DE1792748B2 - Process for the production of glucose isomerizing enzyme - Google Patents

Description

The invention relates to a process for the production of glucose isomerizing enzyme by cultivation r> a microorganism of the genus Streptomyces.

The alkali processes may already be used to convert glucose into fructose of anion exchangers and the enzymatic process became known. In practical application however, both types of process have shown disadvantages.

The alkali processes such as those described in US Pat. Nos. 2,356,664, 2,487,121 and 2,746,889 cause the loss of sugar through decomposition. In addition, the conversion ratio is insufficient.

The enzymatic process for converting glucose to fructose is based on the activity of enzyme. A well-known enzyme is that produced by bacteria glucose isomerizing enzyme.

S. Mitsuhashi et al., Were the first to discover that Lactobacillus pentosus derived enzyme preparation is able to convert glucose into fructose to a limited extent js (). Biol. Chem. 204,1011-1018 [1953]).

M a rs hai I and co-workers, posed in Pseudomonas hydrohila, an enzyme that can convert glucose into fructose in the presence of arsenate (cf. Science, 125, 648-649 [1957]). It was then reported that the same enzyme was found in Aerobacter cloacae of S. Tsumura et al. (Agr. Biol. Chem., [Japan], 25, 616-625 [1961]). This isomerizing to glucose Enzymes of bacterial origin have all or some of the following deficiencies relating to Production method, the amount and the quality of the yield.

1. In producing a glucose isomerizing enzyme from bacteria, the culture medium requires the inclusion of expensive pure D-xylose. ^{> 0}

2. The yield of enzyme is low.

3. The isomerization reaction due to the use of the enzyme requires the presence of harmful arsenate and / or Mn ++.

4. The glucose isomerizing enzyme obtained lacks too much that for industrial use required constancy of quality.

Because of this deficiency, they have not been usable for industrial production and utilization until now. Therefore, the isomerization reaction using these enzymes has not been made technical up to now Implemented in practice.

From the publication “A Study on Isomerization b5 of Dextrose by a Streptomyces Strain "of the Agricultural Chemical Society of Japan through the annual meeting in Sapporo in July 1964, p. 92,93, is also the use of a Sreptomyces strain for the production known from glucose isomerase. However, this known method is only concerned with Assimilating a Streptomyces microorganism in the presence of xylose, resulting in the above Brings disadvantages.

The object of the present invention is a process for the preparation of a glycose isomerizing enzyme which is free from the above-mentioned defects and sufficient possesses industrial applicability.

According to the invention, glucose isomerizing enzyme is obtained by assimilating a xylan and glucose isomerizing enzyme-forming strain of Streptomyces in a xylan or xylan-related Medium containing material grows. Such strains of Streptomyces are Streptomyces flavovirens (ATCC No. 3320), Streptomyces achromogenus (ATCC No. 12 767), Streptomyces echinatus (ATCC No. 21 133) and Streptomyces albus (ATCC No. 21 230).

The assimilation of xylan means the production of xylanase, which xylan or derivatives that D-xylose units in xylanhaitigem material, can hydrolyze.

Xylyn consists mainly of D-xylose units and is one of the main components of hemicellulose, which occurs in practically all land plants. D-xylose is technically made by acid hydrolysis of xylan manufactured.

The xylan-containing materials useful in the present invention include all xylan-containing land plants, such as straw, wheat, bran, corn on the cob, corn husk, rice bran, and wood as well Derivatives thereof, including the effluent resulting from the processing of such land plant materials, such as the various types of pulp waste and waste liquor and, in particular, corn steep liquor. Of the As used herein, the term "xylan-containing material" refers to any material that Contain substances which, after hydrolysis by the microorganism to be used according to the invention produced xylanase produce such substances as D-xylose, which for the production of glucose isomerizing Lead enzyme.

It has been found that a glucose isomerizing enzyme according to the present invention is remarkably high Yield is generated.

The culture for producing the glucoseisomerisierenden enzyme is used usually in an aqueous medium, usually at about 30 ⁰ C under aerobic conditions is carried out.

The addition of cobalt salt remarkably increases the yield of glucose isomerizing enzyme. Therefore, usually 1 χ ΙΟ- ³ moles of cobalt salt are added to the medium.

When actually cultured, the glucose isomerizing enzyme usually appears in the cells. Therefore, after the completion of the cultivation, the cells, which are a rich source of the enzyme, are obtained from the Separated broth. The soluble enzyme can also be used as a source of enzyme.

