SU797582A3 - Method of enzymatic transformation of glucose to fructose - Google Patents

Abstract

Continuous enzymatic isomerization of a glucose syrup with a glucose concentration of 30-55% by weight containing less than about 10-3M calcium, less than about 10-2M of Mg++, the Mg++ being in a concentration, whereby the molar ratio of magnesium to calcium is greater than 5, the isomerization taking place at a pH 7.8-8.6 with a total contact time between enzyme and syrup less than about 3.5 hours, preferably less than 2 hours. A convenient temperature range for isomerization is 60-85.degree.C., preferably 60-70.degree.C. The syrup has no colbalt added thereto. A preferred practice involves a syrup with very little added magnesium. Post isomerization ion exchange treatment can be avoided. The enzyme is a particulate preparation derived from B. coagulans, preferably by glutaraldehyde reaction with homogenized microorganism cells.

Description

(54) METHOD OF ENZYMATIC TRANSFORMATION OF GLUCOSE IN FRUCTOSIS

ept

five

The invention relates to methods for the enzymatic conversion of glucose to fructose and can be used in the food and adjacent areas.

Enzymatic isomerization of glucoa into fructose is based on the use of the enzyme glucose isomerase, which is activated in the presence of Co and Md ions.

However, this greatly increases the cost of syrup conversion, since any amount of cobalt ions added to the original glucose syrup must be removed from the glucose-fructose product using relatively expensive ion exchange methods. Magnesium ions, which are not as toxic as cobalt, can be contained in small amounts in the resulting product. However, a decrease in the use of magnesium ions in the process of converting glucose to fructose also leads to a cheaper process due to a reduction in the cost of refining the resulting product.

Currently, most enzymatic conversion methods

Ch: yukoza in fructose provides for the process in the presence of. With and.

For example, there is a known method of enzymatic conversion of glucose to fructose by passing a solution containing 5-80 wt.% Glucose and Co and Md ions in an amount of 10 M and M, respectively, through a column with an immobilized glucose isomerase at 20-85 ° C and pH at the inlet of an alkaline 6-9. The total contact time varies widely, and Streptomyces sp. Is used as immobilized glucose isomerase. ATCC 21175 immobilized on an intercell-anion exchange cellulose carrier or anion exchange resin (no particle size is indicated) li.

0

However, a product is obtained containing unacceptable impurities of the used Cd ions and. This necessitates the additional purification of the target product by ion exchange methods using additional equipment and costs. In addition, the immobilized glucose isomerase used in the process destroys the temperature of the isomerization process. Performing the process at a pH close to 9 (with a long contact time) causes undesirable staining of the obtained product, which is also a disadvantage of the known Method. The goal of the inventors is to simplify the process and improve the quality of the target product. This goal is achieved in that the method of enzymatic conversion of glucose into fructose uses a syrup containing Md ions in an amount, and the content of Md is interconnected with the concentration of ions, which are additionally contained in the amount in the solution in the amount in the solution: molar ratio of concentrations Hg Vca is 5-500 when the concentration of ions is greater than KGM and 5-10 when the concentration of ions is Md less or equal, immobilized with glutamic acid is used as the immobilized glucose isomerase. ldegida glucose isomerase you. coagulans with a size of 0.01-1 cm, and the process is carried out continuously. Preferably, the iono-Md concentration is 10 -5x10 m, and the concentration of Ca ions is 5 X 10 M and at a concentration ratio (5-10): 1. Usually, the process uses a glucose solution with a concentration of 4045 wt.%, The contact time of the initial solution with the immobilized glucose isomerase is 10 minutes to 2 hours, and the process is preferably carried out at 60-70 ° C. Thus, a preferred implementation of the method involves treatment with such a small amount of added magnesium that the subsequent isomerization ion exchange can be eliminated. In the process, immobilized glucose isomerase obtained from You cells is used. coagulans, which is different from stability and high activity. Specifically, as a drug of immobilized glucose isomerase, the cells of microorganisms Vaz are used. cqagularis, cross-linked with glutaraldehyde, which undergo significant degradation. Preferably, the enzyme preparation is prepared from cells that have been homogenized prior to starting the reaction with glutaraldehyde. The method of obtaining enzymatically active, physically stable, water-insoluble glucose isomerase from cells of microorganisms You. coagulans involves concentrating and homogenizing microbial cells to produce a homogenized cell concentrate containing destroyed cells and with a dry content of substances. VA 3-30 wt.%, the interaction of these cells concentrate with 0,011, 0 weight.h. glutaraldehyde to a portion of the dry matter to thereby create a coherent solid product and then remove the water and disintegrate the coherent product. The method of producing glucose isomerase involves the cultivation under aerobic conditions of atypical you. coagulans producing glucose isomerase, and you are indicated. coagylans can grow only on inorganic nitrogen sources as nitrogen sources when on a nutrient medium containing a nitrogen source, a carbon source, with possible inclusion of xylose, small amounts of inorganic salts, at pH 5–9 and temperature 40–65 ° C, after which thus obtained glucose isomerase. Biochemical needs of immobilized glucose isomerase enzymes from you. The coagulans in cobalt can be completely satisfied without the addition of cobalt during the isomerization process. As a result of excluding Co from the glucose syrup, the performance of the isomerization process and the stability of the enzyme do not deteriorate, and in some cases improve, the biochemical requirements of the glucose isomerase enzyme in magnesium are much less than previously thought. In fact, the addition of Md can generally be eliminated if the Ca concentration is low. The requirements for the activation of glucose isomerase by ions are completely satisfied at a pH of 7.8 or more. when using less than 10M. Calcium ions in syrup are inhibitors, probably more than has been established so far. Obviously, the role of the ions in the syrup is mostly limited to inhibiting inhibition. The low calcium content in the syrup reduces the magnesium content. The Md-Vca-syrup ratio should exceed 5: 1 in a molar ratio, mostly this ratio should exceed 10: 1, but not 500: 1 if the concentration of ions is Md. . The reaction of enzymatic .. isomerization of glucose at a pH of more than 8.0 causes staining of the product. . This is avoided with conventional isomerization carried out at a pH below 8.0 in order to reduce color formation. Removing the color of the resulting syrup, for example, by treatment with activated carbon is included in the cost of processing. However, the degree of staining.

