CA1060824A - Heavy metal ion removal from dextrose solutions - Google Patents
Heavy metal ion removal from dextrose solutionsInfo
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- CA1060824A CA1060824A CA236,962A CA236962A CA1060824A CA 1060824 A CA1060824 A CA 1060824A CA 236962 A CA236962 A CA 236962A CA 1060824 A CA1060824 A CA 1060824A
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Abstract
ABSTRACT
A dextrose containing solution is treated with a chelating reagent capable of inactivating heavy metal ions and thereafter isomerizing at least a portion of the dextrose to levulose in the presence of a dextrose isomerase enzyme preparation.
A dextrose containing solution is treated with a chelating reagent capable of inactivating heavy metal ions and thereafter isomerizing at least a portion of the dextrose to levulose in the presence of a dextrose isomerase enzyme preparation.
Description
~ his invention relates to the production of levulose from dextrose and more in particular it relates to the production of a greater amount of levulose from a dextrose containing solution. Dextrose containing solution, such as those made by dissolving crystalline dextrose in water may be used as the isomerization feedstock. However, it is economically advantageous to utilize as the feedstock a starch hydrolysate syrup, also referred to a starch saccharizate, which has a high dextrose content.
Starch hydrolysates having a high dextrose content are produced today by what is known as the enzyme - enzyme process~
A starch slurry is digested with an alpha-amylase enzyme prepara-tion to produce low molecular weight fragments. The resulting material is then saccharified with a gluco-amylase enzyme preparation to produce the dextrose containing solution.
The solution produced in this manner contains certain impurities such as ash, color bodies, cations, etc. which are generally removed. The usual purification treatment involves passing the liquor through a series of ion exchange columns.
Typical purification methods include a two column method, i.e., a strongly acidic cation exchange resin followed b~ a weakly basic anion exchange resin. A four column method involves a repeat treatment using the above two resins. It is also possible to follow the two column purification with a third column containing a mixture of a strongly acidic cation exchange resin and a strongly basic anion exchange resin.
It has been found that the starch saccharizate solution also contains trace amounts of various heavy metal ions. These heavy metal ions, and in particular Zn++ and Pb ~ inhibit the effectiveness of the enzyme preparation used to isomerize the dextrose to levulose.
1- ~
1~6~82~
Dextrose is generally converted to its isomer, levulose, by the use of an enzyme preparation called glucose isomerase. This enzyme preparation is produced by a number of microorganisms known in the art~
The glucose isomerase enzyme preparations are quite.
expensive and it is economically important to produce the maximum quantity of levulose from each unit of enzyme. A unit of glucose isomerase (G.I.) is defined as that amount of enzyme which will produce one micromole of levulose per minute at 60~C and a pH of 7.5.
The effectiveness of the enzyme preparation may be measured in terms of the isomerization ratio. This is generally expressed as a percent and is calculated as:
levulose le wlose & dextrose It can be seen that the higher the isomerization ratio, the greater the levulose content of the final syrup.
The method of this invention produces a feedstock which will produce a final levulose bearing product having a greater isomerization ratio per G.I. unit of enzyme~
~(~6~3Z4 The production of a levulose bearing syrup is rapidly increased by the method of this invention. The industrial production of levulose is commonly carried out in the following way. Starch is liquefied with a mineral acid or with a liquefy-ing enzyme and then is saccharified with a saccharlfying enzyme~
Further, 20 - 50% of dextrose contained in this saccharified solution is converted into levulose with a glucose isomerase enzyme preparation. After that, the levulose-bearing syrup may be purified and concentrated.
The isomerizing reaction by an isomerizing enzyme may be conducted industrially by the batch method of adding the microbila cells which contain glucose isomerase. Recently continuous isomerization using a fixed isomerizing enzyme prepared by having glucose isomerase immobilized on a special carrier, such as an anion exchange resin, porous glass beads, ox other insoluble material capable of adsorbing or uniting with the enzyme has become the preferred method.
As the feedstock for the production of levulose, a saccharified starch solution or a water solution of purified or crystalline dextrose is used, but the material used industrially at present is mainly the starch saccharizate for economic reasons.
However, when the isomerizing reaction was conducted with a certain amount of glucose isomerase, the isomerization ratio is much lower when the starch saccharizate was used as the material, than when the water solution of crystalline dextrose was used as the material. The difference is particularly remarkable in the case of the continuous isomerizing reaction conducted by using an immobilized isomerizing enzyme.
~6~82a~
It has been found that the isomerization ratio is greatly influenced by certain impurities in the starting materialO
When dextrose is isomerized with glucose isomerase, if certain heavy metal ions such as zinc are present, its isomerizing power is considerably inhibited. The isomerization ratio is also influenced by the purity of the glucose isomerase used. In the case of glucose isomerase extracted from microbial cells and purified, it is influenced more easily by heavy metal ions than in the case of glucose isomerase not extracted and not purified. As the amount of heavy metal ions contained in the starch saccharizate is by far greater than that of heavy metal ions contained in the water solution of crystalline dextrose, the isomerization ratio when the starch saccharizate is used as the feedstock material is lower than that when the water solution of crystalline dextrose is used as the material feed-stock.
Generally, base metal ions such as Ca derived from the starch suspending water, a liquefying enzyme and a saccharifying enzyme, various heavy metal ions such as those of Zn, Pb, Fe and Cu are present in the saccharified solution. Though base metal ions such as Na, K and Ca are removed almost completely by conventional ion exchange resin treatment, heavy metal ions are scarcely removed. Accordingly, the isomerizing reaction is inhibited by these heavy metals, and a large amount of glucose isomerase is necessarily required. These inhibitory materials may be partially removed by crystallizing the pure dextrose and redissolving it in distilled water. However, this method requires large expenses for the crystallizing process and i9 thus poor in practicality. Further, although the dextrose is crystallized, it is impossible to remove the above-mentioned heavy metal ions completely. The cost becomes expensive in 608~4 both the method of isomerizing the starch saccharizate as it is without crystallizing the dextrose and that of conducting its isomerization after the dextrose contained has been crystallized once and a~ain dissolved.
