TITLE OF INVENTION CHEMICAL SUBSTANCE FOR PROTECTION OF BRAIN NERVE CELL AND METHOD FOR PRODUCING THEREOF TECHNICAL FIELD This invention relates to chemical substance for protection of nerve cell and method for producing thereof. In more detail, this invention focuses on chemical substance for protection of nerve cell with new activated prevention function and method for producing thereof, by classifying and identifying Streptomyces sp. 60910 strain, which produces complestatin, an aromatic peptide chemical compound with protection activation of brain nerve cell, from which the prevention active material is separated and purified, and its physicochemical structure and biochemical activity is measured.

BACKGROUND ART In general, apoplexy (palsy, paroxysm) is a disease, which causes regional neurologic disturbances induced by bleeding into ventricle tissue and subarachnoid intracavitary from cerebral artery of intracranial portion caused by sudden blocking of cerebral blood flow or trauma, and also which accompanies successive brain. dysfunction and its equivalent neurologic and mental symptoms. It was reported that about 75% of those apoplexy cases were caused by occlusion of cerebral artery, about 11% by cerebral hemorrhage, and 5% by bleeding in subarachnoid intracavitary [Boulton et al, Neuromethods, Vol. 22 (1992)]. Every year many apoplexy patients were

broken out and about one third of them consequently died. But development activities for ischemic apoplexy therapeutics were not sufficient.

Therefore, researches on apoplexy treatment have been carried out and showed that up to now some materials have treatment effects, including neurotrophic factor such as BFGF (basic fibroblast growth factor), NMDA (N-methyl-D-aspartate), non-NMDA receptor antagonist, lazaroid-series drug such as 21-aminosteroid, free radical scavanger, nitric oxide inhibitor, interleukin-l, 9 inhibitor, polyamine binding area receptor existing in NMDA receptor area, antisense oligonucleotide for glutamate receptor, body temperature depression, or ganglioside. Among them, excitatory amino acid receptor such as NMDA and non-NMDA were known to be closely related to brain nerve cell damages caused by ischemic apoplexy, delayed paroxysm, hypoglycemia-or hypoxia-induced ischemia, and acute brain trauma.

On the other hand, brain nerve cell kill from brain disease such as apoplexy is caused by glutamate, a nerve transmitter substance. In normal state, glutamate secreted at optimum concentration executes the function as a nerve transmitter substance, while in cerebral ischemic state it excessively secretes and thus causes diseases such as apoplexy by inducing excitotoxicity, which kills nerve cell. It is known that, because of apoplexy, brain nerve cell excessively secretes glutamate, which activates ion channel connected to NMDA receptor and non-NMDA receptor and flows extracellular Ca2+ into cell, thus killing nerve cell. The excitotoxicity is caused by excessively secreted glutamate, which. activates glutamate-receptor, NMDA, and AMPA/Kainate receptor, through those receptors large amount of Ca2+ flows into cells. That is, Ca2+ ion flowed excessively into cell activates Ca2+- dependent enzymes such as phospholipase A2 (PLA2), nitric oxide synthetase (NOS), protease, and endonuclease within cell, and those activated enzymes

create excessively active oxygen such as superoxide and hydroxyl radical inside cell, thus breaking the oxidation reduction balance, and this active oxygen randomly destroys DNA, protein, and lipid, thus consequently killing brain nerve cell.

Based on those facts, many researches on chemical substance for protection of brain nerve cell against brain diseases have been carried out and reported NMDA-type receptor antagonist such as MK-801 (Buchan et al, Stroke 21: 9,1990), Ketamine, PCP, D-AP5, and CPP, AMPA/KA-type receptor antagonist such as CNQX, DNQX, NBQX, and LY215490, metabolism control-type receptor antagonist such as L-AP3. For some of those chemical compounds, pre-clinical researches have been carried out and showed that MK-801 is not suitable for clinical treatment due to its safety problem and development was suspended since all caused serious toxicity.

To develop treatment drug for brain disease caused by excitotoxicity of brain nerve cell, therefore, chemical substance for protection of brain nerve cell and glutamate-receptor antagonist without toxicity and side effects are necessary to be developed. Because of those necessities, searches for chemical substance protecting brain nerve cell are now under progress by largely applying two methods. First method is for searching glutamate receptor antagonist as a blocking material to directly cut off excitotoxicity of nerve cell, and second one for searching antioxidant chemical compound to block oxidative toxicity induced within cell.