glucose syrup increases with increasing ptl.

A study of the trend towards coloration shows that the rate of staining at all pH levels is a function of time. In short, more or less, the staining rate at ipH 8.0 can be leveled by faster isomerization. As a result of limiting the total rejection or. the time of contacting the glucose syrup in the enzyme conversion reactor to less than 3.5 hours, the syrup is obtained in a suitable color.

If the duration of contact is limited to less than 1 hour, then treatment to absorb the color of the resulting syrup can be omitted. Thus, the proposed method has the following features: for isomerization, no Co% is added for isomerization. needing to be added in large quantities, ion exchange is not required after isomerization to remove harmful ions, the enzyme has excellent heat resistance, the performance of the method is improved, the method is suitable for the continuous operation of the column, the pressure drop per unit length of the column is small, the conversion level is 40-45% ..

Thus, the method consists in carrying out the isomerization of a glucose syrup as a continuous process at a pH of the supplied syrup in the range of 7.8-8.6 with a contact time of less than 3.5 hours, preferably less than 2 hours. With a continuous process of 30-55% (weight The weight of the glucose syrup is converted to a syrup containing the desired level of fructose, at least 40%, usually about 45%. The conversion temperature is 60-85 0, preferably B0-7CP. The pH range is 7.8-8.6 of the incoming syrup. by adjusting with alkali (NaOH or) and, if necessary, By re-adjusting at selected points in the conversion reactor, the pii of the isomerized exit syrup is lower than the pH of the incoming syrup. Typically, the difference between the pH of the incoming syrup and the pH of the exit syrup is from 0.2 to 0.6. The pH is important in the implementation of the proposed method. If the pH is too high, for example, higher than v8.6, then undesirable staining occurs (if the duration of contact is long). If the pH is too low, e.g. below 7.6, the addition of Co is necessary to maintain the activity of the enzyme.

The optimum pH for the enzyme is from you. coagulans is 8.5. Industrial batch process

Usually carried out with prolonged contact time due to the use of relatively small enzyme concentrations. For this reason, the pH should be maintained IAa | In order to avoid staining, the enzyme activity at this pH will be significantly lower than at optimum pH. Addition is necessary. With the continuous method, the contact time may be kept low and the pH may be high. Isomerization can be carried out at a very close to optimal pH of the enzyme. Co can be H- added. Thus, the continuous method has advantages over the batch process.

The amount of Md added to the supplied syrup is less than t, preferably less than 5x10 M. However, the tolerance is low Md levels, the content should be monitored. calcium in the incoming syrup to less than 10 M, which, for example, can be done by forming a syrup from water with a low calcium content or by appropriate processing of the glucose syrup to limit calcium to less than 2.5 x 10 M. Anyway calcium is present, then enough magnesium is added to the syrup to create an excess of Md ions in relation to its calcium content up to a molar ratio of MgVca greater than 5: 1, preferably a molar ratio of 10: 1.

