U.S. patent application number 09/835241 was filed with the patent office on 2002-08-29 for process for the preparation of gluconic acid and gluconic acid produced thereby.
Invention is credited to Lantero, Oreste J., Shetty, Jayarama K..
Application Number | 20020119538 09/835241 |
Document ID | / |
Family ID | 25490878 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119538 |
Kind Code |
A1 |
Lantero, Oreste J. ; et
al. |
August 29, 2002 |
Process for the preparation of gluconic acid and gluconic acid
produced thereby
Abstract
An enzymatic system comprised of glucose oxidase and a catalase
of the same or different sources to result in the complete
conversion of glucose to gluconic acid at a glucose concentration
greater than 25% (w/w) ds. The resultant gluconic acid, which is
essentially free from impurities normally associated with the
fermentation process, is then spray granulated to produce a
low-dust dry product.
Inventors: |
Lantero, Oreste J.; (Goshen,
IN) ; Shetty, Jayarama K.; (Pleasanton, CA) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1155 Avenue of the Americas
New York
NY
10036-2711
US
|
Family ID: |
25490878 |
Appl. No.: |
09/835241 |
Filed: |
April 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09835241 |
Apr 12, 2001 |
|
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|
08950815 |
Oct 15, 1997 |
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Current U.S.
Class: |
435/136 ;
562/587 |
Current CPC
Class: |
C07C 51/42 20130101;
B01J 2/006 20130101; C12P 7/58 20130101; C07C 51/42 20130101; B01J
2/04 20130101; C07C 59/105 20130101 |
Class at
Publication: |
435/136 ;
562/587 |
International
Class: |
C12P 007/40; C07C
059/10 |
Claims
What is claimed:
1. A process for the enzymatic conversion of glucose to gluconic
acid comprising providing a solution of glucose; and adding to the
solution, in the presence of an oxygen source, from about 25 to
about 30 glucose oxidase units of soluble glucose oxidase/gram
dissolved solids (ds.) of glucose in the solution and at least 1200
catalase units of soluble catalase/gram dissolved solids (ds.) of
glucose in the solution.
2. The process of claim 1, further comprising the solution of
glucose having from about 25% weight/weight (w/w/) ds. of glucose
to about 60% (w/w) ds. of glucose.
3. The process of claim 1, further comprising the solution of
glucose having from about 30% (w/w/) ds. of glucose to about 50%
(w/w) ds. of glucose.
4. The process of claim 1, wherein about 27 GOU of glucose
oxidase/gram ds. of glucose is added to the solution.
5. The process of claim 1, wherein at least about 1279 CU of
catalase/gram ds. of glucose is added to the solution.
6. The process of claim 1, wherein at least about 1559 CU of
catalase/gram ds. of glucose is added to the solution.
7. The process of claim 1, wherein at least about 1999 CU of
catalase/gram ds. of glucose is added to the solution.
8. The process of claim 1, wherein the glucose oxidase and the
catalase are added to the solution of glucose in two equal doses,
the first dose being added at the start of the reaction and the
second dose being added halfway through the total intended time of
the reaction.
9. The process of claim 1, wherein the catalase is naturally
produced by a strain of the species Aspergillus niger.
10. The process of claim 1, wherein the solution of glucose is
maintained at a pH of from about 5 to about 7.
11. The process of claim 10, wherein the solution of glucose is
maintained at a pH of about 6.
12. The process of claim 1, wherein the temperature of the solution
of glucose is maintained at from about 25.degree. C. to about
40.degree. C.
13. The process of claim 12, wherein the temperature of the
solution of glucose is maintained at from about 30.degree. C. to
about 35.degree. C.
14. The process of claim 1, wherein the pressure of the solution of
glucose is maintained at about 1 bar.
15. The process of claim 1, further including maintaining an air
flow through the solution during the reaction of about 1 volume gas
per volume of reaction medium per minute (vvm).
16. A process for the enzymatic conversion of glucose to gluconic
acid comprising providing a solution of glucose having about 25% of
weight/weight dissolved solids glucose to about 60% weight/weight
dissolved solids glucose; and adding to the solution, in the
presence of an oxygen source, from about 25 to about 30 glucose
oxidase units of soluble glucose oxidase/gram dissolved solids
glucose in the solution and from about 1279 to about 1999 catalase
units of soluble catalase/gram dissolved solids of glucose in the
solution while maintaining the solution of glucose at a pH of from
about 5 to about 7, a temperature of from about 25.degree. C. to
about 40.degree. C., a pressure of about 1 bar and maintaining an
air flow of about 1 volume gas per volume of reaction medium per
minute through the solution.
17. A spray-granulated gluconic acid granulate produced by the
process of claim 16.
18. The process of claim 1, wherein the catalase is naturally
produced by a microbial or mammalian source. pg,29
19. The process of claim 1, wherein the catalase is naturally
produced by a microbial source.
20. The process of claim 1, wherein the catalase is naturally
produced by a strain of Micrococcus lysodeikticus.
21. The process for the production of a low-dust spray-granulated
gluconic acid, comprising the steps of: (a) obtaining a gluconic
acid-containing solution wherein said solution is produced by the
enzymatic conversion of glucose to gluconic acid, said conversion
comprising providing a solution of glucose and adding to the
solution from about 25 to about 30 glucose oxidase units of soluble
glucose oxidase/gram dissolved solids of glucose in the solution
and at least 1200 catalase units of soluble catalase/gram dissolved
solids of glucose in the solution; (b) obtaining gluconic acid
crystals from the solution; and (c) spray-coating the gluconic acid
crystals with liquid sodium gluconate in a spray-dryer, whereby a
spray-granulated gluconic acid is obtained.
22. The process of claim 21, wherein the gluconic acid crystals are
obtained from the gluconic acid-containing solution broth by
concentrating and filtering the gluconic acid-containing
solution.
23. The process of claim 21, wherein the catalase is naturally
produced by a strain of Micrococcus lysodeikticus.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to processes for the enzymatic
conversion of glucose to gluconic acid, the gluconic acid produced
thereby, the preparation of a granular gluconic acid product and
the granular product so produced.
