to enzyme production.
  • the composition (constituent enzymes) of the lytic enzyme systems can be manipulated by the use of different inducers and by altering the dilution rate.
  • the existence of lytic enzyme systems with different component activities has a potential for their use in specialized applications. Such applications include production of food grade protein and intracellular enzymes, isolation of intracellular recombinant protein produced in yeast, digestion of cell wall polysaccharides, production of protoplasts, as anticaries agents, in the study of cell wall structure, in the treatment of fungal diseases and as an essential tool for cell fusion, transformation and genetic engineering of yeast.
  • GB 1,048,887 discloses a process for producing an enzyme complex having strong proteolytic activity.
  • the organism used to produce this enzyme complex is a species of the genus Cytophaga.
  • Media compositions and conditions are described for culturing Cytophaga NCIB 9497 in a batch system. Centrifugation of the crude product of the culture and precipitation from aqueous solution with a protein precipitant are described as methods of recovery.
  • GB 1,179,935 discloses a process for producing cytolytic enzymes which are capable of lysing the living cells of microorganisms.
  • the system is a batch fermentation of Cytophaga johnsonii as well as other species of the genus Cytophaga.
  • GB 1,186,998 discloses another process for producing cytolytic enzymes of microorganisms in a batch fermentation.
  • the presence of cells of other microorganisms or decomposites thereof in the nutrient medium results in accelerated production of the cytolytic enzymes.
  • Cytophaga johnsonii as well as other Cytophagas may be cultured under the disclosed invention.
  • US 3,716,452 discloses an enzyme for lysing yeast cell walls.
  • the enzyme is produced in a batch fermentation of microorganisms belonging to Arthrobactgr luteus nov.sp.
  • the enzyme has activity for lysing cell walls of yeast dead or alive and at any stage of growth.
  • Ecky, et al. J. appl. Chem. Biotechnology, vol. 24, pp. 677-686, (1974)
  • Levels of beta (1 ⁇ 3) glucanase in the fermentation broth increased sharply as the dilution rate was decreased below 0.20 h -1 .
  • Continuous culture has a big advantage over batch culture in relation to enzyme production.
  • concentration of the lytic enzymes In continuous culture studies it has been possible to increase the concentration of the lytic enzymes by more than one order of magnitude compared to the batch production. It is also possible to regulate the ratio of glucanase to protease in the lytic enzyme system by using different inducers or altering the dilution rate.
  • the present invention is a method for preparing a yeast cell lytic enzyme system having desired beta (1 ⁇ 3) glucanase and protease activities.
  • a suitable bacterium which produces the lytic enzyme system is inoculated onto an appropriate growth medium containing predetermined amounts of a sugar as the carbon source (for example, sugar) and an inducer of lytic enzyme activity.
  • the bacterium is continuously fermented aerobically at an appropriate dilution rate, the dilution rate being varied to obtain the desired beta (1 ⁇ 3) glucanase and protease activities.
  • the resulting lytic enzyme system is recovered.
  • the bacterium may be one or more of the group consisting of Cytophaga, Qerskovia or Arthrobacter.
  • Fig. 1 Cell and Enzyme Concentrations in Cytophaga NCIB 9497 Continuous Culture as a function of dilution rate (h -1 ).
  • Fig. 2 Specific Enzyme Activity in Continuous Culture of Cytophaga sp. (activity/g cells) as a function of dilution rate (h -1 ).
  • the medium is comprised of 5 g/L glucose and 10 g/L yeast extract. Values are for yeast lytic activity (YLA, % decrease in OD 6 7 0 after 15 min./g cells, (A)); protease (P, u/g cells, ( ⁇ )); beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L/g cells, ).
  • Fig.3. Specific Rate of Enzyme Synthesis in Continuous Culture of Cytophaga sp. (activity/g cells h).
  • the medium is comprised of 5 g/L glucose and 10 g/L yeast extract. Values are for beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L/g cells h, ); protease (P, u/g cells h, ( ⁇ )); yeast lytic activity (YLA, % decrease in OD/g cells h, ( ⁇ )).
  • Fig. 4 Productivity in Continuous Culture of Cytophaga sp. (activity/L h).
