DE1517783A1 - New and useful amylase preparation and process for making same - Google Patents

Description

Taisho Pharmaceutical Co., Ltd.

724, 3-Cliome, Takataminamicho, Toshimaku, Tokyo / Japan

"New and useful amylase preparation and method for

Manufacture of the same "

This invention relates to a new and useful Amy laser. preparation and method of making same.

In particular, this invention relates to an acid-active one and -stable amylase preparation derived from a new strain microorganisms associated with Paecilomyces subglobosum originates as a new and usable product and beyond Process for making the new enzyme.

According to the prior art, different types of micro '

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Organisms known to produce lower than normal Types of amylolytic enzymes, and among them there are some strains that are widely used in the industry are used, for example the yellow-greenish Aspergilli group, like Aspergillus oryzae and the blackish Aspergilli group, like Aspergillus usamii, Aspergillua awamori and Aspergillus niger, a particular strain of the Rhizopus group and bacteria such as Racillus subtilis.

In general there were some differences in the enzyme properties found between the above microbial amylase preparations.

This can come into particular consideration if, according to their characteristic properties, for example, they are more effective pH range, pH stability, like acid and alkali stability, thermal stability and amylolytic Kind to be used.

In terms of amylolytic effectiveness, many amylase preparations originating from various microorganisms have been shown to have two types of typical activities have, namely dexfcrinogenic and saccharifying Functions versus strength.

Some amylase preparations that have this acid activity unti Acid stability of dextrinogenic and saccharifying

809085/1307 ~ ³ ~

BATH ORIGINAL

Features are for different areas in the Traps suitable where they are to be used under acidic condition, especially suitable as a digestive enzyme, which works against dietary starch in the stomach where the gastric juice creates highly acidic conditions.

Akaboli and others (Symposium of Enzymatic Chemistry in Japan, Volume 7, page 107, 1952) report that a Amylaseherstellung which originates from Aspergillus oryzae is active at a pH of 4.0 to 7 _f 0 reioht in dextrinogener activity and that it is easily inactive at a pH below 3.0, on the other hand ur.d Hakko Kogaku Zasshi (J.Fermentation Technology i ⁿ Japan), Sakamoto and other weiaen after that the Rhizopusgruppe produces an amylase, which the optimal effective pH Has a value close to pH 5.0 in terms of dextrinogenic activity.

According to the Minoda report (J. Agr. Chem. Soc. Japan, 3and 291 Page 115 (1955), Volume 35, Pages 479 and 482 (1961, Japanese Patent Published Specification No. 35-5046 and Agr. Biol. Chem. Vol. 37, Kr. 11, page 809 (1963)) it has already been pointed out that there are Amy lases of strains which belong to the Aspergillus niger and that among the amylase produced in this way the dextrinogenic activity it is mainly indicated at values higher than pH 4.0 and that it is sufficient under acidic conditions

^are · ßAD ORIGINAL

909885/1307

However, commercially available forms of amylaah show those made by the methods described above and others described later in the prior art neither desirable amylolytic activity nor stability under acidic conditions below pH 4 »0 (compare drawings 1, 2, 5 and 4).

In contrast, our new strains, Paecilomycee subglobosum TPR-3810 and TPR-3811 an acid-active and acid-stable amylase production produce which in terms of dextrinogenic and saocharifying activities at the acidic pH of more than 3.0 more potent and stable, and moreover, shows desirable activities in this regard at pH 2.5 (see tables described on page 18)

Therefore, it can be stated as a result here that our new Amylaseherstellung is effective at a pH ranging from 2.5 to 6.0, in particular more than other preparations at pH values ranging from 2.5 to 3.5 5 bie 9t0 is sufficient, in particular more stable than others at pH values ranging from 3.5 to 5.0, in terms dextrinogener and aaccharifizierender efficiencies (see figures 1, Z |, and as stable that of Figure 3 is at a pH , -j and 4).