The conversion of glucose to fructose is done by putting this enzyme on the Let the glucose solution take effect. The enzyme produced by the microorganism of the genus Streptomyces is Effective over a wide range of pH, temperature and glucose concentration and therefore shows itself to be effective particularly advantageous for use in technical glucose isomerization.

If desired, the glucose isolation can be carried out in to such an extent that up to about 50% of the initial glucose is in fructose be convicted.

This results in the production of a syrup that is convenient has the sweetening properties of invert sugar.

The isomerization by the enzyme prepared according to the invention can be carried out at a pH between about 5.5 and about 8, preferably between about 6.8 and about 7.2, 7 being the optimum value. The reaction temperature may suitably be in the range between about 45 ° C and about 80 ⁰ C., with the range between about 60 ⁰ C and about 70 ° C preferably \ tl

The following examples illustrate the invention.

example 1

A pre-sterilized medium of 400 ml containing 3% wheat bran (with a xylan content of 21%), 4% rice mash water, 0.1% MgSO ₄ · 7 H ₂ O and 0.3% NaCl was treated with Streptomyces albus, strain YT -No. 44, inoculated and cultivated with shaking for 20 to 30 hours at a temperature of 30 ^{° C.} The seeds obtained from this batch were transplanted into 10 liters of medium of the above composition and further cultivated with aeration with a shaking fermenter (aeration value 7.5 liters / min; 200 rpm; 30 ^° C.). The activity of the glucose isomerizing enzyme in the cells reaches its maximum within 20 to 25 hours. The activity of the glucose isomerizing enzyme was determined with the following reaction mixture:

0.2 m phosphate buffer solution (pH 7.5) 0.5 ml
1 mglucose solution 0.2 ml

0.1 M MgSO ₄ · 7 H ₂ O solution 0.1 ml

Enzyme solution 0.2-0.3 ml

The above approach was made up to a total volume of 2 ml with water and ^{incubated at 70 °} C. for 1 h. The reaction was stopped by adding 2 ml of 0.5 M perchloric acid. The fructose content was determined by the cysteine-carbazole method. The amount of enzyme which produces 1 mg of fructose per hour at 70 ° C. and the above test conditions from glucose is defined as one enzyme unit.

The cell mass is separated from 50 ml of the above culture solution by centrifugation, with water washed, suspended in water and sonicated for 15 min at 10 kHz. After the sound treatment will be Obtained 30 ml of the supernatant solution and used as a source of enzyme. The enzyme activity was 623 Units.

50 g of glucose, 5 ml of 0.1 M MgSO ₄ · 7 H ₂ O, 25 ml of 0.2 M phosphate buffer solution (pH 7.5) and water were then added to this enzyme solution. The total volume was adjusted to 100 ml with water. The mixture was incubated at 65 ⁰ C for 50 h, while the pK value was maintained during the reaction between 6.8-7.2. After the reaction was finished

ίο the precipitate is removed by centrifugation and the supernatant liquid is treated with an ion exchange resin to remove the inorganic salt and protein and then evaporated in vacuo.
A sweet syrup consisting of 54% glucose and 46% fructose was obtained. Complexes of fructose and calcium were separated from this mixture using conventional methods by adding milk of lime. The obtained fructose was further decolorized and, after concentration, crystallized by reducing the pressure to remove solvent. 11 g of fructose were obtained.

The product was examined for fructose by paper chromatography, phenol: water (4: 1) or Pyridine: butane! : Water (4: 6: 3) were used as a solvent. A resorcinol reagent which is a red color generated specific for ketosis was used as a means of identification. This became found that the Rf value corresponds exactly to the Rf value of comparative fructose. The twist of a Solution of the product was also exactly equal to the value of a standard fructose solution.

Example 2

In this example, pure xylan was used as the carbon source of the medium and Streptomyces flavovirens ATCC No. 3320, Streptomyces achromogenus ATCC No. 12,767, Streptomyces echinatus NIHJ 180 (= ATCC 21 133) and Streptomyces albus YT no. 4 (= ATCC 21 230) is used.