Any dextrose containing solution may be used in the purification process of this invention, i.e., refined or unrefined starch saccharizates, redissolved crystalline dextrose, or the various solutions, known as hydrol, remaining after crystallizing the dextrose.
It has been found that the inhibitory effects of the heavy metal ions may be greatly reduced by adding to the dextrose containing solution a chelating reagent which is capable of complexing these ions. In this fashion, the isomerization ratio is increased, leading to a more economical use of the glucose isomerase enzyme preparation.
The useful chelating reagents to be added to the - --dextrose containing solution of this invention include ethylene-di-amine-tetra-acetic acid (EDTA), nitrilotriacetic acid (~TA), glycol-ether-tri-acetic acid (GEDTA) diethylene-tri-amine-p~nta-acetic acid (DTPA) and their salts. Salts of EDTA are preferred. Other chelating agents for heavy metal ions known in the art may also be used.
The chelating reagent can be added at any time prior to the addition of the glucose isomerase enzyme preparation to the dextrose containing solution but it is most suitable to add it immediately prior to the isomerizing reaction. When using the saccharified starch solution, it is preferred to add the chelating reagent to the already purified solution.
106~1~324 The amount of chelating agent utilized is varied according to the amount ofheavy metal ions and the present quality of the glucose isomerase being used. Generally, the amount of chelating reagent will be in the range of from about 10 to about 500 ppm. It is most convenient if the chelating reagent is dissolved in water prior to adding it to the dextrose solution. In the case of purified saccharified starch solutions, it is particularly preferred to add from about 100 to about 300 ppm of the chelating reagent to the solution.
The dextrose containing solution in admixture with the chelating reagent can be isomeri~ed both by either a conven-tional batch method and a continuous method. When using a batch method, the purified saccharified starch solution mixed with the chelating agent and glucose isomerase is isomerized at about 60 - 70~ while its pH is maintained at about 6 to about 7.
106~8~
When using a continuous method, the ~accharified starch solution mixed with the chelating reagent at a pH of about 7 to about 9 is passed through a column which is packed with an immobilized glucose isomerase enzyme preparation which is prepared by having the enzyme adsorbed on or united with an insoluble carrier. The method of this invention is parti-cularly favorable in the case of the continuous isomerization.
The chelating reagent added may be removed, after isomerization of the dextrose, by usual levulose purification methods; for instance, using ion exchange resins after the iso-merizing reaction has completed. The le~ulose may then be de-colorized and concentrated as desired.
When the solution containing dextrose is isomerized using this method, the isomerization ratio is substantially higher than when using a conventional method. Thus, a certain isomerization ratio can be obtained by the use of far less glucose isomerase enzyme than under the conventional method.
When the saccharified starch solution is used as the feedstock material for the production of levulose, the effect of this method is especially beneficial. If the glucose isomerase enzyme preparation used at this time has been extracted from microbial cell~ and purified, the effect of this method is generally doubled. Glucose isomerase extracted from microbial cells and purified is commonly used for the continuous iso-merization processes.
Thus in accordance with the invention there is provided a method for remo~al of heavy metal ions from a dextrose contain-ing solution comprising introducing into said solution a soluble chelating reagent to inactivate ~aid heavy metal ions.
In a particular aspect of the invention there is provided a continuous method for the enzymatic isomerization of dextrose to levulose comprising: (a) intrcducing into a - 7 _ 10608Z~
solution containing dextrose a soluble chelating reagant to selectively inactivate heavy metal ion~ in said solution; (b) passing the resulting solution through a bed comprising a dextrose isomerase enzyme preparation, and (c) removing said chelating reagent including said inactivated heavy metal ions.
In order to provide a better understanding of the invention, the follo~ing exemplary and non-limiting examples are provided.
Example 1 A saccharified solution, obtained from corn starch by conventional means was treated with an ion exchange resin in the usual way. With 4.5, 45 and 450 ppm of tetra sodium EDTA
added, the desalted saccharified solution was permitted to undergo batch isomerization. Two different kinds of glucose isomerase enzyme preparations were used in thi~ test, i.e., whole cell enzyme and purified enzyme. Whole cell enzyme was obtained by ~0~i~8Z4 centrifugation of the culturing broth streptomyces olivochromoqenes which is an actinomycetes. The purified enzyme was obtained by digesting the whole cell enzyme with lysozyme, and subsequent purification with isopropyl alcohol. Two G.I. uni~s of glucose isomerase enzyme preparation were added to the desalted sacchari-fied solution per gram of dextrose in the solution. The iso-merizing reaction was conducted for 48 hours by the batch method at pH 6.5 + 0.3 and at 68C with MgC12.6H2O added to the materia~.
The isomerization ratio of each material after 48 hours is shown in Table 1.
Table l -Glucose Isomerase Whole Purified Substrate Cell -Purified Saccharified Solution 34~6% 18.2% -Purified Saccharified Solution with 4.5 ppm of EDTA (4 ~a) 38.7 22.1 Purified Saccharified Solution with ~5 ppm of EDTA (4 Na) 41.5 41.8 Purified Saccharified Solution 20with 450 ppm of EDTA (4 ~a) 41.9 42.6 From the results of Table 1, it is clear that the rate of isomerization is raised when EDTA-4~a is added to the dextrose solution.
Example 2 The substrates were prepared in the same manner as in Example 1, and their pH values were adjusted to 8.5 with ~aOH.
The continuous isomerization of these materials was conducted by passing them through a glass column at 60UC, space velocity of 2, packed with IRA-9Q4, a strongly basic anion exchange resin (produced by Tokyo Yukikagaku), on which a glucose isomerase solution obtained as in Example 1 had been adsorbed. The 1060~Z4 adsorbed enzyme corresponded to 200 G.I. units of enzyme per 1 ml of the resin. The change of isomerization ratio with time for each substrate is shown :in Table 2.