A new one was previously found by the applicant

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to produce acid-unstable digestive enzyme using a known strain, that of the Paecilomycea varioti group associated therewith is [(Japanese Patent No. 421,345t Published Patent Application No. 38-25280 and U.S. Application Serial No. 322,583, Yakuzaigaku (The Achives of Practical Pharmacy) Volume 22, pages 111 to 129 and Chem. Abstracta Volume 58, 4770 b)], but it could not satisfactorily serve the purpose of acid-active and acid-stable Amylase preparation (see drawing 9 and 10).

As a result of the investigations, two new microorganisms that produce the above amylae preparation have now been which at an acidic pH of 2.5 to 4 »5 with the optimum acts at pH 4.0 to 4.5, among various microorganisms found which belong to the Paeoilomyces subglobosum group belong.

New microorganisms different from Paecilomyces varioti, were designated by the names Paecilomyces subglobosum TPR-3810 and TPR-3811, which were previously were not used.

It is an object of this invention the new microorganisms which the acid-active and acid-stable amylase preparation generate, isolate.

The main object of this invention is the excellent

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To obtain enzyme, which has the above-mentioned acid-active and has stable amylolytic activity, by using the new microorganisms Paecilomyces subglobosum TPR-3810, TPR-3811 and other strains that share the same biological properties as TPR-3810 and TPR-3811.

It is another object of this invention to easily obtain a new and useful amylase preparation.

The novel microorganisms of this invention are isolated from the wild fungi by the following assay procedure, which satisfactorily serves our purposes.
Procedure for isolating the logs.

First stage

Petri dishes in which a modified Gzapek-Dox agar culture medium, as a carbon source 0.5% soluble starch, nitrogen source 0.5% peptone, 0.1% potassium dihydrogen phosphate, 0.05% potassium chloride, 0.05% Magnesium sulfate, 0.001% ferrous sulfate, adjusted to pH 3>, exposed to the indoor and outdoor atmosphere or various soil samples collected in downtown Tokyo for a period of time without covering and then cultured.
Second step
500 tribes are selected from many mutes who are on "

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then 50 strains which produce amylaae that function at pH 3 are selected from 500 strains which are grown on the modified Czapek-Dox culture medium, carbon source 3 $ glycose, nitrogen source 2? 5 peptone, inorganic salt 0.1% potassium dihydrogen phosphate, 0.05 $ potassium chloride, magnesium aulfat 0.05 # 0.001 # iron (II) sulfate were cultured at 3O ⁰ C for 3 days with shaking.

Third step

Among 50 desired strains that produce such acid-active and acid-stable amylase that works at pH 3 will be two microorganisms which produce the more acidic and more acidic amylase, further pure for use selected in this invention.

From morphological observations it can be said that the two new phyla belong to certain identical species and also have completely identical properties, see Iioh

1. the activities and properties of amylase, which they produce in a liquid culture medium,

2. the production of the same pale brown pigments in the culture medium

3. the availability of kitrites.

The morphological properties of the newly found micro ■

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Organisms Paecilomyces subglobosum TPR-3810 and TPR-3811 are the following.

Morphology of the new microorganisms

Colony observations of the microorganisms were made for colonies grown on agar-compatible slides.

Detailed, morphological studies have been carried out on the Myeeliawaehstum performed on microscope slides.

1. Colonies

Surface flat

Color greenish or yellowish brown

2. Spore forming part

similar to the genus Paecilomyces, Sterigmata alternating, partly standing individually, partly 2 to 4 of the same forming a whorl, with some extraordinarily dense growth, which is irregular, penicillus-like Appearance.

3 * sterigmata

ample-like, usually with a narrowing in the middle, measuring 21 times 3 _f 5 u on average, slightly rough on the surface.

4. Konidia

form a long chain from the top of the sterigmata, conical or incompletely spherical, (glo-

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boae or sub-globose), sometimes elliptical.

Averaging 5.5 times 3.6 μ , a little rough on the surface.

5. Penicillus

no such typical ones were found.

