JPS5918989B2 - Process for producing biologically active substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention utilizes Bordetella (B.
The present invention relates to a method for producing biologically active substances using microorganisms belonging to the genus Ordella.

Microorganisms belonging to the genus Bordetella include Bordetella pertussis, Bordetella pertussis, and Bordetella bronchiseptica, which produce various biologically active substances.

For example, a culture of pertussis bacteria (culture medium and bacterial cells) produces an insulin secretion-enhancing active substance (l5le), which is expected to be developed as a treatment or preventive drug for various types of diabetes.
t activating protein I
AP) and leucocytosis factor (Leucocytosis factor), which is attracting attention as a vaccine component for Bordetella pertussis.
ytosis Promoting Factor (hereinafter abbreviated as LPF), medically effective biologically active substances can be obtained.

However, pertussis II phase bacteria tend to undergo phase changes and are difficult to stably culture, resulting in the problem that the antigenicity, pathogenicity, LPF-producing ability, or IAP-producing ability of the bacteria differs markedly depending on the culture conditions.

Attempts have been made in the past to solve these problems.

For example, Rowatt et al.
n, Microbiology) Volume 17, 279
296 and 297-326, 1957), the following factors are listed as factors that inhibit the cultivation of Bordetella microorganisms, particularly pertussis bacteria.

(1) Colloidal sulfide or sulfur obtained by heating (autoclaving) cysteine.

(2) Hydrogen peroxide or organic peroxide obtained by autoclaving a casein hydrolyzate.

(3) Unsaturated fatty acids, especially oleic acid, produced secondarily by bacteria.

Additives to the culture medium that counteract these suppressive effects include albumin, red blood cells or their crushed products, activated carbon, and ion exchange resin for (1), catalase, hemin, and FeSO4 for (2), and catalase, hemin, and FeSO4 for (3). starch,
Amylose, dextrin, etc. are mentioned, but the effects of these additives are unstable when the number of inoculated bacteria is 1 x 106 or less.

Addition of adsorbents such as activated carbon, ion-exchange resins, and amylose are also said to be effective, but they tend to form non-uniform areas in the culture medium (although they are not necessarily sufficient).

Additives such as red blood cells and albumin are not suitable for preparing a stable medium suitable for storage because they tend to change in composition (and are easily denatured) depending on the lot.

In recent years, 5tainer (5tainer) and Scholte (
A synthetic medium for large-scale culture of this pertussis bacteria was developed by J. Cholte (Journal of General Microbiology J).

gen, Microbiol) Volume 63, 211
-220 pages, 1971) This Stenner-Sholte medium (hereinafter abbreviated as SS medium) does not contain additives that may vary between lots, such as blood and polypeptone derived from natural products, so the culture medium composition of bacteria can be strictly controlled. It is possible to perform culture without causing changes in bacterial properties, and to prevent contamination with unnecessary proteins from other species during the separation and purification of biologically active substances such as IAP or LPF mentioned above. In recent years, it has been widely used to produce pertussis vaccines and biologically active substances from Bordetella pertussis on an industrial scale. In particular, when the inoculation size is 10 7 colonies/ml or less, stable growth characteristics cannot be obtained in terms of LPF production ability, etc., which is a drawback.

In addition, an agar medium (hereinafter abbreviated as SSA medium) obtained by adding 1.2% agar to SS medium and solidifying it,
It has a major defect in that colony formation is not observed when seeding 103 colonies or less.

The present inventors have arrived at the present invention as a result of intensive research aimed at improving the drawbacks of the prior art and stably and efficiently obtaining biologically active substances from microorganisms belonging to the Bordetella layer.

That is, the present invention cultivates microorganisms belonging to the Bordetella layer in a medium containing cyclodextrin or its derivatives,
A method for producing a biologically active substance, which is characterized by collecting a substance having hemagglutination activity from a culture (culture medium and bacterial cells), and a medium used in the process.

Microorganisms belonging to the Bordetella family, such as Bordetella pertussis, produce two types of hemagglutinin (HA), which are antigens that protect against pertussis infection.
It is known to produce

One is filamentous hemagglutinin (F-HA) and the other is L
PF-Hemagglutinin (LPF-HA).

Filamentous hemagglutinin is a protein with a molecular weight of approximately 130,000 that exists on the surface of bacterial cells, and although this substance itself is not toxic to living organisms,
It has the property of adsorbing to the cell membranes of red blood cells and various animal cells, leading to cell aggregation.

Since the molecular form of this HA has a fibrous structure of 2 × 40 nm, Filamentous-Hemag
It is called glutinin (F-HA).

LPF-hemagglutinin is a particulate protein with a molecular weight of approximately 110,000, and this highly purified and crystallized HA exhibits various unique biological activities in addition to hemagglutination activity when administered to living organisms.

For example, this protein is 0.1μ? , when inoculated into the tail vein of mice, the number of white blood cells, especially small lymphocytes, in the peripheral blood increases markedly after 2 to 3 days, resulting in so-called leukocytosis Leu.
kocytosis (or Lymphocytos
is ) is derived.