Table 2 Substrate Reaction Time ~days) % % % % % %
Purified Saccharified Solution 49.032.02'2.3 4.8 - -Purified Saccharified Solution with 4.5 ppm of EDTA ~4 ~a) 49O5 48.8 42.8 33.0 2307 13.6 Purified Saccharified Solution 10with 45 ppm of EDTA (4 Na) 49.9 48.9 46.5 42.4 33.7 25.6 Purified Saccharified Solution with 450 ppm of EDTA (4 Na) 50.1 49.9 49.3 46.7 41.1 34.4 As is clear from Table 2, the rate of isomerization of the purified saccharified solution alone dro~ped to half of the maximum value in about 7 days but in the case of the purified saccharified solution with 450 ppm tetra sodium EDTA salt, 31 days passed before the isomerization ratio dropped to half of the maximum value. It is thus seen that continuous isomerization with the addition of a chelating reagent can effectively maintain a high isomerization ratio for a greater length of time.
Example 3 A solution was prepared by dissolving anhydrous crystal-line dextrose in water to a concentration of Bx 50, and its pH
value adjusted to 8.5~ 4.5, 45 and 450 ppm of tetra sodium EDTA salt were added. The isomerization conditions were the same as Example 2. The change in isomerization ratio .. . ..
~L060~ 4 during the continuous i~omerization are as shown in Table 3.
Table 3 _ Reaction Time (daysL __ Substrate 1 10 20 30 40 50 % % % % % %
Aqueous Dextrose Solution 50.6 45.2 35.6 30,1 18~7 12.8 Aqueous Dextrose Solution with 4.5 ppm of EDTA (4Na)50.9 50.4 40.3 37~5 28.1 17.7 Aqueous Dextrose Solution with 45 ppm of EDTA (4 Na) 51.0 50.9 42.4 39.4 30.5 21.3 A~ueous Dextrose Solution with 450 ppm of EDTA (4 ~a) 51.2 51.1 45.6 41,2 34.5 24.9 From the above re~ults, it may be ~een that when using aqueous solutions of dextrose as the feedstock the addition of the chelating reagent effectively maintains a high rate of isomerization for a substantial period of timea Example 4 Sacchari~ied ~tarch ~olution was obtained by lique~yi~g and saccharifying corn starch with an alpha-amylase (Kleistase L-l), trademark, from Daiwa Kaei, and a commercial glucoamylase saccharifying enæyme lSumizyme 800, trademark from Shinnihon Kagaku). The saccharified solution was filtered under reduced pressure using diatomaceou3 earth as a filter aid. Ten liters of filtrate were obtained.
Next, this saccharified solution wa3 purified in the usual way, by pa~sing it successively through a series of ion exchange columns. The first wa~ packed with 370 mls of Amberlite IR-120 B, a polystyrene based gel type resin with a sulphonic acid active group, trademark from Tokyo Yukikagaku Kogyo), hydrogen form. This strongly acidic cation exchange resin was regenerated with HCL. The second wa~ packed with 400 mls Amberlite IRA-93, a styrene/divinyl benzene copolymer with amine active groups (trademark from Tokyo Yukikagaku Kogyo), 82~
in hydroxide form. This weakly basic anion exchange resin was regenerated with ~aOH. The third column contained a mixed bed of 50 mls Amberlite 200 (trademark) of a macroreticular styrene di-vinyl benzene copolymer resin with a ~ulphonic acid active group, 100 mls~Amberlite IRA-411 ~trademark) a styrene divinyl benzene copolymer resin with a quarternary amine active group (both pro-duced by Tokyo Yukikagaku Kogyo), Amberlite 200, us~d in the hydrogen form is a strongly acidic cation exchange resin which was regenerated with ~aOH. The quality of the saccharified solution obtained was as follows:
Sugar Concentration: 33.7 (Bx) DE: 97.1 Dextrose: 94.6 (%) Color Value (O.D. at 420 mu) 0.012 (light path 1 cm) Total Amount of Salt~: 15 mg (in terms of CaC03 ~/1 pH; 5.10 Next, batch isomeriæation wa~ conducted by adding 10 g of MgC12.6H20, adjusting the pH value to 6.5 with ~aOH, and add-ing 450 mg of tetra sodium EDTA and whole cell glucose isomerase~
The cells were obtained by centrifugation from the liquid medium culture of St. olivochromoqenes. The amount added corresponded to 2 G.I. units of glucose isomerase per 1 g of dextrose in the saccharified starch solution. The isomerization was conducted for 48 hours in a 15 liter reactor equipped with a heater and stirrer, at 68C. During the reaction, the solution was stirred 910wly and its pH value was controlled with 5% ~aH~O3 so that it remained between 6.3 and 6.7. The isomerization ratio was 42.2%
after the reaction had been completed.
For comparison, ~he saccharified solution was i50--merized without adding tetra sodium EDTA in otherwise the same manner as above. The isomerization ratio was 34.6%.
, ~,'"
6~3Z4 Example 5 The batch isomerization using 10 liters of the purified saccharizates was conducted by adding 10 g of MgC12.6H2O, adjusting its pH value to 6.5 with NaOH and adding 450 mg of tetra sodium EDTA and a glucose isomerase solution. The enzyme solution was prepared by the lysozyme of St. olivochromo~enes obtained on centrifugation of the liquid medium in which they had been cultured for-about 50 hours. The extract was purified with isopropyl alcohol. 120 units of glucose isomerase per 1 g of dextrose were the same as those in Example 4. When the reaction had been completed, the isomerization ratio was 42.8%.
