DE1642581C3 - Process for the production of alpha-D-galactosidase - Google Patents

Description

The invention relates to a process for the production of (i-galactosidase by aerobically growing a Microorganism.

A process for the hydrolysis of raffinose using α-D-galactosidase from an Actinomyces strain (Strain No. 26) is known (Chemical Abstracts, Vol. 63, 1965, column 10 3351).

It has not yet been possible to break down raffinose on a large scale by means of this α-n-galactosidase To evaluate style industrially. Until now, this decomposition could only be carried out in the laboratory. the The reasons for this are that a strong α-D-galactosidase activity has not yet been achieved could and that, as was found, the activity of invertase is still so strong that sucrose a Suffering from decomposition. In addition, in Chemical Abstracts 1. c. used trunk not with depository and deposit number given.

The object of the invention is to propose a method for producing an a-D-galactosidase bring, with which the yield of beet sugar is increased. Furthermore, a decomposition of Rafrinose take place. This object is achieved according to the invention in a method of the type mentioned at the outset solved by using the strain Mortierella vinacea var. Raffinose-Verwerter ATCC 20 034 in a synthetic, a «-D-galactosidase inducer containing nutrient medium and that the α-D-galactosidase in the form of the mycelium this fungus or in the form of a preparation isolated from the mycelium of this fungus.

The mold used according to the invention was isolated from soil in Chiba / Japan. Although its morphological characteristics are those of Mortierella vinacea. described by Dixon-Stcwart (Manual of Soil Fungi. G i 1 ma η η: Second Edition. The Iowa State Collage Press-Ames, Iowa, USA), it generates α-D-galactosidase almost without invertasc. On the basis of this, the fungus Mortierella vinacea var. Raffinose "recycler" ATCC 20 034 was named. The properties of this Mortierella vinacea var. Raffinose "recycler" are as follows:

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1. A microscopic observation of the fruit bodies grown on a malt extract agar medium shows branched sporangiophores with a diameter of 3 to 4 μ, light brown Sporangia that are roughly spherical in shape a diameter of 10 to 20 μ, which are free of Colluroella, as well as irregular, angular ones Sporangiobores with a size of 2.7 to 5 μ.

2, Macroscopic observation on different agar media:
Malt extract agar:

The mycelium shows a thick, felt-like appearance and changes in color from white to Light brown or dark orange when the spores mature. No soluble pigment will be in that Medium generated. Potato Glucose Agar:

The mycelium changes color from white to light brown as the spores mature. No soluble pigment is produced in the medium.
Yeast Extract-Malt Extract Agar:
The mycelium will show a change in color from white to beige as the spores mature. No soluble pigment is produced in the medium.

Further details of the invention can be found in Examples 1 to 5 below.

The uD-galactosidase activity is measured as follows: An enzymatic reaction is performed at 40 ⁰ C for 2 hours with addition of 1 cm ³ of a mycelial suspension to a mixture of 0.5 cm ³ of a 0.06-M-melibiose and 0.5 cm ^{3 of} a 0.1 M phosphate buffer (pH 5.2). After completion of the reaction, the reaction mixture is kept in boiling water for 5 minutes to inactivate α-D-galactosidase. 1 cm ^{3 of} a 1.8% Ba (OH ₂ ) _{· 8H 2} O solution and 1 cm ^{3 of} a 2% ZnSO ₄ · 7H ₂ O solution are added. After the mixture is centrifuged, the amount of glucose in the above part is quantified by the glucostat method. The activity of α-D-galactosidase which liberates 1 ag of glucose under these reaction conditions is referred to as a unit. Since the amount of exposed glucose and the enzyme concentration are linearly related up to an amount of 1000; xg glucose, the enzyme solution is previously diluted so that the amount of enzyme is within the aforementioned relationship. The enzymatic reaction is then carried out. Finally, the amount of glucose exposed is multiplied by a dilution factor e.i.

example 1

An M 10 phosphate buffer with 9.75% milk sugar. 2% glucose, 1.8% peptone, 1.8% meat extract. 0.2% magnesium sulfate and 0.2% potassium chloride is inoculated with the fungus. The mushroom is cultivated with shaking for 72 hours. After the cultivation is completed, the mycelium is filtered out, carefully washed with water, and weighed in a certain amount for use as an enzyme. Α-D-galactosidase was obtained in an amount of 3500 units per 1 cm ^{3 of} filtrate and in an amount of 28,400 units in the mycelium obtained from 1 cm ^{3 of} medium.

