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`knowledge and belief. This translation is performed and edited by Japanese to
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`
`Executed this _l__8_T day of June, 2012.
`
`
`
`Sayuri (Saya) Rupani-Hayes
`Translation Department
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`. STATE 0 NEW YORK
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`EXHIBIT 1010
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`
` Legal Advantage
`
`Legal Advantage LLC
`Washington DC
`10411 Motor City Drive
`Suite 750, Bethesda, MD 20817
`Phone: 30l—637~6180
`Fax: 800—804—4220
`
`CERTIFICATION OF TRANSLATION
`
`Original text full file name: JP58170492A.
`Translation full file name: JP58170492A En.
`
`This is to certify that the attached document in English language with the
`above title is a true and accurate translation, and has the same meaning as the
`text of the original document of the Japanese language to the best of our
`knowledge and belief. This translation is performed and edited by Japanese to
`English professional translators (ATA).
`
`Executed this 1_8_"j day of June, 2012.
`
`
`
`Translation Department
`
`Sayuri (Saya) Rupani—Hayes
`
`Legal Advantage LLC uses all available measures to ensure the accuracy of each translation, but shall
`not be held liable for damages due to error or negligence in translation or transcription.
`
`

`
`(19) Japan Patent Office (JP)
`
`(11) Japanese Unexamined Patent
`Application Publication Number
`
`(12)Japanese Unexamined Patent
`Application Publication (A)
`
`S58-170492
`
`(51) int. Cl,3
`
`c12r> 19/14
`19/22
`
`identification codes
`
`JPO file numbers
`
`7258-4B
`725848
`
`(54) METHOD FOR MANUFACTURlNG
`OLlGOSACCHARlDE BY AMYLASE G4, 5
`(21) Application Number S57-37536 (1982)
`(22) Filing date March 9, Showa 57 (1982)
`(72) inventor
`Yoshiyuki Takasaki
`
`(43) Publication date: October 7, Showa 58
`(1983)
`Number of inventions 1
`
`Request for examination Requested
`(Total of 7 pages)
`
`1-1—3 Higashi, Yatabe—Cho, Tsukuba-Gun.
`lbaraki Prefecture
`
`(71) Applicant
`
`(74) Designated Agent
`
`c/o Microbiological Technology Research
`Laboratory, Agency of Industrial Science
`and Technology
`The President of Agency of
`Industrial Science and Technology
`The President of Microbiological
`Technology Research
`Laboratory, Agency of industrial
`Science and Technology
`
`Description
`
`1. Title of the invention
`
`METHOD FOR
`
`MANUFACTURINGOLlGOSACCHARlDE BY
`
`AMYLASE G45
`
`2. Claims
`
`decomposing manners on starch have been known
`
`and used for the manufacture of glucose or maltose.
`
`Currently, the development of manufacturing
`
`technology for more high-molecular—weight
`
`oligosaccharide, such as maltotriose (G3),
`
`maltotetraose (G4), maltopentaose (G5), and
`
`Method for manufacturing a maltotetraosev and
`
`maltohexaose (G6), has been demanded. These
`
`maltopentaose—containing sugar solution by amylase
`
`ollgosaccharides are considered to be widely used as
`
`G4,5 which is characterized in that amylase G4, 5 of
`
`sweeteners, fillers, excipients, and clathration agents
`
`genus Bacillus which produces maltotetraose and
`
`in a variety of food, pharmaceutical compounds and
`
`maltopentaose from starch and its partial degradation
`
`industrial products; however, the manufacturing
`
`products is acted on starch, arnylose, amylopectin or
`
`methods thereof are yet to be developed.
`
`partial degradation products thereof.
`
`3. Detailed description ofthe invention
`
`The inventor of the present invention has widely
`
`searched for bacteria in the soil to find bacteria having
`
`The present invention relates to a method for
`
`a strong ability to produce these oligosaccharides. As
`
`manufacturing sugars whose major components are
`
`a result, it was found that the bacteria of the genus
`
`maltotetraose and maltopentaose from starch.
`
`Bacillus can produce a significant amount of
`
`Conventionally, amylase, such as or-amylase, [3~
`
`exobacterial amylase which decomposes starch into
`
`amylase, and glucoamylase, having different
`
`maltotetraose (G4), and maltopentaose (G5).