To develop treatment drug for brain disease accompanying excitotoxicity of nerve cell, this invention is about glutamate receptor antagonist, a chemical substance for protection of brain nerve cell, produced through primary cultivation of mouse cortical neuron. The glutamate receptor antagonists known up to now are mostly synthetic chemical compounds with

strong toxicity, and thus there have been many problems in developing those as treatment drug for apoplexy. But this invention found out glutamate receptor antagonist from Streptomyces sp. culture solution, by primary cultivating, injecting, and surveying low toxic or nontoxic chemical substance for protection of brain nerve cell and glutamate-receptor antagonist extracted from microorganism metabolic products or natural substance into mouse cortical neuron and chick telencephalic neuron. And this invented chemical substance was identified as complestatin, by finding out its chemical structure and physicochemical characteristics, by establishing manufacturing methodology, and by identifying pharmacological activation. Also this invention showed that, through new modus operandi, this chemical substance has special features in suppressing brain nerve cell kill caused by excitotoxicity of nontoxic NMDA and AMPA/KA and brain nerve cell kill caused by hypoxia and anoxia.

In this invention, complestatin, with special features of nontoxically and completely suppressing brain nerve cell kill caused by glutamate through new modus operandi [Kaneko, I., Kamoshida, K. and Takahashi, S.

J. Antibiotics, 42: 236-241, (1989)], is an aromatic peptide chemical compound reported as containing anticomplement activation, anti-HIV antibiotics, and new vasculogenesis inhibition activation.

This invention succeeded in separating and purifying complestatin, a low toxic chemical substance for protection of brain nerve cell with new mechanism to be reported yet, which acts as antagonist to glutamate-receptor including both NMDA-and AMPA/KA-receptor and suppresses brain nerve cell kill caused by hypoxia and anoxia, by effectively separating complestatin from Streptomyces sp. containing protection activation for brain nerve cell and by identifying activation mechanism.

When manufacturing drugs including complestatin compound invented here as effective ingredient, they could be injection or oral inoculation such as tablet, pill, granulum, soft-hard capsule, pulvis, granum, power, emulsion, syrup, pellet, and so on. Pharmaceutical preparation containing complestatin compounds as an effective ingredient can also be easily done in accordance with commercial laws.

The objective of this invention, therefore, is to provide Streptomyces sp. 60910 strain, which produces chemical substance for protection of brain nerve cell. Another objective of this invention is to provide chemical substance for protection of brain nerve cell produced by the above Streptomyces sp. 60910 strains and its method for producing thereof.

DETAILED DESCRIPTION OF THE INVENTION The objectives of this invention were accomplished by identifying and cultivating Streptomyces sp. 60910 strain with protection activation of brain nerve cell, then analyzing the structure and characteristics of chemical substance for protection of brain nerve cell produced by the strain after separating this substance from centrifuged equivalent liquid and bacterial cell, and then measuring the inhibitory effect of this active material against nerve cell kill caused by excitotoxin such as NMDA (N-methyl-D-aspartate), AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionate), and Kainate and caused by oxygen-glucose, deprivation as ischemic brain disease model.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the graph generated by LDH measurement on the

death rate of mouse brain nerve cell after 24 hours of treating 20 11 M NMDA, 40 11 M Kainate (KA), and 30 11 M AMPA and chemical substance invented here for protection of brain nerve cell at the same time, after cultivating mouse cortical neuron.

Figure 2a shows the graph generated by LDH measurement on the death rate of mouse brain nerve cell after 24 hours of treating 10-1000 11 M NMDA and chemical substance invented here for protection of brain nerve cell at the same time, after cultivating mouse cortical neuron.

Figure 2b shows the graph generated by LDH measurement on the death rate of mouse brain nerve cell after 24 hours of treating 10-100 ii M Kainate (KA) and chemical substance invented here for protection of brain nerve cell at the same time, after cultivating mouse cortical neuron.

Figure 2c shows the graph generated by LDH measurement on the death rate of mouse brain nerve cell after 24 hours of treating 10-100 11 M AMPA and chemical substance invented here for protection of brain nerve cell at the same time, after cultivating mouse cortical neuron.

