CA2240294A1 - Process for the preparation of gluconic acid and gluconic acid produced thereby - Google Patents
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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.
Description
W O 97/24454 PCTAUS96n~480 PROCESS FOR THE PREPARATION OF GLUCONIC ACID
AND GLUCONIC ACID PRODUCED T~R~RY
FIELD OF THE INVENTION
The present invention relates to processes for the el~ylllaLic conversion of glucose to gluconic acid, the gluconic acid produced thereby, the ~l~pa.~Lion of a gr~mll~r gluconic acid product and the granular product so produced.
BACKGROUND OF THE INVENTION
Gluconic acid, an oxidation product of glucose, has been ~Lel~sively used in applications as varied as metal cle~nin~ operations in the dairy industry; ~lk~lin~
bottle washing operations; ~lk~lin~o derusting operations in the m~t~ lrgic industry;
and iron deposition ~l~v~lllion in the textile industry. FulLl~ llore, the sodium salt of gluconic acid is used as an additive in cement ~ Ul~;;S.
The production of gluconic acid from glucose may be achieved by the use of processes which may be broadly cl~ iecl as being either l~ ol~ial fe~-nt~tion, electrochf mi~l, cl~ ir~l and enzymatic (wherein el~ylllaLic systems are employed s~al~Lely from their source microorganism(s)). While microbial ferm~nt~tion has perhaps been the most widely employed of these m~ho~l~, it nonetheless suffers many drawbacks, including those associated with process conditions required for the ferment~tion microo~ t~ used, which has limited its commercial applicability.
The enzymatic conversion of glucose to gluconic acid involves treating a glucose bearing m~teri~l with an enzyme preparation having glucose oxidase and c~t~ e 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.
To insure that the glucose oxidase functions at its most effective level,25 t 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 n~cess~ry to continuously regulate W O 97/24454 PCT~US96/20480 the pH of the reaction media throughout the enzymatic conversion. Generally, if the pH is m~int~in~cl between about 4.2 and about 7 (pxeferably, between about 5and about 6), the conversion proceeds s~ti~f~rtorily. A common method of regulating the pH involves the continuous addition of an aLkali, such as sodium S hydroxide. The alkali neutralizes the gluconic acid to a corresponding gluconate, e.g., sodium gluconate.
Examples of el~yllla~ic processes for the production of gluconic acid from glucose using a glucose oxidase/cat~l~ce enzyme system can be found in, for example, United States Letters Patent No. 2,651,592 and ]~om~ni~n Patent No.
92,739.
While being useful for ~eir particular purposes, these el~yl.lalic processes suffer from several drawbacks.
A primary drawback associated with e~yll~dLic processes is that the reaction process generally results in the crude reaction broth cO~ g gluconic acid along with other illl~ulilies inrlll~lin~ biomass. This reaction broth must then be purified by multi-step processes including biomass separation (filtration),carbon tre~tment (decolorization), e~,apoldLion (cc..~rG~ ;on) and cryst~lli7~ti~ n(purification) to provide a final product with high purity.
Another dldwl,ack is the ~l~se.lce of residual mother liquid in the reaction broth which must be either recycled, further purified and/or disposed of, there by adding to the problems and costs of such el~yllla~ic conversion processes.
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 co,.l;....il.~..l in the gluconic acid solution produced thereby. In order to reduce or elimin~t~ such unconverted glucose from the final product, costly dOwll~LI~alll, separation,recovery and purification steps must be employed.
To alleviate problems associated with incomplete conversion, resort has been had to limiting the glucose collcellLldLions of the starting m~trri~l to less than 30% (w/w) dissolved solids (ds.). Ul~llui~lely, such low glucose concentrations in the starting material are ~ c~ r~rtory in that they greatly reduce the efficiency of the process, negatively imp~rting on its commercially desirability.
W O 97/24454 PCTAUS96~0480 Alternatively, resort has been made to intei~ul"ing the process prior to the comple~ion thereof. For example, in the processes disclosed in the aforesaid United States Patent, the reaction was stopped after conv~lLillg only 50 % of the glucose to gluconic acid. Nonetheless, the reaction llliklul~ still needs to be ~ 5 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 ~liffi1elllt and costly, especially where the glucose is still present.
Another drawback associated with enzymatic processes is the use of hydrogen peroxide (H202) as a source of oxygen. As an acid, the presence of H202 neces~ s constant mo.lilo~ g of the pH of the reaction solution, as well as the employment of a buffer (such as calcium carbonate in the form of lime) tomAint~in 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 qll~ntiti~s of yet more hydrogen peroxide and acids, nPcec~ l;,.g the use of yet more ~Al~es (to convert the hydrogen peroxides formed into water and oxygen) and pH neutralizers.
Accordingly, it can be seen that there ie,llains a need for the provision of e,~y,nalic processes for the production of gluconic acid from glucose where in dissolved glucose solid concentrations of 30 % (w/w) ds. (dissolved solids) and 15 higher may be used while still obtaining high conversion rates, wL~lein reduced qll~ntities of buffers, such as sodium hydroxide, need to be employed, wherein the use of ~nsive and/or expensive dowlls~le~ll, separation, l~cov~ly and/or puri~lr~tion processes and/or a~L,alaLuses need not be employed and which permits the production of spray-dried and e~nti~lly pure gluconic acid grAmlles without employing crystSIlli7~tion processes.
~ SUMMARY OF THE INVENTION
It is a primary object of the present invention to provide an enzymatic process for the production of gluconic acid from glucose wherein dissolved solidconcentrations 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 W O 97/24454 PCT~US96/20480 require extensive expensive do~ separation, recovery and/or pllri~lc~tion1 which L)ellnils the production of' spray-dried and essentially pure granular gluconic acid without employing cryst~lli7s~tion processes.
It is a further object of.the present invention to provide such a process 3Q
wherein the final gluconic acid solution produced there~y, has a low concentrations of i...~,uliLies, including residual unconverted glucose.
It is a still further object of.the present invention to provide such a process wherein low (reduced~ ql-~ntiti.os of.buffers, such as sodium hydroxide, need to be employed.
A yet further object of.the present invention is to provide processes for producing a dry, low-dust, granular gluconic acid product without the n~cescity of employing a cryst~lli7~tion process.
In another aspect of the present invention, it is an object herein to provide a ~ub~L~ lly pure gluconic acid solution.
A still yet further object of the present invention is to provide a dry, low-dust, granular gluconic acid product.
In accol-lance with the te~hing~ of the present invention, disclosed herein is a process for the ~ymatic conversion of glucose to gluconic acid. This process includes providing a solution of glucose. The process further in~ les adding to the solution, in the presence of an oxygen source, from about 25 to about 30 GOU of glucose oxidase/gram ds. of glucose in the solution and at least1200 CU of c~t~ o/gram ds. of glucose in the solution. In this fashion, the glucose is e~y~ lly converted to glll~oni~ acid.
Preferably, the solution of glucose has from about 25 % ~wlw) ds. of glucose to about 60 % (w/w) ds. of glucose and, more preferably, from about 30 % (wlw) ds. of glucose to about 50 % (w/w) ds. glucose.
In a ~lc~llcd emb~(1im~nt, about 27 GOU of glucose oxidase/gram ds. of glucose in the solution is added to the solution.
Preferably, from about 1279 to about 1999 CU of c~t~T~elgram ds. of glucose in the solution is added to the solution. r In one ~.efelled embo~lim~.nt7 at least 1279 CU of c~t~ çlgram ds. of glucose in the solution is added to the solution.
WO 97124454 PCT/~JS96f20480 In a second preferred embo-lim~rlt at least 1559 CU of c~t~l~ce/gram ds.of glucose in the solution is added to the solution.
In a third ylcr~lr~d embodiment, at least 1999 CU of c~t~l~ce/gram ds. of glucose in the solution is added to the solution.
Preferably, the glucose oxidase and the c~t~ P 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 tota}
time of the intended reaction.
In another p.lc~llcd embodiment, the glucose oxidase and the c~t~ e 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-thirdsthrough the total time of the reaction.
Preferably, the c~t~l~se is naturally produced by a strain of the species Aspergillus niger.
Preferably, the solution of glucose is m~int~in~cl at a pH of from about 5 to about 7 throughout the reaction and, more yl~relred~ the solution of glucose is 7. at a pH of 6 throughout the reaction.
Preferably, the tempel~Lul~, of the solution of glucose is Ill~ i..P~l at from about 25~C to about 40~C throughout the reaction, and, more p.~cfellc;d, the solution of glucose is m~int~in~.~l at from about 30~C to about 35~C throughout the reaction.
Preferably, the ync~.~.ul~ of the solution of glucose is ~-~A;~ d at about 1 bar throughout the reaction.