  • the medium is comprised of 5 g/L glucose and 10 g/L yeast extract. Values are for yeast lytic activity (YLA, % decrease in OD/L h, ( ⁇ )); protease (P, u/L h, ( ⁇ )); beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L/L h, ); Cells (Cells, OD 670 /L h, (0)).
  • Fig. 5 Cell and Enzyme Concentrations in Oerskovia xanthineolytica Continuous Culture as a function of dilution rate (h -1 ).
  • Fig. 6 Cell and Enzyme Concetration in Oergkovia xanthineolytica Continuous Culture as a function of dilution rate (h -1 ).
  • the medium is comprised of 8 g/L glucose and 0.5 g/L glucan or 2 g/L glucose and 2 g/L glucan. Values are for beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L, protease (P, u, ( ⁇ )).
  • Fig. 8 Specific Enzyme Activity in Continuous Culture of Oerskovia sp. (activity/g cells) as a function of dilution rate (h -1 ).
  • the medium is comprised of 2 g/L glucose and 2 g/L glucan. Values are for beta (1 ⁇ 3) glucanase (B(1 ⁇ 73), U/L/g cells, protease (P, u/g cells, ( ⁇ )); mannanase (M, U/L/g cells, ; yeast lytic activity (YLA, % decrease in OD/g cells,
  • FIG. 9 Specific Enzyme Activity in Continuous Culture of Oerskovia sp. (activity/g cells) as a function of dilution rate (h -1 ).
  • the medium is comprised of 8 g/L glucose and 0.5 g/L glucan. Values are for beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L/g cells, ⁇ protease (P, u/g cells, ( ⁇ )); yeast lytic activity (YLA, % decrease in OD/g cells, ( ⁇ )).
  • Fig. 10 Specific Rate of Enzyme Synthesis in Continuous Culture of Oerskovia sp. (activity/g cells h) as a function of dilution rate (h -1 ).
  • the medium is comprised of 2 g/L glucose and 2 g/L glucan. Values are for protease (P, u/g cells h, ( ⁇ )); mannanase (M, U/L/g cells h, beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L/g cells h, yeast lytic activity (YLA, % decrease in OD/g cells h, ( ⁇ )).
  • Fig. 11 Specific Rate of Enzyme Synthesis in Continuous Culture of Oerskovia sp. (activity/g cells h) as a function of dilution rate (h -1 ).
  • the medium is comprised of 8 g/L glucose and 0.5 g/L glucan. Values are for protease (P, u/g cells h, ( ⁇ )); beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L/g cells h, yeast lytic activity (YLA, % decrease in OD/g cells h, ( ⁇ )).
  • Fig. 12 Productivity in Continuous Culture of Oerskovia sp. as function of dilution rate (h -1 ).
  • the medium used was comprised of 2 g/L glucose and 2 g/L glucan. Values are for yeast lytic activity (YLA, % decrease in OD/L h, ( ⁇ )); beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L/L h, protease (P, u/L h, ( ⁇ )); mannanase (M, U/L/L h, (O)); Cells (Cells, (#/mL) x 10 9 , (O).
  • Fig. 13 Productivity in Continuous Culture of Oerskovia sp.
  • the medium used was comprised of 8 g/L glucose and 0.5 g/L glucan. Values are for yeast lytic activity (YLA, % decrease in OD/L h, ( ⁇ )); beta (1 ⁇ 3) glucanase (B(1 ⁇ 3), U/L/L h, ; protease (P, u/L h, ( ⁇ )); dry weight (D.W., g/L/L h (0)).
  • the present invention is a method for preparing a yeast cell lytic enzyme system having desired beta (1 ⁇ 3) glucanase and protease activities.
  • a suitable bacteriurn which produces the lytic enzyme system is inoculated onto an appropriate growth medium containing predetermined amounts of carbon source and an inducer of lytic enzyme activity.
  • the bacterium is continuously fermented aerobically at an appropriate dilution rate, the dilution rate being varied to obtain the desired protease and beta (1 ⁇ 3) glucanase activities.
  • the resulting lytic enzyme system is recovered.
  • the bacterium may be one or more of the group consisting of Cytophaga, Oerskovia or Arthrobacter.
