JPS62135492A - Novel killer toxin - Google Patents

Abstract

NEW MATERIAL:The compound of the formula. It has following physical and chemical properties: molecular weight: 10,700; thermal stability: 75% residual activity after heat treatment at 120 deg.C for 10min; stable at pH: 4-11. USE:Antimycotic. It has high thermal resistance and high stability to pH. PREPARATION:A yeast in Hansenula, e.g., Hansenula mrakii (IFO 0895) is cultured in an enriched medium which the yeast assimilate by the shaking culture at 30 deg.C for 24hr, then the culture mixture is concentrated. The concentrate is subjected to the column chromatography filled with Sepharose to which the anti-K9 killer toxin monoclonal antibody is activated and immobilized. The eluate fractions are collected, dialyzed in distilled water to give the objective substance.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel killer toxin produced by yeast of the genus Hansenula.

[Conventional technology]

Makovrer and Pepin (B6v)
An) discovered that some yeast bacteria contain bacteria that kill yeast of the same species [Proc.
, Int, Congr, Genet,) XI
1゜202 pages, 1963]. The protein toxins produced by these yeasts are called killer toxins, and are called killer toxins.
ung) et al., the pattern of mutual killing
Too classified [Antonie VanLeauwenhoek]
ek) Vol. 44, pp. 59-77, 1978].

″′70: Lra Muraki (Hansenula mra)
Killer toxins derived from KII) are classified into Type 9 (K9 killer toxins) by Young et al.

Ultraviolet filtration method, molecular sieve/L/
By F' filtration method and ion exchange chromatography method, the molecular weight was 30.
000 or more and molecular weight a, 9o.

Two types of killer toxins were isolated [Agriculture and φ Biologica μ Chemistry (A
gric, Biol, Chem,) Volume 47, 29
pp. 53-2955, 1983, and JP-A-60-13
No. 0599].

The latter substance acts on the cell membrane and is thought to have a yeast-lethal effect (Agric, Biol, Che
m.

47, pp. 2953-2955, 1983).

[Problem that the invention seeks to solve]

An object of the present invention is to provide a novel killer toxin that is stable to temperature and pH and useful as an antibacterial substance.

[Means for Solving the Problems] To summarize the present invention, the present invention relates to a novel killer toxin represented by the following structural formula CI).

Gly Asp Gly Tyr Leu 1. le
Met Cys Lys AsnCys Asp Pr
o Asn Thr Gly Ser Cys Asp
TrpLys Gln Asn Trp Asn T
hr Cys Val Gly l1eGly Ala
Asn Val His Trp Met Val
Thr Gly - (T) Gly Ser Thr
Asp Gly Lys Gin Gly Cys
AlaThr Engineering 1e Trp Glu Gly
8er Gly Cys Val GlyArg
Ser Thr Thr Met Cys Cys
Pro Ala AsnThr Cys Cys
Asn Ile Asn Thr Gly Phe
Tyrlle Arg 8er Tyr Arg A
rg VaI Glu (in the formula, Gly is glycine, Asp
is aspartic acid, Tyr is tyrosine, Leu is leucine, Ile is isoleucine, Met is methionine, Cy
sit, cysteine, Lys is lysine, Asn is asparagine, Pro is proline, Thr is threonine, 8e
(r means serine, Trp means tryptophan, Gin means glutamine, Mal means valine, Ala means afnin, Hls means histidine, Argtiamine, Phe means phenylalanine, and Glu means glutamic acid). The inventors discovered that a new killer toxin belonging to the Crab type was produced and that this substance was useful as an antifungal agent, and completed the present invention.

Examples of the novel killer toxin-producing bacteria of the present invention include H. Muraki IFO0895.

In the method of the present invention, the strain is usually cultured in a medium containing nutrients that can be used by yeast.

As a nutrient source, any known nutrient source conventionally used for culturing Hansenula yeast can be used, preferably a minimal medium supplemented with amino acids and nucleic acids [α67% Difco Yeast Nitrogen Base (Difco Yeast Nitro
gen Ba5e ) vr10 amino acid group, 2 tiglucose, 400q adenine sulfate, 24119 ufusil, 24η tryptophan, 24 ■ histidine, 24! l
v arginine, 24 ■ methionine, 56q tyrosine, 5
6Wi leucine, 56η isoleucine, 361NI lysine, 1001n? Asbafugic acid, 150■valine, 2
00 ■ Threonine, 60 ■ Feniμ alanine 7t p
H5,5] is used. When this strain is cultured with shaking at 50°C for one day, the new killer toxin is mainly produced outside the bacterial cells.