Preferably, the air flow through the solution of glucose is m~int~in~1 at about 1 vvrn throughout the reaction.
In a particularly plc~llcd embodiment disclosed herein, ~e process for the y~ ic conversion of glucose to gluconic acid includes providing a solution of glucose having from about 25 % (w/w) ds. of glucose to about 60 %15(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 ~t~l~cPlgram W O 97/24454 PCTrUS96/20480 ds. glucose in the solution while, throughout the reaction,m~int~ining the solution of glucose at a pH of about 5 to about 7, at a temperature of about 25~C to about 40~C, at a ~lcs:iule of about I bar and at an air flow of about 1 vvm.
Finally, and in another aspect of the present invention disclosed herein is a S process for the ~l~aldtion of a dry, spray-gr~n~ gluconic acid product and the product produced thereby.
In this regard, the gluconic acid-cont~ining 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 sodiumgluconate in a spray-dryer, whereby a spray-gr~mll~t~d gluconic acid is obtained.
DESCRIPTION OF Pl~EFERRED EMBODIMENTS
The disclosed process for the el~ylllalic conversion of glucose to gluconic acid ~.llliL~ the el~y,ll~ic conversion of glucose solutions having a dissolved(glucose) solids content of greater than 25 % (w/w) ds. of glucose without the res~lltin~ build-up of residual unconverted glucose in the gluconic acid solution produced ~llel~y and with the use of reduced qll~ntiti~s of buffers, such as sodium hydroxide, which must be employed.
Furthermore, use of the principles disclosed herein permits the fashioning of such processes which do not re~uire the use of e~Lell~iv~: and/or t~ nsiv~
dow~ Galll, sepal~tion, recovery and/or pnrifir~tif~n processes and/or a~?a.dluses and which permit the production of spray-dried and ess~onti~lly pure gluconic acid without employing cryst~lli7~tion processes and which pC.lllilS the formation ofspray-dried granulation of the gluconic acid obtained thereby.
The processes of the present invention produce spray-gr;ln~ t~l gluconic acid and pure glllconir acid wilh~u~ the need to employ cryst~ 7~tion or purifir~tio~ processes.
The process of the present invention pe~.llil~ 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 concell~latiolls 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 %15(w/w) ds. of W O 97/24454 PCT~US96/tO480 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.
The process further inrl~ s adding to the solution, in the presence of an oxygen source, from a~out 25 to about 30 GOU of glucose oxidase/gram ds.
S glucose in the solution. Preferably, about 27 GOU to about 29 GOU of glucose oxi~ e/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.
As used herein, one glucose oxidase unit is defined as being the amount of enzyme required to oxidase one micromole D-glucose per minute under the assay con-litions 25~C and pH 7Ø
The process further includes adding to the solution, in the presence of an oxygen source, and at least 1200 CU of c~t~ e/gram ds. of glucose in the solution. Preferably, from about 1279 to about 1999 CU of c~t~ e/gram ds. of glucose in the solution is added to the solution. In particular p~r~ ,d embo~limrnt~, at least 1279, 1559 and 1999 CU of r~t~ e/gram ds. of glucose in the solution is added to the solution.
As used herein, one c~t~ e unit is defined as being the amount of enzyme required to decompose 1u mole of hydrogen peroxide per minute under the assay 35 conditions 25~C and pH 7Ø
Any suitable c~t~ e may be employed in the process of the present invention. ~t~l~sPs which are n~tllr~lly produced by (or derived from) strains of Aspergillus niger and Micrococcus lyso-leiktir3l~, as well as c~t~ es produced by m~mili~n sources, such as bovine sources, may be employed. In this context, we have found herein that the c:lt~l~se which is naturally produced by stains of the species Aspergillus niger is particularly efficacious.
Preferably, the glucose oxidase and the ç~t~ e are added to the solution of ~ glucose in equal doses. In this regard, in one embodiment herein, the glucose oxidase and the c~t~ e are added to the solution of glucose in two equal doses,the ~ 30 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 intton~P~l reaction. Toillustrate 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.
Further in this regard, in a second embodiment, the glucose oxidase and 15 the c~t~ e 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 int~n~ l reaction and the thirddose being added two-thirds (66.6 %) through the total time of the in~n-led reaction. To illustrate this point, if the reaction is to proceed for 24 hours then, in 20 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 ninth hour and the third dose would be added at the start of the 17th hour.
The solution of glucose may be m ~int~in~cl at any pH which permits the reaction to occur. However, it is pr~r~lled herein that the solution of glucose be m ~int~in~d at a pH of from about S to about 7 throughout the reaction. Most ~r~rellcd is m ~int~ining the solution of glucose at a pH of 6 throughout the re~cti~,l The solution of glucose may be m ~int~in~(l at any ~ln~el~Lule which permits the reaction to occur. However, in this regard, it is prert;ll~d that the L~ elaL~ , of the solution of glucose is ~ d at from about 25~C to about 40~C throughout the reaction. Most pr~felled is m ~int~inin~ the solution of glucose at from about 30~C to about 35~C throughout the reaction.
While the solution of glucose may be m ~int~in~o~ at any pressure which permits the reaction to occur, it is plc~r~lled that the ~ ure of the solution of 35 glucose is m~int~inP(l at about I bar throughout the reaction.
While the solution of glucose may be m ~int~in~-l by passing air through the glucose solution at any flow rate which permits the reaction to occur, it is plefel~d herein that the rate of air flow through the solution of glucose be m ~int~in~l at about I vvm throughout the reaction.
Having thus described the processes of the present invention for the enzymatic c~llvc;l~ion of glucose to gluconic acid, the gluconic acid produced thereby and processes for producing a spray-gr~n~ te~ gluconic acid product,lt:fel~llce is now made to the following examples which are pleselll~d W O 97/244~4 PCTrUS96/2~480 herein for the purposes of illustration only and are neither meant to be, nor should they be read as being, restrictive.
Unless otherwise specified herein, the Lxamples of the present invention were performed with the use of glucose oxidase from Aspergillus niger (sold by S SOLVAY ENZYMES, GmbH, Germany), c~t~ e from Micrococcus lysodeikticus sold under the tr~ om~rk MicroC~t~ e I~1000 (SOLVAY
ENZYMES, Inc., Elkhart, Tn~ n~) and crystalline glucose ~ kr~d under the name StaleyDex 333 R (Staley, USA~.
Example 1: Effect of pH
The effect of pH on the conversion of glucose to gluconic acid by glucose oxidase-c~t~ e enzyme system according to the method of the present invention,was studied at a glucose conce~ lion of 40 % (w/w) ds.
8 liters of 40 % (w/w) ds. glucose solution was prepared and introduced into a 10 liter ~~, .,.~,.lol (Chempec, Inc., New Jersey, USA). The pH was a~l~ste~l to pH 4.0 with dilute acid (i.e., SN sulfuric acid) or alkali (i.e., 4N
sodium hydroxide), as n~oce~
The reaction was carried out at 35~C with a ~les~u.e at I bar and air was bubbled at a rate of I VVM. The pH was m~int~in~<l at the specified pH during the reaction by the addition of 50 % (w/w) sodium hydroxide. Foam was controlled by the addition of allLiroal,l MAZU DF6000 (PPG/MAZER
CHLMICALS) (80-120 ppm).
Doses of 12.25 glucose oxidase units (Gou) per gram of dissolved solids of glucose of the solution and 726.8 c~t5~ e urlits (CU) per gram of dissolved solids of glucose of the solution were added to the fermentor at log 0 and at log 12 hours.
The conversion of glucose to gluconic acid was measured by ~lete~nining the millieq~livalent of sodium hydroxide con~l~m~ocl and/or by analyzing the samples using high pressure liquid chromatographic (HPLC) method.
HPLC analysis was carried out using an HPLC system col.si~,ng of Beckman 112 Solvent Delivery Module (BECKMAN, USA) fitted to a RI detector Model ERC-7515A, (The ANSPEC Co., Inc., USA). Glucose and gluconic acid W O 97/24454 PCT~US96/20480 were separated using an HPX 87-C column (Bio-Rad, USA) at 80~C and 0.01M
calcium acetate (pH 5.5) was used as the mobile phase at a flow rate of 1 ml/min.
The color illl~ulilies were determined by measuring the absorbance(optical density) of 37.5 % (w/w) solution gluconic acid in 5 % (w/w) NaOH solution at 470nm.