  • the method may consist of inoculating Oerskovia onto a growth medium containing glucose as the carbon source and yeast wall glucan as the inducer.
  • the growth medium may contain up to 20 g/L of glucose and 20 g/L of yeast wall glucan.
  • the growth medium may contain 8 g/L glucose and 0.5 g/L yeast wall glucan.
  • the dilution rate for the continuous fermentation may be selected so it provides high beta (1 ⁇ 3) glucanase activity.
  • the dilution rate may be between about 0.05 and about 0.20 -1 for high beta (1 ⁇ 3) glucanase activity.
  • the dilution rate may also be selected so it provides high protease activity.
  • the dilution rate may be between about 0.02 and about 0.10h -1 for high protease activity.
  • the dilution rate may be selected so it provides high beta (1 ⁇ 3) glucanase activity and low protease activity.
  • the dilution rate may be between about 0.15 and about 0.20h -1 for high beta (1 ⁇ 3) glucanase activity and low protease activity.
  • Another growth medium for Oerskovia may contain 2 g/L glucose as the carbon source and 2 g/L yeast wall glucan as the inducer.
  • the dilution rate for the continuous fermentation may be selected so it provides high beta (1 ⁇ 3) glucanase activity.
  • the dilution rate may be between about 0.05 and about 0.14h -1 for high beta (1 ⁇ 3) glucanase activity.
  • the dilution rate may also be selected so it provides high protease activity.
  • the dilution rate may be between about 0.02 and about 0.14h -1 for high protease activity.
  • the dilution rate may also be selected so it provides high beta (1 ⁇ 3) glucanase and low protease activity.
  • the dilution rate may be between about 0.15 and about 0.20h -1 for high beta (1 ⁇ 3) glucanase activity and low protease activity.
  • the amount of inducer present may be an amount sufficient to produce high levels of beta (1 ⁇ 3) glucanase activity.
  • Bacterial strains suitable for the method of this invention include wild type strains of Cytophaga, Oerskovia and Arthrobacter and derivatives thereof.
  • the dilution rate which is suitable for the present invention may be between about 0.02 and about 0.40 h -1 .
  • the continuous culture may be maintained at a pH and temperature suitable for preparing a yeast cell lytic enzyme system.
  • Another aspect of the invention may consist of inoculating Cytophaga onto a growth medium containing glu cose as a carbon source.
  • the growth medium may contain 5 g/L glucose.
  • the dilution rate for the continuous fermentation may be selected so it provides high beta (1 ⁇ 3) glucanase activity and high protease activity.
  • the dilution rate may be between about 0.05 and about 0.10h -1 for high beta (1 ⁇ 3) glucanase and high protease activity.
  • the pH for this method may be about 7.0 and the temperature may be about 29°C
  • Synthesis of enzymes may be inducible, semi-constitutive or constitutive. Inducible enzymes are synthesized at a low level in the absence of inducers. The uninduced, basal level of enzyme synthesis can vary considerably. When the basal enzyme activity is high, it is classified as semi-constitutive. Constitutive enzymes are synthesized maximally in the presence or absence of an inducer.
  • Cytophaga NCIB 9497 has been considered to be constitutive for yeast lytic enzyme synthesis.
  • the specific activities of the beta (1 ⁇ 3) glucanase and yeast lytic activity are high at dilution rates between 0.05 and 0.10h -1 . In this range of dilution rates the level of reducing sugars in the fermentation broth is low. Results indicate dual control of beta (1 ⁇ 3) glucanase synthesis by induction and catabolite repression.
  • catabolite repression is minimal because the low growth rate of the organism does not lead to an accumulation of repressing catabolites. Synthesis is regulated by the amount of inducer present. Growth rate increases with increasing dilution rate and as a result the rate of enzyme synthesis increases. At a dilution rate between 0.07 and 0.1 h -1 the concentration of the repressor has reached a level high enough to cause significant catabolite repression. As the dilution rate increases the growth rate increases and catabolite repression becomes dominant over induction and the rate of enzyme production drops.