K. Purification of novel killer toxin from culture medium.

Satscharomyces cerevisiae (
Saccharomyces cerevisiae)
5059 strain is used, and conventionally known methods for purifying antibacterial proteins are used, but preferably the method of Köhler and Milstein [Keller Mi/l/V Utahin] is used.
Kδhler, Milstein), Nature (N
A monoclonal antibody against a novel killer toxin, which was prepared by J. At, Ur., Vol. 256, p. 476 (1975), is used. The purification method will be described in detail below.

After culturing H. Murakie FO0895, it was sterilized and concentrated 400 times using Amicon YM5 (manufactured by Amicon).

This concentration'g! 4- Sephadex
Engineering) 050 column (Famacia 2.6X100c
Wl) and eluted with distilled water at 10-7 hours, and the A2g6 and killer activity of each fraction were measured. Killer activity was determined using the method of Bepin et al.
General Microbiology (J, Gen, Mic
[YPAD plate containing LO03% methylenebutylene (2
Chig course, 2 Chipolybedeton, 1ts yeast extract,
004% adenine sulfate, 2-chi agar) 1 strain of 5059
067p/deV-) was applied, a cup with a diameter of 8 gourd was placed, 100 μt of a fractionated solution obtained by Seven Fadex G50 was added, and the mixture was incubated at 30° C. for 2 days, and the diameter of the inhibition circle was measured.

(Creation of monoclonal antibody) Killer activity fraction 12' fractionated with Sephadex G50
Mice (BAL, B/C) were immunized with 5 μl in the abdomen, and 1 month later, 50 μg was injected into the tail vein.

Cell fusion was carried out by the method of O.L. et al. [Immunoglobulin-producing hybrid cell system, selected method in cellular immunity
hybrid cell 1ines, In 5el
ected methods 1ncellular
Immunology) Michelle, B.
andShiigL S, M, 1B, Free
and Co., pp. 551-572, 1980]. Monoclonal antibodies obtained by cell fusion were subjected to primary screening using a solid phase method. The solid phase method uses killer activity fraction 50 fractionated with Sephadex G50.
μt (10 μg/− in physiological saline) was solidified on a polyvinyl microtiter plate (manufactured by Costar) at 4°C.
It was left overnight. After washing the plate three times with saline, 0
．． 100 µt of 25% tallow serum containing 1 t NaN3 was added and kept at room temperature for 1 hour. Next, after washing 5 times with physiological saline, 50μt of monoclonal antibody obtained by cell fusion
was added and held at 37°C for 3 hours. After washing the plate, use horseradish, μ-ocintase, and anti-mouse IgG.
50 μt was added, and the mixture was reacted at 37° C. for 3 hours, washed four times with physiological saline, and then colored with 0-phinidinediamine. -A secondary screening was performed on the antibodies obtained in the secondary screening using the ability to neutralize the killer activity as an index as follows.

After diluting the killer toxin and the monoclonal antibody obtained in the primary screening in YPAD liquid medium,
5 μt each in a 96-well microplate (manufactured by NANC)
The mixture was mixed inside and kept at 37°C for 1 hour. Furthermore, 50 μt of stationary phase cells of the S. cerevisiae strain 5059 diluted in YPAD liquid medium were added to the wells at a concentration of 4×10′ cells/L//− (finally 2000 cells/1//well), and the cells were incubated at 30°C. Incubate the mixture overnight and examine the growth of S. cerevisiae strain 5059. As a control, a monoclonal antibody (anti-HBs) unrelated to killer toxin was used.

When 250μ97ml of antibody (anti-HBs) was reacted with dog killer toxin, the killer activity was reduced to 0.5μg/-. In contrast, the antibody was added at 250μg/Gi
25μg/mt and λ5μg/-, 10μ
g/- or more, 2.5 μg/ld, and 0.5 μs/fR
They were found to have neutralizing activity against the T killer toxin.

As described above, the antibody obtained in the sequential screening was found to be an anti-dog killer toxin monoclonal μ antibody that has the ability to neutralize dog killer activity.

(Purification of Killer Toxin by Affinity Lamb Chromatography) An anti-4-kiotoxin monoclonal antibody was immobilized on activated Sepharose 4B at a concentration of 10 m/- ((L
9X15cM 5ml bed volume) H, A Raki I
FO089517) Pour 200 times concentrated culture solution 3-
I took the bath at 7-7 o'clock. After neutralizing the elution section with NaOH,
The active fractions were collected and dialyzed in distilled water to obtain a new killer toxin.

(Physical and chemical properties of novel killer toxin) (1) Amino acid composition and amino acid sequence The amino acid composition is shown in Table I.

The amino acid composition is based on reduced pyridylethylation (R
PE), hydrolyzed in the presence of 6NHC/, 1% thioglyco-/l' acid at 110°C for 24 hours in a vacuum sealed tube,
Amino acid analysis was performed using a Hitachi high-speed amino acid spectrometer model 855.

Reductive pyridylethylation was performed as follows.