Further, for comparison, the saccharified solution without the tetra sodium EDTA-4 addition was isomerized under the same conditions as in Example 4. The isomerization ratio was 18~/o~
Example 6 100 liters of the purified saccharified solution, obtained in the same way as Example 4, was submitted to continuous isomerization by adding 100 g of MgC12.6H2O adjusting its pH value to 8.5 and adding 4.5 g of the tetra sodium DTA. The continuous isomerization was conducted by passing the saccharified solution through a jacketed glass column (height: 12 cm: diameter: 2.1 cm) packed with 15 ml of Amberlite IRA-904 on which 200 units of glucose isomerase per milliliter of resin had been adsorbed.
Amberlite IRA-904 used in the chloride form is a strongly basic anion exchange resin (produced by Tokyo Yukikagaku Kogyo). Conditions were SV 2 and 60UC by the descending method.
During the continuous isomerization, the changes of isomerization ratio was as follows:
Table 4 Time (days) 1 5 10 15 20 ~5 Isomerization Ratio (%) 49.9 48.9 46.5 42.4 33.7 25.6 * trademark ~6~824 Further, for comparison, the continuous isomerization of the saccharified solution without the tetra sodium EDTA
addition was conducted under the same conditions. The change of the isomeriæation ratio was as follows:
Table 5 Time tdays) 1 5 10 15 Isomerization Ratio (%) 49rO 32~0 22~3 4~8 Example 7 100 liters of an aqueous solution of dextrose was obta~ned by dissolving 61. 3 kg of crystalline dextrose in 61 liters of desalted water. The quality of this solution was as follows:
Sugar Concentration: 50~1 (Bx) Dextrose: 99.7 (%) Total Amount of Salts: Trace pH: 4.8 The aqueous solution of crystalline dextrose was continuously isomerized by adding 100 g of MgC12.6H2O, adjusting its pH to 8.5 with ~aOH and further adding 450 mg o~ tetra sodium EDTA as in Example 6. During this continuous isomeri-zation, the change of the isomerization ratio was as follows:
Table 6 Time (days) 1 10 20 30 40 50 Isomerization Ratio (%) 50~9 50~4 41~3 37~5 28~1 17~7 For comparison, a similar solution of dextrose without tetra sodium EDTA was isomerized continuously in a similar manner. The change of the isomerization ratio was as foilows:
~ 14 _ 1~6~3Z~
Table 7 Time (days~ 1 10 20 30 40 50 Isomerization Ratio (%) 50.9 45~2 35.6 30.1 18.7 12.8 Example 8 A saccharified starch solution was obtained by liquefy-ing corn starch with "Kleistase L-l" (produced by Daiwa Kasei), a commercial liquefying enzyme, and saccharifying it with "Sumizyme 800" (produced by Shinnihon Kagaku~, a commercial saccharifying enzyme. The saccharified solution was filtered using diatomaceous earth as a filter aid. 100 liters of filtrate were obtainedO The quality of this saccharified solution was as follows:
Sugar Concentration: 35.0 (Bx) DE: 95 nextrose: 93.2 (%) Color Value (O.D. at 420 mu) 0.086 (light path lcm) Total Salt: 665 mg (in terms of caCo3)/
pH: 4.8 ~ext, this saccharified solution was isomerized continuously by adding 100 g of MgC12.6H2O adjusting its pH
value to 8.5 with NaOH and further adding 45 g of tetra sodium EDTA as in Example 6. During the continuous isomerization, the change of isomerization was as follows:
Table 8 Time (days) 1 5 10 15 20 Isomerization Ratio (%) 47.747.644.2 40.5 33.2 * trade mark ~6~8Z~
For comparison, the saccharified solution was iso-merized continuou~ly without adding tetxa sodium EDTA under other-wise the same conditions. The change of the isomerization ratio was as follows:
Table 9 :
Time (days) 1 5 10 15 30 Isomerization Ratio (%) 44.0 14~9 3.1 - -The enzymes referred to-in this disclosure have been assigned the following enzyme numbers:
alpha-amylase 3.2.1,1, glucoamylase 3.2,1.3.
glucose isomerase 5.3.1.180 The enzyme that catalyzes the isomerization of glucose to levulose is also referred to as dextrose isomerase, and is intended to embrace other enzymes that have this property, In this specification the name glucose isomerase is to be understood as embracing the enzyme xylose isomerase which has been assigned the enzyme number 5.3.1.5. The enzyme from Streptomyces olivochromogenes employed in the examples is a xylose isomerase. The enzyme xylose isomerase is a non-specific enzyme which isomerizes glucose to levulose. In the art the terms xylose isomerase and glucose isomerase are often used interchangeably.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any variations, used, or adaptions of the invention following, in general, the principles of the invention including such departures from the present disclosure as come within known and customary practice in the art to which the invention pertains, and as may be applied to the essential features hereinbefore set forth, and as fall within the scope of the invention.
Starch hydrolysates having a high dextrose content are produced today by what is known as the enzyme - enzyme process~
A starch slurry is digested with an alpha-amylase enzyme prepara-tion to produce low molecular weight fragments. The resulting material is then saccharified with a gluco-amylase enzyme preparation to produce the dextrose containing solution.
The solution produced in this manner contains certain impurities such as ash, color bodies, cations, etc. which are generally removed. The usual purification treatment involves passing the liquor through a series of ion exchange columns.
Typical purification methods include a two column method, i.e., a strongly acidic cation exchange resin followed b~ a weakly basic anion exchange resin. A four column method involves a repeat treatment using the above two resins. It is also possible to follow the two column purification with a third column containing a mixture of a strongly acidic cation exchange resin and a strongly basic anion exchange resin.
It has been found that the starch saccharizate solution also contains trace amounts of various heavy metal ions. These heavy metal ions, and in particular Zn++ and Pb ~ inhibit the effectiveness of the enzyme preparation used to isomerize the dextrose to levulose.
1- ~
1~6~82~
Dextrose is generally converted to its isomer, levulose, by the use of an enzyme preparation called glucose isomerase. This enzyme preparation is produced by a number of microorganisms known in the art~
The glucose isomerase enzyme preparations are quite.
expensive and it is economically important to produce the maximum quantity of levulose from each unit of enzyme. A unit of glucose isomerase (G.I.) is defined as that amount of enzyme which will produce one micromole of levulose per minute at 60~C and a pH of 7.5.