6. Metulae or branches

not so demarcated or differentiated, if available, to the extent of 12 times 3 »5 μ on average.

7. Conidiophores (Conidiophores)

about size 20 by 4 u on average.

8. Macrospores

sometimes present in the medium, incompletely spherical (subglobular) mostly 8 times 10 u.

9. Sclerotia, perithecia or ascospores none.

10. Funicular myceria and gelatin rising into the air Exudation similar to that of the culture Paeoilomyces varioti does not observe any.

To indicate that the microorganisms under consideration are similar to the species Paeoilomyces varioti, kläi In-depth studies reveal the differences to the same.

Morphological differences between the strains of Paecilomyces found varioti

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809885/1307

-ΙΟΙ. Konidia

Shape more spherical, not ao elliptical surface a little rougher.

2. Sterigmata

Shape a little indented in the middle, not

so slightly pointed. Surface a little rougher.

3. Regularity of the spore-forming parts

show irregular, sterigmata sometimes more densely distributed Penieillus-like appearance.

From the properties given above, it is concluded that the microorganisms found belong to the genus Paecilomyces and that as a species they do not fall under the concept of Paecilomyces varioti.

They are therefore classified as a new species in the genus Paeoilomycea, and they are referred to as Paecilomyces sublobosum IPR-3810 and TPR-3811, respectively.

Process for the production of the acid-stable amylase.

According to the invention, the novel and useful amylase can be produced are obtained by cultivating the newly found microorganisms, Paecilomyces subglobosum IPR-3810 and TPR-3811, in a suitable nutrient medium under the same conditions as are customary.

809865/1307 bad original. _n _.

A modified culture method was used in this invention found and introduced which gives good results due to the fact that the available quantity and Amylase activity can be significantly influenced by the Components of the culture medium used.

Essentially, the culture medium contains a carbon source, a nitrogen source and traces of inorganic elements, each in a suitable amount as nutrient substances.

1. Liquid culture medium

Examples of suitable carbon sources in the culture medium 55 * different types of starch or 5 # maltose, but it is Glucose, sucrose, dextrin, glycerin, and wheat flour can also be used in amounts of 5% each in place of Starch or maltose.

Suitable nitrogen sources are 3 ^ peptone and in addition, raw soybean meal, defatted soybean, dried yeast, corn steep liquor in an amount of yf »can be used. Suitable inorganic elements are:

Magneaium Eulfate 0.05 $

. Eiaen (II) sulfate 0

Potassium chloride 0.1 Potassium dihydrogen phosphate 0.2

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The liquid culture medium is prepared by misoing the above ingredients with water (these ingredients are called main liquid culture medium or liquid culture medium used for koji vaccination).

and then it is desirable to adjust the pH of the medium to pH 6.0 after each of these substances is added to the medium.

The maximum yield and high amylolytisohe effectiveness of amylase available should be under adequate condi- · conditions for the crop treatments, the following may be performed:

In culture "he keeps

Ventilation speed 4/3 vol./vol./min.

Stir 350 ohms. per minute

Temperature 30 ⁰ C

Culture time approx. 90 to 94 hours

The medium prepared and treated in this way is sterilized and inoculated with Paecilomyees eubglobosum TPR-3810 or 5PR-38i1 inoculation cold medium, which is incubated for 70 hours on the medium containing the same components as above, and the next step is the cultivation stage (submerged culture ) Performed 90 to 94 hours with ventilation.

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2. Pest's culture medium

The solid culture medium base is produced using the following components, wheat bran: Häoksel: Waeser in a ratio of 5: 3 * 4. The solid culture medium thus prepared is sterilized and inoculated with Paecilomycea subglobosum TPSS-3810 or 3811 TPJR-seed culture medium, then the thus inoculated medium is kept in the stacked state and aunäohst at 3O ⁰ C incubated about 70 hours. (In the same way, these components are used as the main solid culture medium or as a solid culture medium for the koji seeds or brood.)