Protein Table I Asp 4.46911.08 (11) 12Th
r A214 7.97 (8) 9Set 1
．． 854 4.60(5) 5Glu 1.54
3 582 (4) aPro O, 5611,
39(1) 2Gly! L21812.94(
13) 15Ala 1.53a A32(5)
3Val 1.986 4.92 (5)
5Met 1.200 2.97 (5) 311
e 1.964 4.87(5) 5Leu
Q, 410 1.02(1) I
Tyr 1.206 2.99(3) 3Phe
Q, 454 1.13(1) ILys O
,9982,47(3) 3His O,51?
0.79(i) IArg 1.615 4
．． 00(4) 4Trp 1.250 3115
(3-4) 4(2) Amino acid sequence The amino acid sequence determination method is as follows.

500μg of killer toxin (0.13M) Lis-HC
L (pH 7,6), 6M guanidine hydrochloride, 10mM
37°C in the presence of DTT (reaction volume-ii 500 μt)
Restored overnight. Next, add 4-vinylpyridine to a final volume of 50m
Proteins were separated by reverse-phase HPLC under an acetonitrile gradient of 0 to 50 cm (Yamamura Kagaku).

Decomposition with various enzymes was performed as follows.

<Tosylaminopheni p-ethylchloromethyp-ketone (T
PCK)-) Lipsin〉 RP E-Evening 7 Baku 1'! rNH4HCO3(
pH 8) Soluble in 200 μt, almost insoluble) TP
Add 5 μg of CK-1-ribusin (F, /S11-10
0) Digested at 37°C for about 24 hours.

After digestion, the precipitate is centrifuged, the supernatant remains as it is, and the precipitate is 0.1
It was dissolved in TFA/H20 and separated by HPLC.

<Lysyl endobedetidase> 100μt of 0.2M in 100μt of unprocessed protein
2-amino-2-methiμm 1,5-propanedio-/L
'(pH 9,5), add enzyme CLO5AU and add 37
Digested at ℃ for 24 hours.

After digestion, 5 μL of IMDTT was added and the mixture was reduced at 90° C. for 5 minutes, followed by separation by HPLC.

<Staphylococcus aureus (Staphylococcus aureus) V
8 Protease> RPE-protein was dissolved in 200 μt of 105M ammonium acetate buffer (pH 4,0), 5 μl of enzyme was added, and the mixture was digested at 37° C. for 24 hours. After digestion, it was directly separated by HPLC.

<α-Chymotrypsin> RPE-protein is completely dissolved in a small amount of α1% TFA) Then 200 μt of 1 NH4HCO3 (p
H8) was added. (Becomes a suspension because it is insoluble) Here α
- 5 μg of chymotrypsin was added and the mixture was digested at 37° C. for 6 hours.

After the digestion, the precipitate was completely dissolved and was then separated by HPLC.

The amino acid sequences of the fragments separated by HPLC were determined using an ABTSID Biosystems 470A protein sequencer.

The amino acid sequence of the novel killer toxin determined by the above method is shown in formula [I].

□ Trypsin □ □ Lysi μ endopeptidase □ 1←--Chymotrypsis ζ Gly Ser Thr Asp Gly Lys G
ln Gly Cys Ala-lysiμ endopeptidase → ←-1 trypsin □ chymotrysin Thr Ile Trp Glu Gly Ser G
ly Cys Val Gly□Trypsin□ Chymotrypsin→1 1←--V, Protease□T
hr Cys Cys Asn Ile Asn
Thr Gly Phe TyrV, protease □ 2) Molecular weight: 1 to 700 3) Temperature stability: After treatment at 120°C for 10 minutes, residual activity is approximately 75%.

4) pH stability Stable at room temperature and pH 4-11.

A new killer toxin exhibits antibacterial activity against fungi.

The antifungal spectrum is shown in Table 3 below.

Table ■ 1% yeast extract, pH 6.5) was used.

As mentioned above, the new killer toxin has an antibacterial effect on pathogenic fungi and is therefore useful as an antifungal agent.

The novel killer toxin of the present invention has antifungal activity by inhibiting the biosynthesis of β-1,3-glucan, which is a cell wall component of the above-mentioned fungi.

〔Example〕

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples.

Example 1 1) Culture Killer Yeast H, Muraki IFO0895 in a minimal medium supplemented with amino acids and nucleic acids (0.67% Difco Yeast Nitrogen Base V10 amino acid group, 2% glucose, 400η adenine sulfate, zaη Urashi A/, 24 mg ) Ridetophan,
24 ■ Histidine, 24 Misaki Apginine, 24 ■ Methionine, 36 ■ Tyrosine, 56q Leucine, 56Wq Isoleucine, 361N! Lysine, 100η aspartic acid,
3 containing 1 t of 1501111 valine, 200 ■ threonine, 60 η pheniμ alanine / 1 pH a 5) was cultured with shaking at 30°C for 1 day in a volumetric flask, and after sterilization, the culture solution was incubated with Amicon YM-5 (manufactured by Amicon) for 2 days. It was concentrated 1,000 times to obtain a 5-culture solution.