The effectiveness of the enzyme preparation may be measured in terms of the isomerization ratio. This is generally expressed as a percent and is calculated as:
levulose le wlose & dextrose It can be seen that the higher the isomerization ratio, the greater the levulose content of the final syrup.
The method of this invention produces a feedstock which will produce a final levulose bearing product having a greater isomerization ratio per G.I. unit of enzyme~
~(~6~3Z4 The production of a levulose bearing syrup is rapidly increased by the method of this invention. The industrial production of levulose is commonly carried out in the following way. Starch is liquefied with a mineral acid or with a liquefy-ing enzyme and then is saccharified with a saccharlfying enzyme~
Further, 20 - 50% of dextrose contained in this saccharified solution is converted into levulose with a glucose isomerase enzyme preparation. After that, the levulose-bearing syrup may be purified and concentrated.
The isomerizing reaction by an isomerizing enzyme may be conducted industrially by the batch method of adding the microbila cells which contain glucose isomerase. Recently continuous isomerization using a fixed isomerizing enzyme prepared by having glucose isomerase immobilized on a special carrier, such as an anion exchange resin, porous glass beads, ox other insoluble material capable of adsorbing or uniting with the enzyme has become the preferred method.
As the feedstock for the production of levulose, a saccharified starch solution or a water solution of purified or crystalline dextrose is used, but the material used industrially at present is mainly the starch saccharizate for economic reasons.
However, when the isomerizing reaction was conducted with a certain amount of glucose isomerase, the isomerization ratio is much lower when the starch saccharizate was used as the material, than when the water solution of crystalline dextrose was used as the material. The difference is particularly remarkable in the case of the continuous isomerizing reaction conducted by using an immobilized isomerizing enzyme.
~6~82a~
It has been found that the isomerization ratio is greatly influenced by certain impurities in the starting materialO
When dextrose is isomerized with glucose isomerase, if certain heavy metal ions such as zinc are present, its isomerizing power is considerably inhibited. The isomerization ratio is also influenced by the purity of the glucose isomerase used. In the case of glucose isomerase extracted from microbial cells and purified, it is influenced more easily by heavy metal ions than in the case of glucose isomerase not extracted and not purified. As the amount of heavy metal ions contained in the starch saccharizate is by far greater than that of heavy metal ions contained in the water solution of crystalline dextrose, the isomerization ratio when the starch saccharizate is used as the feedstock material is lower than that when the water solution of crystalline dextrose is used as the material feed-stock.
Generally, base metal ions such as Ca derived from the starch suspending water, a liquefying enzyme and a saccharifying enzyme, various heavy metal ions such as those of Zn, Pb, Fe and Cu are present in the saccharified solution. Though base metal ions such as Na, K and Ca are removed almost completely by conventional ion exchange resin treatment, heavy metal ions are scarcely removed. Accordingly, the isomerizing reaction is inhibited by these heavy metals, and a large amount of glucose isomerase is necessarily required. These inhibitory materials may be partially removed by crystallizing the pure dextrose and redissolving it in distilled water. However, this method requires large expenses for the crystallizing process and i9 thus poor in practicality. Further, although the dextrose is crystallized, it is impossible to remove the above-mentioned heavy metal ions completely. The cost becomes expensive in 608~4 both the method of isomerizing the starch saccharizate as it is without crystallizing the dextrose and that of conducting its isomerization after the dextrose contained has been crystallized once and a~ain dissolved.
Any dextrose containing solution may be used in the purification process of this invention, i.e., refined or unrefined starch saccharizates, redissolved crystalline dextrose, or the various solutions, known as hydrol, remaining after crystallizing the dextrose.
It has been found that the inhibitory effects of the heavy metal ions may be greatly reduced by adding to the dextrose containing solution a chelating reagent which is capable of complexing these ions. In this fashion, the isomerization ratio is increased, leading to a more economical use of the glucose isomerase enzyme preparation.
The useful chelating reagents to be added to the - --dextrose containing solution of this invention include ethylene-di-amine-tetra-acetic acid (EDTA), nitrilotriacetic acid (~TA), glycol-ether-tri-acetic acid (GEDTA) diethylene-tri-amine-p~nta-acetic acid (DTPA) and their salts. Salts of EDTA are preferred. Other chelating agents for heavy metal ions known in the art may also be used.
The chelating reagent can be added at any time prior to the addition of the glucose isomerase enzyme preparation to the dextrose containing solution but it is most suitable to add it immediately prior to the isomerizing reaction. When using the saccharified starch solution, it is preferred to add the chelating reagent to the already purified solution.
106~1~324 The amount of chelating agent utilized is varied according to the amount ofheavy metal ions and the present quality of the glucose isomerase being used. Generally, the amount of chelating reagent will be in the range of from about 10 to about 500 ppm. It is most convenient if the chelating reagent is dissolved in water prior to adding it to the dextrose solution. In the case of purified saccharified starch solutions, it is particularly preferred to add from about 100 to about 300 ppm of the chelating reagent to the solution.
The dextrose containing solution in admixture with the chelating reagent can be isomeri~ed both by either a conven-tional batch method and a continuous method. When using a batch method, the purified saccharified starch solution mixed with the chelating agent and glucose isomerase is isomerized at about 60 - 70~ while its pH is maintained at about 6 to about 7.
106~8~
When using a continuous method, the ~accharified starch solution mixed with the chelating reagent at a pH of about 7 to about 9 is passed through a column which is packed with an immobilized glucose isomerase enzyme preparation which is prepared by having the enzyme adsorbed on or united with an insoluble carrier. The method of this invention is parti-cularly favorable in the case of the continuous isomerization.