BACKGROUND OF THE INVENTION
[0002] Gluconic acid, an oxidation product of glucose, has been
extensively used in applications as varied as metal cleaning
operations in the dairy industry, alkaline bottle washing
operations; alkaline derusting operations in the metallurgic
industry, and iron deposition prevention in the textile industry.
Furthermore, the sodium salt of gluconic acid is used as an
additive in cement mixtures.
[0003] The production of gluconic acid from glucose may be achieved
by the use of processes which may be broadly classified as being
either microbial fermentation, electrochemical, chemical and
enzymatic (wherein enzymatic systems are employed separately from
their source microorganism(s)). While microbial fermentation has
perhaps been the most widely employed of these methods, it
nonetheless suffers many drawbacks, including those associated with
process conditions required for the fermentation microorganisms
used, which has limited its commercial applicability.
[0004] The enzymatic conversion of glucose to gluconic acid
involves treating a glucose bearing material with an enzyme
preparation having glucose oxidase and catalase activity. This
reaction is performed in the presence of a free oxygen source, such
as hydrogen peroxide. Generally, the glucose-bearing material is in
the form of an aqueous solution.
[0005] To insure that the glucose oxidase functions at its most
effective level, during enzymatic conversion the pH of the reaction
media is controlled so as to favor the desired reaction In the
glucose oxidase conversion of glucose, acid (gluconic) is
continuously formed. Thus, it is necessary to continuously regulate
the pH of the reaction media throughout the enzymatic conversion.
Generally, if the pH is maintained between about 4 2 and about 7
(preferably, between about 5 and about 6), the conversion proceeds
satisfactorily. A common method of regulating the pH involves the
continuous addition of an alkali, such as sodium hydroxide. The
alkali neutralizes the gluconic acid to a corresponding gluconate,
e.g., sodium gluconate. Examples of enzymatic processes for the
production of gluconic acid from glucose using a glucose
oxidase/catalase enzyme system can be found in, for example, U.S.
Pat. No. 2,651,592 and Romanian Patent No. 92,739.
[0006] While being useful for their particular purposes, these
enzymatic processes suffer from several drawbacks.
[0007] A primary drawback associated with fermentation processes is
that the reaction process generally results in the crude reaction
broth containing gluconic acid along with other impurities
including biomass. This reaction broth must then be purified by
multi-step processes including biomass separation (filtration),
carbon treatment (decolorization), evaporation (concentration) and
crystallization (purification) to provide a final product with high
purity.
[0008] Another drawback is the presence of residual mother liquid
in the reaction broth which must be either recycled, further
purified and/or disposed of, thereby adding to the problems and
costs of such fermentation conversion processes.
[0009] A further drawback associated with enzymatic processes is
that they have a low conversion efficiency. This feature results in
incomplete conversion of glucose to gluconic acid leaving residual
unconverted glucose as a contaminant in the gluconic acid solution
produced thereby. In order to reduce or eliminate such unconverted
glucose from the final product, costly downstream, separation,
recovery and purification steps must be employed.
[0010] To alleviate problems associated with incomplete conversion,
resort has been had to limiting the glucose concentrations of the
starting material to less than 30% weight to weight (w/w) dissolved
solids (ds.). Unfortunately, such low glucose concentrations in the
starting material are unsatisfactory in that they greatly reduce
the efficiency of the process, negatively impacting on its
commercial desirability.
[0011] Alternatively, resort has been made to interrupting the
process prior to the completion thereof. For example, in the
processes disclosed in the aforesaid U.S. patent, the reaction was
stopped after converting only 50% of the glucose to gluconic acid.
Nonetheless, the reaction mixture still needs to be subjected to
electrodialysis to separate and recover the gluconic acid from the
residual unconverted glucose and purification of the gluconic acid
produced by such processes remains difficult and costly, especially
where the glucose is present.
[0012] Another drawback associated with enzymatic processes is the
use of hydrogen peroxide (H.sub.2O.sub.2) as a source of oxygen. As
an acid, the presence of H.sub.2O.sub.2 necessitates constant
monitoring of the pH of the reaction solution, as well as the
employment of a buffer (such as calcium carbonate in the form of
lime) to maintain the solution in a pH range which is acceptable
(about 5-6) for the conversion reaction. Furthermore, the use of
hydrogen peroxide as the oxygen source results in the by-product
formation of large quantities of yet more hydrogen peroxide and
acids, necessitating the use of yet more catalases (to convert the
hydrogen proxides formed into water and oxygen) and pH
neutralizers.
[0013] Accordingly, it can be seen that there remains a need for
the provision of enzymatic processes for the production of gluconic
acid from glucose wherein dissolved glucose solid concentrations of
30% (w/w) ds. (dissolved solids) and higher may be used while still
obtaining high conversion rates, wherein reduced quantities of
buffers, such as sodium hydroxide, need to be employed, wherein the
use of extensive and/or expensive downstream, separation, recovery
and/or purification processes and/or apparatuses need not be
employed and which permits the production of spray-dried and
essentially pure gluconic acid granules without employing
crystallization processes
SUMMARY OF THE INVENTION
[0014] It is a primary object of the present invention to provide
an enzymatic process for the production of gluconic acid from
glucose wherein dissolved solid concentrations of glucose of 30%
(w/w) ds. and higher may be used while still enjoying high
conversion rates (such as those approaching 100%), which does not
require extensive expensive downstream separation, recovery and/or
purification, which permits the production of spray-dried and
essentially pure granular gluconic acid without employing
crystallization processes.
[0015] It is a further object of the present invention to provide
such a process wherein the final gluconic acid solution produced
thereby, has a low concentrations of impurities, including residual
unconverted glucose.
[0016] It is a still further object of the present invention to
provide such a process wherein low (reduced) quantities of buffers,
such as sodium hydroxide, need to be employed.
[0017] A yet further object of the present invention is to provide
processes for producing a dry, low-dust, granular gluconic acid
product without the necessity of employing a crystallization
process.