Therefore, this protein is known as leukocytosis factor (Leukocytosis factor).
tosis-Promting-Factor:
Also called LPF.

In addition, this substance is a histamine sensitizing factor (Hist-am
ine-S ensitizing-Factor
: Also called HSF).

This is because mice, rats, etc. treated with this substance become significantly more sensitive to histamine, serotonin, etc., which are toxic amines in living organisms, and can easily die from histamine inoculation at about i/100 of the normal lethal dose. It's for a reason.

This substance also contains eyelet activation protein (I5le
t-Activating-Protein:
Also called IAP).

In other words, it is known that the insulin concentration in the peripheral blood of animals administered with this protein increases, and this is said to be because this substance stimulates the β cells of the islets of Langerhans in the pancreas and promotes insulin secretion.

In the present invention, the microorganisms belonging to the Bordetella layer refer to Bordetella pertussis, Bordetella pertussis, and Bordetella bronchiseptica.

In the present invention, Bordetella pertussis is preferably used, and Bordetella pertussis is particularly preferred.

Regarding the mycological properties and culture conditions of microorganisms belonging to the Bordetella Formation, see Bergys Manusl of D.
eterminative Bacteriology
, 8th edition, 1974, The Williams &
WillkinsCo, Published by J, Exp Me
d, vol. 129, pp. 523-550, 1969 or bacteriology practice summary, 3rd edition, pp. 80 et seq., 1939.
Published by Maruzen in 1997, it is already publicly known.

Cyclodextrin is a starch or starch hydrolyzate produced by Bacillus maceraus amyla.
It is a crown-shaped molecule in which 6 to 10 D-glycopyranose groups are bonded in a ring through α-1,4 glycosidic bonds, and is obtained by the action of se (transglycosylase) or the like.

The main one consists of 6.7 or 8 D-glycopyranose groups and is called α-1β-1γ-cyclodextrin, respectively.

The cyclodextrin in the present invention refers to the above α, β,
Refers to cyclodextrin such as γ or a mixture thereof.

Since cyclodextrin molecules have a large number of primary and secondary hydroxyl groups, various derivatives can be obtained by applying reactions widely used for monosaccharides.

Cyclodextrin derivatives in the present invention mean derivatives obtained by such methods, such as aminated derivatives such as aminocyclodextrin and aminodeoxycyclodextrin, esterified derivatives such as acetylcyclodextrin and nitrocyclodextrin, methylcyclodextrin, There are etherified derivatives (etherified cyclodextrin) such as ethercyclodextrin, propylcyclodextrin, and carpoquinelmethelcyclodextrin.

Preferred in the present invention are etherified cyclodextrins, among which hexakis(2,6-0-dimethyl)α-cyclodextrin (Meα-CD) and heptakis(2,6-0-dimethyl)β-cyclodextrin (
Methylcyclodextrins such as Meβ-CD) are particularly preferred.

In the present invention, the medium means a conventionally known liquid medium such as bouillon or peptone water, or a conventionally known solid medium made by adding agar, gelatin, egg white, serum, etc. to a liquid medium, but preferred is SS. These are a medium and an SS medium in which about 1 to 2% (w/v) agar is added and solidified.

SS medium contains per 11, sodium daltamate, ■
-proline, sodium chloride, potassium dihydrogen phosphate,
Potassium chloride, magnesium chloride, calcium chloride, and trishydroxymethylaminemethane were added at 10% each.
7.0.24.2.5.0, 5.0.2.0.1.0.
The aqueous solution containing 02.1.5251 was adjusted to pH 7.6 with concentrated hydrochloric acid.
l-cystine, ferrous sulfate,
11 ascorbic acid, niacin, and reduced glutathione
4.1.2.0.4.101, respectively, using a Millipore filter (0.45μ) to sterilize the solution, and add a supplementary solution to the basal medium at 1.0% (V/V'). It is obtained by adding in the proportion of .

In the present invention, the amount of cyclodextrin or its derivative added to the medium depends on the amount of bacteria to be inoculated, but for example, when the number of bacteria to be inoculated is 106 to 105 colonies/rJl, 10-50001 in the medium
n9/m11 preferably 100-1000μ? /rrt
1 of cyclodextrin or a derivative thereof is added and mixed to obtain the medium used in the present invention.

A medium supplemented with such a cyclodextrin or a derivative thereof,
In particular, the added SS medium allows bacteria to grow stably and efficiently, so IA is expected to have a medicinal effect as a diabetes treatment drug.
P, L expected to be a pertussis vaccine component
It is an extremely advantageous medium for producing active substances such as PF and bacterial vaccines.

The method and conditions for culturing microorganisms belonging to the genus Bordetella using such a medium are not particularly limited, and conventionally known methods and conditions can be adopted, but shaking and
Cultivation is more preferable, and the culture temperature is approximately 35° C. and the culture time is appropriate for 10 to 100 hours.

The method and means for collecting the produced biologically active substance from the culture (culture medium and bacterial cells) are not particularly limited, and known methods and means can be used.