The chelating reagent added may be removed, after isomerization of the dextrose, by usual levulose purification methods; for instance, using ion exchange resins after the iso-merizing reaction has completed. The le~ulose may then be de-colorized and concentrated as desired.
When the solution containing dextrose is isomerized using this method, the isomerization ratio is substantially higher than when using a conventional method. Thus, a certain isomerization ratio can be obtained by the use of far less glucose isomerase enzyme than under the conventional method.
When the saccharified starch solution is used as the feedstock material for the production of levulose, the effect of this method is especially beneficial. If the glucose isomerase enzyme preparation used at this time has been extracted from microbial cell~ and purified, the effect of this method is generally doubled. Glucose isomerase extracted from microbial cells and purified is commonly used for the continuous iso-merization processes.
Thus in accordance with the invention there is provided a method for remo~al of heavy metal ions from a dextrose contain-ing solution comprising introducing into said solution a soluble chelating reagent to inactivate ~aid heavy metal ions.
In a particular aspect of the invention there is provided a continuous method for the enzymatic isomerization of dextrose to levulose comprising: (a) intrcducing into a - 7 _ 10608Z~
solution containing dextrose a soluble chelating reagant to selectively inactivate heavy metal ion~ in said solution; (b) passing the resulting solution through a bed comprising a dextrose isomerase enzyme preparation, and (c) removing said chelating reagent including said inactivated heavy metal ions.
In order to provide a better understanding of the invention, the follo~ing exemplary and non-limiting examples are provided.
Example 1 A saccharified solution, obtained from corn starch by conventional means was treated with an ion exchange resin in the usual way. With 4.5, 45 and 450 ppm of tetra sodium EDTA
added, the desalted saccharified solution was permitted to undergo batch isomerization. Two different kinds of glucose isomerase enzyme preparations were used in thi~ test, i.e., whole cell enzyme and purified enzyme. Whole cell enzyme was obtained by ~0~i~8Z4 centrifugation of the culturing broth streptomyces olivochromoqenes which is an actinomycetes. The purified enzyme was obtained by digesting the whole cell enzyme with lysozyme, and subsequent purification with isopropyl alcohol. Two G.I. uni~s of glucose isomerase enzyme preparation were added to the desalted sacchari-fied solution per gram of dextrose in the solution. The iso-merizing reaction was conducted for 48 hours by the batch method at pH 6.5 + 0.3 and at 68C with MgC12.6H2O added to the materia~.
The isomerization ratio of each material after 48 hours is shown in Table 1.
Table l -Glucose Isomerase Whole Purified Substrate Cell -Purified Saccharified Solution 34~6% 18.2% -Purified Saccharified Solution with 4.5 ppm of EDTA (4 ~a) 38.7 22.1 Purified Saccharified Solution with ~5 ppm of EDTA (4 Na) 41.5 41.8 Purified Saccharified Solution 20with 450 ppm of EDTA (4 ~a) 41.9 42.6 From the results of Table 1, it is clear that the rate of isomerization is raised when EDTA-4~a is added to the dextrose solution.
Example 2 The substrates were prepared in the same manner as in Example 1, and their pH values were adjusted to 8.5 with ~aOH.
The continuous isomerization of these materials was conducted by passing them through a glass column at 60UC, space velocity of 2, packed with IRA-9Q4, a strongly basic anion exchange resin (produced by Tokyo Yukikagaku), on which a glucose isomerase solution obtained as in Example 1 had been adsorbed. The 1060~Z4 adsorbed enzyme corresponded to 200 G.I. units of enzyme per 1 ml of the resin. The change of isomerization ratio with time for each substrate is shown :in Table 2.
Table 2 Substrate Reaction Time ~days) % % % % % %
Purified Saccharified Solution 49.032.02'2.3 4.8 - -Purified Saccharified Solution with 4.5 ppm of EDTA ~4 ~a) 49O5 48.8 42.8 33.0 2307 13.6 Purified Saccharified Solution 10with 45 ppm of EDTA (4 Na) 49.9 48.9 46.5 42.4 33.7 25.6 Purified Saccharified Solution with 450 ppm of EDTA (4 Na) 50.1 49.9 49.3 46.7 41.1 34.4 As is clear from Table 2, the rate of isomerization of the purified saccharified solution alone dro~ped to half of the maximum value in about 7 days but in the case of the purified saccharified solution with 450 ppm tetra sodium EDTA salt, 31 days passed before the isomerization ratio dropped to half of the maximum value. It is thus seen that continuous isomerization with the addition of a chelating reagent can effectively maintain a high isomerization ratio for a greater length of time.
Example 3 A solution was prepared by dissolving anhydrous crystal-line dextrose in water to a concentration of Bx 50, and its pH
value adjusted to 8.5~ 4.5, 45 and 450 ppm of tetra sodium EDTA salt were added. The isomerization conditions were the same as Example 2. The change in isomerization ratio .. . ..
~L060~ 4 during the continuous i~omerization are as shown in Table 3.
Table 3 _ Reaction Time (daysL __ Substrate 1 10 20 30 40 50 % % % % % %
Aqueous Dextrose Solution 50.6 45.2 35.6 30,1 18~7 12.8 Aqueous Dextrose Solution with 4.5 ppm of EDTA (4Na)50.9 50.4 40.3 37~5 28.1 17.7 Aqueous Dextrose Solution with 45 ppm of EDTA (4 Na) 51.0 50.9 42.4 39.4 30.5 21.3 A~ueous Dextrose Solution with 450 ppm of EDTA (4 ~a) 51.2 51.1 45.6 41,2 34.5 24.9 From the above re~ults, it may be ~een that when using aqueous solutions of dextrose as the feedstock the addition of the chelating reagent effectively maintains a high rate of isomerization for a substantial period of timea Example 4 Sacchari~ied ~tarch ~olution was obtained by lique~yi~g and saccharifying corn starch with an alpha-amylase (Kleistase L-l), trademark, from Daiwa Kaei, and a commercial glucoamylase saccharifying enæyme lSumizyme 800, trademark from Shinnihon Kagaku). The saccharified solution was filtered under reduced pressure using diatomaceou3 earth as a filter aid. Ten liters of filtrate were obtained.