[0018] In another aspect of the present invention, it is an object
herein to provide a substantially pure gluconic acid solution.
[0019] A still yet further object of the present invention is to
provide a dry, low-dust, granular gluconic acid product.
[0020] In accordance with the teachings of the present invention,
disclosed herein is a process for the enzymatic conversion of
glucose to gluconic acid. This process includes providing a
solution of glucose. The process further includes a e of an oxygen
source, from about 25 to about 30 GOU of glucose oxidase/grams ds.
of glucose in the solution and at least 1200 CU of catalase/grams
ds. of glucose in the solution. In this fashion, the glucose is
enzymatically converted to gluconic acid.
[0021] Preferably, the solution of glucose has from about 25% (w/w)
ds. of glucose to about 60% (w/w) ds. of glucose and, more
preferably, from about 30% (w/w) ds. of glucose to about 50% (w/w)
ds. glucose.
[0022] In a preferred embodiment, about 27 GOU of glucose
oxidase/gram ds. of glucose in the solution is added to the
solution.
[0023] Preferably, from about 1279 to about 1999 CU of
catalase/gram ds. of glucose in the solution is added to the
solution.
[0024] In one preferred embodiment, at least 1279 CU of
catalase/gram ds. of glucose in the solution is added to the
solution.
[0025] In a second preferred embodiment, at least 1559 CU of
catalase/gram ds. of glucose in the solution is added to the
solution.
[0026] In a third preferred embodiment, at least 1999 CU of
catalase/gram ds. of glucose in the solution is added to the
solution.
[0027] Preferably, the glucose oxidase and the catalase are added
to the solution of glucose in two equal doses, the first dose being
added at the start of the reaction (log 0 hours) and the second
dose being added halfway (50%) through the total time of the
intended reaction
[0028] In another preferred embodiment, the glucose oxidase and the
catalase are added to the solution of glucose in three equal doses,
the first dose being added at the start of the reaction (log 0
hours), the second dose being added one-third through the total
time of the reaction and the third dose being added two-thirds
through the total time of the reaction.
[0029] Preferably, the catalase is naturally produced by a strain
of the species Aspergillus niger.
[0030] Preferably, the solution of glucose is maintained at a pH of
from about 5 to about 7 throughout the reaction and, more
preferred, the solution of glucose is maintained at a pH of 6
throughout the reaction.
[0031] Preferably, the temperature of the solution of glucose is
maintained at from about 25.degree. C. to about 40.degree. C.
throughout the reaction, and, more preferred, the solution of
glucose is maintained at from about 30.degree. C. to about
35.degree. C. throughout the reaction.
[0032] Preferably, the pressure of the solution of glucose is
maintained at about 1 bar throughout the reaction.
[0033] Preferably, the air flow through the solution of glucose is
maintained at about 1vvm throughout the reaction.
[0034] In a particularly preferred embodiment disclosed herein, the
process for the enzymatic conversion of glucose to gluconic acid
includes providing a solution of glucose having from about 25%
(w/w) ds. of glucose to about 60% (w/w) ds. of glucose. The process
further includes adding to the solution, in the presence of an
oxygen source, from about 25 to about 30 GOU of glucose
oxidase/gram ds. glucose in the solution and from about 1279 to
about 1999 CU of catalase/gram ds. glucose in the solution while,
throughout the reaction, maintaining the solution of glucose at a
pH of about 5 to about 7, at a temperature of about 25.degree. C.
to about 40.degree. C., at a pressure of about 1 bar and at an air
flow of about 1 vvm.
[0035] Finally, an in another aspect of the present invention
disclosed herein is a process for the preparation of a dry,
spray-granulated gluconic acid product and the product produced
thereby.
[0036] In this regard, the gluconic acid-containing solution,
produced as described above, is concentrated and filtered, whereby
gluconic acid crystals (as sodium gluconate) are obtained. The
crystals are then sprayed-coated with liquid sodium gluconate in a
spray-dryer, whereby a spray-granulated gluconic acid is
obtained.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0037] The disclosed process for the enzymatic conversion of
glucose to gluconic acid permits the enzymatic conversion of
glucose solutions having a dissolved (glucose) solids content of
greater than 25% (w/w) ds. of glucose without the resulting
build-up of residual unconverted glucose in the gluconic acid
solution produced thereby and with the use of reduced quantities of
buffers, such as sodium hydroxide, which must be employed.
[0038] Furthermore, use of the principles disclosed herein permits
the fashioning of such processes which do not require the use of
extensive and/or expensive downstream, separation, recovery and/or
purification processes and/or apparatuses and which permit the
production of spray-dried and essentially pure gluconic acid
without employing crystallization processes and which permits the
formation of spray-dried granulation of the gluconic acid obtained
thereby.
[0039] The processes of the present invention produce
spray-granulated gluconic acid and pure gluconic acid without the
need to employ crystallization or purification processes.
[0040] The process of the present invention permits the efficient
enzymatic conversion of glucose to gluconic acid. This process
includes providing a solution of glucose. As taught herein, this
process is useful with solutions having glucose concentrations
greater than 25% (w/w) ds. of glucose. Preferably, the solution of
glucose has from about 25% (w/w) ds. of glucose to about 60% (w/w)
ds. of glucose. Particularly good results are obtained by use of
glucose solutions having from about 30% (w/w) ds. of glucose to
about 50% (w/w) ds. of glucose.
[0041] The process further includes adding to the solution, in the
presence of an oxygen source, from about 25 to about 30 GOU of
glucose oxidase/gram ds. glucose in the solution. Preferably, about
27 GOU to about 29 GOU of glucose oxidase/gram ds. of glucose in
the solution is employed. In one embodiment about 28.6 GOU of
glucose oxidase/gram ds. of glucose in the solution is
employed.
[0042] As used herein, one glucose oxidase unit is defined as being
the amount of enzyme required to oxidase one micromole of D-glucose
per minute under the assay conditions 25.degree. C. and pH 7.0.