  • protease enzyme synthesis does not appear to be under dual control by induction and catabolite repression in the range of dilution rates studied (between 0.03 and 0.30h -1 ). Protease synthesis is subject to catabolite repression but is not induced.
  • Reducing sugars in the fermentation broth described above accumulated at dilution rates greater than 0.15 h -1 .
  • the reducing sugar content was 6.9 g/L, indicating that most of the measured reducing sugars were glucose.
  • washout did not occur.
  • the accumulation of reducing sugars at dilution rates above 0.15 h -1 indicates the presence of a second limiting substrate. This substrate is unknown. Dissolved oxygen was never limiting.
  • Protease synthesis does not appear, from continuous culture studies, to be inducible. The activity levels therefore are similar in late batch culture and in continuous culture at low dilution rates. Enzyme levels in continuous culture at high dilution rates (when glucose accumulates) are similar to those in batch culture when approximately half the reducing sugars have been metabolized. It appears that the proteolytic activity is made up of activity from more than one protease enzyme and that the syntheses of these enzymes are not regulated together. In carbon limited media, two peaks of specific rate of enzyme synthesis exist; one at dilution rates above 0.15 h -1 and one at dilution rates below 0.15 h -1 .
  • the appreciable rate of enzyme synthesis at high dilution rates is evidence that not all the proteolytic enzymes are catabolite repressed. There is also evidence that one of the proteases may be induced by the second, unknown limiting substrate. The peak at low dilution rates indicates that one of the proteases may be weakly induced.
  • Oerskovia xanthineolytica Another suitable bacterium of the disclosed method is Oerskovia xanthineolytica. Continuous cultures were carried out using two different media; one with 2 g/L of glucose and 2 g/L of glucan and the other with 8 g/L of glucose and 0.5 g/L of glucan.
  • Beta (1 ⁇ 3) glucanase specific activities in the two continuous cultures there is a sharp peak of beta (1 ⁇ 3) glucanase specific activity at low dilution rates.
  • the maximum specific activity is more than three times that on the medium of 8 g/L glucose and 0.5 g/L of glucan.
  • Beta (1 ⁇ 3) glucanase specific activity in the medium containing 2 g/L glucose and glucan appears to be controlled by both induction and catabolite repression. Protease does not appear to be induced at low dilution rates but is subject to catabolite repression.
  • Cytophaga sp. NCIB 9497 was obtained f rom the National Collection of Industrial Bacteria (Aberdeen, Scotland) .
  • Oerskovia xanthineolytica LL-G109 was a gift f rom M. Lechavalier, Rutgers University, New Jersey, USA.
  • Arthrgbacter sp. GJM-1 was obtained f rom C. Ballou, University of California, Berkeley, USA.
  • Saccharomyces cerevisiae NCYC 1006 was f rom the National Collection of Yeast Cultures, Norwich, UK.
  • Saccharomyces carlsbergensis was a gift f rom the Stroh Brewing Company, Michigan, USA and the bakers yeast used was Red Star active dried yeast f rom the Universal Food Corporation, Wisconsin, USA.
  • Yeast extract - "Ardamine Yep”, (autolysed yeast extract powder), was supplied by Yeast Products Inc., New Jersey, USA.
  • Glucose (used in fermentation media) - technical grade, Sigma Chemical Company, Missouri, USA.
  • Glucan prepared by alkali extraction from bakers yeast, (Manners et al., 1973a) was a gift from J. Hunter, Department of Chemical Engineering and Applied Chemistry, Columbia University, New York, USA. It was sieved and the fraction smaller than 177 microns (mesh #80) was used.
  • Yeast cell walls (used as an inducer) were prepared from Saccharomyces carlsber ⁇ ensis disrupted in a Dyno mill (Willy A. Bachofen AG, Switzerland) for 20 minutes
  • OD optical density
  • DW dry weight
  • viable cell count colony count
  • Optical Density was measured in a Spectronic 21 spectrophotometer (Bausch and Lomb, New York, USA). Samples were diluted before reading to fall within the range from 0 to 0.6 on the absorbance scale. When used to estimate growth optical densities were read at 670 nm.