Next, this saccharified solution wa3 purified in the usual way, by pa~sing it successively through a series of ion exchange columns. The first wa~ packed with 370 mls of Amberlite IR-120 B, a polystyrene based gel type resin with a sulphonic acid active group, trademark from Tokyo Yukikagaku Kogyo), hydrogen form. This strongly acidic cation exchange resin was regenerated with HCL. The second wa~ packed with 400 mls Amberlite IRA-93, a styrene/divinyl benzene copolymer with amine active groups (trademark from Tokyo Yukikagaku Kogyo), 82~
in hydroxide form. This weakly basic anion exchange resin was regenerated with ~aOH. The third column contained a mixed bed of 50 mls Amberlite 200 (trademark) of a macroreticular styrene di-vinyl benzene copolymer resin with a ~ulphonic acid active group, 100 mls~Amberlite IRA-411 ~trademark) a styrene divinyl benzene copolymer resin with a quarternary amine active group (both pro-duced by Tokyo Yukikagaku Kogyo), Amberlite 200, us~d in the hydrogen form is a strongly acidic cation exchange resin which was regenerated with ~aOH. The quality of the saccharified solution obtained was as follows:
Sugar Concentration: 33.7 (Bx) DE: 97.1 Dextrose: 94.6 (%) Color Value (O.D. at 420 mu) 0.012 (light path 1 cm) Total Amount of Salt~: 15 mg (in terms of CaC03 ~/1 pH; 5.10 Next, batch isomeriæation wa~ conducted by adding 10 g of MgC12.6H20, adjusting the pH value to 6.5 with ~aOH, and add-ing 450 mg of tetra sodium EDTA and whole cell glucose isomerase~
The cells were obtained by centrifugation from the liquid medium culture of St. olivochromoqenes. The amount added corresponded to 2 G.I. units of glucose isomerase per 1 g of dextrose in the saccharified starch solution. The isomerization was conducted for 48 hours in a 15 liter reactor equipped with a heater and stirrer, at 68C. During the reaction, the solution was stirred 910wly and its pH value was controlled with 5% ~aH~O3 so that it remained between 6.3 and 6.7. The isomerization ratio was 42.2%
after the reaction had been completed.
For comparison, ~he saccharified solution was i50--merized without adding tetra sodium EDTA in otherwise the same manner as above. The isomerization ratio was 34.6%.
, ~,'"
6~3Z4 Example 5 The batch isomerization using 10 liters of the purified saccharizates was conducted by adding 10 g of MgC12.6H2O, adjusting its pH value to 6.5 with NaOH and adding 450 mg of tetra sodium EDTA and a glucose isomerase solution. The enzyme solution was prepared by the lysozyme of St. olivochromo~enes obtained on centrifugation of the liquid medium in which they had been cultured for-about 50 hours. The extract was purified with isopropyl alcohol. 120 units of glucose isomerase per 1 g of dextrose were the same as those in Example 4. When the reaction had been completed, the isomerization ratio was 42.8%.
Further, for comparison, the saccharified solution without the tetra sodium EDTA-4 addition was isomerized under the same conditions as in Example 4. The isomerization ratio was 18~/o~
Example 6 100 liters of the purified saccharified solution, obtained in the same way as Example 4, was submitted to continuous isomerization by adding 100 g of MgC12.6H2O adjusting its pH value to 8.5 and adding 4.5 g of the tetra sodium DTA. The continuous isomerization was conducted by passing the saccharified solution through a jacketed glass column (height: 12 cm: diameter: 2.1 cm) packed with 15 ml of Amberlite IRA-904 on which 200 units of glucose isomerase per milliliter of resin had been adsorbed.
Amberlite IRA-904 used in the chloride form is a strongly basic anion exchange resin (produced by Tokyo Yukikagaku Kogyo). Conditions were SV 2 and 60UC by the descending method.
During the continuous isomerization, the changes of isomerization ratio was as follows:
Table 4 Time (days) 1 5 10 15 20 ~5 Isomerization Ratio (%) 49.9 48.9 46.5 42.4 33.7 25.6 * trademark ~6~824 Further, for comparison, the continuous isomerization of the saccharified solution without the tetra sodium EDTA
addition was conducted under the same conditions. The change of the isomeriæation ratio was as follows:
Table 5 Time tdays) 1 5 10 15 Isomerization Ratio (%) 49rO 32~0 22~3 4~8 Example 7 100 liters of an aqueous solution of dextrose was obta~ned by dissolving 61. 3 kg of crystalline dextrose in 61 liters of desalted water. The quality of this solution was as follows:
Sugar Concentration: 50~1 (Bx) Dextrose: 99.7 (%) Total Amount of Salts: Trace pH: 4.8 The aqueous solution of crystalline dextrose was continuously isomerized by adding 100 g of MgC12.6H2O, adjusting its pH to 8.5 with ~aOH and further adding 450 mg o~ tetra sodium EDTA as in Example 6. During this continuous isomeri-zation, the change of the isomerization ratio was as follows:
Table 6 Time (days) 1 10 20 30 40 50 Isomerization Ratio (%) 50~9 50~4 41~3 37~5 28~1 17~7 For comparison, a similar solution of dextrose without tetra sodium EDTA was isomerized continuously in a similar manner. The change of the isomerization ratio was as foilows:
~ 14 _ 1~6~3Z~
Table 7 Time (days~ 1 10 20 30 40 50 Isomerization Ratio (%) 50.9 45~2 35.6 30.1 18.7 12.8 Example 8 A saccharified starch solution was obtained by liquefy-ing corn starch with "Kleistase L-l" (produced by Daiwa Kasei), a commercial liquefying enzyme, and saccharifying it with "Sumizyme 800" (produced by Shinnihon Kagaku~, a commercial saccharifying enzyme. The saccharified solution was filtered using diatomaceous earth as a filter aid. 100 liters of filtrate were obtainedO The quality of this saccharified solution was as follows:
Sugar Concentration: 35.0 (Bx) DE: 95 nextrose: 93.2 (%) Color Value (O.D. at 420 mu) 0.086 (light path lcm) Total Salt: 665 mg (in terms of caCo3)/
pH: 4.8 ~ext, this saccharified solution was isomerized continuously by adding 100 g of MgC12.6H2O adjusting its pH
value to 8.5 with NaOH and further adding 45 g of tetra sodium EDTA as in Example 6. During the continuous isomerization, the change of isomerization was as follows:
Table 8 Time (days) 1 5 10 15 20 Isomerization Ratio (%) 47.747.644.2 40.5 33.2 * trade mark ~6~8Z~
For comparison, the saccharified solution was iso-merized continuou~ly without adding tetxa sodium EDTA under other-wise the same conditions. The change of the isomerization ratio was as follows:
Table 9 :
Time (days) 1 5 10 15 30 Isomerization Ratio (%) 44.0 14~9 3.1 - -The enzymes referred to-in this disclosure have been assigned the following enzyme numbers:
alpha-amylase 3.2.1,1, glucoamylase 3.2,1.3.