2) Purification The 200-fold concentrated solution of IFo 0895 strain obtained in 1) was purified by Affinity chromatography using the monoclonal μ antibody shown below.

(Immunization) Transfer the concentrated solution 4WIl obtained in 1) to Sephadex G-5.
0 column (2.6 x 100 c1n) with distilled water.
Each fraction (5
-) A280 value and killer activity were measured. Killer activity was determined by YPAD plate containing 0.003% methylene blue (
2 Tig μ course, 2% polypeptone, 1 T yeast extract,
0.04% adenine sulfate, 2% agar) and 106 cells/I of cerevisiae 5059 strain (killer-susceptible diploid strain).
//After coating the plate, place a cup with a diameter of 8 gourds, add 100 μt of Sephadex G-50 fractionated solution, and add 30 μt of fractionated solution.
After culturing at ℃ for 2 days, the diameter of the inhibition zone was measured. Mice (BALB/C) were immunized abdominally with 125 μl of the killer active fraction fractionated with Sephadex G-50, and 50 μg was injected into the tail vein one month later.

(Cell Fusion) Five days after the final immunization, the mouse pancreas was taken out and a single cell suspension was prepared. This lX10' pancreatic cells and lX10
' mouse myeloma cells were fused using 50 tipolyethyl glycol (molecular weight 1500). Cells were distributed into four 96-well microplates. After 24 hours, half of the supernatant was replaced with HAT medium (hypoxanthymine/aminopterin/thymidine) and HAT-resistant cells were observed to proliferate after 2 weeks.

(Hamanuki of anti-killer toxin monoclonal antibodies) Monoclonal μ antibodies obtained by cell fusion were primarily screened by solid-phase method. The solid phase method was performed as follows. 50 μt (10 μg/ml) of the killer activity fraction fractionated with Sephadex G-50 column chromatography was solid-phased on a polyvinyl microtiter plate (manufactured by Costar),
It was left standing at 4°C overnight. After washing the plate five times with physiological saline, 100 μt of 25-tuber fat serum containing 0.1% sodium azide was added and kept at room temperature for 1 hour. Next, after washing three times with physiological saline, 50 μt of the monoclonal antibody obtained by cell fusion was added and maintained at 37° C. for 3 hours. After washing the plate, 50 µt of anti-mouse IgG coated with horseradish peroxidase was added and reacted at 37°C for 3 hours, washed, and colored with 0-phenylenediamine.

-Secondary screening was performed on the antibodies obtained in secondary screening using the ability to neutralize killer activity as an index as follows.

After diluting the killer toxin and the monoclonal antibody obtained in the next screening with YPAD liquid medium,
25 μt portions were mixed in a 96-well microplate (manufactured by NANC) and held at 37° C. for 1 hour. Furthermore, in this large medium, S, Cereviche 5059 diluted with YPAD liquid medium was added.
Add 4 x 1 o4cells/m1oaK cells (2000 cells/well at R end) and overnight at 50°C. i feed, 50
The growth of 59 strains was investigated. As a control, a monoclonal antibody (anti-HBs) without killer toxin was used.

(Killer toxin affinity chromatography made by Yuzu) A column (11
IF obtained in 1) on 9Xi 5m, 3-bed volume)
1.51 Rt of a 200-fold concentrated solution of O0895 strain (containing 210 μl of killer toxin) was added and eluted at 2.7 d/hour. Washing was (115M NaCl, elution was 0.2M
Each reaction was carried out with glycine-HCl (pH 2, 2), and the washing section was left as is, and the elution section was neutralized with sodium hydroxide solution, and the amount of killer toxin (μm1/rnt) was measured based on a calibration curve determined in advance. . The protein content was also measured according to the method of Lo'vry et al. As a result, killer toxin was obtained for 157 μs.

〔Effect of the invention〕

As explained in detail above, the present invention provides a structurally novel killer toxin with excellent heat resistance and pH stability.

Claims (1)

[Claims] 1. Structure of the following formula [1]: ▲There are mathematical formulas, chemical formulas, tables, etc.▼... [I] (In the formula, Gly is glycine, Asp is aspartic acid, T
yr is tyrosine, Leu is leucine, He is isoleucine, Met is methionine, Cys is cysteine, Ly
s is lysine, Asn is asparagine, Pro is proline, Thr is threonine, Ser is serine, Trp is tryptophan, Gln is glutamine, Val is valine, A
A killer toxin characterized in that la means alanine, His means histidine, Arg means arginine, Phe means phenylalanine, and Glu means glutamic acid.