Figure 3a is the graph showing measurements on cytotoxicity after 80 minutes of cultivation from adding chemical substance for protection of nerve cell invented here 110 11 M, NMDA receptor antagonist MK-801 10 11 M or AMPA/kainate receptor antagonist CNQX 20 p M into culture medium, when in vitro cultivation of nerve cell with oxygen-glucose deprivation.

Figure 3b is the graph showing measurements on cytotoxicity after 20 hours of cultivation from adding chemical substance for protection of nerve cell invented here 0.1,1,5,10 li M and apoptosis inhibitor Z-VAD, when cell kill was induced by hypoxia after endothelial cell was cultivated in vitro.

Figure 4a shows measurements on Ca2+ amount flowed into cell by fura-2 dye after inducing Ca2+ flow into cell by treating 100 11 M NMDA to

mouse cortical neuron cultivated.

Figure 4b shows measurements on Ca2+ amount flowed into cell by fura-2 dye after inducing Ca2+ flow into cell by treating 100 li M NMDA and chemical substance for protection of brain nerve cell invented here simultaneously to mouse cortical neuron cultivated.

Figure 4c shows measurements on Ca2+ amount flowed into cell by fura-2 dye after inducing Ca2+ flow into cell by treating 100 11 M NMDA, and treating chemical substance for protection of brain nerve cell invented here before NMDA treatment to mouse cortical neuron cultivated.

BEST MODES FOR CARRYING OUT THE INVENTION The composition and function of this invention are described in detail, as following. This invention is composed of several stages; stage for classification and identification of Streptomyces sp. strain with protection activity for brain nerve cell and thus identification of new StreptonZyces sp.

60910 strain producing chemical substance for protection of brain nerve cell and analyses of cultivation condition and characteristics of the strain; stage for separation of active material from equivalent liquid and bacterial cell by centrifugation after cultivating Streptomyces sp. 60910 in G. S. S culture medium (glucose 10g, soluble starch 20g, soybean meal 25g, meat extract lg, yeast extract 4g, NaCl 2g, K2HP04 0. 25g, CaCO3 2g, pH 7.2, D. W 1L); stage for identifying the active material as complestatin, an aromatic peptide chemical compound by surveying UV and IR spectrum absorption, by measuring molecular weight of this active material through FAB and ESI- mass measurement, and by deciding molecular structure from NMR spectrum; stage for excitotoxicity inhibition capability of chemical substance for

protection of brain nerve cell by quantifying amount of lactate dehydrogenase (LDH) after treating NMDA of acute toxicity, non-NMDA excitotoxin AMPA with prolonged neurotoxicity and Kainate, and chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 strain simultaneously after cultivating chick telencephalic neuron separated from embryo of fertilized egg and mouse cortical neuron separated from mouse embryo; stage for measuring nerve cell protection activity of chemical substance for protection of brain nerve cell by conducting oxygen-glucose derivation experimentation as in vitro ischemia model and by conducting in vivo mouse retinal ischemia model; stage for surveying if nerve cell protection activation of this invented chemical compound is induced by antioxidation activation, from measurement of antioxidation activation after treating Fe 50 u M, H202 150 ii M, BSO 10 mM to mouse cortical tissue of primary cultured mouse; and stage for surveying effect of this chemical substance for protection of brain nerve cell on Ca2+ accumulation into cell caused by excitotoxin such as NMDA, by using Fura-2, a Ca2+-binding dye.

The specific methods of this invention are described in detail by citing practical application examples as following, but right scope of this invention is not limited only to them.

EXAMPLE 1: Classification and identification of the strain producing chemical substance for protection of brain nerve cell The Streptomyces sp. strain showing protection activation for brain nerve cell was classified and identified, by surveying its cultural, morphologic, and physiologic characteristics. For cultural characteristics, color of aerial hyphae, color and viability of basal hypha, and water soluble pigment

formation were surveyed every 7 days, while cultivating for 21 days on yeast extract-malt extract agar medium, oatmeal agar medium, inorganic salts- starch agar medium, glycerol-asparagine agar medium, peptone-yeast extract- iron agar medium, tyrosine agar medium, glucose-asparagine agar medium, Bennet's agar medium, and nutrient's agar medium. For morphologic characteristics, shape and size of spore, shape of exosporium, and shape of spore chain were surveyed by optical and electron microscope. For physiologic characteristics, melanin formation, gelatin liquefaction capability, starch hydrolysis, and carbon source use were surveyed.