`
`521
`
`

`
`It is known that the amylase produced by
`
`Besides the above enzymes, oi-amylase produced
`
`Pseudomonas stutzeri is an enzyme which can
`
`by Bacillus subtilis can produce a mixture containing
`
`produce maltotetraose from the non~reducing terminal
`
`various sugars, such as maltotetraose and
`
`of amylose or amylopectin (Archive of Biochemistry
`
`maltopentaose; however, this enzyme is not suitable
`
`and Biophysics Volume 145 page 105 — 114 (1971)).
`
`for the manufacture of maltotetraose or
`
`However, this enzyme does not produce
`
`maltopentaose as they are not the major components
`
`maltopentaose.
`
`Meanwhile, the composition of sugars produced
`
`Meanwhile‘ it has been reported that the heat-
`
`from starch by the enzyme according to the present
`
`reslstant oi—amylase produced by Bacillus
`
`invention may depend on the DE of starch used and
`
`licheniformis can produce sugars containing
`
`the reaction conditions of the decomposition (the
`
`maltopentaose (G5) as the major component from
`
`concentration of starch, the pH, and the concentration
`
`starch. However, the producing ability of
`
`ofthe enzyme); however, the products generally
`
`maltotetraose of this enzyme is significantly low; it is
`
`contain 15 to 35% of maltotetraose and 15 to 30% of
`
`as low as ‘A of the producing ability of maltopentaose.
`
`maltopentaose. Thus, the total of these sugars may
`
`Additionally, this enzyme also produces
`
`reach 30 to 60%. The typical sugar composition is
`
`oligosaccharides of G1 to G12 at the same time
`
`shown in Table 1.
`
`(Archive of Biochemistry and Biophysics Volume 155
`
`page 290 — 2.98 (1973)).
`
`J
`
`_
`_
`Table 1
`Content
`Type of sugar___
`4.1
`G1 T
`_
`13.0
`G2 __
`13.8
`G3
`36.2
`G4
`20.7
`G5 M
`1.8
`G5
`10.4
`’
`other
`As described above. the enzyme according to the
`
`_
`g_A_”_
`
`present invention is an enzyme to produce sugars
`
`containing maltopentaose and maltotetraose as the
`
`major components, The enzymes with these
`
`characteristics are not known to date; therefore this
`
`enzyme is novel and the inventor named it “amylase
`
`G4, 5". The Characteristics of this enzyme are
`described below
`
`(1) Action. This enzyme decomposes glucans,
`
`such as starch, amylose, amylopectin and
`
`dextriii by the units of maltotetraose and
`
`maliopentaose to produce sugars containing
`
`these as the major components.
`
`522
`
`(2) Range of reaction temperature and optimum
`
`reaction temperature: This enzyme can
`
`catalyze the reaction at the temperature up
`
`to 70 “C and the optimum reaction
`
`temperature is 50 to 55 ‘C (reaction of 30
`
`minutes under 1% of starch concentration
`
`and optimum reaction pH, Figure 1(a)).
`
`(3) Optimum pH range and optimum reaction
`
`pH: This enzyme can react under the pH
`
`range of4.5 to 11, and the optimum reaction
`
`pH is 6.5 to 7.5 (Flgure1 (b)).
`
`(4) Thermal stability: When heated in 0.05lVl Tris
`
`buffer (pH 7.0), this enzyme is deactivated
`
`by 70 to 90% after heating it to 50 “C for1O
`
`minutes (Figure 1 (0))
`
`(5)
`
`pH stability: Each residual activity of the
`
`enzyme in 0.05l\/l buffer solution was
`
`measured after setting it aside for3 hours.
`
`

`
`As a result, the enzyme was stable within the
`
`pH range of about 6 to about 10 (Figure 1
`(d))-
`Stabilizationr An increase in the thermal
`
`(5)
`
`stability of the enzyme was found in the
`presence of calcium ions.
`
`Inhibitor: This enzyme was deactivated by
`
`95% or more with 5x1o‘3ivi of HgClg, Z|'lSOa.
`CuSO;-.. FeSO4, CoCl2, /-\gNO3 and the like.