On the other hand, sulfuric acid becomes 10 g beet mash (Raffinose content 1.088 g) was added to adjust the pH to 5.2. This is followed by

Addition of M / IQ phosphate powder (pH 5.2) as well as a setting of the resulting solution with the help of water to 20 ° Brix,

The mycelium is then added to the dilute beet rolasse solution prepared in this way. At 50 ⁰ C for 24 hours, the enzyroatische reaction is carried out with shaking. After the completion of the reaction, the amount of raffinose and the increased amount of sucrose in the filtrate are quantitatively determined by an elution method of paper chromatography. When the mycelium having an enzyme activity of 450,000 units is used, the degree of decomposition of raffinose and the increased amount of sucrose are 70.4% and 364 mg, respectively. If a mycelium with 900,000 units of enzyme activity is used, the corresponding values are 81.5% and 544 mg.

Example 2

The fungus is cultivated under the same conditions as described in the example. A certain amount of the mycelium thus obtained is used as an enzyme.

On the other hand, sulfuric acid and phosphate buffer (pH 5.2) become 10 g of beet molasses (raffinose content 1.088 g) are added and the mixture is adjusted to 36 ° Brix with water. To the diluted Beet molasses solution is then added to 1,700,000 units of the enzyme to make the enzymatic To effect a reaction, shaking at 37 "C and for a period of 24 hours. After the completion of the reaction, the reaction mixture is filtered. In the filtrate, the degree the breakdown of raffinose and the increased amount of sucrose were measured. K I a careful one Washing the mycelium with water it is dried for repeated use in different, dilute beet molasses solutions with the same composition. The one here Results obtained with repeated use are shown in Table 1 below.

turning of 1 750 000 units of the knocked down Part as an enzyme - in the same way as described in Example 2 - carried out here The results obtained are contained in Table 2 below.

Table 2

frequency
the
use
¹⁰ des
Low
blow Enlarged
Amount of
Sucrose
(mg) degree of
Decomposition of
Raffinose
(%) Remaining
Activity from
nD-Galacio-
sidase
(%) 1
15 2
3 262
244
244 67.3
65.4
50.9 61
44
29

Table 1

Often wedge

the

use
of the mycelium

Enlarged
Amount of
Sucrose

(possibly

372
426
390

degree of

Decomposition of

Raffinose

67.3 72.7 67.3

Example 3

Remaining

Activity from

ii-ij-galacto-

sidase

98

84

55 The decomposition of raffinose is also carried out by concentrating the aforementioned part under reduced pressure, adding 1,750,000 units of aD-galactosidase of this concentrate to the beet molasses (raffinose content 1,088 g), and adjusting the resulting mixture to one pH of 5.2 and

36 ° Brix each with water and sulfuric acid. The whole is then left at 37 ° C. for 24 hours stand. It is found that the degree of decomposition of raffinose is 65.3%, while the increased Amount of sucrose is 260 mg.

B e i s η i e 1 4

It becomes a medium with 1.5% lactose. 0.5% glucose, 1% corn steep liquor, 0.1% urea. 0.1% ammonium sulfate. 0.3% KH ₂ PO ₄ . 0.2%

MgSO ₁₁ 7H ₂ O and 0.2% NaCl prepared. After the medium has been sterilized, the spores of the fungus are inoculated and cultivated at 30 ° C. for 72 hours. In the mycelium, which is obtained from 1 cm ^{3 of} the medium, 28,000 units of ii-D-galactosidase are present.