The strain producing chemical substance for protection of brain nerve cell grew very well on yeast extract-malt extract agar medium, oatmeal agar medium, inorganic salts-starch agar medium, glycerol-asparagine agar medium, tyrosine agar medium, glucose-asparagine agar medium, Bennet's agar medium, and nutrient's agar medium, and gray-color aerial hypha were formed in abundance. The color of basal hypha was light yellow and did not form water-soluble pigment. The spore was cylinder-like shape and its size was 0.7-0.8 x 0.8-0.9 um, with the smooth surface. The spore chain was spiral and formed with 10-20 resting spores. It did not form melanin and showed negative reaction to milk peptonization and nitric acid reduction, but positive to milk agglutination and hydrolysis of starch and skim milk. The diaminopymeric acid of cell wall was L, L shape. The survey of carbon source usability showed that D-glucose, D-mannitol, raffinose, and D-galactose used it very well, D-xylose, L-rhamnose, sucrose, cellulose used it normally, and L-arabinose, inositol, and D-fructose did not use it.

Based on the above taxonomic characteristics, it was known that the strain identified as above belonged to Streptomyces sp. Inventors of this invention deposit this strain at Korean Collection for Type Cultures (KCTC)

with deposit no. of KCTC 0843 BP on July 28,2000 and named this strain as Streptomyces sp. 60910.

EXAMPLE 2: Culture of the above Streptomyces sp. 60910 producing chemical substance for protection of brain nerve cell, and isolation and purification of chemical substance for protection of brain nerve cell First step: Culture of Streptomvces sp. 60910 producing chemical substance for protection of brain nerve cell To culture Streptomyces sp. 60910 strain producing protection active material for brain nerve cell as the above example 1, G. S. S (glucose lOg, soluble starch 20g, soybean meal 25g, beef extract lg, yeast extract 4g, NaCI 2g, K2HP04 0.25g, CaCO3 2g, pH 7.2, D. W 1L) culture medium was used.

The G. S. S culture medium of 100 mt was split into the 500 mt Erlenmeyer flask, in which Streptomyces sp. 60910 strain producing protection active material for brain nerve cell as above was inoculated and pre-cultivated for 2 days (28°C, 150 rpm).

The above pre-cultured solution of 3 me was inoculated in 30 baffle Erlenmeyer flask of 500 m, in which same culture medium of 100 m was already split, and then cultured for 5 days at same condition as above.

Second step: Isolation and identification of chemical substance for protection of brain nerve cell The protection active material for brain nerve cell was isolated from the above Streptomyces sp. 60910 strain and purified.

The culture solution in 2nd step, 3L, was centrifuged into equivalent liquid and bacterial cell. The equivalent liquid was adjusted at pH 4 by using

IN HC1, and then extracted by ethyl acetate and solvent layer was concentrated, then silica gel column chromatography was applied by using CHCl3-MeOH (10: 1-1: 4) as developing solvent. The activated fraction was stored for one day at low temperature and then sediment generated was filtered and purified to get active material separated. The bacterial cell resulted from centrifugation of the above material was extracted by 70% acetone, put under decompressed concentration, adjusted to be at pH 4 by applying IN HC1, and then extracted by ethyl acetate through solvent extraction. The ethyl acetate was put under decompressed concentration, dissolved in MeOH, and stored at low temperature, and consequently white sediment was generated. This sediment was filtered and purified, and then same active material as compound separated from the above equivalent liquid was isolated.

EXAMPLE 3: Physicochemical characteristics of complestatin, chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 The compound of the above example 2 showed the physicochemical characteristics as following. It was yellow powder with molecular weight of 1325 determined by FAB and ESI-mass measurement, and its molecular formula was determined as C6lH45N7015Cl6 from Mass data and 1H, 13C NMR spectrum, and NMR spectrum for other structural interpretation. The measurement of its UV spectrum by using MeOH as solvent showed that maximum absorption rate was at 280 and 290 nm. Also, survey of its solubility showed that it dissolved well in DMSO, but did not dissolve in other solvent. Its Rf value on TLC was measured at 0.09, when

using 40% of ACN and 60% of citrate buffer (0. 05M, pH6.0) as developing solvent.

