CS207702B2 - Method of enzymatic transformation of the glucose to the fructose - Google Patents

Description

(54) A method of enzymatic conversion of glucose to fructose

The invention relates to a process for the enzymatic conversion of glucose to fructose, in particular to the enzymatic conversion of part of the glucose in solution to fructose.

A number of methods are known for converting glucose into fructose or for producing a solution containing fructose. These methods can be divided into three broad categories. In the first category, sucrose is converted to glucose and fructose by acid or invertase.

In the second category, glucose is converted to fructose by the action of alkaline catalysts. There are a number of publications and patents relating to various alkaline catalysts and their use to convert glucose to fructose. Examples of such processes are those disclosed, for example, in U.S. Patent Nos. 2,487,121, 2,746,889,

354,665, 3,285,245, 3,285,776, 3,383,245 a

However, isomerization under alkaline conditions is associated with a number of disadvantages. For example, the process is not selective, and for this reason a number of undesirable by-products are produced, for example large amounts of differently colored compounds and substances of an acidic nature. Thus, the products of this process need to be purified to remove these undesirable by-products, which methods are complex and very expensive.

A third category for the production of fructose-containing solutions is the enzymatic conversion of glucose, in particular a glucose solution such as corn syrup into fructose. A variety of known microorganisms which produce glucose isomerase can be used for this purpose. For example, in Science, Vol. 125, pp. 648-649 (1957) discloses that the enzyme produced by Pseudomonas hydrophilla causes glucose isomerization to fructose. Takasaki, British Patent 1,103,394 and Japanese Patent 7428 (1966) disclose that microorganisms belonging to the genus Streptomyces such as Streptomyces flavovirens, Streptomyces achromogenes, Streptomyces eohinatus and Streptomyces albus produce glucose isomerase. There are a number of other microorganisms that are also capable of producing this isomerase, for example, Aerobacter cloacae, Bacillus megaterium, Acetobacter suboxydans, Acetobacter melainogenus, Acetobacter roseus, Acetobacter oxydans, Bacillus fructose and Lactobacillus fermenti.

In order to make the process for converting glucose by isomerase economical, it is necessary to use isomerase under conditions that achieve maximum fructose yields using a minimum amount of glucose isomerase. In addition, the conditions for isomerization should be

2D7702 to produce as few undesirable by-products as possible.

Glucose isomerase is produced predominantly intracellularly by the above microorganisms. This means that a major proportion of this isomerase is found inside and / or in the wall of the microorganism: In conventional methods in which these cells are used to isomerize glucose to fructose, isomerase is released during isomerization or extracted from the cells. In both these cases it basically gets into solution. It would be very expensive and difficult to recover the isomerase from the solution in order to use it for further isomerization reactions.

Tsumury et al., Published in Nlppon Shokuhin Kogyo Gakkaishi, 14 (12) (1967), was a glucose isomerase produced by Streptomyces phaechromogemes. bound to DEAE-Sephadex. The enzyme thus bound was used in the form of a column through which a glucose solution continuously flowed. When flowing through the column, glucose came into contact with the bound enzyme, thereby producing continuous frutose production. Although the method disclosed in this publication seems to partially solve the problem of both reusing glucose isomerase, it has been carried out on such a small scale that the values obtained cannot be translated into commercial practice. It would therefore be difficult, if not impossible, to simply convert this process to an industrial scale to produce a solution containing glucose and fructose while maintaining the desired quality.

For this reason, there is still a need for a continuous process of enzymatic conversion of g) to fructose on an industrial scale. '. The present invention provides a method for the enzymatic conversion of glucose to fructose by maintaining a glucose solution having a viscosity of 5 to 10 bar, a pH of 6 to 9, containing 5 to 80% of glucose, to keep the temperature warm. 2'0 to 80 ^e C., and passed through a ..isomerázy glucose 'tick .mu.Ci of said solution passes through the layer' isomerases glucose. attached to a carrier which is. a cellulose derivative or ion exchange resin and isomerase having an activity of 3 to 10,000, preferably at least 20 IGIU per cm ² layer and a stability of 50 to 1,200 hours, preferably at least 300 and especially at least 400 hours.

. The various terms and terms used throughout the application are defined as follows:

Stability

Stability is determined by placing a sufficient amount of bound glucose isomerase in a column such that the column contains 1000 to 4000 IGIU. A 3 M glucose solution at pH 6.5 is passed through the column, the solution still containing 0.001 M CO 2 and 0.005 M MgSO 4, at a rate of 10 to 200 mL / hr. The column temperature was maintained at 60 ° C.