`
`(8)
`
`Method of purification and molecular weight:
`This enzyme can be purified to a single
`
`material with respect to a chromatography,
`by fractioning the filtrate of a liquid cultivation
`
`with ammonium sulfate and purifying the
`fraction with a chromatography, such as
`
`DEAF: sepharose column chromatography
`(gradient elution with 0.2 to O.6Mot KCI),
`
`Sephadex G-200 column chromatography,
`Sephadex G100 column chromatography,
`and the like. This enzyme is divided into
`isomers having molecular weights of 40,000
`and 110,000, respectively; however, the
`
`isomer having the smaller molecular weight
`
`(2)
`
`(3)
`(4)
`
`(5)
`
`(5)
`
`(7)
`
`(8)
`
`Spore The spore cell is swollen and forms a
`sphere ~ oval spore.
`
`Gelatin The bacterium will liquefy.
`
`Meat Juice agar: The bacterium grows very
`well therein and shows yellowish light brown
`color.
`
`Glucose meat juice agar: This bacterium
`grows poorly and shows light yellow color.
`
`Glucose nitrate agar: This bacterium barely
`grows therein and shows translucent color.
`
`Meat juice: This bacterium will get muddy and
`cloudy and settle out therein.
`
`Sodium chloride meat juice: This bacterium
`can grow under 1 to 10% of sodium chloride
`
`concentration; and the growth is facilitated
`under 1 to 5% of sodium chloride
`concentration.
`
`(9)
`
`Milk: Milk does not decompose the bacterium
`
`so strongly but the bacterium will solidify and
`become peptonized. Litmus is reduced.
`
`(10) Potato: This bacterium grows normally
`therein and does not produce any pigments.
`
`is the major component. These two isomers
`have almost same enzymatic activities.
`
`(9) Measuring method of potency: An
`appropriate amount of enzyme was added to
`O.5mL of 1% soluble starch (pH 7.0) solution
`
`in 0.1M phosphate buffer, water was added
`to make the total volume 1mL and the
`
`reaction was performed at 40C. The amount
`of enzyme which can produce a reducing
`ability corresponding to 1mg of glucose
`within 1 hour was defined as one unit.
`
`The exemplary bacterium of the genus Bacillus to
`
`produce this enzyme may include Bacillus circulans
`G45. The mycological characteristics of this Strain are
`the as follows and this strain was deposited at
`
`Microbiological Technology Research Laboratory,
`
`Agency of Industrial Science and Technology as
`FERM-P Number 6237.
`
`(1) Configuration: Rod-shaped bacterium; Size
`0.7 to 0.8p of width x 2.5 to 5p of length; there
`are many bacteria formed—~from 2 to 3
`
`individual organisms; Non-motile bacterium;
`Gram-negative.
`
`(11) Tyrosine agar: This bacterium grows
`considerably well therein and shows light
`yellow color. Negative tyrosinase activity.
`(12) Glucose asparagine agar: This bacterium
`can hardly grow therein.
`
`(13) lndole: This bacterium does not produce
`indole.
`
`(14) Acetylmethylcarbinol: Not produced.
`(15) Hydrogen sulfide: A little amount is produced.
`(16) Reduction of nitrate: Positive.
`(17) Urease: Not produced.
`(18) Catalasez Positive.
`(19) Hydrolysis of starch: Positive.
`(20) Utilization of carbohydrate: This bacterium
`can utilize glucose, fructose, mannose,
`galactose, D—xylose, L—arabinose, sucrose,
`maltose, L—sorbose, mannitol and starch and
`
`thereby produces acids but does not produce
`gas. Only little or no amount of acids is
`produced from lactose, ratfinose, sorbitol and
`inulin.
`
`(21) Methylene blue: This bacterium reduces
`methylene blue.
`
`523
`
`

`
`Citric acid: This bacterium does not utilize
`(22)
`citric acid.
`
`The characteristics of the pullulanase produced by
`this bacterium are as follows:
`
`(23)
`
`Growth temperature: Optimum growth
`
`temperature is about 30°C, and maximum growth
`temperature is 50 to 57"C.
`(24)
`Thermal death point: This bacterium does
`not die after heating at 100°C for 10 minutes.