The content and Ca is controlled and regulated so that as a result of the removal of ion exchange from the glucose syrup, if necessary, to avoid obtaining co-concentration of ions in a quantity greater than 10 M, and ions in a quantity greater than 10. If the glucose syrup contains a lower concentration, then the added amount of Md can be reduced in an appropriate ratio, such that it is maintained in the range of 5-500 for more than 10 and is higher than 5 for MDO (see drawing) . Thus, preferably, the syrup contains less than 2.5 x 10 M and 5 x M Mg

Almost the glucose syrups that the invention relates to are obtained from starch hydrolyzate. Frequently, the syrup is obtained by suspending the starch in hard water with wired water, and therefore there is Ca.

H ... one hundred starch hydrolysis and acidification of starch hydrolyzate are carried out enzymatically, using Sa-activated enzymes. Therefore, it is rarely absent in glucose syrups (besides TRX, which is obtained under laboratory conditions directly from crystalline dextrose using deionized water), it is necessary to monitor that 4To6ii Ca content exceeded 10 M. In continuous isomerization processes, the practical size of the enzyme particles is determined by processing of syrup (and equipment. For a method using a column that is preferred for the invention, particles are more y (are preferred (since the column is prone to clogging) If small particles are used. In thin layer processes (e.g. isomerization in a filter press reactor) particles of any size can be used. In the following examples of specific embodiments of the invention, the activity of the immobilized enzyme is measured in IGI C units {international unit ), 1 IGIC is determined by the amount of enzyme that catalyzes the isomerization of glucose to fructose, with an initial rate of 1 / mol / min under normal conditions, i.e. at 40 weight,% glucose, pH 8.5 at the inlet, and 4 X 10 M Ca, in a column with a discontinuous packed bed, column size 2.5 X 40 cm. In the examples, the enzyme productivity during a specified time (h 7 is determined by as the amount of glucose in kg, which can be converted into a mixture of fructose and glucose with a conversion rate of 0.45 per kg of enzyme with an initial activity of 100 IGlC / r,. Uses of the enzyme preparations have an activity from 150 to 250 IGlC / r, For comparison, the results from preparations with different activity all performance values are recalculated for an enzyme with activity 1 00 IGlC / r, In the examples, the contact time corresponds to an enzyme with an activity of 100 IGlC / r. For example, a contact time of 1 hour to form an enzyme of 200 IGlC / r activity corresponds to the duration of contact 2 with the usual 100 IGlC / r activity, Example 1, Isomerization in the absence and in the absence of Co, All isomerization is carried out in the form of continuous reactions in the stream with the use of plugs in the form of a compacted packing. Enzyme you. coagulans are prepared as follows, 1, preparation of the concentrate and its immobilization. Cultured you cells. coagulan strain NRRL B 5656, then the cells are extracted from the fermentation broth by centrifuging with a self-cleaning chamber at a pH close to 6.3, the Concentrate contains approximately 10% dry matter and about 40% of intact cells, 38 ml are added to 1 kg of concentrate commercial 50% glutaraldehyde with vigorous stirring to thoroughly mix glutaraldehyde with cell concentrate. Thereafter, the reaction mass is left alone at ambient temperature. After 1 hour, the reaction mixture thickens into a homogeneous mass of the curd consistency. This mass is broken weakly by stirring and washed with two volumes of deionized water, after which the water is drained. The gel particles are then transferred to vacuum, drum-dried, in which approximately 1 kg of the product is dehydrated to a weight of about 160 g. During the dehydration process, the soft gel-like pieces are transformed into hard pieces of material of constant size. The dehydrated samples are ground further to particles with a size of less than 1 mm in diameter. Regeneration of the enzyme from loading to loading will vary from 50 to 60% of its initial activity. However, about 15% by weight of the final product is extremely fine particles of material (1-70 ° CJ. 2, Immobilization by freezing. The procedure of Example 1 is repeated until the mixture of cell concentrate and glutaraldehyde is obtained and a gel-like mass of the inactive preparation is obtained. After that the gel the container) is placed in a deep cold and left there overnight. The next day, the frozen gel is thawed to ambient temperature and turns into a water mass. More water is added with stirring, after which it is drained. The resulting product is dried in a rotary dryer to a weight of about 160 g. The final product consists of nopiic particles that look somewhat like flakes in appearance. The restoration of the initial activity from loading to loading is 60-70%, there are virtually no small particles. varies from 150 to 2800. The enzyme preparation is pre-soaked in 40% w / w glucose syrup at room temperature for 1 hour. This previously soaked glucose isomerase is loaded into various water jacketed columns. The flow of glucose syrup, which has a certain concentration, pH and other characteristics, is given in Table 1, and is passed up through the enzyme material. The flow rate is adjusted to the target. obtain conversion rate output per 100 IGlC / r