After 4 hours, the proportion of glucose that is converted to fructose is determined to show that the bound Isomerase layer is in equilibrium. The bound glucose isomerase activity index is calculated as follows: potency = = (R / E) log [0.504 / (0.504-1)], where I is the glucose fraction converted to fructose, R is the flow rate in ml per hour and E is the number IGIU, originally present in the column.

The activity index is determined periodically and the time for which the efficiency index drops to half its original value is stability in hours.

Colored units

The color was determined spectrophotometrically by measuring the absorption at 450 nm and 600 nm in appropriately diluted liquid in 1 cm cuvettes and compared to the value obtained for water. The Beckman DK-2A manufactured by Beckman Instrument C-ompany was used as spectrophotometry. The color was calculated according to the following formula:

_n,. , (109) (A450 - ΑδΟΟ)

Color units = --- _n 'Ai5 (! = Absorption at 450 nm ...............

Αβοο = absorption at 600 nm C = concentration in g / 100 ml.

Content of fructose in solution after isomerization

The fructose content of the solution after isomerization is determined by changing the specific rotation due to isomerization. Specific rotation was measured with a Bendix Corporation NPL Model 969 Automatic Polarimeter. Rotation was determined at a concentration of 2.5 g / 100 ml in a glass cuvette, at a thermostatically maintained temperature of 25 ° C. The cuvette path was 50. The specific rotation was determined by 11a the onset of isomerization after decanting all components of the solution. The change in fructose content was determined by a second measurement of specific rotation after isomerization. The change in fructose content was calculated according to the following formula:

Percentage of fructose = - ¹ · - · o, y - -.

Aj = specific solution rotation after isomerization,

Ao = specific rotation of the glucose solution prior to isomerization.

In the formula, a factor of -138.9 means a change in the specific rotation that would occur when glucose was completely converted to fructose.

IGIU

IGIU stands for International Glucose

Isomerization Unit, i.e., an amount of eh207702 zyme that converts 1 coin of glucose to fructor in a solution containing 2 moles of glucose, 0.02 moles of magnesium sulfate and 0.001 moles of cobalt chloride, at pH 6.84-6, per liter. 85 (0.2 M sodium maleate) at 60 ^J C.

The process of the invention has a number of distinct advantages. This is a process which can be easily and economically carried out on an industrial scale. In addition, the process is very effective in using glucose isomerase to produce a solution containing glucose and fructose with minimal discoloration, low ash content and psicosis. Furthermore, this process can be carried out continuously, which is a great advantage.

The properties of a solution that contains glucose are relatively important in determining precise conditions for isomerization. A variety of methods are known for the production of glucose-containing solutions. These are, for example, methods which are commonly used industrially and in which corn starch is broken down into glucose. These methods can be divided into three groups. The first group comprises processes in which starch is hydrolyzed to glucose with a dilute acid solution. In the second group, the methods are enzymatic, which are carried out under acidic reaction, thereby converting starch to glucose. In the third group two consecutive enzymes are used, the first enzyme liquefying the starch and the second enzyme converting the liquid starch into glucose. In carrying out the process of the invention, it is preferable to use the glucose solution produced according to the last two methods, since these solutions usually contain a greater amount of dextrose in dry matter, a small amount of acid, less intense coloration and less oligosaccharides. The glucose-containing solution may be purified, if desired, by conventional means prior to its use in the process of the invention.

The viscosity of the glucose solution should be in the range of 0.0005 to 0.1 Pa, preferably 0.002 to 0.2 Pa. If the viscosity of the solution is too high, the pressure exerted by the solution on the bound isomerase layer is too high. The pressure reduction also results in a decrease in the flow rate so that the residence time of the solution in the isomerase layer may be too long due to the efficient use of the enzyme. Since the time the solution is kept under isomerization conditions may be too long, a too saturated color develops and psicosis is produced.

The glucose concentration in the solution should be in the range of 5 to 80% by weight, preferably 40 to 60% by weight.

The glucose solution should have a pH in the range of 6 to 9, preferably 6, 5 to 8, especially 7 to 7.5. It is important to maintain the pH of the glucose solution within this range during isomerization, otherwise isomerase is rapidly inactivated and / or large amounts of undesirable byproducts such as dyes and psicosis are formed. The glucose solution may contain various metal ions as activators and / or stabilizers for iso6-merase, for example soluble cobalt, magnesium salts and the like.