`(25)
`Optimum growth pH: 7.5 to 8.5
`Based on the mycological characteristics described
`above and by
`referring to Bergey's Manual of
`
`Determinative Bacteriology, 7”‘ and 8"‘ Edition (The
`Williams & Wilkins Company, 1957 and 1974),
`this
`bacterium was
`identified as
`a bacterium closely
`related to Bacillus circulans
`
`This bacterium strain produces pullulanase
`besides amylase G4, 5; the both enzymes collaborate
`to produce maltotetraose and maltopentaose from
`
`substrates having branched bonds ((1-1, 6—glucoside
`bond) with a high yield.
`
`(1) Action: This enzyme produces maltotriose by
`cleaving oi-1, 6—glucoside bonds contained in
`
`pullulan. Also, this enzyme cleaves ci—1, 6-
`glucoside bonds in starch, amylopectin,
`
`glycogen or the derivatives thereof.
`(2) Range of reaction temperature and optimum
`reaction temperature: This enzyme can
`
`catalyze the reaction under the temperature
`up to about 70“C and the optimum reaction
`temperature is 50 to 55°C (reaction of 30
`minutes under 1% of pullulan and 0.05M Tris
`
`buffer solution).
`
`(3) Optimum pH range and optimum reaction pH:
`This enzyme can catalyze the reaction at the
`pH range of about 5 to 9, and the optimum
`reaction pH is around 7 (reaction under 1% of
`pullulan and 0.05M Tris buffer solution at
`40°C).
`
`(4) Thermal stability: This enzyme was heated in
`
`chromatography such as a DEAE sepharose
`
`0.05M Tris buffer (pH 7.0) at each
`
`column chromatography (gradient elution with
`
`temperature for 10 minutes and each residual
`
`0.2 to 0.6Mof KCl) Thereafter, this enzyme
`
`activity was measured. As a result the activity
`
`can be purified to a homogeneous state with
`
`of this enzyme was deactivated by 73% after
`
`respect to a chromatography by Sephadex G-
`
`being heated at 50°C for 10 minutes, and by
`
`200 column chromatography.
`
`97% after being heated at 55°C for 10
`minutes.
`
`(5) pH stability; This enzyme was stable at the
`
`pH range of about 6 to about 9 (the enzyme
`
`was dissolved into 0.1M acetate buffer
`
`solution or phosphate buffer solution at room
`
`temperature (25"C) and the residual activities
`
`were measured).
`
`(6)
`
`inhibitor‘ This enzyme was strongly inhibited
`
`by cu"? Zn”, Agfi Hg”, Fe” and the like.
`
`(7) Stabilization: Thermal stability of the enzyme
`
`was increased by calcium ion.
`
`(8) Method of purification: This enzyme can be
`
`separated from amylase G4, 5 by fractioning
`
`the filtrate of liquid cultivation with ammonium
`
`sulfate and subjecting the fraction to a
`
`524
`
`(9) Molecular weight: The molecular weight
`
`according to a Sephadex G—200 gel filtration
`
`method was about 70.000.
`
`(10) Measuring method of potency: An appropriate
`amount of enzyme was added to 0.5mL of 1%
`pullulan (pH 70) solution in 0.1 M phosphate
`buffer, water was added to make the total
`
`volume 1mL and the reaction was performed
`under 40°C. The amount of enzyme which
`
`can produce a reducing ability corresponding
`to 1mg of maltotriose within 1 hour was
`defined as one unit.
`
`Generally used solid medium and liquid
`medium were used for the cultivation to
`
`produce the enzymes according to the
`present invention. The nitrogen source for the
`medium for the fluid culture may include
`
`peptone, meat extract, yeast extract, casein,
`corn steep liquor, and the like;
`
`

`
`the carbon source may include starch, dextrin,
`maltose, glucose. sucrose and the like. Besides the
`
`aforementioned sources, the medium containing the
`supplementary nutrients such as inorganic nitrogen
`source, phosphate salts, magnesium salts, metal salts
`may be used. ’l he cultivation was performed
`aerobically at pH range of 6 to 9 and at the
`temperature of 25 to 550 for 2 to 3 days. Since the
`amylase G4, 5 and pullulanase are produced outside
`of bacterial organism, after the cultivation, these
`
`enzymes can be precipitated by adding an organic
`solvent or a sait to the supernatant solution which was
`
`separated by a filtration or a centrlfugation, or they
`can be purified by the purification methods as
`described above.
`
`Next, the condition for the sugar formation from
`starch shall be described.