Residual activity

60 ml

% Duration of contact per 100 IGlC / r, 90-140 90-150 min. Productivity per 100 IGlC / r350 320 Residual activity.

1 l

Duration of contact per 100 IGIC / g, min. Table. 2 (and in the similar tables below, where several values are indicated for the duration of contact, the first value corresponds to the beginning of the experience with high activity, and the last value indicates the end of the experience with lower activity. From Table 2 we can draw the following conclusions: performance at 100 IGlC / r and the residual activity is better without Co than with the addition of Co, if the pH is maintained at the recommended level, if the pH is below the recommended level, the performance at 100 I01 C / g and the Residual activity will be better with using Co than without it, but values using Co will be less than the values obtained

table 2

410

420

375

395

52

42

28

41

90-130 90-155 75-125 75-170 75-170 75-170 75-145 75-180 75-270 75-185 440 370 400 without Со, and at pH within the recommended level. Example 2. Isomerization in the presence and absence of .Md. Isomerization is carried out as indicated in Example 1. The following parameters are kept constant during the experiments: Glucose concentration, wt.% 40 pH inlet Adding Co Ca, M Concentration Less than 2, 5x1: 6 Temperature, ° С65 Adding magnesium varies from O to G X 10 M. After 450 hours of isomerization, the experiments are interrupted. The residual activity and productivity per 100 IGlC / r are determined and the duration of contact is calculated. On 100 IGlC / r. The results are indicated in the table. 3

Performance on

100 IGlC / r400 419 424 415

60 ml Residual activity,

Duration of contact at 100 IGI C / g, min

Performance on

100 IGlC / r410 435 435 450

Residual activity.

1 l

Contact duration at 100 IGlC / r,

min

Example 3, Isomerization c. varying ratios of md and ca.

Isomerization is carried out as described in Example 1.

At the same time, 5 columns of 60 ml each are used, one with a constant addition of MD to the incoming syrup and without the addition of Ca, and the other with varying ratios of MD and Ca in the glucose syrup.

Use the following parameters:

The results for the table. 4 show that the ratio is important for the lifetime of the enzyme; the ratio should exceed 5, preferably 10.

Table 3

420

50

50

52

48

75-150 75-150 75-150 75-155 75-145

440

60

62

60

62

75-130 75-125 75-125 72-125 78-125

The activity in the control column (without Ca) is measured using. parameters shown in parentheses. After working for 800 hours, the enzyme in the control column loses activity up to 25% of its initial activity. The numbers in the table. 4 represents the% activity relative to the enzyme activity of the control column after operation for the same number of hours.

Table 4

Example 4. The dependence of the isomerization on the glucose concentration in the incoming syrup.

The isomerization of the wastes as described in Example J. The following parameters are used: Glucose concentration, wt.% 40-50 RP at the inlet8.4 Temperature, c65 No MD, M 0.004 Ca, M Less than 2.5x10 In the table. 5 capacity, residual activity and duration of contact are indicated for operation for 450 hours in 60 ml columns. Table Operating Characteristics Indicators for glucose, wt.% Ticks 40 I 45 I 50 Production capacity per 100 IGIC / g 430 410 380 Residual activity 48 50% Contact duration, min / 100. IGIC-75-15085-17095rN

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

Actin-

% 5 15 45 60 70 85 95 100 95 90 Example 6. The dependence of activity and stability on temperature. 35 Isomerization was carried out as described in example 1, using glucose isomerase, prepared as described in example 1. 7 columns are operated in an isothermal mode at different temperatures in the range BS-EO C. Initial activity is measured, and also half-time as the time required to reduce the activity by 50% of the initial activity). The following parameters are used: Glucose concentration, wt.% 40 pH8.5 Temperature Variable No M0.004 M less than 2.5 x 10 The following results are obtained, shown in that one & l. 7. Table 75-150 56-110 Y5