The properties of the bound glucose isomerase layer are very important for the quality of the future fructose and glucose solution to be produced and for the commercial use of the method of the invention. The layer should contain at least 3 IGIU isomerase per cm ² , preferably at least 20 IGIU. in cm ² . If the layer contains less than 3 of these units per cm &^lt; ^{2 &gt};, it is necessary to use too large a layer to achieve an isomerization of an equivalent amount of dextrose. This may lead to other problems, such as the possibility of a higher pressure, the need for a longer residence time of the solution in the layer and hence higher costs, since it is necessary to use a much larger device. In addition, the greater the depth of the layer, there is also a greater tendency to sinter due to the higher pressure that must be used to ensure the solution passes through the layer. The relatively low layer has no possibility of sintering to a greater extent, while the deep layers have a much higher sintering tendency. Once sintering occurs, there will be a greater pressure drop across the layer, so that exceptionally high pressures must be used to achieve the glucose passage, so that a conventional design device can no longer be used to store the layer due to the high pressure.

The stability of the bound glucose isomerase should be at least 50 hours, preferably at least 300 hours, in particular at least 400 hours.

The glucose isomerase layer can be produced by any conventional method, for example, binding the isomerase to inert carriers such as diethylaminoethyl cellulose or the like with very good results. If isomerase binding is to be achieved, the isomerase must be released from the cells of the producing microorganism and no interfering substances must be present during binding. The binding may be carried out in an aqueous medium or in a sugar solution, for example corn syrup. Thus, the isomerase may be bound to inert carriers either together with the cellular material or in a relatively pure state. Various polymeric substances may be used for this purpose, but these materials must have a porosity such that glucose can come into contact with the isomerase.

When the isomerase is bound to a finely divided carrier such as DEAE-cellulose, it is preferred that the glucose solution pass through a relatively low layer of this material. The thickness of the bound isomerase layer thus ranges, for example, from 2.5 * to 13 cm. In this case, however, the cross-section of the layer will be large. It is preferred that the ratio of layer depth to layer width be in the range of 0.01 to 0.1, preferably 0.02 to 0.05. This has the advantage that the pressure drop through the layer is small and the sintering of the layer is minimal. However, since the layer is relatively shallow, there is a greater tendency to form channels. Glucose isomerase is underused when channels are formed. In the case of two layers in succession, preferably six layers in succession, while the solutions obtained when passing through one layer are mixed before passing through the next layer, the formation of channels will not adversely affect the efficiency of the process according to the invention.

An apparatus which can be used for this purpose is the known pressure filter laminate. This filter consists of a set of flat filter elements which do not extend vertically or horizontally in a cylindrical vessel. The filter components may be circular or rectangular in shape and the filter surface is located on both sides. The cylindrical tank may have a longitudinal axis mounted horizontally or vertically. One filter layer may consist of a fluted plate, over which the filter medium itself, such as a fabric or a fine wire mesh, is attracted. The isoS glucose merase bound to an inert carrier can be suspended in a glucose solution and the suspension is then passed through the above filter so that each filter component is uniformly coated with the bound enzyme. The applied solution pressure maintains the bound isomerase on the filter components in the case of vertical flow. The glucose solution can then be passed through the filter continuously to produce glucose isomerization. The amount of fructose depends on the time period during which the glucose solution is in contact with the bound enzyme.

The exact composition of the glucose solution will vary according to the process conditions of the invention. The essential properties of the glucose solution after isomerization are shown in Table I below.

φ me

Characteristics of unpurified fructose solutions (percentage of drying)

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2.07702

The invention will be illustrated by the following examples, which are not intended to limit the invention in any way.

Example 1

This example illustrates the use of DEAE-cellulose-linked glucose isomerase to continuously convert glucose to fructose.

The Streptomyces ATCC 21175 strain is grown in submerged aerobic culture and then separated from the culture medium by filtration. 1 kg of the filter cake is introduced in suspension in 5 liters of deionized water, to which 50 ml of 0.1 M CO 2 is added, and 8 g of a cationic detergent (Arquad 18-50, Armor Industrial Chemical Co.) is maintained. The mixture was stirred for 3.75 hours, then the suspension was vacuum filtered through Whatman # 1 filter paper. The filtrate was concentrated by evaporation in vacuo to 30 IGIU / ml.

