US3652400A - Process for the preparation of amylase - Google Patents

Process for the preparation of amylase Download PDF

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Publication number
US3652400A
US3652400A US531816A US3652400DA US3652400A US 3652400 A US3652400 A US 3652400A US 531816 A US531816 A US 531816A US 3652400D A US3652400D A US 3652400DA US 3652400 A US3652400 A US 3652400A
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Prior art keywords
amylase
percent
paecilomyces
dextrinogenic
tpr
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US531816A
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English (en)
Inventor
Jiro Sawada
Tetsuo Misaki
Masao Okabe
Hajime Yasui
Kazunori Hanada
Tadayasu Okazaki
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Taisho Pharmaceutical Co Ltd
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Taisho Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C12N9/242—Fungal source
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/932—Paecilomyces

Definitions

  • FIG. 1 A first figure.
  • this invention is concerned with an acidactive and stable amylase derived from a new strain of microorganism belonging to Paecilomyces subglobosum as novel and useful product and moreover with a process for the preparation of the said novel enzyme.
  • strains of the yellow-greenish Aspergilli group such as Aspergillus oryzae and of the blackish Aspergilli group such as Aspergillus usamii, Aspergillus awamori and Aspergillus niger, a certain strain of the Rhizopus group, and bacterium such as Bacillus substilis.
  • each characteristic property for example, active pH range, pH-stability such as acid-stability and alkali-stability, heat-stability and amylolytic mode.
  • amylolytic activity With respect to amylolytic activity, many amylase preparations derived from various microorganisms have been found to possess two kinds of typical activities, namely, dextrinogenic and saccharifying functions against starch.
  • amylase preparations possessing acid-activity and acid-stability of dextrinogenic and saccharifying functions are suitable for various uses under acidic conditions, being especially suitable as digestive enzyme which functions against dietary starch in the stomach where the gastric juice provides a strongly acidic condition.
  • amylases are derived from strains belonging to Aspergillus niger and among thus-obtained amylase preparations, the dextrinogenic activity is mainly indicated at pH above 4.0, the preparations being sufficiently stable under the acidic conditions.
  • the new strains, Paecilomyces subglobosum TPR-38ll employed in this invention can produce an acid-active and acid-stable amylase preparation which is stronger and more stable in dextrinogenic and saccharifying activities at an acidic pH of more than 3.0 and furthermore exhibits the desirable activities thereof at pH 2.5 (cf. tables hereinafter described).
  • the present invention provides new amylase preparations which are active at pH ranging from 2.5 to 6.0, in particular are stronger than the prior preparations at pH ranging from 2.5 to 3.5 and also are stable at pH ranging from 3.5 to 9.0, in particular more stable than the prior preparations at PH ranging from 3.5 to 5.0 in dextrinogenic and saccharifying activities (cf. FIGS. 1, 2, 3 and 4).
  • a new acid-stable digestive enzyme had heretofore been prepared by using a strain (TPR-220; ATCC No. 14572) belonging to the Paecilomyces varioti group, but it could not satisfactorily serve our purpose to obtain an acid-active and stable amylase preparation (cf. FIGS. 9 and 10).
  • the main object of this invention is to obtain excellent enzyme having the above acid active and stable amylolytic activity by employing the new microorganisms, Paecilamyces subglobosum TPR-38 l0, TPR-381 l and other strains possessing the same biological characteristics as TPR-38l0 and TPR-38l 1.
  • the new microorganisms of this invention are isolated from wild fungi by the following screening which serves satisfactorily our purpose.
  • Petri-dishes in which a modified Czapek-Dox agar culture medium:
  • the morphological characteristics of the new discovered microorgansims, Paecilomyces subglobosum TPR-38l0 and TPR-38l l are as follows:
  • novel and useful amylase can be produce by culturing the new microorganisms, Paecilomyces subglobosum TPR-38l0 and TPR-38l l, in an appropriate nutrient medium in per se conventional manner.
  • the medium contains a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source, a carbon source,
  • Liquid culture medium examples of more suitable carbon sources in the modified culture medium are percent of various kinds of starch or 5 percent of maltose, but glucose, sucrose, dextrine, glycerol and wheat flour in amount of 5 percent each can also be used instead of starch or maltose.
  • Suitable nitrogen sources are 3 percent of peptone, and subsidiarily there can be employed raw soybean flour, defatted v soybean, dried yeast, corn steep liquor in amount of 3 percent.
  • Suitable inorganic elements are:
  • Liquid culture medium is made up by mixing the above components with water. (These components are employed as main liquid culture medium or liquid culture medium for seed koji).
  • the medium so prepared and treated is sterilized and inoculated with Paecilamyces subglobasum TPR-38I0 or TPR- 3811 seed culture medium pre-incubated upon the medium containing the same components as above for 70 hours, after which the cultivating (submerged culture) step is carried out for 90 to 94 hours with aeration.
  • Solid culture medium-base is made up by the following components, wheat bran:chaff:water in the ratio of 5:314.
  • the solid culture medium so prepared is sterilized and inoculated with Paecilomyces subglobosum TPR38 10 or TPR38l 1 seed culture medium, the medium so inoculated is laid in state of pile and then cultivated at 30 C for about 70 hours. (Similarly, these components are employed as main solid culture medium or solid culture medium for seed koji.)
  • adsorbents such as DEAE-Sephandex-ion exchanger, DEAE-Cellulose-ion exchanger and the like may advantageously be employed.
  • a cultured medium or extracted solution of the amylase is adsorbed on the ion-exchanger.
  • the thus-adsorbed material is eluted with pH 4.0, 0.25 M sodium citrate buffer solution.
  • amylolytic activity of amylase prepared by this invention is compared with that of the following commercial amylase preparations A, B, C and D.
  • FIG. 1 illustrates the activity of dextrinogenic power of the aforesaid compared amylases at various pH levels. Rec. time: 30 minutes. Rec. temperature: 40' C.
  • FIG. 2 shows the activity of saccharifying power of the aforesaid compared amylases at various PH levels. Rec. time: 2 hours. Rec. temperature: 40 C.
  • FIG. 3 shows the stability of dextrinogenic power of the aforesaid compared amylases at pH 4.5, 40 C, for 30 minutes, after pre-treatment at various pH levels, 40 C, for 30 minutes.
  • FIG. 4 shows the stability of saccharifying power of the aforesaid compared amylases at pH 4.5, 40 C, for 30 minutes, afterpretreatment at various pH levels, 40 C, for 2 hours.
  • FIG. 5 shows the effect of rec. temperature on the dextrinogenic power of the aforesaid compared amylases. Rec. pH: 4.5. Rec. time: 30 minutes.
  • FIG. 6 shows the effect of rec. temperature on the saccharifying power of the aforesaid compared amylases. Rec. pH: 4.5 Rec. time: 2 hours.
  • FIG. 7 sets forth graphically the effect of temperature on the stability of dextrinogenic power of the aforesaid compared amylases, after pre-treatment at pH 6.0, at various temperatures, for 30 minutes.
  • FIG. 0 sets forth graphically the effect of temperature on the stability of saccharifying power of the aforesaid compared amylases, after pretreatment at pH 6.0, at various temperatures, for 2 hours.
  • FIGS. 9 to 12 inclusive sets forth comparative results obtained with amylase of this invention (Example 2, infra) vis-avis an amylase preparation derived from Paecilomyces varioti TPR-220.
  • FIG. 9 shows the comparative results re activity of dextrinogenic power at various pH levels. Rec. temperature: 40 C. Rec. time: 30 minutes.
  • FIG. 10 shows the comparative results re activity of saccharifying power at various pH levels. Rec. temperature: 40 C. Rec. time: 2 hours.
  • FIG. 11 shows the effect of temperature on the dextrinogenic power. Rec. pH: 4.5. Rec. time: 30 minutes.
  • FIG. 12 shows the effect of temperature on the saccharifying power. Rec. pH: 4.5. Rec. time: 2 hours.