Separation of the desired amylase

The separation of a highly purified and effective Amylase from the cultured medium and the extracted solution should also be carried out after following procedures a) and b) while adjusting the pH to approximately 6.0

a) Resignation proceedings

The excretion of the desired amylase is facilitated by the addition of hydrophilic, organic solvents like ethanol and acetone or ammonium sulfate.

If desired, the designated separation avoidance

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Drive to use the subsequent treatments lead to good results; Adding ethanol to $ 65 at the final concentration or adding acetone to $ 55 at the final concentration when using acetone and in the case of ammonium sulfate, adding it to 0.8 its Saturation at the final concentration,

b) Adsorption process

If it is desired to use this process, adsorbents such as DEAE Sephadex ion exchangers, DEAE cellulose ion exchanger (registered trademark manufactured by "Pharmacia" in Sweden) and the like can be advantageously used.

A cultured medium or an extracted solution of amylase is adjusted with pH 7.0, 0.005 M phosphate buffer solution.

After the solution has been adsorbed by the ion exchanger, the elution of the adsorbed material is further adjusted with pH 4.0, 0.25 M sodium citrate buffer solution. _;

This adjusted elution is carried out according to the same procedure treated as in elimination procedure (a) above, to get the desired enzyme.

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Determination of the amylolytic effectiveness

The amylolytic effectiveness of the new Amy laser manufacturing is compared with the following, commercially available amylase preparations A, B, C and D.

Amylase Production A, an amylase marketed under the registered trademark "Sanactase" originating from Aapergillus niger (Japanese Patent No. 35.5046) by Meiji Seika Go. in Japan.

Amylase production B A Amy lase, marketed under the Registered Trade Mark "Stalaae", derived from Ton Aapergillus aures (Japanese Patent No. 39-12284) by Hoda Sangyo Co. in Japan.

Amylase production C is an Amy lässe marketed under the registered trademark "Myläse 100" originating from Aspergillus oruzae duroh Wallerstein Co. in America.

Amylase production D is an amylase marketed under the registered trademark "Taka Diastase" originating from Aapergillus oryzae by Sankyo Co. in Japan.

These results of determination are in the accompanying Figures 1 to 12 and the following paragraphs.

1. pH activity curves of amylase

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The digestive activity of the new amylase preparation, as shown in FIGS. 1 and 2, was determined at different pH values. The amylaae originating from Paecilomyces subglobosum TPR-3810 and TPR-3811 possesses dextrinogenic and saccharifying powers at pH ranges from about pH 2.5 to 6.0, particularly more excellent activity at pH range from 3.0 to 6.0 , much stronger activities than the others at pH ranges of 2.5 to 3.5 and their optimal pH around 4.0 to 4.5.

On the other hand, the above commercially available amylase outputs are effective optimal conditions at pH 5.0.

As a result of these determinations, it was found that the activity of the new amylase generation was more excellent in acidic conditions than the others.

2._jgH "Jgtability curves from A myla ae

The new dextrinogenic and saccharifying powers of the new Aaylaase formulations are well stable at pH ranges of about pH 3.5 to 9.0, compared to the above, commercially available amylase formulations, which only exist in the pH range of 5 to 9 , 0, as shown in Figures 3 and 4 a ul ge.

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Finally, the new amylase consultations show more excellent ones Stability and activity in acidic conditions.

3. Optimal temperature for amylolytic activity pH 4.0 to 4.5

new amylase products 50 ⁰ C

commercially available

Amylase production approx. 40 C

4. Thermal stability of the amylolytic activity at pH 4.0

to 4.5

All Amylaseheratellungen commercially available are stable under 40 ⁰ G, but the new Amylaseheratellungen are more stable than the commercially available Amylaseherstellungen at 5O ⁰ C.

In addition, further attempts were made to the amylolytic activities and properties of the new strains and of Paecilomyces varioti TPR-220 in comparison that are largely similar in terms of their morphologies.