  • the colony count method was used to estimate growth in fermentations containing insoluble substances where optical density and dry weight were not useful. Serial dilutions of the culture borth were made in sterile distilled water. 0.1mL of the appropriate dilutions were plated in duplicate. After 72 hours the colonies were counted and the number of cells per unit volume calculated.
  • insoluble inducers When insoluble inducers were used they were added directly to the medium resevoir which was stirred throughout the fermentation. The tubing (silicone) was checked daily for buildups of insoluble material.
  • Culture purity was checked daily by plating two 0.1 mL sampels of culture borth on nutrient agar plates.
  • the sample to be used for enzyme assays was filtered (membrane filter, 0.45 microns) and frozen. When larger volumes of enzyme were needed the supernatent was centrifuged at 2000 rpm for 15 minutes, separated and the enzyme preparation frozen.
  • the method used to estimate the overall lytic activity was decrease in optical density in an incubation of whole yeast cells, lytic enzyme and buffer.
  • Beta (1 ⁇ 3) Glucanase activity assay equal volumes of enzyme, laminarin (a 3% w/v solution) and buffer (50 mM Tris-HCl, pH 7.3) were incubated at 37 C. The reaction was terminated by boiling in a water bath for 5 minutes. Reducing sugars released were assayed using the Nelson-Somogyi method (Nelson, 1944) or the Micro Nelson method (Spiro, 1966). Standard curves were prepared using glucose.
  • Mannanase activity was assayed in the same way Beta (1 ⁇ 3) glucanase except that the substrate used was a 0.5% solution of mannan.
  • Glucanase and mannanase activities are expressed in international units, 1 U is that amount of enzyme which releases 1 umole of glucose per minute under specified conditions.
  • Protease activity was measured by the method of Rowley and Bull (1977).
  • the substrate used was azocasein at a concentration of 2.5 g/L. Equal volumes of substrate and enzyme were incubated at 37 C for 20 minutes. The reaction was terminated by the addition of 2 mL of 3 M TCA (trichloroacetic acid). After centrifugation at 2000 rpm for 30 minutes the absorbance of the supernatant was read at 400 nm. Various blanks and controls were run. The activity is expressed in units (u) where lu equals 1% hydrolysis in 20 min. in the range from 0% to 30%. A unit corresponds to the solubilization of 0.00125 mg. protein per min. per ml. of enzyme solution.
  • Glucose concentrations were determined by the PGO (Peroxidase Glucose Oxidase) method, Sigma Technical Bulletin No. 510.
  • Cytophaga NCIB 9497 was grown in continuous culture.
  • the medium contained 10 g/L of yeast extract and 5 g/L of glucose.
  • the pH was controlled at 7 and the temperature at 29°C.
  • Steady state conditions at dilution rates from 0.03 h -1 to 0.30 h -1 were analyzed for cell concentration, beta (1 ⁇ 3) glucanase, protease, mannanase and yeast lytic activities as well as reducing sugars present in the fermentation broth.
  • the level of glucose (reducing sugars) in the fermentation broth was between 0.5 and 0.8 g/L up to a dilution rate of 0.15 h -1 . At dilution rates above 0.15 h -1 glucose accumulated and another substrate became limiting.
  • beta (1 ⁇ 3) glucanase is repressed by glucose.
  • Yeast lytic activity is mainly attributable to production of proteolytic enzymes. Activities of beta (1 ⁇ 3) glucanase, protease, mannanase and overall yeast lytic activity are similar to those obtained in batch culture. The beta (1 ⁇ 3) glucanase and protease activities are highest at a dilution rate of about 0.07 h -1 where there is a corresponding peak in yeast lytic activity.
  • the enzyme system has a high beta (1 ⁇ 3) glucanase/protease ratio.
  • Specific activity (activity/g cells) is calculated using a dry weight per unit optical density correlation of 0.93 g per liter per unit optical density.
  • beta (1 ⁇ 3) glucanase activity exhibits a peak of specific activity at dilution rates between about 0.05 and about 0.07 h -1 . At dilution rates above and below this range the specific activities drop significantly. At dilution rates below 0.07 h -1 catabolite repression is minimal and the rate of glucanase synthesis is dependent mainly upon the concentration of inducer present. Catabolite repression increases with increasing dilution rate until it becomes dominant over induction at dilution rates of about 0.07 h -1 and above. Cytophaga NCIB 9497 has in the past been classified as constitutive for enzyme synthesis. It appears from these continuous culture studies that enzyme synthesis in Cytophaga NCIB 9497 is regulated by induction and catabolite repression.