Four additional experiments were then conrl~lr-t~l 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 8Ø
The effects of dir~l~ll pHs on the efficiency of the conversion of glucose to gluconic acid are ~ rl below in Table 1 W O 97/24454 PCTrUS96/20480 Table 1 Effect o~ pH on Conversion of Glucose to Gluconic Acid using Glucose Oxidase-Catalase Enzyme System Reaction Per Cent Conversion (Hours) pH 4.0 pH 5.0pH 6.0 pH 7.0pH 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 0 4 4.6 29.0 30.3 26.7 10.0 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 4.8 55.9 57.9 16 19.5 55.3 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 20 14 4.8 78.5 78.1 77.1 29.0 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 25 19 4.8 99.3 100.0 94.9 41.2 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 30 24 4.8 100.0 100.0 100.0 51.9 " Table 1 shows that, 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.
CA 02240294 l998-07-08 W O 97/24454 PCT~US96/20480 Example 2: Enzyme Dosa~e - Sin~le Addition and Multiple Addition The effect of adding the glucose oxidase and c~tzlz~e 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 ~iel~ P~1 by pelrollllh2g S three experim~nt~ wherein the protocols and process conditions were mzint,-in the same, with the exceptions of timing and n~z~ el of the dosing of the enzymes.
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 ex~ermPnt.~ were lliz~ Pfl at pH 6.0; and the qllz~ntitiPs and timing of the enzyme ~litinn~ were varied. In that regard, the total concel~ lion of glucose oxidase and c~tz~ls~e added was m~intZinP(l constant (at 27 glucose oxidase units per gram of dissolved glucose solids and 1599 c~tz~ e units per gram of dissolved glucose solids) but were added in either one, two or threedoses.
As regards the quantity and time of the enzyme addition in the ~lrst 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 ~elilllent, 9 GOU/gram ds.
and 533 CU/gram ds. were added at log 0, log 6 and log 12 hours.
The effect of adding the glucose oxidase and c~t~ e in either one or several doses throughout the reaction on the conversion of glucose to gluconic acid are ~ d below in Table 2:
Tabl~2 PerCentC~ .biull Time(Hours) FY1~ l#1 E~ lL#2 E~ 3 O O O O
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 38.6 36.7 27.7 0 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 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 74.5 83.3 82.7 16 74.9 8g.0 88.0 17 74.9 94.3 93.2 18 75.9 99.2 97.9 19 75.9 lOO.O 99.7 75.9 lOO.0 IOO.O
21 75.9 lOO.O lOO.O
22 75.9 lOO.O lOO.O
23 75.9 100.0 lOO.O
Exarnple 3: Role of (~t~l~se on the Oxidation of Glucose to Gluconic Acid by Glucose Oxidase The effect of various concentrations of c~t~ e on the conversion of glucose to gluconic acid according to the method of the present invention was ~e~;....i..f d by performing five experiments wll~leill the protocols and process W O 97/24454 PCT~US96/20480 conditions were rll~int:~in~d the same with the exception of the ç~tz~ e concentrations added.
The conversion of glucose to gluconic acid was carried out in five s~ardLe experiments using the same protocol and under the same process conditions as S described above in Example 2, but with the following exceptions:the temperatures of the reaction mixtures were m~int~in~ at 40~C; the concellLlaLioll of glucose oxidase added was 28.6 GOU/gram ds.; the c~ cellllaLion of the cat~l~ee added was varied as will be described below; and all of the c~t~ e and glucose oxidasewas added at log 0 time.
As regards to the concentration of the c~t~l~ce added, the concentration of c~t~ e added was varied, as follows: in the first experiment, 0 c:lt~ e units/gram ds. were added; in the second experiment, 959 c~t~ ce units/gram ds.were added; in the third e~ i,llcll~, 1279 ~t~ e units/grams ds. were added;
in the fourth f~x~ , 1559 c~t~ e units/gram ds. were added; and in the fif~
experiment, 1999 ç~t~l~ce units/grams ds. were added.
The effect of ~dl,yill,~ c~t~l~ce collce~ dlions on the c~ vt;l~ion of glucose to gluconic acid under i~lentir~l conditions are ~.."-,-.~.i;~ed below in Table 3:
W O 97124454 PCT~US96/20480 Table3 PerCentCu~ O
Reaction Time O 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 ~.6 3.4 4.5 3.4 4.6 2 5.1 13.4 13.2 13.6 14.0 3 63. 22.6 22.3 22.3 22.8 4 6.9 30.7 31.5 30.9 31.3 0 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 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 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 7.1 59.9 68.7 75.9 82.9 21 7.1 59.9 68.7 75.9 82.9 The results of Table 3 show that, in the method of the present invention, the inactivation of glucose oxidase by hydrogen peroxide is .~ignifif'~ntly reduced by the addition of c~t~ e. In the absence of c~t~ e, 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 increasingconcentration of c~t~ e and a m~ximllrn conversion of 80% was reached even at high concellLlalion of ç~t~ e The residual unconverted glucose could be due to the stability (half-life) associated with glucose oxidase under the experimental conditions.
W O 97/24454 PCTrUS96/20480 ;Example 4: Effect of Temperature on Oxidation of Glucose to Gluconic Acid Usin~ Glucose Oxidase-c~t~l~ce EnzYme System.
The effect of various temperatures on the conversion of glucose to gluconic acid according to the m~thofl of the present invention was ~l~L~....i"P(1 by performing four e~e~ ents wherein the protocols and process contlitions were rn~int~inPrl the same with the exception of the tempcldlulc of the reaction.
The conversion of glucose to gluconic acid was carried out in four separate e~e,illlents using the same protocol and under the same process con~1itionC as described above in Example 2, but with the following exceptions: the tempeldLu,~s of the reaction l~ Lul~,S were varied, as will be described below;50 % of the glucose oxidase units (13.5 glucose oxidase units) and 50 % of the 10 c~t~ e units (799.5 c~t~ e units) were added at log 0 hours and 50 % of the glucose oxidase units (13.5 glucose oxidase UIUtS) and 50 % of the catalase units (799.5c~t~l~se units) were added at log 9 hours.
As regards to the lem~eldLures employed, the tempc.dL~ s employed were varied, as follows: in the first e~clJ,l~llt, the l~nl~c~ was 25~C; in the second e~c.illlent, the telll~c.dLul~, was 30~C; in the third e~elill~llL, the ,peldLure was 35~C; and in the fourth experiment, the telll~e.ature was 40~C.
The effect of the dirr~r~llL ~ dlul~s on the conversion of glucose to gluconic acid under i~lenti~ conditions are ~ulmllaliGed below in Table 4:
CA 02240294 l998-07-08 W O 97/244S4 PCTrUS96/20480 Table 4 Per Cent Conversion Reaction Time25~C 30~C 35~C 40~C
AND GLUCONIC ACID PRODUCED T~R~RY
FIELD OF THE INVENTION
The present invention relates to processes for the el~ylllaLic conversion of glucose to gluconic acid, the gluconic acid produced thereby, the ~l~pa.~Lion of a gr~mll~r gluconic acid product and the granular product so produced.
BACKGROUND OF THE INVENTION
Gluconic acid, an oxidation product of glucose, has been ~Lel~sively used in applications as varied as metal cle~nin~ operations in the dairy industry; ~lk~lin~
bottle washing operations; ~lk~lin~o derusting operations in the m~t~ lrgic industry;
and iron deposition ~l~v~lllion in the textile industry. FulLl~ llore, the sodium salt of gluconic acid is used as an additive in cement ~ Ul~;;S.
The production of gluconic acid from glucose may be achieved by the use of processes which may be broadly cl~ iecl as being either l~ ol~ial fe~-nt~tion, electrochf mi~l, cl~ ir~l and enzymatic (wherein el~ylllaLic systems are employed s~al~Lely from their source microorganism(s)). While microbial ferm~nt~tion has perhaps been the most widely employed of these m~ho~l~, it nonetheless suffers many drawbacks, including those associated with process conditions required for the ferment~tion microo~ t~ used, which has limited its commercial applicability.
The enzymatic conversion of glucose to gluconic acid involves treating a glucose bearing m~teri~l with an enzyme preparation having glucose oxidase and c~t~ e 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.
To insure that the glucose oxidase functions at its most effective level,25 t 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 n~cess~ry to continuously regulate W O 97/24454 PCT~US96/20480 the pH of the reaction media throughout the enzymatic conversion. Generally, if the pH is m~int~in~cl between about 4.2 and about 7 (pxeferably, between about 5and about 6), the conversion proceeds s~ti~f~rtorily. A common method of regulating the pH involves the continuous addition of an aLkali, such as sodium S hydroxide. The alkali neutralizes the gluconic acid to a corresponding gluconate, e.g., sodium gluconate.
Examples of el~yllla~ic processes for the production of gluconic acid from glucose using a glucose oxidase/cat~l~ce enzyme system can be found in, for example, United States Letters Patent No. 2,651,592 and ]~om~ni~n Patent No.