glucose isomerase 5.3.1.180 The enzyme that catalyzes the isomerization of glucose to levulose is also referred to as dextrose isomerase, and is intended to embrace other enzymes that have this property, In this specification the name glucose isomerase is to be understood as embracing the enzyme xylose isomerase which has been assigned the enzyme number 5.3.1.5. The enzyme from Streptomyces olivochromogenes employed in the examples is a xylose isomerase. The enzyme xylose isomerase is a non-specific enzyme which isomerizes glucose to levulose. In the art the terms xylose isomerase and glucose isomerase are often used interchangeably.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any variations, used, or adaptions of the invention following, in general, the principles of the invention including such departures from the present disclosure as come within known and customary practice in the art to which the invention pertains, and as may be applied to the essential features hereinbefore set forth, and as fall within the scope of the invention.
Claims (11)
1. A method for removal of heavy metal ions from a dextrose containing solution comprising introducing into said solution a soluble chelating reagent to inactivate said heavy metal ions.
2. A method in accordance with claim 1, wherein the chelating reagent is selected from the group consisting of EDTA, NTA, GEDTA, DTPA and the salts thereof.
3. A method in accordance with claim 1 or 2, wherein the dextrose containing solution is a starch saccharizate.
4. A method in accordance with claim 1 or 2, wherein the chelating reagent is added in an amount sufficient to produce a concentration in the range of about 10 to about 500 ppm.
5. A method in accordance with claim 1 or 2, wherein the chelating agent is dissolved in water and the resulting aqueous solution is introduced into the dextrose containing solution.
6. A continuous method for the enzymatic isomerization of dextrose to levulose comprising:
(a) introducing into a solution containing dextrose a soluble chelating reagent to selectively inactivate heavy metal ions in said solution;
(b) passing the resulting solution through a bed comprising a dextrose isomerase enzyme preparation;
and (c) removing said chelating reagent including said inactivated heavy metal ions.
(a) introducing into a solution containing dextrose a soluble chelating reagent to selectively inactivate heavy metal ions in said solution;
(b) passing the resulting solution through a bed comprising a dextrose isomerase enzyme preparation;
and (c) removing said chelating reagent including said inactivated heavy metal ions.
7. A method in accordance with claim 6, wherein said dextrose isomerase enzyme preparation is immobilized on an insoluble substrate.
8. A method in accordance with claim 6, wherein the chelating reagent is selected from the group consisting of EDTA, NTA, GEDTA, DTPA and the salts thereof.
9. A method in accordance with claim 6, 7 or 8, wherein the chelating reagent is added in an amount sufficient to produce a concentration in the range of about 10 to about 500 ppm.
10. A method in accordance with claim 6, 7 or 8, wherein the dextrose containing solution is a starch saccharizate.
11. A method in accordance with claim 6, 7 or 8, wherein the chelating agent is dissolved in water and the resulting aqueous solution is introduced into the dextrose containing solution.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11399974A JPS5141451A (en) | 1974-10-04 | 1974-10-04 | ISEIKATONOSEIZOHOHO |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1060824A true CA1060824A (en) | 1979-08-21 |
Family
ID=14626515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA236,962A Expired CA1060824A (en) | 1974-10-04 | 1975-10-03 | Heavy metal ion removal from dextrose solutions |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS5141451A (en) |
| CA (1) | CA1060824A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9783860B2 (en) | 2011-12-30 | 2017-10-10 | Renmatix, Inc. | Compositions comprising C5 and C6 oligosaccharides |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL206678A0 (en) | 2010-06-28 | 2010-12-30 | Hcl Cleantech Ltd | A method for the production of fermentable sugars |
| US9617608B2 (en) | 2011-10-10 | 2017-04-11 | Virdia, Inc. | Sugar compositions |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5641233B2 (en) * | 1972-09-05 | 1981-09-26 |
-
1974
- 1974-10-04 JP JP11399974A patent/JPS5141451A/en active Granted
-
1975
- 1975-10-03 CA CA236,962A patent/CA1060824A/en not_active Expired
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9783860B2 (en) | 2011-12-30 | 2017-10-10 | Renmatix, Inc. | Compositions comprising C5 and C6 oligosaccharides |
| US9797021B2 (en) | 2011-12-30 | 2017-10-24 | Renmatix, Inc. | Compositions comprising C5 and C6 oligosaccharides |
| US10487369B2 (en) | 2011-12-30 | 2019-11-26 | Renmatix, Inc. | Compositions comprising C5 and C6 oligosaccarides |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5649119B2 (en) | 1981-11-19 |
| JPS5141451A (en) | 1976-04-07 |
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