The differences and the superiority of the new amylase preparations can be seen from the following tables and from the fact that all of the amylase producers

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lungs made according to the invention have better dextrinogenic and saccharifying effects Have efficacies, as shown in FIGS. 9 to 12.

5 «differences in pH activity and effective temperature

pH activity

Effect temp.

, virksamer area h opt. pH (optimal temp.)

new amylase

(according to example 2) pH 2.5 - 7.0 pH 4.0 -4.5

new amylase

(according to example) pH 2.5 - 7.0 pH 4.0 -4.5

Amylase from Paecilomyces pH 3.0 - 7.0 pH 5.0 varioti TPR-220

50 ⁰ C

50 ⁰ C

50 - 60 *

DEXTHiHOGENEOUS KRAPT SACCHARIFICATING ^ FORCE

(at pH 4.0-4.5) (at pH 4.0-4.5)

new amylase

(according to example 2) 70,000 unit./g 110,000 unit./g

new Amy read

(according to example 1) 200,000 unit / g 220,000 unit / g

Amylase from

Paecilonycea 4,000 unit./g

varioti TPR-220

13,000 units / g

885/1307

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Amylolytic Activities

dextrinogenic power aaco-hardening Kr.

PH 2.5 pH 3.0 , pH 2.5 pH 3.0 unit./g unit./g unit / g unit / g

new amylase

(as per example 1) 80,000 160,000 60,000 120,000

Amylase is A <10,000 ^ - 12,000

Amylase produced .B < 5,000. <10,000

Amylase first, C < 20,000 <10,000

Amylase produces D < 4,000 <. 7,000

It should also be noted that the new microorganisms Advantageously, the acidic, active and stable ones Amylase treatments, both in liquid culture medium and auoh solid culture medium, while in contrast the Paeoilomyoes rarioti TPR-220 Amylaae only in can generate solid culture medium (no generation of amylase in liquid culture medium).

Calculation of the dextrinogenic and saccharifying forces

In this invention, the dextrinogenic and saooharifying Forces according to the following provisions he ~ calculates.

1. Starch-dextrinogenic power: test according to the blue value method

909885/1307 " ² °"

1 ml of a Amyläselösung a 10 ml 2 # aqueous soluble starch solution was added, which amount of 1% / 1O M sodium citrate buffer contained and shut off for 30 minutes at 4O ⁰ C.

To a 1 ml sample of the mixture was added 1 ml of 1N HCl was added to the mixture to stop the continued reaction, and 10 ml of iodine solution was further added.

The sample was tested for optimal density (wavelength 660 mp, absorption layer t 1 cm).

A null test was performed by the same procedure using distilled water in place of the enzyme solution.

Dextrinogenic power unit "

Nominal value - test value _ * _ηΛΛ _ 1, 1000 g ^{x 100} ° ^x TG ^x T

where ^

η = mg of the enzyme preparation used . _ ■■■ ->.

■ Γ '. * I - I *

In this determination, the unit is based on the dextrino \! Ü * - the strength that is required to decrease the blue color * f \ { reaction in 10 ml of 2 # leelioher starch to O ₉ 1 ^ pro

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9ÖS665 / 1307

ORIGINAL »NSPECTED

Minute b * i 40 O, from where the f «rt« nreehnet becomes ala tine Unit uaA practical * ι »$ · 1ι« ΐΜβ1) 1ι becomes duroh di · unit 4 per grwnn used

2. Saoo-tariffing power t test through the iodine experience.

1 ml of a Aaylaeelöeung a 20 ml solution of 2 ^ löeliohen Stärkelöeung was added gebtn which a half amount of 1/10 H sodium citratpufferlö · ling contained and held for 2 hours at 4O ⁰ C.

20 ml of N / 10 iodine solution and 20 ml of V / 5 S * 0H were added and then let out at room temperature.

Naoh 20 Miauttn standing were 4 ft! "I I-S ^ SO. Added, and free iodine was titrated with 0.1 Ν-Λι ^ Β ^ Ο.