[0043] The process further includes adding to the solution, in the
presence of an oxygen source, and at least 1200 CU of catalase/gram
ds. of glucose in the solution Preferably, from about 1279 to about
1999 CU of catalase/gram ds. of glucose in the solution is added to
the solution. In particular preferred embodiments, at least 1279,
1559 and 1999 CU of catalase/gram ds. of glucose in the solution is
added to the solution.
[0044] As used herein, one catalase unit is defined as being the
amount of enzyme required to decompose 1 .mu. mole of hydrogen
peroxide per minute under the assay conditions 25.degree. C. and pH
7.0.
[0045] Any suitable catalase may be employed in the process of the
present invention. Catalases which are naturally produced by (or
derived from) strains of Aspergillus niger and Micrococcus
lysodeikticus, as well as catalases produced by mamilian sources,
such as bovine sources, may be employed. In this context, we have
found herein that the catalase which is naturally produced by
stains of the species Aspergillus niger is particularly
efficacious.
[0046] Preferably, the glucose oxidase and the catalase are added
to the solution of glucose in equal doses. In this regard, in one
embodiment herein, the glucose oxidase and the catalase are added
to the solution of glucose in two equal doses, the first dose being
added at the start of the reaction (log 0 hours) and the second
dose being added halfway (50%) through the total time of the
intended reaction. To illustrate this point, if the reaction is to
proceed for 24 hours then, in that event, the first dose would be
added at the start of the reaction and the second dose would be
added at the start of the 13th hour.
[0047] Further in this regard, in a second embodiment, the glucose
oxidase and the catalase are added to the solution of glucose in
three equal doses, the first dose being added at the start of the
reaction (log 0 hours), the second dose being added one-third
(33.3%) through the total time of the intended reaction and the
third dose being added two-thirds (66.6%) through the total time of
the intended reaction. To illustrate this point, if the reaction is
to proceed for 24 hours then, in that event, the first dose would
be added at the start of the reaction and the second dose would be
added at the start of the nineth hour and the third dose would be
added at the start of the 17th hour.
[0048] The solution of glucose may be maintained at any pH which
permits the reaction to occur. However, it is preferred herein that
the solution of glucose be maintained at a pH of from about 5 to
about 7 throughout the reaction. Most preferred is maintaining the
solution of glucose at a pH of 6 throughout the reaction.
[0049] The solution of glucose may be maintained at any temperature
which permits the reaction to occur. However, in this regard, it is
preferred that the temperature of the solution of glucose is
maintained at from about 25.degree. C. to about 40.degree. C.
throughout the reaction. Most preferred is maintaining the solution
of glucose at from about 30.degree. C. to about 35.degree. C.
throughout the reaction.
[0050] While the solution of glucose may be maintained at any
pressure which permits the reaction to occur, it is preferred that
the presssure of the solution of glucose is maintained at about 1
bar throughout the reaction.
[0051] While the solution of glucose may be maintained by passing
air through the glucose solution at any flow rate which permits the
reaction to occur, it is preferred herein that the rate of air flow
through the solution of glucose be maintained at about 1 vvm
throughout the reaction.
[0052] Having thus described the processes of the present invention
for the enzymatic conversion of glucose to gluconic acid, the
gluconic acid produced thereby and processes for producing a
spray-granulated gluconic acid product, reference is now made to
the following examples which are presented herein for the purposes
of illustration only and are neither meant to be, nor should they
be read as being, restrictive.
[0053] Unless otherwise specified herein, the Examples of the
present invention were performed with the use of glucose oxidase
from Aspergillus niger (sold by SOLVAY ENZYMES, GmbH, Germany),
catalase from Micrococcus lysodeikticus sold under the trademark
MicroCatalase L- 1000 (SOLVAY ENZYMES, Inc., Elkhart, Indiana) and
crystalline glucose marketed under the name StaleyDex 333 R
(Staley, U.S.A.).
Example 1: Effect of pH
[0054] The effect of pH on the conversion of glucose to gluconic
acid by glucose oxidase-catalase enzyme system according to the
method of the present invention, was studied at a glucose
concentration of 40% (w/w) ds.
[0055] 8 liters of 40% (w/w) ds. glucose solution was prepared and
introduced into a 10 liter fermentor (Chempec, Inc., N.J., U.S.A).
The pH was adjusted to pH 4.0 with dilute acid (i e., 5N sulfuric
acid) or alkali (i.e., 4N sodium hydroxide), as necessary.
[0056] The reaction was carried out at 35.degree. C. with a
pressure at 1 bar and air was bubbled at a rate of 1 VVM. The pH
was maintained at the specified pH during the reaction by the
addition of 50% (w/w) sodium hydroxide. Foam was controlled by the
addition of antifoam MAZU DF6000 (PPG/MAZER CHEMICALS) (80-120
ppm).
[0057] Doses of 12.25 glucose oxidase units (GOU) per gram of
dissolved solids of glucose of the solution and 726.8 catalase
units (CU) per gram of dissolved solids of glucose of the solution
were added to the fermentor at log 0 and at log 12 hours.
[0058] The conversion of glucose to gluconic acid was measured by
determining the milliequivalent of sodium hydroxide consumed and/or
by analyzing the samples using high pressure liquid chromatographic
(HPLC) method.
[0059] HPLC analysis was carried out using an HPLC system
consisting of Beckman 112 Solvent Delivery Module (BECKMAN, U.S.A.)
fitted to a RI detector Model ERC-7515A, (The ANSPEC Co., Inc.,
U.S.A.) Glucose and gluconic acid were separated using an H:PX 87-C
column (Bio-Rad, U.S.A.) at 80.degree. C. and 0.001 M calcium
acetate (pH 5 5) was used as the mobile phase at a flow rate of 1
ml/min.
[0060] The color impurities were determined by measuring the
absorbance (optical density) of 37.5% (w/w) solution gluconic acid
in 5% (w/w) NaOH solution at 470 nm.