EXAMPLE 4: Chemical structure of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 To find out chemical structure of the above chemical substance for protection of brain nerve cell, one dimensional NMR including lH, l3C, and DEPT, and two dimensional NMR spectrum including COSY and HMBC were measured. Based on those NMR data and physicochemical characteristics of practical application example 3, chemical structure of the above protection active material for brain nerve cell was interpreted and consequently protection active material for brain nerve cell was identified as complestatin, an aromatic peptide chemical compound. Its structural chart was as general formula shown below.

EXAMPLE 5: Bio-activation examination of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910

First step: Activation measurement of chemical substance for protection of brain nerve cell produced by Streptomvces sp. 60910 through LDH assay To measure activation of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 as the above practical application example 1, chick telencephalic neuron separated from embryo of fertilized egg and mouse cortical neuron separated from mouse fetus were cultivated. The chick telencephalic neuron, separated from embryo of fertilized egg, was cultivated in 48 well microplate for 5 days at concentration of 5x 105 cell and then 300 p M Kainate inducing brain nerve cell kill and complestatin, a protection active material for brain nerve cell were treated simultaneously. Then 24 hours later, viable cells were counted per unit area, by surveying kill inhibition degree of nerve cell through optical microscope.

Also, mouse cortical neuron, separated from mouse fetus of 15 day-pregnant, was cultivated for 12-14 days and then NMDA (N-methyl-D-aspartate), AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionate), and Kainate, all inducing nerve cell kill, and complestatin, a protection active material for brain nerve cell were treated simultaneously. After 24 hours of treatment, viability of cell was surveyed by optical microscope and LDH assay was conducted to quantify the amount of lactate dehydrogenase (LDH), and their results were shown in Figures 1 and 2.

When treating excitotoxins such as NMDA and Kainate, and complestatin into primary mouse cortical neuron, and surveying toxin inhibition capability of complestatin, a protection active material for brain nerve cell, it was shown that swelling of cortical neuron caused by 20 M of NMDA and 40p M of Kainate was completely impeded by 10p M of chemical substance for protection of brain nerve cell (refer to Figures 2a, 2b, and 2c). Also, when activation of chemical substance for protection of brain

nerve cell invented here was surveyed against excitotoxicity of NMDA and AMPA/Kainate, nerve cell kill induced by NMDA and Kainate/AMPA was inhibited at concentration of 3-20 li M, and excitotoxicity of NMDA and Kainate/AMPA was completely impeded at concentration of 3 Oil M (Figure 1). In primary mouse cortical neuronal culture, cytotoxicity of this chemical compound was very lower, compared to that of other NMDA, AMPA/kainate receptor antagonist reported up to now.

To determine whether nerve cell protection activation of chemical substance for protection of brain nerve cell invented here was competitive or non-competitive type, 10-10001l M of NMDA (short term), 10-10001 M of Kainate and 3-10001l M of AMPA were treated and 24 hours later activation of chemical substance for protection of brain nerve cell was measured. The result showed that this material had strong protection activation for nerve cell against NMDA, Kainate, and AMPA of diverse concentration identically, and from those results activation of chemical substance for protection of brain nerve cell of this compound was found out to be of non-competitive type.

Second step : Activation measurement of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 using In vitro and in vivo ischemia model To find out if chemical substance for protection of brain nerve cell with excitotoxicity inhibition effectiveness invented here protect brain nerve cell in real ischemia model, oxygen-glucose deprivation experiment used as an in vitro ischemia model was carried out.

In general, if brain nerve cell was put under oxygen-glucose deprivation condition for 60-80 minutes, then most nerve cells were killed by ischemia type. But, if 1011 M of complestatin was added, nerve cell kill

caused from oxygen-glucose deprivation was inhibited and the inhibition capability was strong protection activation for nerve cell, similar to simultaneous treatment effects of MK-801 (NMDA antagonist) and CNQX (AMPA/kainate antagonist) combined (Figure 3a). Also, this compound completely inhibited death of nerve cell as well as endothelial cell induced by hypoxia (Figure 3b).