92,739.
While being useful for ~eir particular purposes, these el~yl.lalic processes suffer from several drawbacks.
A primary drawback associated with e~yll~dLic processes is that the reaction process generally results in the crude reaction broth cO~ g gluconic acid along with other illl~ulilies inrlll~lin~ biomass. This reaction broth must then be purified by multi-step processes including biomass separation (filtration),carbon tre~tment (decolorization), e~,apoldLion (cc..~rG~ ;on) and cryst~lli7~ti~ n(purification) to provide a final product with high purity.
Another dldwl,ack is the ~l~se.lce of residual mother liquid in the reaction broth which must be either recycled, further purified and/or disposed of, there by adding to the problems and costs of such el~yllla~ic conversion processes.
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 co,.l;....il.~..l in the gluconic acid solution produced thereby. In order to reduce or elimin~t~ such unconverted glucose from the final product, costly dOwll~LI~alll, separation,recovery and purification steps must be employed.
To alleviate problems associated with incomplete conversion, resort has been had to limiting the glucose collcellLldLions of the starting m~trri~l to less than 30% (w/w) dissolved solids (ds.). Ul~llui~lely, such low glucose concentrations in the starting material are ~ c~ r~rtory in that they greatly reduce the efficiency of the process, negatively imp~rting on its commercially desirability.
W O 97/24454 PCTAUS96~0480 Alternatively, resort has been made to intei~ul"ing the process prior to the comple~ion thereof. For example, in the processes disclosed in the aforesaid United States Patent, the reaction was stopped after conv~lLillg only 50 % of the glucose to gluconic acid. Nonetheless, the reaction llliklul~ still needs to be ~ 5 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 ~liffi1elllt and costly, especially where the glucose is still present.
Another drawback associated with enzymatic processes is the use of hydrogen peroxide (H202) as a source of oxygen. As an acid, the presence of H202 neces~ s constant mo.lilo~ g of the pH of the reaction solution, as well as the employment of a buffer (such as calcium carbonate in the form of lime) tomAint~in 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 qll~ntiti~s of yet more hydrogen peroxide and acids, nPcec~ l;,.g the use of yet more ~Al~es (to convert the hydrogen peroxides formed into water and oxygen) and pH neutralizers.
Accordingly, it can be seen that there ie,llains a need for the provision of e,~y,nalic processes for the production of gluconic acid from glucose where in dissolved glucose solid concentrations of 30 % (w/w) ds. (dissolved solids) and 15 higher may be used while still obtaining high conversion rates, wL~lein reduced qll~ntities of buffers, such as sodium hydroxide, need to be employed, wherein the use of ~nsive and/or expensive dowlls~le~ll, separation, l~cov~ly and/or puri~lr~tion processes and/or a~L,alaLuses need not be employed and which permits the production of spray-dried and e~nti~lly pure gluconic acid grAmlles without employing crystSIlli7~tion processes.
~ SUMMARY OF THE INVENTION
It is a primary object of the present invention to provide an enzymatic process for the production of gluconic acid from glucose wherein dissolved solidconcentrations 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 W O 97/24454 PCT~US96/20480 require extensive expensive do~ separation, recovery and/or pllri~lc~tion1 which L)ellnils the production of' spray-dried and essentially pure granular gluconic acid without employing cryst~lli7s~tion processes.
It is a further object of.the present invention to provide such a process 3Q
wherein the final gluconic acid solution produced there~y, has a low concentrations of i...~,uliLies, including residual unconverted glucose.
It is a still further object of.the present invention to provide such a process wherein low (reduced~ ql-~ntiti.os of.buffers, such as sodium hydroxide, need to be employed.
A yet further object of.the present invention is to provide processes for producing a dry, low-dust, granular gluconic acid product without the n~cescity of employing a cryst~lli7~tion process.
In another aspect of the present invention, it is an object herein to provide a ~ub~L~ lly pure gluconic acid solution.
A still yet further object of the present invention is to provide a dry, low-dust, granular gluconic acid product.
In accol-lance with the te~hing~ of the present invention, disclosed herein is a process for the ~ymatic conversion of glucose to gluconic acid. This process includes providing a solution of glucose. The process further in~ les adding to the solution, in the presence of an oxygen source, from about 25 to about 30 GOU of glucose oxidase/gram ds. of glucose in the solution and at least1200 CU of c~t~ o/gram ds. of glucose in the solution. In this fashion, the glucose is e~y~ lly converted to glll~oni~ acid.
Preferably, the solution of glucose has from about 25 % ~wlw) ds. of glucose to about 60 % (w/w) ds. of glucose and, more preferably, from about 30 % (wlw) ds. of glucose to about 50 % (w/w) ds. glucose.
In a ~lc~llcd emb~(1im~nt, about 27 GOU of glucose oxidase/gram ds. of glucose in the solution is added to the solution.
Preferably, from about 1279 to about 1999 CU of c~t~T~elgram ds. of glucose in the solution is added to the solution. r In one ~.efelled embo~lim~.nt7 at least 1279 CU of c~t~ çlgram ds. of glucose in the solution is added to the solution.
WO 97124454 PCT/~JS96f20480 In a second preferred embo-lim~rlt at least 1559 CU of c~t~l~ce/gram ds.of glucose in the solution is added to the solution.
In a third ylcr~lr~d embodiment, at least 1999 CU of c~t~l~ce/gram ds. of glucose in the solution is added to the solution.
Preferably, the glucose oxidase and the c~t~ P 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 tota}
time of the intended reaction.
In another p.lc~llcd embodiment, the glucose oxidase and the c~t~ e 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-thirdsthrough the total time of the reaction.
Preferably, the c~t~l~se is naturally produced by a strain of the species Aspergillus niger.
Preferably, the solution of glucose is m~int~in~cl at a pH of from about 5 to about 7 throughout the reaction and, more yl~relred~ the solution of glucose is 7. at a pH of 6 throughout the reaction.
Preferably, the tempel~Lul~, of the solution of glucose is Ill~ i..P~l at from about 25~C to about 40~C throughout the reaction, and, more p.~cfellc;d, the solution of glucose is m~int~in~.~l at from about 30~C to about 35~C throughout the reaction.
Preferably, the ync~.~.ul~ of the solution of glucose is ~-~A;~ d at about 1 bar throughout the reaction.
Preferably, the air flow through the solution of glucose is m~int~in~1 at about 1 vvrn throughout the reaction.
In a particularly plc~llcd embodiment disclosed herein, ~e process for the y~ ic conversion of glucose to gluconic acid includes providing a solution of glucose having from about 25 % (w/w) ds. of glucose to about 60 %15(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 ~t~l~cPlgram W O 97/24454 PCTrUS96/20480 ds. glucose in the solution while, throughout the reaction,m~int~ining the solution of glucose at a pH of about 5 to about 7, at a temperature of about 25~C to about 40~C, at a ~lcs:iule of about I bar and at an air flow of about 1 vvm.
Finally, and in another aspect of the present invention disclosed herein is a S process for the ~l~aldtion of a dry, spray-gr~n~ gluconic acid product and the product produced thereby.
In this regard, the gluconic acid-cont~ining 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 sodiumgluconate in a spray-dryer, whereby a spray-gr~mll~t~d gluconic acid is obtained.
DESCRIPTION OF Pl~EFERRED EMBODIMENTS
The disclosed process for the el~ylllalic conversion of glucose to gluconic acid ~.llliL~ the el~y,ll~ic conversion of glucose solutions having a dissolved(glucose) solids content of greater than 25 % (w/w) ds. of glucose without the res~lltin~ build-up of residual unconverted glucose in the gluconic acid solution produced ~llel~y and with the use of reduced qll~ntiti~s of buffers, such as sodium hydroxide, which must be employed.
Furthermore, use of the principles disclosed herein permits the fashioning of such processes which do not re~uire the use of e~Lell~iv~: and/or t~ nsiv~
dow~ Galll, sepal~tion, recovery and/or pnrifir~tif~n processes and/or a~?a.dluses and which permit the production of spray-dried and ess~onti~lly pure gluconic acid without employing cryst~lli7~tion processes and which pC.lllilS the formation ofspray-dried granulation of the gluconic acid obtained thereby.
The processes of the present invention produce spray-gr;ln~ t~l gluconic acid and pure glllconir acid wilh~u~ the need to employ cryst~ 7~tion or purifir~tio~ processes.
The process of the present invention pe~.llil~ 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 concell~latiolls 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 %15(w/w) ds. of W O 97/24454 PCT~US96/tO480 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.
The process further inrl~ s adding to the solution, in the presence of an oxygen source, from a~out 25 to about 30 GOU of glucose oxidase/gram ds.