`
`Before the sugar formation, amylose, amylopectln
`or starch is liquefied. This liquefacation may be
`performed by usual methods using acids or Ct-
`
`amylase; as the degree of liquefacation can
`significantly affect the yield of maltotetraose and
`maltopentaose, the preferred degree of liquefacation
`of starch, DE (the abbreviation for the dextrose
`
`equivalent, and is the amount of reducing sugar in a
`solid material that is expressed in terms ofthe amount
`
`of glucose), is 15 or less. In case in which this kind of
`starch is used, the yield of maltotetraose and
`maltopentaose will be about 40 to 60%.
`The substrate concentration is usually about 5 to
`40%. The pH at the time of sugar formation
`particularly affects the yield of maltopentaose. That is
`to say, when the pH at the time of sugar formation is
`about 7 or lower, the maltopentaose produced first is
`easily decomposed into maltose and maltotriose. it is
`
`preferred to maintain the pH of the reaction mixture at
`about 7.5 or more to suppress the decomposition
`
`reaction. Usually, the reaction is carried out at 40 to
`60°C.
`
`The stability of this enzyme is significantly
`
`Since the pullulanase was also produced in the same
`
`increased by the presence of calcium ion; therefore
`
`medium, the fractions which were precipitated by 90%
`
`for example, about 5 X 104M to 2 x 1O'2M of calcium
`
`chloride is added to the reaction mixture at the time of
`
`reaction.
`
`of ammonium sulfate were collected. The fractions
`
`were dialyzed with distilled water, adsorbed on a
`
`DEAE sepharose column which was buffered by
`
`Next, the detail of the present invention shall be
`
`0.025M Tris buffer solution containing 0.2M KCl, and
`
`explained by referring to the embodiments.
`
`the amylase fraction free of pullulanase was obtained
`
`Embodiment 1
`
`by eluting the column with a gradient ofeluent of from
`
`Four hundred mL of liquid medium (pH 7.0)
`
`O.2l\/l to 0.5M KCl (67 units/OD25om,,). Twenty units of
`
`consisting of polypeptone 8 (made of soybean,
`
`enzyme solution and 5 x 1O"3l\/l of calcium chloride
`
`available from Wako Pure Chemical industries Ltd.,)
`
`were added to 19 of starch having DE of 8.05 and the
`
`4%, K2HPO4 0.3%, l\/lgSO4/'/H20 0.1%, soluble starch
`
`total volume was adjusted to 10mL and the reaction
`
`1%, and ammonium sulfate 01% was placed in a 2-L
`
`was carried out for 24 hours at 50“C. The sugar
`
`Erlenmeyer flask and the medium was disinfected
`
`composition and components of the resulting
`
`according to a conventional method. Then Bacillus
`
`decomposition products of starch were analyzed and
`
`circulans G45 (PERM-P 6237) was seeded onto the
`
`separated by using a high—performance liquid
`
`medium and cultivated for 2 days at 30°C. After the
`
`chromatography (column: NH peak J~411
`
`cultivation, the bacteria were removed by a
`
`manufactured by Showa Denko KK, eluent.
`
`centrifugation, and the resulting supernatant solution
`
`acetonitrile 85°/o+water 35%). As a result, the sugar
`
`was measured in terms of amylase G4, 5. As a result,
`
`composition was as follows:
`
`6.1 units of amylase were found in 1mL of medium.
`
`525
`
`

`
`glucose 4.5%, maltose 10.7%, maltotriose 11.2%,
`
`Embodiment 3
`
`maltotetraose 23.6%, maltopentaose 28%, and
`
`Amylase G4, 5 and pullulanase which were
`
`maltohexaose 60%.
`
`Embodiment 2
`
`prepared by the same manners as described in
`
`Embodiment 1 were used. Twenty units of said
`
`Potato starch was liquefied by d—amylase of
`
`amylase G4, 5, 30 units of said pullulinase and 1x1O’2
`
`Genus Bacillus (Kleistase KM, manufactured by
`
`M amount of calcium chloride were added to liquefied
`
`Daiwa Fine Chemicals Co, Ltd.) to form liquefied
`
`starch solution having a DE of 4.3. And the pH of
`
`starch solutions (100mg as dried weight) having DE of
`
`mixture was adjusted to 7.5 and the total volume was
`
`238,425, 5.81. 8.05, 12.6 and 27.2, respectively.