  • FIGS. 1 to 8 inclusivem 0 represents the new amylase according to Example I
  • O- 0 represents amylase preparation
  • A represents amylase preparation B
  • A- "A represents amylase preparation C
  • [:l l:l represents amylase preparation D
  • FIGS. 9 to 12 inclusive represents the new amylase according to Example 2 represents amylase from Paecilomyces varioti TPR-220 INTERPRETIVE EXPLANATIONS 1. pH activity curves of amylase The digestive activity of the compared amylase preparations has been determined as shown in FIGS. 1 and 2.
  • amylase preparations derived from Paecilomyces subglobosum TPR-38 l0 and TPR-38ll possess dextrinogenic and saccharifying powers at pH ranging from about pH 2.5 to 6.0, in particular more excellent activity at pH ranging from pH 3.0 to 6.0, substantially stronger activities than the other compared preparations at pH ranging from 2.5 to 3.5 and their optimum pH approximately at pH 4.0 to 4.5.
  • pH stability curves of amylase The dextrinogenic and saccharifying powers of the new amylase preparations are of good stability at pH ranging from about pH 3.5 to 9.0 in comparison with the above commercial amylase preparations which require pH 5 to 9.0 as shown in FIGS. 3 and 4.
  • the new amylase preparations show more excellent stability and activity under acidic conditions.
  • New amylase preparations 50 C Commercial amylase preparations about 40 C 4. Heat stability of amylolytic activity at pH 4.0 to 4.5
  • Starch dextrinogenic power assaying by blue value method I ml. of an amylase solution was added to 10 ml. of 2 percent soluble starch solution containing a half quantity of 1/10 M sodium citrate buffer solution and kept at 40C for 30 minutes.
  • the sample was assayed by optical density (wave length: 660 mp., absorption layer: 1 cm.).
  • the unit is based on dextrinogenic power necessary for decreasing the blue color reaction in ml. of 2 percent soluble starch to 0.1 percent per minute at 40 C., whereby the value is calculated as one unit and it is practically exchanged by the unit per g. of the used amylase preparation.
  • Saccharifying power assaying by iodine method 1. ml. of an amylase solution was added to 20 ml. of 2 percent soluble starch solution containing a half quantity of l/ 10 M sodium citrate buffer solution and kept at 40 C for 2 hours.
  • This unit is based on the power necessary for saccharifying 20 ml. of 2 percent soluble starch at 40 C, whereby the value is calculated as one unit when 1 mg. of glucose is produced when using 1 g. of the amylase in iodine titration.
  • the medium is sterilized at C for 10 minutes.
  • the seed koji pre-incubated by 1st step is inoculated upon the main liquid culture medium and cultivated in the culturing tank under the following conditions: temperature: 30 C; aeration rate: liters/min; stirring: 350 r.p.m.; cultivating time: 94 hours.
  • the cultured medium is centrifuged to separate fungi and 60 liters of supernatant containing an acid-active and stable amylase are obtained with the centrifuging.
  • the precipitate is thoroughly washed with ethanol and dried in vacuo at 30 C.
  • 180 g. of powdered amylase preparation are obtained from the preparation, which has 200,000-205,000 unit/g. in dextrinogenic power at pH 4.0 to 4.5 and 210,000-220,000 unit/g. in saccharifying power at pH 4.0 to 4.5.
  • Solid culture medium for seed koji Wheat bran 500 g. Chall' 300 g. Water 400 ml.(milliliters) 2nd step:
  • the medium is sterilized at 125 C for 30 minutes.
  • the seed koji pro-incubated by 1st step is inoculated upon the main solid culture medium and layed to cultivate at 30 C for 70 hours.
  • the precipitate is washed with ethanol and dried in vacuo at 30 C.
  • 900 g. of powdered amylase preparation are obtained from the precipitate, which has 70,000 unit/g. in dextrinogenic power at pH4.0 to 4.5 and 110,000 unit/g. in saccharifying power at pH 4.0 to 4.5.
  • the acid-active and acid-stable amylases of the present invention are useful, by virtue of their superior amylolytic, dextrinogenic and saccharifying activities in extended fields, for example as digestive for medicaments, for pre-treatment of materials for processing foods, as enzymes for ripening materials during production, as clarificant of fermentative foods of 95 per- C in order to obtain a precipitasuch as beer, sake (Japanese wine), vinegar and the like, as a tenderizer of beef or corn before cooking, as well as for other purposes for which amylases are conventionally used.