[0061] Four additional experiments were then conducted using the
same protocol, and under the same process conditions, as described
above with the exception that the pH of the reaction mixture was
for each experiment to, respectively, pH 5.0, pH 6.0, pH 7.0 and pH
8.0.
[0062] The effects of different pHs on the efficiency of the
conversion of glucose to gluconic acid are summarized below in
Table 1.
1TABLE 1 Effect of pH on the Conversion of Glucose to Gluconic Acid
using Glucose Oxidase-Catalase Enzyme System Reaction Time Percent
Conversion (Hours) pH 4.0 pH 5.0 pH 6.0 pH 7.0 pH 8.0 1 2.6 6.8 5.1
5.0 3.3 2 3.7 14.2 14.9 13.1 5.6 3 5.2 22.4 32.1 20.2 8.0 4 4.6
29.0 30.3 26.7 10.0 5 4.8 35.1 36.7 32.5 11.8 6 4.8 40.6 42.5 34.1
13.4 7 4.8 45.3 47.2 40.5 15.0 8 4.8 49.1 51.3 45.5 16.5 9 4.8 52.5
54.8 48.9 17.9 10 4.8 55.9 57.9 55.3 19.5 11 4.8 61.8 60.8 61.2
22.0 12 4.8 67.6 64.8 66.6 24.4 13 4.8 73.2 71.7 71.7 26.7 14 4.8
78.5 78.1 77.1 29.0 15 4.8 84.0 83.8 81.1 31.2 16 4.8 88.8 89.0
84.8 33.4 17 4.8 93.4 94.3 87.9 35.5 18 4.8 97.4 99.2 91.5 39.3 19
4.8 99.3 100.0 94.9 41.2 20 4.8 99.6 100.0 98.2 45.2 21 4.8 99.9
100.0 99.9 46.9 22 4.8 99.9 100.0 100.0 48.5 23 4.8 100.0 100.0
100.0 50.2 24 4.8 100.0 100.0 100.0 51.9 Table 1 shows that, at pHs
of between 5 and 7, using the method of the present invention
achieves a 100% conversion of glucose to gluconic acid in less than
24 hours.
Example 2 Enzyme Dosage--Single Addition and Multiple Addition
[0063] The effect of adding the glucose oxidase and catalase in
either one or several doses throughout the reaction on the
conversion of glucose to gluconic acid according to the method of
the present invention was determined by performing three
experiments wherein the protocols and process conditions were
maintained the same, with the exceptions of timing and manner of
the dosing of the enzymes.
[0064] The conversion of glucose to gluconic acid was carried out
in three separate experiments using the same protocol and under the
same process conditions as described above in Example 1, but with
the following exceptions: the reaction mixtures of all experiments
were maintained at pH 6 0, and the quantities and timing of the
enzyme additions were varied. In that regard, the total
concentration of glucose oxidase and catalase added was maintained
constant (at 27 glucose oxidase units per gram of dissolved glucose
solids and 1599 catalase units per gram of dissolved glucose
solids) but were added in either one, two or three doses.
[0065] As regards the quantity and time of the enzyme addition in
the first experiment 27 GOU/gram ds. and 1599 CU/gram ds. were
added at log 0 hours, in the second experiment, 13.5 GOU/grams ds.
and 799 5 CU/gram ds. were added at log 0 and at log 12 hours; and
in the third experiment, 9 GOU/gram ds. and 533 CU/gram ds. were
added at log 0, log 6 and log 12 hours.
[0066] The effect of adding the glucose oxidase and catalase in
either one or several doses throughout the reaction on the
conversion of glucose to gluconic acid are summarized below in
Table 2.
2TABLE 2 Percent Conversion Time (Hours) Experiment #1 Experiment
#2 Experiment #3 0 0 0 0 1 3.4 5.1 4.5 2 13.6 14.9 11.5 3 22.3 23.1
17.6 4 30.9 30.3 23.1 5 38.6 36.7 27.7 6 45.6 42.5 31.8 7 51.9 47.2
37.3 8 59.1 51.3 43.8 9 64.7 54.8 49.6 10 69.5 57.9 54.9 11 72.3
60.8 60.5 12 73.3 64.8 65.4 13 74.1 71.7 71.6 14 74.5 78.1 77.1 15
74.5 83.3 82.7 16 74.9 89.0 88.0 17 74.9 94.3 93.2 18 75.9 99.2
97.9 19 75.9 100.0 99.7 20 75.9 100.0 100.0 21 75.9 100.0 100.0 22
75.9 100.0 100.0 23 75.9 100.0 100.0
Example 3: Role of Catalase on the Oxidation of Glucose to Gluconic
Acid by Glucose Oxidase
[0067] The effect of various concentrations of catalase on the
conversion of glucose to gluconic acid according to the method of
the present invention was determined by performing five experiments
wherein the protocols and process conditions were maintained the
same with the exception of the catalase concentrations added.
[0068] The conversion of glucose to gluconic acid was carried out
in five separate experiments using the same protocol and under the
same process conditions as described above in Example 2, but with
the following exceptions the temperatures of the reaction mixtures
were maintained at 40.degree. C.; the concentration of glucose
oxidase added was 28.6 GOU/gram ds.; the concentration of the
catalase added was varied as will be described below; and all of
the catalase and glucose oxidase was added at log 0 time.
[0069] As regards to the concentration of the catalase added, the
concentration of catalase added was varied, as follows: in the
first experiment, 0 catalase units/gram ds. were added; in the
second experiment, 959 catalase units/gram ds. were added; in the
third experiment, 1279 Catalase units/grams ds. were added; in the
fourth experiment, 1559 catalase units/gram ds. were added; and in
the fifth experiment, 1999 catalase units/grams ds. were added.
[0070] The effect of varying catalase concentrations on the
conversion of glucose to gluconic acid under identical conditions
are summarized below in Table 3.