`
`adjusted to 10mL. The reaction was carried out for 24
`
`Two units of enzyme and 5 x 1O‘3M amount of calcium
`
`hours at 50°C. After the reaction was completed, the
`
`chloride were added to the each resulting solution,
`
`resulting composition of sugar products was as shown
`
`and the each total volume was adjusted to 1mL. The
`
`in Table ‘1_[KF1] .
`
`reaction was carried out at 50°C for 24 hours. After
`
`Table 1
`
`the reaction was completed, the sugar composition of
`
`each product was quantified. The results are shown in
`,
`,
`,
`Figure 2. As clearly understood from Figure 2, it was
`f
`d t f b t I
`'
`'
`t
`f
`a
`o a ou
`or ower IS appropria e or
`oun
`the preparation of maltotetraose and maltopentaose.
`
`Glucose
`Maltose____
`; Manomose
`l
`Maltotetraose
`Mahopemaose
`Other
`
`W
`
`l
`;
`,l
`
`Comm’
`_
`22%
`10.2
`124
`
`2176
`Q39
`
`with addition
`__N____A_c_>f_p_ullu|anase
`W 4.0%
`16.8
`159
`
`I
`
`229
`6.2
`
`and Technology [Seal of the
`designated agent]
`
`As clearly understood from Table, by performing
`the reaction under the presence of pullulanase, the
`sugar product containing large amounts of
`maltotetraose and maltopentaose was obtained.
`
`4. Brief description of drawings
`
`Figures 1 (a), (b), (c), and (d) are showing the
`optimum temperature, optimum pH, thermal stability,
`and pH stability of the decomposition reaction of
`
`starch by amylase G4, 5, respectively.
`Figure 2 shows the contents of (31 (glucose), G2
`(maltose). G3 (maltotriose), G4 (maltotetraose), and
`G5 (maltopentaose) in the sugar formation product
`resulting from the sugar formation reaction of liquefied
`potato starch having a variety of DE by amylase G.
`
`Patent applicant
`
`Designated agent
`
`Seiichi lshizaka, the President of
`Agency of Industrial Science and
`Technology
`
`The‘[KF2] President of
`Microbiological Technology
`Research Laboratory,
`Agency of industrial Science
`
`526
`
`

`
`H6. 2
`
`<%’
`§
`
`.3
`
`
`
`[Seal of JPO dated July 30. Showa
`57 (1982))
`
`Title of the invention on page 1 of the description,
`
`“METHOD FOR MANUFACTURING
`OLIGOSACCHARIDE BY AMYLASE G45” is
`corrected to “METHOD FOR MANUFACTURING
`OLIGOSACCHARIDE BY AMYLASE G4, 5".
`
`FlG.1
`
`ta)
`
`( b)
`
`5
`
`6
`
`B
`
`8
`
`7
`TH
`
`(d)
`
`100
`
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`Amendment (Formaiity)
`
`July 29, Showa 57 (1982)
`To Director~Generai of Japan Patent Office
`1.
`Case identification
`Showa 57 Patent
`
`2.
`
`3.
`
`4.
`
`5
`
`6.
`
`7.
`
`8.
`
`Application Number 37536
`Title of the invention
`METHOD FOR MANUFACTURING
`OLIGOSACCHARIDE BY AMYLASE G4,
`5
`
`Party making the amendment
`Relationship to the case,
`Patent applicant
`Address 1-3-1 Kasumigaseki, Chiyoda~Ku
`Tokyo
`
`Seiichi Ishizaka,
`(114)
`Name
`the President of Agency of industrial Science
`and Technology
`Designated agent
`1-1~3 Higashi, Yatabe—Cho,
`Address
`Tsukuba—Gun, ibaraki Prefecture
`Name
`Yoshikazu Takahara, the
`President of Microbiological Technology
`Research Laboratory, Agency of industrial
`Science and Technology [Seal of designated
`agenfl
`Date of the amendment directive
`Showa 57 (1982)
`(Date of shipment
`Showa 57 (1982))
`Number of invention increased by the
`amendment
`None
`
`June 11,
`
`June 29,
`
`Object of the amendment
`The column of “Titie of the invention" in
`
`the Description
`Content of the amendment
`
`As per the attached sheet
`
`527