Dilute 500 ml of the concentrated filtrate to 1500 ml with deionized water. 5 g DEAE cellulose (Cellex-D manufactured by Bio-Rad Laboratories) was added to the filtrate, the mixture was stirred for 1 hour and filtered again through Whatman # 1 under vacuum. The filter cake is washed with water on the filter and the wash water is added to the filtrate. The filter cake now contains almost only ballast material. The purified filtrate contains 9.2 IGIU / ml.

g DEAE cellulose is suspended in 1500 ml of water, stirred vigorously and the suspension is allowed to settle for 30 to 45 minutes. The upper part of the fluid is decanted to remove too fine DEAE-cellulose particles. This is repeated four times, after which the suspension is suction filtered. 20 g of DEAE-cellulose filter cake were added to the purified filtrate containing 9.2 IGIU / ml and the mixture was stirred at room temperature for 2 hours. The suspension is filtered and the wet filter cake containing DEAE-cellulose with adsorbed isomerase is isolated. The filter cake contains 215 IGIU / g. 6.25 g of this cake were suspended in 50 ml of an aqueous solution containing 0.001 moles of COCl2 per liter, 0.005 M MgSO3 and 0.1 M NaCl per liter. This suspension is applied to the top of a 1 cm diameter column and allowed to settle. The bottom of the column contains a shallow layer of glass wool to hold DEAE-cellulose .: The bottom exit of the column was opened and the height of the DEAE-cellulose layer was 25 cm. Thereafter, 350 ml of an aqueous solution containing 0.001 M COCl 2, 0.005 M MgSO 3 and 0.1 M NaCl were added to the column to remove colored materials and other impurities from DEAE-cellulose. |

Then, a glucose solution at pH 6.5 containing 3 moles of glucose, 0.001 moles of COCl2 and 0.005 moles of MgSO4 was passed through the column at a rate of 0.2 ml per minute. The column temperature was maintained at 60 ° C. The conversion of glucose to fructose after 6 hours was 49.6 percent and after 186 hours 45.0%. The stability was 198 ° / o.

Example 2 This example illustrates the use of glucose isomerase which is bound to a (porous synthetic anion exchange resin) to continuously convert glucose to fructose.

Glucose isomerase was obtained as in Example 1.

100 g of Amber Lite IRA-938 resin (Rohm and Haas) were placed in a 2.6 cm diameter column. The height of the resin layer was 37 cm, the column temperature was maintained at 60 ° C. 500 ml of 1.5 N NaOH, 1000 ml of deionized water, 1000 ml of 2M HCl and 1000 ml of deionized water are passed through the column successively. To 1480 ml of the partially purified glucose isomerase solution of Example 1 was added at 50 ° C 100 g of Amberlite IRA-938 resin. The solution was stirred for 2 hours, filtered and the filtrate was passed through the resin bed at a rate of 3 ml / minute. The layer was washed with 100 ml of deionized water, then containing 3430 IGIU.

Then a glucose solution of pH 6.5 containing 3 moles of glucose, 0.001 moles of CO 2 and 0.005 moles of MgSO 3 was passed through the resin bed at a rate of 3.6 ml / min, maintaining the column temperature at 60 ° C. The degree of conversion of glucose to fructose was 21.9% at 41 hours and 19.5% at 210 hours. Stability was 600 hours.

Claims (5)

SUBJECT A process for the enzymatic conversion of glucose to fructose, wherein a glucose solution having a viscosity of 5 to 1000 Pas, a pH of 6-9 containing 5 to 80% by weight glucose is maintained at a temperature of 20 to 80 ° C while passing through a layer of glucose isomerase characterized in that the solution passes through a carrier-bound glucose isomerase layer which is a cellulose derivative or an anion exchange resin and an isomerase, has an activity of 3 to 1000, preferably at least 20 IGIU per cm ² layer and a stability of 50 to 1200 hours, preferably at least 300 and especially at least 400 hours. 2. The method of claim 1, wherein: OF THE INVENTION that glucose isomerase is bound to diethylaminoethylcellulose. Method according to claim 2, characterized in that the depth to width ratio of the cellulosic carrier layer is 0.01 to 0.1, preferably 0.02 to 0.05. 4. The method of claim 3 wherein the glucose-containing solution is passed through two to twelve layers of glucose isomerase bound to a cellulose carrier. 5. The method of claim 1, wherein the glucose isomerase is bound to a column-type synthetic anion exchange resin.