3TABLE 3 Percent Conversion Reaction Time 0 CU 959 CU 1279 CU 1559
CU 1999 CU (Hours) per g ds. per g. ds. per g. ds. per g. ds. per g
ds 1 1.6 3.4 4.5 3.4 4.6 2 5.1 13.4 13.2 13.6 14.0 3 6.3 22.6 22.3
22.3 22.8 4 6.9 30.7 31.5 30.9 31.3 5 7.0 38.5 39.7 38.6 39.1 6 7.0
45.7 47.2 45.6 46.7 7 7.0 51.7 53.6 51.9 53.7 8 7.0 56.1 59.3 59.1
59.6 9 7.0 57.8 64.0 64.7 65.5 10 7.1 58.6 66.1 69.5 71.0 11 7.1
59.1 67.4 72.3 75.5 12 7.1 59.4 68.1 73.3 79.1 13 7.1 59.9 68.4
74.1 80.3 14 7.1 59.9 68.4 74.5 81.4 15 7.1 59.9 68.7 74.5 81.8 16
7.1 59.9 68.7 74.9 81.8 17 7.1 59.9 68.7 74.9 82.4 18 7.1 59.9 68.7
75.9 82.4 19 7.1 59.9 68.7 75.9 82.4 20 7.1 59.9 68.7 75.9 82.9 21
7.1 59.9 68.7 75.9 82.9
[0071] The results of Table 3 show that, in the method of the
present invention, the inactivation of glucose oxidase by hydrogen
peroxide is significantly reduced by the addition of catalase. In
the absence of catalase, the oxidation of glucose to gluconic acid
by glucose oxidase proceeded for 5 hours with only 7%. conversion.
The percent conversion of glucose to gluconic acid was increased
with increasing concentration of catalase and a maximum conversion
of 80% was reached even at high concentration of catalase. The
residual unconverted glucose could be due to the stability
(half-life) associated with glucose oxidase under the experimental
conditions.
Example 4: Effect of Temperature on Oxidation of Glucose to
Gluconic Acid Using Glucose Oxidase-Catalase Enzyme System.
[0072] The effect of various temperatures on the conversion of
glucose to gluconic acid according to the method of the present
invention was determined by performing four experiments wherein the
protocols and process conditions were maintained the same with the
exception of the temperature of the reaction.
[0073] The conversion of glucose to gluconic acid was carried out
in four separate experiments using the same protocol and under the
same process conditions as described above in Example 2, but with
the following exceptions the temperatures of the reaction mixtures
were varied, as will be described below, 50% of the glucose oxidase
units (13.5 glucose oxidase units) and 50% of the catalase units
(799.5 catalase units) were added at log 0 hours and 50% of the
glucose oxidase units (13.5 glucose oxidase units) and 50% of the
catalase units (799.5 catalase units) were added at log 9
hours.
[0074] As regards to the temperatures employed, the temperatures
employed were varied, as follows in the first experiment, the
temperature was 25.degree. C., in the second experiment, the
temperature was 30.degree. C.; in the third experiment, the
temperature was 35.degree. C.; and in the fourth experiment, the
temperature was 40.degree. C.
[0075] The effect of the different temperatures on the conversion
of glucose to gluconic acid under identical conditions are
summarized below in Table 4.
4TABLE 4 Percent Conversion Reaction Time (Hours) 25.degree. C.
30.degree. C. 35.degree. C. 40.degree. C. 0 0.0 0.0 0.0 0.0 1 2.6
0.0 6.5 6.1 2 5.3 12.0 14.4 13.8 3 7.9 16.7 21.5 21.5 4 14.0 22.1
28.2 28.3 5 18.0 28.2 34.4 34.6 6 22.0 33.4 39.7 40.0 7 25.2 37.3
44.2 44.4 8 27.5 42.1 48.2 47.6 9 32.8 45.4 54.4 52.5 10 38.6 51.3
61.1 59.9 11 42.4 56.5 67.4 67.1 12 47.9 61.8 73.6 73.9 13 54.0
66.8 79.5 80.4 14 59.6 71.7 84.7 85.1 15 64.8 77.0 89.5 87.2 16
69.5 81.2 93.1 88.0 17 74.2 85.3 95.7 88.5 18 78.1 89.6 96.7 88.7
19 82.9 93.3 97.0 88.8 20 87.0 96.8 97.6 89.0 21 90.9 99.2 97.7
89.0 22 93.7 99.7 98.0 89.0 23 97.2 99.9 98.0 89.0 24 99.1 99.9
98.0 89.0 25 99.1 100.0 98.0 89.0
Example 5: Effect of Glucose Concentration on the Conversion of
Glucose to Gluconic Acid Using Glucose Oxidase-Catalase Enzyme
System
[0076] The effect of various concentrations of glucose on the
conversion of glucose to gluconic acid according to the method of
the present invention was determined by performing six experiments
wherein the protocols and process conditions were maintained the
same with the exception of the glucose. concentrations added.
[0077] The conversion of glucose to gluconic acid was carried out
in six separate experiments using the same protocol and under the
same process conditions as described above in Example 2, but with
the following exceptions: the temperatures of the reaction mixtures
were maintained at 30.degree. C.; the concentration of the glucose
added was varied as will be described below; and all of the
catalase and glucose oxidase was added at log 0 time.
[0078] The effect of glucose concentration (dissolved solids) on
the conversion rate of glucose to gluconic acid by the Glucose
oxidase-catalase enzyme was studied as described above in Example
2, but with the glucose concentration being varied, as follows: in
the first experiment, the glucose concentration was 30% ds.; in the
second experiment, the glucose concentration was 35% ds., in the
third experiment, the glucose concentration was 40% ds.; in the
fourth experiment, the glucose concentration was 45% ds.; in the
fifth experiment, the glucose concentration was 50% ds; and in the
sixth experiment, the glucose concentration was 55% ds.
[0079] The effect of varying glucose concentrations on the
conversion of glucose to gluconic acid under identical conditions
are summarized below in Table 5.