  • the instant amylases are however more especially suitable for use under acidic conditions and particularly as digestive enzyme which functions against dietary starch in the stomach where the gastric juice provides a strongly acidic condition.
  • a culture of Paecilamyces subglobosum, strain TPR-38ll is on deposit at American Type Culture Collection, Rockville, Maryland, under ATCC accession number 16492.
  • a culture of Paecilomyces subglabosum, strain TPR-38 10 is similarly on deposit under ATCC accession number 16493.
  • a process for the preparation of acid-active and stable amylase having superior amylolytic activities under acidic conditions which comprises, in combination, the steps of lst: pre-incubating a microorganisms selected from the group of Paecilomyces subglobosu'm strain TPR-38 l (ATCC 16493) and Paecilamyces subglobosum strain TPR-38ll (ATCC 16492) on a sterilized liquid or solid culture medium for seed koji at 30C for 70 hours under aerobic conditions and thereby obtaining a seed koji of the Paecilomyces subglogosum group; and
  • 2nd inoculating and cultivating the thus-obtained seed koji on a main liquid or solid culture medium at 30C under aerobic conditions until the objective acid-active and stable amylase is substantially accumulated in the culture medium, and recovering the acid-active and stable amylase from the cultured medium.
  • a process according to claim 1, wherein the microorganism is Paecilomyces subglobosum TPR-SB l0 (ATCC 16493).
  • main liquid culture medium and the liquid culture medium for seed koji are aqueous media and contain:
  • a carbon source selected from the group consisting of starch and maltose, in a total amount of 5 percent by weight;
  • carbon source also comprises a member selected from the group consisting of glucose, dextrine, glycerol and wheat flour, the total amount of carbon source not exceeding the said 5 percent by weight
  • nitrogen source also comprises a member selected from the group consisting of raw soybean flour, defatted soybean, dried yeast and corn steep liquor, the total amount of nitrogen source not exceeding the said 5 percent by weight.
  • main solid culture medium or the solid culture medium for the seed koji is constituted by wheat bran, chaff and water in the following ratio:
  • wheat bran:chaff:water 5:3:about 4 by weight.
  • the microorganism is a member selected from the group consisting of Paecilomyces subglobosum strain TPR-38 l0 (ATCC 16493) and Paecilomyces subglobosum strain TPR-38ll (ATCC 16492) and, whereby the obtained amylase is characterized by the following:
  • Aspergillus aureus, As rgillus oryzae and Paecilognyces variotz in the acidic p range from 2.5 to 3.5 the optimum pH ranging from 4.0 to 4.5,

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US531816A 1965-03-04 1966-03-04 Process for the preparation of amylase Expired - Lifetime US3652400A (en)

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CH (1) CH459114A (forum.php)
DE (1) DE1517783A1 (forum.php)
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FR (2) FR1482888A (forum.php)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983001458A1 (en) * 1981-10-26 1983-04-28 Lundell, Ralf Process for the preparation of enzymes

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JPS6034178A (ja) * 1983-08-08 1985-02-21 Daikin Ind Ltd 新規微生物

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US3288683A (en) * 1963-11-07 1966-11-29 Taisho Pharmaceutical Co Ltd Production of acid-stable proteolytic and amyloytic enzyme

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3288683A (en) * 1963-11-07 1966-11-29 Taisho Pharmaceutical Co Ltd Production of acid-stable proteolytic and amyloytic enzyme

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983001458A1 (en) * 1981-10-26 1983-04-28 Lundell, Ralf Process for the preparation of enzymes

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FR7581M (forum.php) 1970-01-12
ES323766A1 (es) 1967-01-16
CH459114A (fr) 1968-07-15
NL6602761A (forum.php) 1966-09-05
DE1517783A1 (de) 1970-01-29
GB1099446A (en) 1968-01-17

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