  • the specific activity profile for the proteolytic enzyme shows a steady decline with no peak. It does not appear to be inducible but is subject to catabolite repression.
  • the specific rate of enzyme synthesis (also called the "specific productivity") is the amount of enzyme activity produced per gram of cells per hour. In the range specific rates of beta (1 ⁇ 3) glucanase and protease synthesis both exhibit peaks. At dilution rates above 0.15 h -1 the rate of glucanase synthesis is low but the rate of protease synthesis shows another peak.
  • Productivity is activity per liter per hour for enzyme measurement and optical density per liter per hour for measurement of biomass growth. At dilution rates above 0.25 h -1 a peak of high productivity but low concentration occurs with proteolytic and yeast lytic activities. Productivity of beta (1 ⁇ 3) glucanase activity is low at this range of dilution rates. The productivity of biomass increases slowly with increasing dilution rate. Between rates of about 0.03 and about 0.15 h -1 , all enzyme activities exhibit a sharp peak in productivity. Productivity of beta (1 ⁇ 3) glucanase activity was 20 times greater and yeast lytic activity 4 times greater than such productivity in batch culture.
  • Oerskovia xanthinedytica LL-G109 was grown in continuous culture.
  • the medium contained 2 g/L glucose as the carbon source and 2 g/L yeast wall glucan as the inducer. Enzyme concentrtion and growth are monitored as well as glucose present in the fermentation broth over a range of dilution rates from 0.03 to 0.38h -1 .
  • the level of glucose in the fermentation broth was about 0.02 g/L throughout the range of dilution rates used.
  • the level of beta (1 ⁇ 3) glucanase activity is low when dilution rates are below 0.05h -1 .
  • the level increases with increasing dilution rate so that activity is high at dilution rates between about 0.05 and about 0.14b -1 . Dilution rates above 0.14h -1 result in low activity.
  • Protease activity is low throughout the range of dilution rates studies, and very low at dilution rates above 0.15h -1 .
  • Specific activity (activity/g cells) is calculated using a dry weight per unit optical density correlation of 0.61 g per liter per unit optical density.
  • the specific activity of beta (1 ⁇ 3) glucanase is high at dilution rates between about 0.03 and about 0.09h -1 , with a peak at a dilution rate of about 0.05h -1 .
  • Specific activities at dilution rates below 0.03h -1 and above 0.09h -1 are low.
  • Protease specific activity is greatest at dilution rates below 0.05h -1 . Increasing dilution rates result in low protease specific activity.
  • the specific rate of enzyme synthesis is the amount of enzyme activity produced per gram of cells per hour. For beta (1 ⁇ 3) glucanase, this value is greatest at dilution rates below 0.15h -1 . At dilution rates above 0.15h -1 the specific rate of enzyme synthesis is low. For protease, this value is highest at low dilution rates but diminishes to a low synthesis rate at dilution rates above 0.10h -1 .
  • Productivity is activity per liter per hour for enzyme measurement, and optical density per liter per hour for measurement of biomass growth.
  • Beta (1 ⁇ 3) glucanase activity exhibits high productivity at low dilution rates (below 0.2h -1 ) with a maximum at about 0.15h -1 .
  • Protease productivity is maximized at a dilution rate of about 0.15 -1 as well.
  • Protease productivity above dilution rate of 0.15h -1 is lower but constant, and below a dilution rate of 0.15h -1 steadily declines.
  • Oerskovia xanthineolytica LL-G109 was grown in continuous culture.
  • the medium contained 8 g/L glucose as the carbon source and 0.5 g/L yeast wall glucan as the inducer. Enzyme concentration and growth are monitored as well as glucose present in the fermentation both over a range of diltuion rates for 0.03 to 0.38h -1 .
  • the glucose level was close to 0 g/L up to a dilution rate of 0.28h -1 , above which it accumulates.