In the same way, a Hullrereuoh became close to the same The process is carried out consistently, with the ensemble solution being followed by the additive the iodine solution, was not added.

This unit is based on a force erfordernoh to Saooharifixierung of 20 ml of 2 £ strength lösHoher strength at 40 ⁰ C, where the value is erreohnet as a solohe unit when 1 mg glucose, using 1 of the amylase g in the Jodtitrierung , found Witt ,. The following examples illustrate the appropriate method by division

ORIGINAL

the Amy read, but do not limit the invention.

Example 1 (first stage)

liquid culture medium for kojlseed

Potato starch 100 g

Peptone 60 g potassium dihydrogen phosphate 4 g

Potassium chloride 2 g Magnesium sulfate 1 g

Isen (II) sulfate 0.2 g

The above materials are dissolved in 2 liters of water and adjusted to pH 6.0 with concentrated HCl.

NaOH sterilizing the medium for 10 minutes at 125 ⁰ C, 423 g Paecilomycea subglobosum TPR-3810 preincubated for 70 hours with running around Sohüttelapparaturen on the above liquid culture medium for Kojisaat (-brut) at 30 ⁰ C.

Pre-incubation results in a Koji seed of Paecilomyces subglobosum TPR-3810.

(Second step)

Main liquid culture medium «, _ Potato starch 5 kg

. P «pton 3 kg - 23 -

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Potassium dihydrogen phosphate 200 g Potassium chloride 100g Magnesium sulfate 50 g Iron (II) sulfate 10 g

Water 100 1

The above materials are mixed and adjusted to pH

6.0 adjusted with concentrated HCl. '

In gleioher, the medium at 125 ⁰ C is sterilized 10 minutes ■. The KoJi seed incubated through the first stage is inoculated on the main liquid culture medium and cultivated in the culture container under the following conditions. Temperature 30 ⁰ C, ventilation "speed"

130 liters / min., Charging 350 rev. per min., cultivation for 94 hours.

Then the cultivated medium is centrifuged to separate the fungi and 60 l of supernatant, with the content of ' Acid-activated and -stable Amy glasses were kept with the centrifugation.

The resulting supernatant is adjusted to pH 6.0 and 130 1 95 £ strength "Ithanol, cooled at -20 ⁰ C, are added in the top can to the Aussoheidung the Amy were reading to obtain.

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909885/1307

The precipitation is sufficiently gewasohen with ethanol and dried in vacuo at 30 ⁰ C.

180 g of powdered amylase manufacture are made from the manufacture obtained, which 200,000 - 205,000 unit./g dextrinogenic potency at pH 4.0 to 4.5 and 210,000 220,000 Unit./g acid-hardening power at pH 4.0 to 4.5 have.

Example 2 (first stage)

Solid culture medium for kojieaat

Wheat bran 500 g

Chaff 300 g

Water 400 ml

The above materials are mixed.

After 30 minutes of sterilization at 125 ⁰ C 120 g Paeoilomyces be subglobosum TPR-3811 preincubated in the above liquid culture medium for the Kojieaat at 30 ⁰ C 70 Stundtn. Pre-incubation results in a Koji seed of, Patoilo myces subglobosum TPR-3811.

(Second step)

solid main culture medium

Wheat bran '50 kg

Chaff 30 kg

Water 40 1 - 25 -

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t ν ■; ■ '

! The above materials are mixed to make the base material cut out for the main solid culture medium.

The medium is sterilized in a similarly quiet manner at 125 ^° C. for 30 minutes

Kojisaat will be pre-incubated through the first stage the main solid culture medium is inoculated and used for cultivation stored at 30 ° 0 for 70 hours.

250 l of water are added to the cultivated medium in order to extract the amylase su.

Haoh. 3 hours of extrusion at 30 ⁰ O and further centrifugation, 150 l of transparent solution with the content

an acid-active and -stable amylase.