On the other hand, 10 g of beet molasses (raffinose content 1.088 g) diluted with water to 15 ° Brix. The resulting solution is brought to a pH of 5.2 adjusted with the help of sulfuric acid. The ver

A mycelium containing 980,000 units of α-D-galactosidase is then added to a thin beet molasses solution. An enzymatic reaction is in each case carried out at 20, 30, 40, 50, 60 and 7O ⁰ C under 24 hours of shaking. Then the decomposition

degree of raffinose measured. The results obtained are shown in Table 2 below.

Table 3

The fungus is cultivated in a medium which has the same composition as that in Medium described in Example 1 to obtain the mycelium. The mycelium is in a homogenizer homogenized and then by means of an ultrasonic generator (10 kHz) during a Treated for an hour.

The mycelium disrupted in this way is then centrifuged to remove the above part and to separate the precipitate. The activity of (i-D-galactosidase is measured in each part. Es there is an enzyme activity of 39% in the above part and 61% in the precipitate. The decomposition of the raffinose is then carried out

^ caktionstcmperiilur /. degree of slaughter by Raffmnse (C) 1%) 20th 42.1 30th 78.2 40 80.1 50 83.5 60 78.0 70 53.9

An enzymatic reaction is also carried out in such a way that 10 g of Rübcnmclasse (Raffinoscgchalt 1.088 g) with a Mcllvainc buffer

^Q 15 Brix be diluted so that the pH is in each case adjusted to 2.2, 3, 4, 5, 6, 7 and 8 that units 980 000 are added to the mycelium and that the resulting solution at 5O ⁰ C during 6 Is shaken for hours. The degree of decomposition of the raffinose is shown in Table 4 below.

Table 4

pH of the reaction liquid Degree of decomposition of raffinose 2.2 11.0 3 61.2 4th 80.5 5 80.7 6th 51.9 7th 38.1 8th 12.0

ben to the decomposition of Rafönose repeatedly under the same conditions as described above. The under repeated use The results obtained are shown in Table f.

Table 5 Example 5

10 g of beet molasses (raffinose content 1.088 g) are diluted with water to 15 ° Brix. Then the Solution adjusted to pH 5.2 with sulfuric acid. The resulting dilute beet molasses solution 980,000 units of the mycelium according to Example 4 are then added. The enzymatic The reaction is carried out at 50 ° C. for 6 hours with shaking. Then the mycelium Carefully washed with water and various diluted beet pulp solutions (same composition as described above) admitted frequency

of use

of the mycelium

1
2
3

Degree of decomposition
of raffinose

77.3 74.6 65.3

Remaining

Activity from

o-galactosidase

in the mycelium

79.4 66.6 58.3

With the actinomycetes strain no. 26 in the reference "Chemical Abstracts" 1 α, which the applicant was available, comparative tests were carried out.

The applicant has found that the u-D-galactosidase of this strain, since it is an extracellular Represents enzyme, in contrast to the method according to the invention, where the mycelium can be used, not repeated and also not in the continuous column process can be used to hydrolyze raffinose from beet juice or beet molasses.

Furthermore, the invention obtainable according to aD-galactosidase, in the raffinose hydrolysis up to a temperature of 70 ° C and up to 60 ° Brix (enzyme of the strain No. 26 in accordance with "Chemical Abstracts" Ie. Up to 30 ⁰ C and up to 7 ° Brix).

Claims (2)

i 642 Patent claims: 1, a process for the production of ap-galactostdase by aerobic cultivation of a microorganism , characterized in that the strain Mortierella vtrmcea var, RafSnose-Verwerter ATCC 20 034 is used in a synthetic nutrient medium containing an aD-galactostdase inducer and that the aD-galactosidase is obtained in the form of the mycelium of this fungus or in the form of a preparation isolated from the mycelium of this fungus. 2. Use of the α-D-galactosidase obtained according to claim 1 for the hydrolysis of raffinose of beet juice or beet molasses in galactose and sucrose.