S glucose in the solution. Preferably, about 27 GOU to about 29 GOU of glucose oxi~ e/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.
As used herein, one glucose oxidase unit is defined as being the amount of enzyme required to oxidase one micromole D-glucose per minute under the assay con-litions 25~C and pH 7Ø
The process further includes adding to the solution, in the presence of an oxygen source, and at least 1200 CU of c~t~ e/gram ds. of glucose in the solution. Preferably, from about 1279 to about 1999 CU of c~t~ e/gram ds. of glucose in the solution is added to the solution. In particular p~r~ ,d embo~limrnt~, at least 1279, 1559 and 1999 CU of r~t~ e/gram ds. of glucose in the solution is added to the solution.
As used herein, one c~t~ e unit is defined as being the amount of enzyme required to decompose 1u mole of hydrogen peroxide per minute under the assay 35 conditions 25~C and pH 7Ø
Any suitable c~t~ e may be employed in the process of the present invention. ~t~l~sPs which are n~tllr~lly produced by (or derived from) strains of Aspergillus niger and Micrococcus lyso-leiktir3l~, as well as c~t~ es produced by m~mili~n sources, such as bovine sources, may be employed. In this context, we have found herein that the c:lt~l~se which is naturally produced by stains of the species Aspergillus niger is particularly efficacious.
Preferably, the glucose oxidase and the ç~t~ e are added to the solution of ~ glucose in equal doses. In this regard, in one embodiment herein, the glucose oxidase and the c~t~ e are added to the solution of glucose in two equal doses,the ~ 30 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 intton~P~l reaction. Toillustrate 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.
Further in this regard, in a second embodiment, the glucose oxidase and 15 the c~t~ e 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 int~n~ l reaction and the thirddose being added two-thirds (66.6 %) through the total time of the in~n-led reaction. To illustrate this point, if the reaction is to proceed for 24 hours then, in 20 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 ninth hour and the third dose would be added at the start of the 17th hour.
The solution of glucose may be m ~int~in~cl at any pH which permits the reaction to occur. However, it is pr~r~lled herein that the solution of glucose be m ~int~in~d at a pH of from about S to about 7 throughout the reaction. Most ~r~rellcd is m ~int~ining the solution of glucose at a pH of 6 throughout the re~cti~,l The solution of glucose may be m ~int~in~(l at any ~ln~el~Lule which permits the reaction to occur. However, in this regard, it is prert;ll~d that the L~ elaL~ , of the solution of glucose is ~ d at from about 25~C to about 40~C throughout the reaction. Most pr~felled is m ~int~inin~ the solution of glucose at from about 30~C to about 35~C throughout the reaction.
While the solution of glucose may be m ~int~in~o~ at any pressure which permits the reaction to occur, it is plc~r~lled that the ~ ure of the solution of 35 glucose is m~int~inP(l at about I bar throughout the reaction.
While the solution of glucose may be m ~int~in~-l by passing air through the glucose solution at any flow rate which permits the reaction to occur, it is plefel~d herein that the rate of air flow through the solution of glucose be m ~int~in~l at about I vvm throughout the reaction.
Having thus described the processes of the present invention for the enzymatic c~llvc;l~ion of glucose to gluconic acid, the gluconic acid produced thereby and processes for producing a spray-gr~n~ te~ gluconic acid product,lt:fel~llce is now made to the following examples which are pleselll~d W O 97/244~4 PCTrUS96/2~480 herein for the purposes of illustration only and are neither meant to be, nor should they be read as being, restrictive.
Unless otherwise specified herein, the Lxamples of the present invention were performed with the use of glucose oxidase from Aspergillus niger (sold by S SOLVAY ENZYMES, GmbH, Germany), c~t~ e from Micrococcus lysodeikticus sold under the tr~ om~rk MicroC~t~ e I~1000 (SOLVAY
ENZYMES, Inc., Elkhart, Tn~ n~) and crystalline glucose ~ kr~d under the name StaleyDex 333 R (Staley, USA~.
Example 1: Effect of pH
The effect of pH on the conversion of glucose to gluconic acid by glucose oxidase-c~t~ e enzyme system according to the method of the present invention,was studied at a glucose conce~ lion of 40 % (w/w) ds.
8 liters of 40 % (w/w) ds. glucose solution was prepared and introduced into a 10 liter ~~, .,.~,.lol (Chempec, Inc., New Jersey, USA). The pH was a~l~ste~l to pH 4.0 with dilute acid (i.e., SN sulfuric acid) or alkali (i.e., 4N
sodium hydroxide), as n~oce~
The reaction was carried out at 35~C with a ~les~u.e at I bar and air was bubbled at a rate of I VVM. The pH was m~int~in~<l at the specified pH during the reaction by the addition of 50 % (w/w) sodium hydroxide. Foam was controlled by the addition of allLiroal,l MAZU DF6000 (PPG/MAZER
CHLMICALS) (80-120 ppm).
Doses of 12.25 glucose oxidase units (Gou) per gram of dissolved solids of glucose of the solution and 726.8 c~t5~ e urlits (CU) per gram of dissolved solids of glucose of the solution were added to the fermentor at log 0 and at log 12 hours.
The conversion of glucose to gluconic acid was measured by ~lete~nining the millieq~livalent of sodium hydroxide con~l~m~ocl and/or by analyzing the samples using high pressure liquid chromatographic (HPLC) method.
HPLC analysis was carried out using an HPLC system col.si~,ng of Beckman 112 Solvent Delivery Module (BECKMAN, USA) fitted to a RI detector Model ERC-7515A, (The ANSPEC Co., Inc., USA). Glucose and gluconic acid W O 97/24454 PCT~US96/20480 were separated using an HPX 87-C column (Bio-Rad, USA) at 80~C and 0.01M
calcium acetate (pH 5.5) was used as the mobile phase at a flow rate of 1 ml/min.
The color illl~ulilies were determined by measuring the absorbance(optical density) of 37.5 % (w/w) solution gluconic acid in 5 % (w/w) NaOH solution at 470nm.
Four additional experiments were then conrl~lr-t~l 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 8Ø
The effects of dir~l~ll pHs on the efficiency of the conversion of glucose to gluconic acid are ~ rl below in Table 1 W O 97/24454 PCTrUS96/20480 Table 1 Effect o~ pH on Conversion of Glucose to Gluconic Acid using Glucose Oxidase-Catalase Enzyme System Reaction Per Cent Conversion (Hours) pH 4.0 pH 5.0pH 6.0 pH 7.0pH 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 0 4 4.6 29.0 30.3 26.7 10.0 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 4.8 55.9 57.9 16 19.5 55.3 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 20 14 4.8 78.5 78.1 77.1 29.0 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 25 19 4.8 99.3 100.0 94.9 41.2 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 30 24 4.8 100.0 100.0 100.0 51.9 " Table 1 shows that, 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.
CA 02240294 l998-07-08 W O 97/24454 PCT~US96/20480 Example 2: Enzyme Dosa~e - Sin~le Addition and Multiple Addition The effect of adding the glucose oxidase and c~tzlz~e 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 ~iel~ P~1 by pelrollllh2g S three experim~nt~ wherein the protocols and process conditions were mzint,-in the same, with the exceptions of timing and n~z~ el of the dosing of the enzymes.
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 ex~ermPnt.~ were lliz~ Pfl at pH 6.0; and the qllz~ntitiPs and timing of the enzyme ~litinn~ were varied. In that regard, the total concel~ lion of glucose oxidase and c~tz~ls~e added was m~intZinP(l constant (at 27 glucose oxidase units per gram of dissolved glucose solids and 1599 c~tz~ e units per gram of dissolved glucose solids) but were added in either one, two or threedoses.
As regards the quantity and time of the enzyme addition in the ~lrst 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 ~elilllent, 9 GOU/gram ds.
and 533 CU/gram ds. were added at log 0, log 6 and log 12 hours.
The effect of adding the glucose oxidase and c~t~ e in either one or several doses throughout the reaction on the conversion of glucose to gluconic acid are ~ d below in Table 2:
Tabl~2 PerCentC~ .biull Time(Hours) FY1~ l#1 E~ lL#2 E~ 3 O O O O
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 38.6 36.7 27.7 0 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 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 74.5 83.3 82.7 16 74.9 8g.0 88.0 17 74.9 94.3 93.2 18 75.9 99.2 97.9 19 75.9 lOO.O 99.7 75.9 lOO.0 IOO.O
21 75.9 lOO.O lOO.O
22 75.9 lOO.O lOO.O
23 75.9 100.0 lOO.O
Exarnple 3: Role of (~t~l~se on the Oxidation of Glucose to Gluconic Acid by Glucose Oxidase The effect of various concentrations of c~t~ e on the conversion of glucose to gluconic acid according to the method of the present invention was ~e~;....i..f d by performing five experiments wll~leill the protocols and process W O 97/24454 PCT~US96/20480 conditions were rll~int:~in~d the same with the exception of the ç~tz~ e concentrations added.