These microorganisms were cultured in a medium of 50 ml at 30 ⁰ C, containing 1% polypeptone, 0.3% K ₂ HPO _4, 0.1% MgSO ₄ · 7H ₂ O and 0.5% xylan. After inoculation, the medium was shaken. After the cultivation, the cells were collected by centrifugation and, after washing with water, the collected cells were suspended in water and sonicated at 10 kHz for 150 min. After this sonic treatment, 20 ml of the supernatant liquid was collected and used as an enzyme solution. The activities of the enzyme solutions obtained are shown in the following table:

Table I. At first- Rest- enzyme protein Cultivate Cultures Xylan Xylan activity the cell Time (g / 50 ml (g / 50 ml
Medium) (Units) (mg) (H) Medium) 0.20 0.25 10.0 66.4 14th St. flavovirens 0.16 ATCC No. 3320 0.25 41.2 76.8 10 St. achromogenus 0.19 ATCC No. 12,767 0.25 32.8 82.3 22nd St. Echinatus NIHJ 180 0.07 (= ATCC 21 133) 0.25 122.8 86.4 34 St. albus YTNr. 4th (= ATCC 21 230)

The enzyme activity shown in Table I represents the amount of fructose produced when 80 ml the 0.1 M glucose solution with 20 ml enzyme solution for 1 h 70 ° C react. Though differences consist in enzyme activity, each xylane assimilating strain gave glucose isoinerizing enzyme.

Example 3

III

A build-up medium (50 ml) composed of 1% xylose, 1% polypepion, 0.3% K ₂ HPO ₄ , 0.1% MgSO ₄ · 7 H ₂ O was placed in a flask and sterilized. Then Streptomyces; .lbus YT-No. 4 (= ATCC 21 230) inoculated into the medium and cultivated at 30 ° C. for 3 d. This culture was then used as the seed for the following experiment.

The medium for producing the enzyme contained 5% rice bran, 3% corn mash liquor, 0.1% MgSO ₄ · 7 H ₃ O and was adjusted to a pH of 7.0. 100 ml of this was put in a 50 ml flask and sterilized. Then they were inoculated with 4 ml of the aforementioned hall and cultured with shaking for 30 hours at 30 ° C. The enzyme solution prepared from 50 ml of this culture had an enzyme activity of 700 units. The cultured cells were then collected by centrifugation. The collected cells were then placed in Suspended water and destroyed by sonication at 10 kHz for 15 minutes. The destroyed cells were centrifuged and the resulting supernatant liquid wedge was used as an enzyme source. This enzyme solution was reacted with 200 g of glucose in the same manner as in the previous example. 49.5% of that as substrate for Glucose (200 g) used in the enzyme was converted to fructose After removing the protein and purifying it using activated carbon and ion exchange resin, the above reaction solution was concentrated to a water content of 25%, and the yield was 195 g as measured as an anhydrous substance.

Example 4

This example illustrates the use of corn hulls and corn cobs as sources of xylan.

The medium (50 ml, pH 7.5), which contained 4% corn mash water, 0.1% MgSO ₄ · 7 H ₂ O, 0.025% CoCl ₂ · 6 H ₂ O and 3% corn husks or 3% corn cobs, which on No. 50 mesh was placed in a 200 m 'flask and sterilized. Then the medium with Streptomyces albus ATCC 21 132 YT-No. 5 (= ATCC 21132) was inoculated and cultured 48 h with shaking at 3O ⁰ C. The activity of the glucose isoinerizing enzymes is as given in Table II.

b0

Tables Enzyme activity
(Units) Xylan source 24.5
481.0
320.5 Blank sample
Miii pods
Corn on the cob

b5

The reason for the production of glucose isomerizing The enzyme used in the all-round test for the xylan source is that corn steep liquor is also a xylan-containing material isl.

The glucose isomerizing enzymes are when using corn husks and corn cobs as Generate xylan source.

The enzyme solution, which has 481 units of enzyme aclivity and which was obtained in the method using corn hulls as the xylan source, became 100 g glucose (anhydrous), 50 ml 0.2 M phosphate buffer solution (pH 7.5) and 10 ml 0 , 1 m MgSO ₄ ■ 7 H ₂ O given. The total volume was made up to 200 ml with water. The mixture was seeded at 70 ⁰ C. During the reaction the pH was kept between 6.8 and 7.2 with NaOH.

After the reaction, protein was removed from the reaction solution and the solution was made using Activated carbon and ion exchange resin cleaned. The solution was then down to a water content of 25% concentrated. The yield obtained by the above procedure was 98.0 g of anhydrous substance.