TABLE OF CHARGE IN MEANS OF MEASURES Example 5. Effect of pH on activity. The effect of pH on the activity of the obtained immobilized glucose isomerase, prepared as described in example 1, is determined in a continuous flow column with a 2.5 x 35 cm plug. The isomerization is carried out as described in example 1. Before measuring the activity, the column is held for 5 hours to achieve equilibrium. The loading pH value is then changed to the next value, after which the column is operated for another 5 hours, and so on. In this case, the following parameters are used: Glucose concentration,% 40 pH = Change from -5.0 to 9.5 Temperature, OG 65 Cof -. No Hg, M0.004 Ca +% MH, less than 2.5 x 10 Duration of contact, min. 50 Table. 6 shows the relative% activity, the maximum activity at pH 8.5 is taken as 100. Continued table. 7 Example 7. Effect of the size of the pic on activity. With the exception of obtaining the enzyme, the omerization is carried out as described in Example 1. In this example, two enzyme preparations are used, indicated by X and Y, respectively. Before m, as the enzyme preparations load the columns, they are classified. In this case, the following parameters are used: glucose concentration, wt.% Temperature, 0 m |, M Caf, M. . Less than 2.5x10 Preparation of preparation X (see in prior 1).

Preparation of the drug.

Immobilization of flocculus.

1550 l of culture broth from fermentation you. coagulans NRRLB 5656 is concentrated by centrifugation at 10 ° C, resulting in a suspension containing about 12 g of dry weight, 100 ml of concentrate.

11 kg of this concentrate (pH 7.9) is left under for 3 hours with gentle stirring in order to carry out autolysis. The pH is maintained at 6.5 with dilute acetic acid. To a suspension containing more than 70% of active units / in soluble form, add 330 ml of a 50% glutaraldehyde solution, resulting in a concentration of glutaraldehyde in the reaction mixture of about 1.4% (weight / volumeT, After 1 hour partially crosslinked The gel is vigorously stirred after adding 20 liters of deionized water. 80 ml of Drewfloc EC 25 solution is added to this suspension to obtain a clear solution. The suspension is then filtered to remove water as far as possible. The filter cake is dried in a vacuum. and 35 ° C. The dried cake is crushed to a particle size of less than 30 microns. The activity is determined by isomerization in the feed with the dried spray with a powder obtained from the concentrate as a comparison. Condition: pH 7.0, 0.1 g CoS047 Hap / l and 2.0 g of MgSQj., 40% glucose (weight / volume) The nutrient medium is purged with nitrogen. The activity of the immobilized enzyme is more than 70% compared to the control. After use, the immobilized enzyme is removed by filtration and reused. This is repeated 5 times. After five reuse, the activity remains at the same level.

The second part of autolyzed cells of the concentrate is treated with 20 ml of 30% Orewfloc EC 25 per kg of suspension before interaction with 1.4% glutaraldehyde (weight / volume. As a result, practically the same product is obtained.

The effect of particle size on the activity of preparations X and Y can be seen from the table. 8 and 9.

Table 8

Continued table. eight

Table

Particle Size Distributions

Aktivpreparatov have isomerase Gluteness, goats, M%

75-250

100 69 48 39 250-354 354-500 500-707 707-1000

34

It is established that stability does not depend largely on the size of the particles. As can be seen from the above values, the activity of some drugs depends more on the particle size than on other parameters.

Example 8. Getting color.

The isomerization is carried out in the same manner as in Example 1, and the b columns are filled with different amounts of enzyme, maintaining the conversion rate and the contact time constant and equal to 45% and 1 hour, respectively.

The following parameters are used:

Concentration

0

45 glucose, wt. pH

8.5

Temperature

Variable

) About No

-four

WITH

Md

8 X 10

m

.-6. +1

five

 M

Less than 2.5 x 10

An increase in Icumsa color is observed, as shown in Table. 10, after vloO h isomerization.

0

Table 10

55

60

Note: Color nq. icun, -4 OL

 ten

sa iu

where ex is OD, j.j (optical density); L - DS in g / ml (dry matter); Y5 s - measured cell length, see

Example 9, Effect of column size (as its size increases).

With the exception of enzyme production, isomerization was carried out as described in Example 1.

The enzyme is prepared according to the procedure for preparing the preparation Y described in Example 7. The particle size is 150-500JLI.

Thus, by increasing the size of the column, starting from 60 ml, the isomerization method can be carried out without any difficulties and with the same performance indicators and residual activity.

Claims (1)

The invention of the invention ft9 .p-2 Ca 2lO