The in vivo experiment was conducted using mouse retinal ischemia model. When intraocular pressure of a mouse was kept high at 160-180 mmHg for 90 minutes, thus ischemia was induced and then this invented compound was treated, chemical substance for protection of brain nerve cell invented here inhibited cell kill, depending on concentration. When mouse retinal ischemia was induced and nerve cell protection activation for inner nuclear layer and ganglion cell layer was measured, about 50-60% of nerve cells in control group not treated with chemical substance for protection of brain nerve cell invented here were killed, because of retinal ischemia.

Meanwhile over 90% of nerve cells in experimental group treated with intravitreal injection of 0.5-1 nM of chemical substance for protection of brain nerve cell survived, depending on the concentration.

Third step: Antioxidation activation measurement of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 against oxidative toxicity To determine the nerve cell protection effect of chemical substance for protection of brain nerve cell by inhibiting oxidative toxicity within cells, primary cultured mouse cortical cell was treated with Fe2+ 501l M, H202 150p M, BSO lOmM, respectively, and then antioxidation activation was measured. The result showed that the oxidative nerve cell death caused by

Fe2+, H202, and BSO treatment was not inhibited by this invented compound, and consequently nerve cell protection activation of this invented compound was found out to be not antioxidation activation.

Activation of Glutamate-receptor caused Ca2+ accumulation within cell, and increased Ca2+ in cell promoted the in-flow of other ions and water molecule, and finally nerve cell kill was induced due to oxidative toxicity.

From these results, it was found out that this compound revealed special protection effect against excitotoxicity.

To clarify this unique mechanism, effect of this compound to Ca2+ accumulation within cell by NMDA was surveyed. To prove Ca2+ accumulation within cell by NMDA, Fura-2, a Ca2+-binding dye within cell was used and this method was to clarify Ca2+ accumulation status determined by excitement rate of Fura-2 combined with Ca2+ and Fura-2 not combined with Ca2+ at 340 nm and 380 nm of UV, respectively, after dissolved by esterase within cell. When 100p M NMDA was applied to cortical cell dyed by Fura-2, C2+ accumulation within cell was induced within a few minutes (Figure 4a). But simultaneous treatment of this invented chemical compound and NMDA, or pre-treatment of this compound inhibited Ca2+ accumulation by NMDA considerably or completely (Figures 4b and 4c).

By electrophysiological test it was clarified that this invented chemical compound acted as a reversible receptor antagonist, and receptor binding activity was tested on AMPA, Kainate, NMDA, glycine, phencyclidine, and polyamineS reported so far as glutamate-receptor agonist but no activation was shown. It was assumed, therefore, that binding region unique to this invented chemical compound existed in nerve cell receptor.

INDUSTRIAL APPLICABILITY As clarified in the above practical application examples, this invention was effective in providing chemical substance for protection of brain nerve cell containing nerve cell protection activation, which inhibited brain nerve cell kill induced by excitotoxin such as NMDA, AMPA, and Kainate, and in providing aromatic peptide chemical compounds containing new brain nerve cell protection activation, not existent before, through chemical modification or diverse alteration of this invented chemical compound. Also, this invented chemical compound had different modus operandi from other chemical compounds already known, and could be of wide and effective use in prevention and treatment drugs for brain disease such as apoplexy, food, and reagent of brain nerve cell-related researches because of no toxicity in drugs. This invention, therefore, would be of great use in health improvement of the general public and pharmaceutical manufacturing industry.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL (PCT Rule 13bis) A. The indications made below relate to the deposited micmonanism or other bioloeical material referred to in the description on page 9-10 line 9: 25-27-10 : 1-2. B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution . Korean Collection for Type Cultures (KCTC) Address of depositary institution (includtng postal code and country) Korean Collection for Type Cultures (KCTC) #52, Aeun-dong, Yoosung-gu, Daejon-city 305-333 Republic of Korea Korea Research Institute of Bioscience and Biotechnology (KRIBB) E Date of deposit Accession Number 2000.7.28. KCTC 0843BP C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE fly the indications are notfor all desigsated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau'later (specify thc seral name of the tnd catiorw e."Aocession Ntunberof Dwit7 For receiving Office use only For International Bureau use only, This sheet was received with the international application This sheet was received by the International Bureau on: Authorized officer Authorized officer