The conversion of glucose to gluconic acid was carried out in five s~ardLe experiments using the same protocol and under the same process conditions as S described above in Example 2, but with the following exceptions:the temperatures of the reaction mixtures were m~int~in~ at 40~C; the concellLlaLioll of glucose oxidase added was 28.6 GOU/gram ds.; the c~ cellllaLion of the cat~l~ee added was varied as will be described below; and all of the c~t~ e and glucose oxidasewas added at log 0 time.
As regards to the concentration of the c~t~l~ce added, the concentration of c~t~ e added was varied, as follows: in the first experiment, 0 c:lt~ e units/gram ds. were added; in the second experiment, 959 c~t~ ce units/gram ds.were added; in the third e~ i,llcll~, 1279 ~t~ e units/grams ds. were added;
in the fourth f~x~ , 1559 c~t~ e units/gram ds. were added; and in the fif~
experiment, 1999 ç~t~l~ce units/grams ds. were added.
The effect of ~dl,yill,~ c~t~l~ce collce~ dlions on the c~ vt;l~ion of glucose to gluconic acid under i~lentir~l conditions are ~.."-,-.~.i;~ed below in Table 3:
W O 97124454 PCT~US96/20480 Table3 PerCentCu~ O
Reaction Time O 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 ~.6 3.4 4.5 3.4 4.6 2 5.1 13.4 13.2 13.6 14.0 3 63. 22.6 22.3 22.3 22.8 4 6.9 30.7 31.5 30.9 31.3 0 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 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 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 7.1 59.9 68.7 75.9 82.9 21 7.1 59.9 68.7 75.9 82.9 The results of Table 3 show that, in the method of the present invention, the inactivation of glucose oxidase by hydrogen peroxide is .~ignifif'~ntly reduced by the addition of c~t~ e. In the absence of c~t~ e, 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 increasingconcentration of c~t~ e and a m~ximllrn conversion of 80% was reached even at high concellLlalion of ç~t~ e The residual unconverted glucose could be due to the stability (half-life) associated with glucose oxidase under the experimental conditions.
W O 97/24454 PCTrUS96/20480 ;Example 4: Effect of Temperature on Oxidation of Glucose to Gluconic Acid Usin~ Glucose Oxidase-c~t~l~ce EnzYme System.
The effect of various temperatures on the conversion of glucose to gluconic acid according to the m~thofl of the present invention was ~l~L~....i"P(1 by performing four e~e~ ents wherein the protocols and process contlitions were rn~int~inPrl the same with the exception of the tempcldlulc of the reaction.
The conversion of glucose to gluconic acid was carried out in four separate e~e,illlents using the same protocol and under the same process con~1itionC as described above in Example 2, but with the following exceptions: the tempeldLu,~s of the reaction l~ Lul~,S were varied, as will be described below;50 % of the glucose oxidase units (13.5 glucose oxidase units) and 50 % of the 10 c~t~ e units (799.5 c~t~ e units) were added at log 0 hours and 50 % of the glucose oxidase units (13.5 glucose oxidase UIUtS) and 50 % of the catalase units (799.5c~t~l~se units) were added at log 9 hours.
As regards to the lem~eldLures employed, the tempc.dL~ s employed were varied, as follows: in the first e~clJ,l~llt, the l~nl~c~ was 25~C; in the second e~c.illlent, the telll~c.dLul~, was 30~C; in the third e~elill~llL, the ,peldLure was 35~C; and in the fourth experiment, the telll~e.ature was 40~C.
The effect of the dirr~r~llL ~ dlul~s on the conversion of glucose to gluconic acid under i~lenti~ conditions are ~ulmllaliGed below in Table 4:
CA 02240294 l998-07-08 W O 97/244S4 PCTrUS96/20480 Table 4 Per Cent Conversion Reaction Time25~C 30~C 35~C 40~C
5(Hours) - O 0.0 0.0 0.0 0.0 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 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 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 20 14 59.6 71.7 84.7 85.1 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 25 19 82.9 93.3 97.0 88.8 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 30 24 99.1 99.9 98.0 89.0 99.1 100.0 98.0 89.0 W O 97/24454 PCT~US96/20480 Ex~ml?le 5: Effect of Glucose Concentration on the Conversion of Glucose to Gluconic Acid usin~ Glucose Oxidase-~t~ e Enzvme Svstem The effect of various concentrations of glucose on the conversion of glucose to gluconic acid according to the method of the 19 S present invention was ~letermin~-1 by performing six experiments wherein the protocols and process conditions were m~int~int-~l the same with the exception of the glucose concentrations added.
The conversion of glucose to gluconic acid was carried out in six separate eA~,elil,lents using the sarne protocol and under the same process conditions asdescribed above in Example 2, but with the following exceptions: the tempclaLu,~s of the reaction l~ lul~s were m~int~in~l at 30~C; the co~cellLlalion of the glucose added was varied as will be described below; and all of the c~t~l~ce and glucoseoxidase was added at log 0 time.
The effect of glucose collcellLLaLion (dissolved solids) on the conversion rate of glucose to gluconic acid by the Glucose oxidase-çz~t~ e enzyme was studied asdescribed above in Example 2, but with the glucose concentration being varied, as follows: in the first experiment, the glucose concellllaLion was 30 % ds.; in the second experiment, the glucose concentration was 35 % ds.; in the third experiment, ~e glucose concentration was 40 % ds.; in the fourth experiment, theglucose concentration was 45 % ds.; in the fifth e~ t, the glucose concentration was $0 % ds.; and in the sixth experiment, the glucose concentration was 55 % ds.
The effect of varying glucose concentrations on the conversion of glucose to gluconic acid under i~lerlti~l conditions are sl~mmz~ri7f ~1 below in Table 5:
W O 97l24454 PCT~US96/20480 TableS
PerCentC~llv~l~ion Re~tion Glucose c~ n~l [in % (w/w)]
Time d (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 0 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 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 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 20 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 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 25 18 100.0 100.0 89.6 70.5 52.9 33.6 19 100.0 100.0 93.3 74.2 55.6 35.1 100.0 100.0 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 30 23 100.0 100.0 99.9 87.7 64.7 41.7 - 24 100.0 100.0 99.9 90.7 66.8 43.2 100.0 100.0 100.0 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 35 28 100.0 100.0 100.0 99.7 75.0 48.8 29 100.0 100.0 100.0 99.8 76.8 50.2 100.0 100.0 100.0 100.0 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 57.5 100.0 100.0 100.0 100.0 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 0 38 100.0 100.0 100.0 100.0 89.8 62.7 39 100.0 100.0 100.0 100.0 90.6 64.0 100.0 100.0 100.0 100.0 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 70.1 100.0 100.0 100.0 100.0 93.7 71.3 46 lO0.0 100.0 100.0 100.0 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 73.7 100.0 100.0 100.0 100.0 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 The results of Table S showed a rem~rk~ble 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 ~le~ulllably responsible for lower rate. However, by adjusting ~e enzyme dosage, it is possible to complete the collvc,~ion within the specified time. The data in the Table S clearly showed that the conce,~ lion of oxygen/available oxygen and W O 97/24454 PCT~US96/20480 length of ~e reaction time (enzyme dosage) greatly influences the overall economics of the process.
Example 6: Evaluation of ~t~ es from Different Sources Effect of c~t~ es from dirr~;lcll~ sources on the conversion of glucose to S gluconic acid by the use of glucose oxidase was studied under ~ ntic~l reaction conditions. The following c~t~l~ees were used: commercial product of bovine c~t~ e (CATALASE L) sold by SOLVAY ENZYMES, Inc., USA; Aspergillus niger c~t~l~ce from SOLVAY ENZYMES, GmbH (Germany) and Micrococcus c~t~
The c~t~ e activity of all samples was measured by s~ec~,ol?hotometrically as described by H. Luck iII Methods of El~ymaLic Analysis (H.U. Bergmeyer, ed.), 1965, pp. 885-894, Verlag Chemie & Academic Press, New York, l_ondon~.
The time taken for the absorbance of 10mM hydrogen peroxide solu~ion(in 0.05M phosphate buffer, pH 7) from 0.45 to 0.40 was measured and used to ~5 calculate activity.
The experiments were carried out using the protocol, and under the conditions described in Example 2, but with the exception t'nat all of the glucose oxidase and the c~t~ e were added to the glucose solution at log 0 hour.