Example 5

A hall medium containing 1% xylose, 1% polypeptide, 0.3% K ₂ HPO _4, and 0.1% MgSO ₄ · _{7H 2} O was placed in a flask and sterilized. Then the medium with Streptomyces albus YT-No. 4 (= ATCC 21230) was inoculated and cultured for 48 h at 30 ^c C to prepare an inoculum. In order to increase cell production, an inoculation solution from the seed stage was inoculated with 50 L of medium, and this was cultured. The latter was used to inoculate 500 liters of medium and these were cultured, and finally they were used to inoculate and cultured 50001 medium. The composition of the media used in this treatment (excluding the seed medium) was 3% wheat dough, 4% maize mash water, 0.1 MgSO ₄ · 7 H ₂ O and 0.025% CoCl ₂ · 6 H 2 O and water. After this medium had been sterilized, the initial pH was adjusted to 6.5 with NaOH, and the cultivation was carried out at 30 ^° C. with aeration in an amount of one third of the volume of the broth per minute and a stirring speed of 200 rpm. The enzyme activity reached a maximum after 24 hours. The enzyme solution prepared from 50 ml of broth had an activity of 750 units.

After the reaction was complete, the wheat bran in the broth was removed using a sieve. The cells were collected using a filter press, whereby 1000 kg of cells were obtained. The collected cells were then suspended in water. The muddy cells were means destroyed by a high pressure homogenizer. The disrupted cells were used as a source of enzyme.

A converted starch syrup of DE = 92.5 in a concentration of 50% (consisting of 87% glucose and 13% disaccharide-containing oligosaccharide) was used as the substrate for isomerization. 15,000 kg (calculated as solid) of the above converted starch syrup was placed in an isomerization tank of 30 m ³ capacity. 0.0025 M Mg (OH) ₂ was added, and after the syrup had been heated to 65 "C, the enzyme source was added. The pH was maintained near 7.0 by means of NaOH. In the drawing this is Conversion ratio, the influence of temperature and the influence of pH during the isomerization process are shown.

After 90 hours, the conversion ratio reached approximately 45%. The reaction solution was then taken Using activated carbon and ion exchange resin cleaned as in the conventional cleaning process. The solution was concentrated to a water content of 25%. The yield of produced Product was 19,600 kg. An analysis of this product showed 29.6% fructose, 36% glucose, 9.4% Disaccharide containing oligosaccharide and 25% water.

Example 6

Using the procedure of Example 5, seed cultivation, intermediate cultivation and Main cultivation carried out successively.

The medium composition contained 3% wheat bran, 4% corn mash liquor, 0.1% MgS (X · 7 H2O and 0.025% CoCl ₂ · _{6H 2} O. After the medium was sterilized, the initial pH of the medium was adjusted with NaOH adjusted to 6.5 and the cultivation at 3O ⁰ C, aeration at one third of the volume of broth per minute and stirring at 200 rpm performed. After 24 h the cell activity reaches a maximum. the cells were collected by means of a filter press, wherein 900 kg of cells were collected, and the collected cells were used as an enzyme source.

A starch syrup in 50% concentration with a DE value ο of 97.0 (consisting of 95% glucose and 5% disaccharide-containing oligosaccharide) was used as the substrate for isomerization. 15,000 kg (calculated as the solids content) of the above-mentioned converted starch syrup was placed in an isomerization tank of 30 ^mJ capacity. 0.0025M MgSO ₄ · _{7H 2} O was added, the syrup heated to 65 ° C and the enzyme source added. The pH was maintained at approximately 7.0 with NaOH.

After 100 hours the conversion ratio was

i) about 47%. The reaction solution was then with Activated carbon and ion exchange resin cleaned as in conventional cleaning processes. The refocusing product obtained to a water content of 25% weighed 19,500 kg. Analysis of this product found: 33.4% fructose, 37.7% glucose, 3.9% disaccharide-containing oligosaccharide and 25% water.

In the original documents of the parent application none of the strains used in the preceding examples with depository and

j "> deposit number given.

Claims (1)

Claim: A method for producing glucose isomerizing enzyme by culturing a microorganism of the genus Streptomyces, characterized in that one assimilates a xylan and glucose isomerizing enzyme-forming strain of Slreptomyces in a xylan or cultured medium containing xylan-containing material.