Piping solution obtained is washed with 330 1 95J (IFEM ithanol, cooled at -20 ⁰ C, gemlsoht to obtain the precipitation of the amylase.

The Aussoheidung is gewasohen with Xthanol in vacuo at 30 ⁰ C getrooknet.

It. 900 g of powdered amylase production from the Auto oh · i dung, which 70,000 JBlnheit./g dextringenic power at pH 4.0 to 4.5 and 110,000 unit./g saoohari he fell end · has power at pH 4.0 to 4 »5 ·

Claims (1)

Pat int ana sayings 1. Acid-effective and acid-stable amylase production characterized by a) amylolytic, dectrinogenic and sacoharifying Activities in a wide pH range of 2.5 to 6.0, essentially excellent activities or stronger activities than others in the acidic pH range of 2.5 to 3.5, where the optimum pH range from 4.0 to 4.5 rows, as in Figures 1, 2, 9 and shown in the drawings, b) excellent or more stable dextrinogenic and saccharifying powers over a wide pH range of 3.5 up to 9.0, as shown in Figures 3 and 4 of the drawings, o) 70,000 to 200,000 units / g of dextrinogenic potency and 110,000 to 220,000 units / g saooharifying agents Force in the acidic pH range of 4.0 to 4.5, as shown in then Figures 1, 2, 9 and 10 of the drawings, ι d) Heat stability at about 50 ⁰ G and excellent Activity at 4O ⁰ C, at a pH range of 4.0 to 4,5 * 2 · Process for the production of acid-active and -etabi-i «n Anylase has excellent amylolytic activity - 27 - 900005/1307 in satire terms characterized in that it comprises a combination of the stages of the a) Vorbebrütena liquid of a novel microorganism of Paecilomyces eubglobosum group on a sterilized "or solid Kulturmedium.für Kojisaat (-brut) for 70 hours at 3O ⁰ C under aerobic conditions and obtain a Kojisaat of Paecilomyces subglobosum- g gruppeι b) lupping and cultivating the Kojisaat of the Paecilomyoes subglobosua group on the liquid or solid main culture medium at 30 ⁰ C under aerobic conditions until the acid-active and stable amylase is essentially collected after about 70 to 90 hours in the culture medium and recovery of the acid-active ones and stable amylase from the cultured medium by excretion or adsorb. 3 «Method according to claim 2, characterized in that { the new microorganisms Paeoilomyoes subglobosum IPR-, 3810, TPS-3811, and the like Bind. 4. The method according to claim 2 and 3, characterized in that that the new microorganisms Paeoilomyoes subglobosum TPß-3810 and TPR-3811 are separated from wild fungi in the outer atosphere or from various soil samples that - 28 - 909885/1307 collected by seven in dtr downtown Tokyo. ' 5. The method according to claim 2, characterized in that the liquid main culture medium or the liquid culture medium for the koji seed from the following suitable ones Sources exists a) Appropriate carbon source selected from various types of starch, maltose, or that contributing glucose, sucrose, dextrin, glycerin and wheat flour are used with the content ratio of 5J (of each in the total amount, b) suitable nitrogen source selected from peptone raw soybean meal, defatted soybean, dried yeast and cereal soaking water with a content never of 5J (of everyone in the total, c) 0.05 $ magnesium sulfate, 0.01 $ iron (II) sulfate, 0.1 # Potassium chloride and 0.2 # potassium dihydrogen phosphate. 6. The method according to claim 2, characterized in that the solid main culture medium or solid culture medium for the koji seed consists of the following suitable components: Wheat bran: chaff: water with a content ratio of 5: 3: 4. - 29 - 909885/1307 • \ · ■ ■ - 29 - 7. Procedure essentially wit in tizitm the apezifieohtn Example · bttohritbtn. 8. SäurtakfeiTt tua, d -s tabl It Anylaethtre division, tofern sit naoh tintn 71rflUirtη, ftmäi tintm the previous test was htrgtatellt. . I. 909886/1307