  • the level of beta (1 ⁇ 3) glucanase activity is high at dilution rates between about 0.03 and about 0.2h -1 .
  • Activity is low at dilution rates above 0.2b -1 .
  • Protease activity is low throughout the range of dilution rates studies, and very low at dilution rates above 0.15h -1 .
  • Specific activity is calculated using a dry weight per unit optical density correlation of 0.61 g per liter per unit optical density.
  • the specific activity of beta (1 ⁇ 3) glucanase is greatest at low dilution rates (below 0.15h -1 ).
  • For protease this value is highest at dilution rates above 0.25h -1 .
  • Productivity is activity per liter per hour for enzyume measurement, and optical density per liter per hour for measurement of biomass growth.
  • Beta (1 ⁇ 3) glucanase activity exhibits high productivity at diltuion rates below 0.2h -1 , with a maximum at about 0.15h -1 .
  • Protease productivity above a dilution rate of 0.15h -1 is lower but constant, and below a dilution rate of 0.15h -1 steadily declines.

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Abstract

Procédé en continu de préparation d'un système d'enzyme lytique de cellules de levure ayant des activités désirées de bêta (1 - 3) glucanase et protéase. Une bactérie appropriée qui produit le système d'enzyme lytique est inoculée sur un support de croissance approprié contenant des quantités prédéterminées d'une source de carbone (par exemple du sucre) et un inducteur d'activité enzymatique lytique. La bactérie subit une fermentation aérobie en continu, tout en faisant varier la vitesse de dilution pour obtenir les activités désirées de bêta (1 - 3) glucanase et protéase. La bactérie peut être une ou plusieurs des bactéries du groupe constitué de Cytophaga, Oerskovia ou Arthrobacter.
EP19860906046 1985-09-04 1986-09-03 Procede de preparation d'un systeme d'enzyme lytique de cellules de levure. Withdrawn EP0236475A4 (fr)

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US772363 1985-09-04

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EP0236475A4 EP0236475A4 (fr) 1989-12-19

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EP0496861A1 (fr) * 1990-08-17 1992-08-05 Her Majesty In Right Of Canada As Represented By The National Research Council Of Canada Production d'adn recombinant de beta-1,3-glucanase
JPH04158784A (ja) * 1990-10-24 1992-06-01 Tax Adm Agency 酵母細胞壁溶解酵素の製造法及びその利用
EP0785995A1 (fr) * 1994-10-14 1997-07-30 Novo Nordisk A/S Nouvelle enzyme a activite beta-1,3-glucanase
US5919688A (en) * 1994-10-14 1999-07-06 Novo Nordisk A/S Enzyme with B-1, 3-glucanase activity
US5637745A (en) * 1995-01-30 1997-06-10 Elf Atochem North America, Inc. Organometallic compounds and polymers made therefrom
EP0904359A1 (fr) 1996-04-12 1999-03-31 Novo Nordisk A/S ENZYME DOTEE D'UNE ACTIVITE DE $g(b)-1,3-GLUCANASE
PL2655644T3 (pl) 2010-12-22 2019-05-31 Basf Enzymes Llc Poprawa sposobów fermentacji i produktów ubocznych
HUE042895T2 (hu) 2013-02-21 2019-07-29 Direvo Ind Biotechnology Gmbh Mikotoxin-kötõ anyagok
DK2958437T3 (da) 2013-02-21 2020-03-30 Direvo Ind Biotechnology Gmbh Præbiotiske dyrefoderprodukter
US20160106122A1 (en) 2013-05-16 2016-04-21 Direvo Industrial Biotechnology Gmbh Animal feed product for monogastric animals
WO2021092589A1 (fr) * 2019-11-08 2021-05-14 The Trustees Of Columbia University In The City Of New York Procédés d'ingéniérie d'agents thérapeutiques et utilisations associées

Non-Patent Citations (3)

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CHEMICAL ABSTRACTS *
See also references of WO8701388A1 *

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DK227587A (da) 1987-07-03
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DK227587D0 (da) 1987-05-04
EP0236475A4 (fr) 1989-12-19
JPS63500772A (ja) 1988-03-24

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