The results are ~ ~ed below in Table 6:
W O 97/244S4 PCT~US96/20480 Table 6 Per Cent Conversion Reaction TimeA. niger Bovine Micrococcus 5(Hours) 1137 CU/g. ds.1600 CU/g. ds.1600 CU/g. ds.
O 0.0 0.0 0.0 8.6 4.3 0.0 2 16.9 10.6 12.0 3 25.1 17.6 16.7 0 4 32.8 23.6 22.1 S 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 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 20 14 94.3 52.2 71.2 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 25 19 lOO.Q 62.0 93.3 100.0 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 30 24 100.0 62.5 99.9 100.0 62.5 100.0 26 100.0 62.5 100.0 As can be seen from Table 6, both A. niger and Micrococcus c~t~l~ces showed a 100% conversion of glucose to gluconic acid but at varying rates.
However, bovine c~t~ e reached only 60% conversion. Even though all ~ree W O 97/244S4 PCT~US96/20480 enzymes were added at the same dosage, but still observed differences in the rate.
This could be due to the difft;~ ces in the stability of c~t~l~.ces against hydrogen peroxide.
~xample 7: Evaluation of Dirr~ L Commercial Glucose Syrups S The effect of difr~,ell~ sources of glucose syrups wi~ varying degrees of purity on the conversion of glucose to gluconic acid according to the method of the present invention was detennin.orT
The conversion of different glucose syrups was carried out in three separate experirnents using the same protocol and process conditions as those ~lçsc.rihe-l above in Examp}e 2.
The different glucose syrups used were commercially available glucose 10 syrup with varying degree of purity, as follows: Commercia+l products of Clintose "L" TM (ADM Corn Procç~ing, USA); Clearsweet TM 99 Refined Liquid Dextrose (Cargill, USA); and Royal R Glucose Liquid, 2637 (Corn Products, USA).
The effects of dirft;~ L glucose syrups on the efficiency of the conversion of glucose to gluconic acid according to the method of the present invention are l i7.~1 below in Table 7:
W O 97/24454 PCTrUS96/20480 Table 7 Per Cent Co~ ioll Reaction TimeClintose Clcal;7~ ,L Royal Glucose S(Hours) O 0.0 0.0 0.0 4.5 3.7 4.5 2 12.5 9.8 10.8 3 20.2 17.1 17.3 0 4 27.2 23.1 23.2 S 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 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 20 14 68.2 51.8 61.5 lS 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 25 19 91.2 74.1 81.9 94.8 76.0 84.6 21 97.9 78.5 84.6 22 99.2 79.9 87.8 23 100.0 81.1 88.7 30 24 100.0 81.8 89.5 100.0 83.4 89.5 As can be seen from Table 7, under the standard conditior~, 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.
W O 97/24454 PCTrUS96/20480 Ex~mple 8: Formation of Dry. Spray-Gr~mll~t~d Gluconic Acid/Gluconate and Comparison With Co~ ;ial Preparations 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 cornmercial gluconic acid/gluconate is produced without any carbon tre~tm~nt or crys~l1i7~ticm Gluconic acid/gluconate obtained from the reactor as described in Experiment 3 of ~xample 2. The gluconic acid so obtained (in the form of liquid sodium gluconate) was then col~cellll~ted and crystals were separated by filtration(micro/ultra/polish) and dried at 37~C. These crystals were then used as a feed to produce a spray gr~lnll~te~l gluconic acid/gluconate.
The spray-dry-er used was a Uni-Glatt with Wurster Laboratory Model fluid bed dryer with a variable air temperature and flow through the bed.A~plox~il,lal~;ly 3/4 of the air holes outside the column were blocked off. The ~layillg nozzle was a twin fluid nozzle (Schlick).
The alonl~tion air cap ol)cl~illg was 0.5 mm around the liquid nozzle and 1 rnm below the c~e~ g of the liquid nozzle. The liquid nozzle opening was 1.2mm. The air flow was sufficient to fl~ the bed and m~int~in flow through the Wurster column (110 C~M m~ximllm~
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~ Brix) was then spray-coated onto the crystals with low inlet airflow due to low buLk density (0.5 grams/cc) of crystals (inlet air temperature of 100-105~C,air outlet te~ c~d~ of 60-65~c~.
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 mimlt.oc The final particles were irregular spheres with smooth surfaces. Mass recovery in the process was 97.5 %.
The above procedure was then repeated using each of the commercially-available gluconic acid ~lc~aralions from the following sources: (I) Sigma Ch~mic~lc (USA); (2) ADM-Decatur (USA); (3) AKZO (USA); (4) Penta W O 97/24454 PC~US96120480 Mfg.(USA); and (5) PMl' (USA). Sample (6) was the gluconic acid preparation p~epared according to this E~ample.
The gluconic acid content of the different l,lc~aldLions was ~letenninPcl by I-[PLC using the protocol and under the same conditions as those described abovein Example 1.
The color .~ ulilies were d~Ltlll~hled by mto~llring the absorbance (optical density) of 37.5 % (w/w) solution gluconic acid in 5 % (wfw) NaOH solution at 470nm.
The results are ~ .t;d below in Table 8:
Table8 S~mple Optic~ Pun~ % ' Density (1) 0.003 lOO.0 (2) 0.029 95.7 (3) 0.029 995 (4) 0.014 98.9 (5) 0.051 100.0 (6) 0.001 100.0 Purity % was 1 ml/min, 80~C, 0.01M Ca Acetate pH 5.5, HPX-87C
The results of Table 8 show that the quality of the spray-gr~nnl~te-l gluconic acid preparations of the present invention without any further puri~lcations, such as carbon treatment and cryst~lli7~tion was superior to the commercially available dry gluconic acid plepaldLions.
The conversion of glucose to gluconic acid was carried out in six separate eA~,elil,lents using the sarne protocol and under the same process conditions asdescribed above in Example 2, but with the following exceptions: the tempclaLu,~s of the reaction l~ lul~s were m~int~in~l at 30~C; the co~cellLlalion of the glucose added was varied as will be described below; and all of the c~t~l~ce and glucoseoxidase was added at log 0 time.
The effect of glucose collcellLLaLion (dissolved solids) on the conversion rate of glucose to gluconic acid by the Glucose oxidase-çz~t~ e enzyme was studied asdescribed above in Example 2, but with the glucose concentration being varied, as follows: in the first experiment, the glucose concellllaLion was 30 % ds.; in the second experiment, the glucose concentration was 35 % ds.; in the third experiment, ~e glucose concentration was 40 % ds.; in the fourth experiment, theglucose concentration was 45 % ds.; in the fifth e~ t, the glucose concentration was $0 % ds.; and in the sixth experiment, the glucose concentration was 55 % ds.
The effect of varying glucose concentrations on the conversion of glucose to gluconic acid under i~lerlti~l conditions are sl~mmz~ri7f ~1 below in Table 5:
W O 97l24454 PCT~US96/20480 TableS
PerCentC~llv~l~ion Re~tion Glucose c~ n~l [in % (w/w)]
Time d (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 0 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 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 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 20 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 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 25 18 100.0 100.0 89.6 70.5 52.9 33.6 19 100.0 100.0 93.3 74.2 55.6 35.1 100.0 100.0 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 30 23 100.0 100.0 99.9 87.7 64.7 41.7 - 24 100.0 100.0 99.9 90.7 66.8 43.2 100.0 100.0 100.0 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 35 28 100.0 100.0 100.0 99.7 75.0 48.8 29 100.0 100.0 100.0 99.8 76.8 50.2 100.0 100.0 100.0 100.0 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 57.5 100.0 100.0 100.0 100.0 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 0 38 100.0 100.0 100.0 100.0 89.8 62.7 39 100.0 100.0 100.0 100.0 90.6 64.0 100.0 100.0 100.0 100.0 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 70.1 100.0 100.0 100.0 100.0 93.7 71.3 46 lO0.0 100.0 100.0 100.0 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 73.7 100.0 100.0 100.0 100.0 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 The results of Table S showed a rem~rk~ble 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 ~le~ulllably responsible for lower rate. However, by adjusting ~e enzyme dosage, it is possible to complete the collvc,~ion within the specified time. The data in the Table S clearly showed that the conce,~ lion of oxygen/available oxygen and W O 97/24454 PCT~US96/20480 length of ~e reaction time (enzyme dosage) greatly influences the overall economics of the process.
Example 6: Evaluation of ~t~ es from Different Sources Effect of c~t~ es from dirr~;lcll~ sources on the conversion of glucose to S gluconic acid by the use of glucose oxidase was studied under ~ ntic~l reaction conditions. The following c~t~l~ees were used: commercial product of bovine c~t~ e (CATALASE L) sold by SOLVAY ENZYMES, Inc., USA; Aspergillus niger c~t~l~ce from SOLVAY ENZYMES, GmbH (Germany) and Micrococcus c~t~
The c~t~ e activity of all samples was measured by s~ec~,ol?hotometrically as described by H. Luck iII Methods of El~ymaLic Analysis (H.U. Bergmeyer, ed.), 1965, pp. 885-894, Verlag Chemie & Academic Press, New York, l_ondon~.