5TABLE 5 Percent Conversion Reaction Time Glucose Concentration [in
% (w/w)] (Hours) 30% 35% 40% 45% 50% 55% 0 0.0 0.0 0.0 0.0 0.0 0.0
1 7.7 4.0 0.0 3.6 2.0 0.0 2 17.2 12.5 12.0 8.2 6.0 4.4 3 26.6 22.5
16.7 13.7 9.7 6.5 4 34.7 31.6 22.1 18.4 13.3 8.4 5 41.9 39.7 28.2
22.5 16.7 10.4 6 47.8 47.1 33.4 25.8 20.1 12.4 7 53.2 53.6 37.3
30.4 23.2 14.5 8 57.8 59.4 42.1 34.4 26.1 16.5 9 61.3 64.4 45.4
37.1 29.0 18.6 10 68.2 71.2 51.3 39.6 31.7 20.5 11 78.5 79.0 56.5
42.6 -- 22.4 12 88.4 86.6 61.8 46.4 36.9 24.1 13 97.1 94.5 66.8
51.0 40.0 25.7 14 99.5 99.3 71.2 55.2 42.7 27.2 15 99.9 99.7 77.0
59.4 45.4 28.5 16 100.0 99.9 81.2 63.1 48.1 30.5 17 100.0 100.0
85.3 67.0 50.5 32.0 18 100.0 100.0 89.6 70.5 52.9 33.6 19 100.0
100.0 93.3 74.2 55.6 35.1 20 100.0 100.0 96.8 77.2 58.0 36.6 21
100.0 100.0 99.2 80.9 60.1 38.3 22 100.0 100.0 99.7 84.3 62.4 39.9
23 100.0 100.0 99.9 87.7 64.7 41.7 24 100.0 100.0 99.9 90.7 66.8
43.2 25 100.0 100.0 100.0 93.4 68.8 44.7 26 100.0 100.0 100.0 96.1
70.9 46.0 27 100.0 100.0 100.0 98.9 72.9 47.4 28 100.0 100.0 100.0
99.7 75.0 48.8 29 100.0 100.0 100.0 99.8 76.8 50.2 30 100.0 100.0
100.0 100.0 78.6 51.8 31 100.0 100.0 100.0 100.0 80.4 53.3 32 100.0
100.0 100.0 100.0 82.1 54.7 33 100.0 100.0 100.0 100.0 83.6 56.1 34
100.0 100.0 100.0 100.0 85.0 57.5 35 100.0 100.0 100.0 100.0 86.5
58.9 36 100.0 100.0 100.0 100.0 87.7 60.1 37 100.0 100.0 100.0
100.0 88.9 61.5 38 100.0 100.0 100.0 100.0 89.8 62.7 39 100.0 100.0
100.0 100.0 90.6 64.0 40 100.0 100.0 100.0 100.0 91.4 65.3 41 100.0
100.0 100.0 100.0 92.0 66.5 42 100.0 100.0 100.0 100.0 92.5 67.7 43
100.0 100.0 100.0 100.0 92.9 69.0 44 100.0 100.0 100.0 100.0 93.4
70.1 45 100.0 100.0 100.0 100.0 93.7 71.3 46 100.0 100.0 100.0
100.0 94.0 72.6 47 100.0 100.0 100.0 100.0 94.2 73.7 48 100.0 100.0
100.0 100.0 94.5 73.7 49 100.0 100.0 100.0 100.0 94.6 73.7 50 100.0
100.0 100.0 100.0 94.8 73.7 51 100.0 100.0 100.0 100.0 94.9 73.7 21
100.0 100.0 100.0 100.0 95.1 73.7 53 100.0 100.0 100.0 100.0 95.2
73.7 54 100.0 100.0 100.0 100.0 95.3 73.7
[0080] The results of Table 5 showed a remarkable beneficial effect
of dissolved solids on the reaction rate using the method of the
present invention. The decrease in the solubility of oxygen with
increasing dissolved solids presumably responsible for lower rate.
However, by adjusting the enzyme dosage, it is possible to complete
the conversion within the specified time. The data in the Table 5
clearly showed that the concentration of oxygen/available oxygen
and length of the reaction time (enzyme dosage) greatly influences
the overall economics of the process.
Example 6:Evaluation of Catalases from Different Sources
[0081] Effect of catalases from different sources on the conversion
of glucose to gluconic acid by the use of glucose oxidase was
studied under identical reaction conditions. The following
catalases were used commercial product of bovine catalase (CATALASE
L) sold by SOLVAY ENZYMES, Inc., U.S.A., Aspergillus niger catalase
from SOLVAY ENZYMES, GmbH (Germany) and Micrococcus catalase.
[0082] The catalase activity of all samples was measured by
spectrophotometrically as described by H. Luck in Methods of
Enzymatic Analysis (H. U. Bergmeyer, ed.), 1965, pp. 885-894,
Verlag Chemie & Academic Press, N.Y., London).
[0083] The time taken for the absorbance of 10 mM hydrogen peroxide
solution (in 0.05 M phosphate buffer, pH 7) from 0.45 to 0.40 was
measured and used to calculate activity.
[0084] The experiments were carried out using the protocol, and
under the conditions decribed in Example 2, but with the exception
that all of the glucose oxidase and the catalase were added to the
glucose solution at log 0 hour.
[0085] The results are summarized below in Table 6.
6TABLE 6 Percent Conversion Reaction Time A. niger Bovine
Micrococcus (Hours) 1137 CU/g. ds. 1600 CU/g. ds 1600 CU/g. ds 0
0.0 0.0 0.0 1 8.6 4.3 0.0 2 16.9 10.6 12.0 3 25.1 17.6 16.7 4 32.8
23.6 22.1 5 40.1 28.9 28.2 6 47.1 34.0 33.6 7 53.7 36.7 37.3 8 59.9
37.9 42.1 9 66.1 38.6 45.4 10 72.6 39.1 51.3 11 78.2 39.3 56.5 12
82.9 41.1 61.8 13 88.7 46.7 66.8 14 94.3 52.2 71.2 15 99.7 56.8
77.0 16 99.9 59.4 81.2 17 100.0 60.7 85.3 18 100.0 61.6 89.6 19
100.0 62.0 93.3 20 100.0 62.3 96.8 21 100.0 62.5 99.2 22 100.0 62.5
99.7 23 100.0 62.5 99.9 24 100.0 62.5 99.9 25 100.0 62.5 100.0 26
100.0 62.5 100.0
[0086] As can be seen from Table 6, both A. niger and Micrococcus
catalases showed a 100% conversion of glucose to gluconic acid but
at varying rates. However, bovine catalase reached only 60%
conversion. Even though all three enzymes were added at the same
dosage, but still observed differences in the rate This could be
due to the differences in the stability of catalases against
hydrogen peroxide.