For example, to obtain LPF, pertussis I phase bacteria (Bordetella patasis Higashihama strain) were mixed with 300 μy'/yd of methyl β-
Culture was performed at 35°C for 18 hours in SS medium containing cyclodextrin, and the centrifuged supernatant (pH 8.6) of the resulting culture was equilibrated with 0.001M phosphate buffer at pH 8.0. Pass through an apatite column.

Then, after adjusting the obtained permeate to pH 6.0, this time it is 0.01M at pH 6.0! The protein fraction is adsorbed onto a hydroxyapatite column equilibrated with J phosphate buffer, and eluted with 0.1M phosphate buffer (pH 7.0) containing 0.5M sodium chloride to obtain a protein fraction.

This protein fraction is adsorbed on affinity chromatography using haptoglobin-Sepharose as a support.
LPF can be obtained by desorption with 0.1M phosphate buffer containing 0.5M Na Cl and 3M potassium thiocyanide.

In addition, the adsorbed material on a hydroxyapatite column with a pH of 8.0 was
A protein fraction is obtained by elution with MIJ acid buffer (pH 7,0).

This protein fraction is passed through affinity chromatography using haptoglobin Sepharose 4B as a support, and F-HA can be obtained from the flow through.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 Bordetella pertu
ssis ) (Bacillus pertussis) Higashihama strain ■ Suspend the freeze-dried bacterial cells of the compatible bacteria in a 1% casamino acid solution, and add defibrinated horse blood for 2 hours.
The cells were cultured at 35° C. for 3 days in Bordet-Gengou medium (hereinafter referred to as BG medium) containing 0% Bordet-Gengou medium.

One platinum loopful of this bacteria was scraped off, and the bacteria was refreshed with BG medium for 24 hours and suspended in SS medium to obtain an inoculated bacterial suspension.

This inoculum suspension was suspended in SS medium containing a predetermined concentration of Meβ-CD at a concentration of 107 colonies/rfLl, and cultured at 35° C. for 18 hours under standing or shaking conditions.

After culturing, the turbidity (0D650) of the culture solution was measured, and then the LPF produced was collected by the following method and its activity was measured.

The centrifuged supernatant (pH 8.6) of the culture solution was added to a 0.
The resulting effluent was passed through a hydroxyapatite column equilibrated with OIMIJ acid buffer and adjusted to pH 60. The protein fraction was adsorbed onto a hydroxyapatite column equilibrated with OIM'J phosphate buffer, and eluted with 0.1M phosphate buffer (pH 7.0) containing 0.5M sodium chloride to obtain a protein fraction.

This protein fraction is adsorbed on affinity chromatography using haptoglobin-Sepharose as a support.
LPF was obtained by desorption with 0.1 M) Lys buffer containing 0.5 M NaCl and 3 M potassium thiocyanide.

LPF activity was determined by the enzyme antibody method (ELISA method) of Sahara et al., 28th Toxin Symposium (July 23-24, 1981)
B, Hachimantai, Iwate Prefecture) Lecture Abstracts, pages 141-144), the activity unit (u) of LPF is OD
400 mμ was drawn at a dilution factor of each sample giving 0.1 per unit volume (ml).

The results are shown in Figure 1.

From FIG. 1, it can be seen that Meβ-CD significantly increases the LPF production capacity per bacterial cell, especially under shaking conditions.

Incidentally, even when Meα-CD was used, similar results were obtained.

Example 2 An inoculum suspension obtained in the same manner as in Example 1 was mixed with Me
500 pieces of β-CD? SS medium 15 containing /r/ll
The cells were suspended in 0TLl at a concentration of 3.3×10 8 /rul, and cultured at 35° C. for a predetermined period of time under shaking conditions.

Culture time, turbidity of culture solution, and amount of LPF produced (L
Figure 2 shows the relationship with PF activity).

Note that the LPF activity was measured in the same manner as in Example 1.

From Figure 2, it can be seen that if the culture time exceeds at least 20 hours, M
Depending on the presence or absence of eβ-CD, the measurement of the culture solution, that is, the absolute amount of bacteria grown, does not differ much, but the amount of LPFO produced differs significantly, and the presence of Meβ-CD significantly increases the LPF production ability of B. pertussis. You can see that

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between the amount of Meβ-CD added to the culture medium, the turbidity of the culture solution, and the amount of LPF produced (LPF activity). FIG. 2 is a diagram showing the relationship between the culture time, the turbidity of the culture solution, and the amount of LPFO in the presence or absence of Meβ-CD addition to the culture medium.

Claims (1)

[Scope of Claims] 1. Biology, characterized in that microorganisms belonging to the Bordetella family are cultured in a medium containing cyclodextrin or its derivatives, and a substance having hemagglutination activity is collected from the culture. Method for producing clinically active substances. 2. The method for producing a biologically active substance according to claim 1, wherein the cyclodextrin derivative is an etherified cyclodextrin. 3. The method for producing a biologically active substance according to claim 2, wherein the etherified cyclodextrin is methylcyclodextrin.