The time taken for the absorbance of 10mM hydrogen peroxide solu~ion(in 0.05M phosphate buffer, pH 7) from 0.45 to 0.40 was measured and used to ~5 calculate activity.
The experiments were carried out using the protocol, and under the conditions described in Example 2, but with the exception t'nat all of the glucose oxidase and the c~t~ e were added to the glucose solution at log 0 hour.
The results are ~ ~ed below in Table 6:
W O 97/244S4 PCT~US96/20480 Table 6 Per Cent Conversion Reaction TimeA. niger Bovine Micrococcus 5(Hours) 1137 CU/g. ds.1600 CU/g. ds.1600 CU/g. ds.
O 0.0 0.0 0.0 8.6 4.3 0.0 2 16.9 10.6 12.0 3 25.1 17.6 16.7 0 4 32.8 23.6 22.1 S 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 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 20 14 94.3 52.2 71.2 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 25 19 lOO.Q 62.0 93.3 100.0 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 30 24 100.0 62.5 99.9 100.0 62.5 100.0 26 100.0 62.5 100.0 As can be seen from Table 6, both A. niger and Micrococcus c~t~l~ces showed a 100% conversion of glucose to gluconic acid but at varying rates.
However, bovine c~t~ e reached only 60% conversion. Even though all ~ree W O 97/244S4 PCT~US96/20480 enzymes were added at the same dosage, but still observed differences in the rate.
This could be due to the difft;~ ces in the stability of c~t~l~.ces against hydrogen peroxide.
~xample 7: Evaluation of Dirr~ L Commercial Glucose Syrups S The effect of difr~,ell~ sources of glucose syrups wi~ varying degrees of purity on the conversion of glucose to gluconic acid according to the method of the present invention was detennin.orT
The conversion of different glucose syrups was carried out in three separate experirnents using the same protocol and process conditions as those ~lçsc.rihe-l above in Examp}e 2.
The different glucose syrups used were commercially available glucose 10 syrup with varying degree of purity, as follows: Commercia+l products of Clintose "L" TM (ADM Corn Procç~ing, USA); Clearsweet TM 99 Refined Liquid Dextrose (Cargill, USA); and Royal R Glucose Liquid, 2637 (Corn Products, USA).
The effects of dirft;~ L glucose syrups on the efficiency of the conversion of glucose to gluconic acid according to the method of the present invention are l i7.~1 below in Table 7:
W O 97/24454 PCTrUS96/20480 Table 7 Per Cent Co~ ioll Reaction TimeClintose Clcal;7~ ,L Royal Glucose S(Hours) O 0.0 0.0 0.0 4.5 3.7 4.5 2 12.5 9.8 10.8 3 20.2 17.1 17.3 0 4 27.2 23.1 23.2 S 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 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 20 14 68.2 51.8 61.5 lS 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 25 19 91.2 74.1 81.9 94.8 76.0 84.6 21 97.9 78.5 84.6 22 99.2 79.9 87.8 23 100.0 81.1 88.7 30 24 100.0 81.8 89.5 100.0 83.4 89.5 As can be seen from Table 7, under the standard conditior~, 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.
W O 97/24454 PCTrUS96/20480 Ex~mple 8: Formation of Dry. Spray-Gr~mll~t~d Gluconic Acid/Gluconate and Comparison With Co~ ;ial Preparations 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 cornmercial gluconic acid/gluconate is produced without any carbon tre~tm~nt or crys~l1i7~ticm Gluconic acid/gluconate obtained from the reactor as described in Experiment 3 of ~xample 2. The gluconic acid so obtained (in the form of liquid sodium gluconate) was then col~cellll~ted and crystals were separated by filtration(micro/ultra/polish) and dried at 37~C. These crystals were then used as a feed to produce a spray gr~lnll~te~l gluconic acid/gluconate.
The spray-dry-er used was a Uni-Glatt with Wurster Laboratory Model fluid bed dryer with a variable air temperature and flow through the bed.A~plox~il,lal~;ly 3/4 of the air holes outside the column were blocked off. The ~layillg nozzle was a twin fluid nozzle (Schlick).
The alonl~tion air cap ol)cl~illg was 0.5 mm around the liquid nozzle and 1 rnm below the c~e~ g of the liquid nozzle. The liquid nozzle opening was 1.2mm. The air flow was sufficient to fl~ the bed and m~int~in flow through the Wurster column (110 C~M m~ximllm~
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~ Brix) was then spray-coated onto the crystals with low inlet airflow due to low buLk density (0.5 grams/cc) of crystals (inlet air temperature of 100-105~C,air outlet te~ c~d~ of 60-65~c~.
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 mimlt.oc The final particles were irregular spheres with smooth surfaces. Mass recovery in the process was 97.5 %.
The above procedure was then repeated using each of the commercially-available gluconic acid ~lc~aralions from the following sources: (I) Sigma Ch~mic~lc (USA); (2) ADM-Decatur (USA); (3) AKZO (USA); (4) Penta W O 97/24454 PC~US96120480 Mfg.(USA); and (5) PMl' (USA). Sample (6) was the gluconic acid preparation p~epared according to this E~ample.
The gluconic acid content of the different l,lc~aldLions was ~letenninPcl by I-[PLC using the protocol and under the same conditions as those described abovein Example 1.
The color .~ ulilies were d~Ltlll~hled by mto~llring the absorbance (optical density) of 37.5 % (w/w) solution gluconic acid in 5 % (wfw) NaOH solution at 470nm.
The results are ~ .t;d below in Table 8:
Table8 S~mple Optic~ Pun~ % ' Density (1) 0.003 lOO.0 (2) 0.029 95.7 (3) 0.029 995 (4) 0.014 98.9 (5) 0.051 100.0 (6) 0.001 100.0 Purity % was 1 ml/min, 80~C, 0.01M Ca Acetate pH 5.5, HPX-87C
The results of Table 8 show that the quality of the spray-gr~nnl~te-l gluconic acid preparations of the present invention without any further puri~lcations, such as carbon treatment and cryst~lli7~tion was superior to the commercially available dry gluconic acid plepaldLions.
Claims (20)
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 GOU of glucose oxidase/gram ds. of glucose in the solution and at least 1200 CU of catalase/gram ds. of glucose in the solution.
2. The process of claim 1, further comprising the solution of glucose having from about 25 % (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 dosebeing added at the start of the reaction and the second dose being added halfwaythrough 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°C to about 40°C.
13. The process of claim 12, wherein the temperature of the solution of glucose is maintained at from about 30°C to about 35°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 vvm.
16. A process for the enzymatic conversion of glucose to gluconic acid comprising providing a solution of glucose having about 25 % (w/w) ds. glucose to about 60 % (w/w) ds. glucose, and 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 l999CU of catalase/gram ds.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°C to about 40°C, a pressure of about I bar and maintaining an air flow of about 1 vvm through the solution.
glucose in the solution and from about 1279 to about l999CU of catalase/gram ds.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°C to about 40°C, a pressure of about I bar and maintaining an air flow of about 1 vvm through the solution.
17. A substantially pure gluconic acid solution produced by the process of claim 1.
18. A process for the production of spray-granulated gluconic acid,comprising the steps of obtaining a gluconic acid-containing broth, obtaining gluconic acid crystals from the broth, whereby gluconic acid crystals are obtained, and sprayed-coating the gluconic acid crystals with liquid sodium gluconate in aspray-dryer, whereby a spray-granulated gluconic acid is obtained.
19. The process of claim 18, wherein the gluconic acid crystals are obtained from the gluconic acid-containing broth by concentrating and filtering the gluconic acid-containing broth.
20. A spray-granulated gluconic acid granulate produced by the process of claim 18
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US57926595A | 1995-12-27 | 1995-12-27 | |
| US08/579,265 | 1995-12-27 | ||
| PCT/US1996/020480 WO1997024454A1 (en) | 1995-12-27 | 1996-12-20 | Process for the preparation of gluconic acid and gluconic acid produced thereby |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2240294A1 true CA2240294A1 (en) | 1997-07-10 |
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ID=29406148
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2240294 Abandoned CA2240294A1 (en) | 1995-12-27 | 1996-12-20 | Process for the preparation of gluconic acid and gluconic acid produced thereby |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2240294A1 (en) |
-
1996
- 1996-12-20 CA CA 2240294 patent/CA2240294A1/en not_active Abandoned
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