Example 7 : Evaluation of Different Commercial Glucose Syrups
[0087] The effect of different sources of glucose syrups with
varying degrees of purity on the conversion of glucose to gluconic
acid according to the method of the present invention was
determined.
[0088] The conversion of different glucose syrups was carried out
in three separate experiments using the same protocol and process
conditions as those described above in Example 2.
[0089] The different glucose syrups used were commercially
available glucose syrup with varying degree of purity, as follows:
Commercia+1 products of Clintose "L" TM (ADM Corn Processing,
U.S.A.); Clearsweet TM 99 Refined Liquid Dextrose (Cargill,
U.S.A.); and Royal R Glucose Liquid, 2637 (Corn Products,
U.S.A.).
[0090] The effects of different glucose syrups on the efficiency of
the conversion of glucose to gluconic acid according to the method
of the present invention are summarized below in Table 7.
7TABLE 7 Percent Conversion Reaction Time (Hours) Clintose
Clearsweet Royal Glucose 0 0.0 0.0 0.0 1 4.5 3.7 4.5 2 12.5 9.8
10.8 3 20.2 17.1 17.3 4 27.2 23.1 23.2 5 32.8 28.6 28.7 6 38.0 33.3
33.3 7 42.5 37.2 37.6 8 46.4 40.4 40.8 9 49.6 42.6 43.7 10 52.0
44.5 46.3 11 54.3 45.7 48.4 12 57.8 46.5 51.1 13 62.8 47.1 56.1 14
68.2 51.8 61.5 15 72.3 57.4 66.4 16 77.3 62.4 70.8 17 82.5 67.1
75.1 18 87.0 71.0 78.4 19 91.2 74.1 81.9 20 94.8 76.0 84.6 21 97.9
78.5 86.4 22 99.2 79.9 87.8 23 100.0 81.1 88.7 24 100.0 81.8 89.5
25 100.0 83.4 89.5
[0091] As can be seen from Table 7, under the standard conditions
of the experiments, a 100% conversion of glucose occurred only with
Clintose and crystalline glucose. However, both Clearsweet and
Royal glucose reached between 80% and 90% conversion.
Example 8: Formation of Dry Spray-Granulated Gluconic
Acid/Gluconate and Comparison With Commercial Preparations
[0092] The method of practicing the present invention is further
illustrated by the following examples wherein the production of
liquid or granular gluconic acid/gluconate of purity equivalent or
better than commercial gluconic acid/gluconate is produced without
any carbon treatment or crystallization.
[0093] Gluconic acid/gluconate obtained from the reactor as
described in Experiment 3 of Example 2. The gluconic acid so
obtained (in the form of liquid sodium gluconate) was then
concentrated and crystals were separated by filtration
(micro/ultra/polish) and dried at 37.degree. C. These crystals were
then used as a feed to produce a spray granulated gluconic
acid/gluconate.
[0094] The spray-dryer used was a Uni-Glatt with Wurster Laboratory
Model fluid bed dryer with a variable air temperature and flow
through the bed. Approximately 3/4 of the air holes outside the
column were blocked off. The spraying nozzle was a twin fluid
nozzle (Schlick).
[0095] The atomization air cap opening was 0.5 mm around the liquid
nozzle and 1 mm below the opening of the liquid nozzle. The liquid
nozzle opening was 1.2 mm. The air flow was sufficient to fluidize
the bed and maintain flow through the Wurster column (110 CFM
maximum).
[0096] Five hundred grams of sodium gluconate crystals (dried) were
taken in the Uni-Glatt Dryer and 8480 grams of clear filtrate of
gluconic acid/sodium gluconate (41.2.degree. Brix) was then
spray-coated onto the crystals with low inlet air flow due to low
bulk density (0.5 grams/cc) of crystals (inlet air temperature of
100-105.degree. C., air outlet temperature of 60-65.degree.
C.).
[0097] Initially, the air flow was 50 cfm (cubic feet per minute)
being increased to 110 cfm as the particle density increased. Total
drying time was 316 minutes.
[0098] The final particles were irregular spheres with smooth
surfaces. Mass recovery in the process was 97.5%.
[0099] The above procedure was then repeated using each of the
commercially-available gluconic acid preparations from the
following sources. (1) Sigma Chemicals (U.S.A.); (2) ADM-Decatur
(U.S.A.); (3) AKZO (U.S.A.); (4) Penta Mfg. (U.S.A.); and (5) PMP
(U.S.A.). Sample (6) was the gluconic acid prepration prepared
according to this Example.
[0100] The gluconic acid content of the different preparations was
determined by HPLC using the protocol and under the same conditions
as those described above in Example 1.
[0101] The color impurities were determined by measuring the
absorbance (optical density) of 37.5% (w/w) solution gluconic acid
in 5% (w/w) NaOH solution at 470 nm.
8TABLE 8 Optical Sample Density Purity %.sup.1 (1) 0.003 100.0 (2)
0.029 95.7 (3) 0.029 99.5 (4) 0.014 98.9 (5) 0.051 100.0 (6) 0.001
100.0 .sup.1Purity % was 1 ml/min, 80.degree. C., 0.01 M Ca Acetate
pH 5.5, HPX-87C
[0102] The results of Table 8 show that the quality of the
spray-granulated gluconic acid preparations of the present
invention without any further purifications, such as carbon
treatment and crystallization was superior to the commercially
available dry gluconic acid preparations
* * * * *