(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2002/0076769 A1
`(43) Pub. Date:
`Jun. 20, 2002
`Brady et al.
`
`US 20020076769A1
`
`(54) REDUCED MOLECULAR WEIGHT
`GALACTOMANNANS OXIDIZED BY
`GALACTOSE OXIDASE
`
`(22)
`
`Filed:
`
`Aug. 2, 2001
`
`Related U.S. Application Data
`
`(76)
`
`Inventors: Richard Brady, Wilmington, DE (US);
`H. N. Cheng, Wilmington, DE (US);
`Alfred Jacques Haandrikman, WX
`Amersfoort (NL); Allison B. Moore,
`Newark, DE (US); Pong-Kuen Kuo,
`Hockessin, DE (US); William T.
`McNabola, Bear, DE (US); Charles R.
`Wheeler, Claymont, DE (US); Zu-Feng
`Xu, Newark, DE (US); Richard James
`Riehle, Wilmington, DE (US); Thyen
`Thanh Nguyen, Wilmington, DE (US);
`Hielke T. Jeerd de Vries, BF ede (NL);
`John Arthur Lapre, HB Ede (NL)
`
`Correspondence Address:
`Gary A. Samuels
`Hercules Incorporated, Hercules Plaza
`1313 N. Market Street
`Wilmington, DE 19894-0001 (US)
`
`(21) Appl. No.:
`
`09/920,694
`
`(63) Non-provisional of provisional
`60/222,869, filed on Aug. 3, 2000.
`
`application No.
`
`Publication Classification
`
`(51)
`
`Int. Cl.7 ........................ .. C12P 19/04; C08B 37/00;
`A61K 31/736
`........................... .. 435/101; 514/54; 536/114
`
`(52) U.S. Cl.
`
`ABSTRACT
`(57)
`Presented are compositions of reduced molecular weight
`galactomannans, particularly guar gum, which have been
`oxidized by the enzyme galactose oxidase. Further,
`the
`invention relates to a process for enzymatically reducing the
`molecular weight of a galactomannan wherein the galacto-
`mannan is simultaneously or subsequently oxidized using
`galactose oxidase, optionally in combination with other
`enzymes including peroxidases and or catalases. This pro-
`cess enables production of high concentrations of oxidized
`galactomannans, which have particular use in the paper
`making industry.
`
`P&G Exhibit 2008
`
`|PR2014-00628
`
`Conopco v. P&G
`
`

`
`US 2002/0076769 A1
`
`Jun. 20, 2002
`
`REDUCED MOLECULAR WEIGHT
`GALACTOMANNANS OXIDIZED BY GALACTOSE
`OXIDASE
`
`[0001] This application is related to U.S. Provisional
`patent application Ser. No. 60/222,869, filed Aug. 3, 2000
`from which priority is claimed.
`
`FIELD OF THE INVENTION
`
`[0002] This invention relates to reduced molecular weight
`galactomannans, particularly guar gum, which have been
`oxidized by galactose oxidase. A particular aspect of the
`invention additionally relates to a novel process for enzy-
`matically reducing the molecular weight of the galactoman-
`nan using mannanase, wherein the galactomannan is simul-
`taneously or subsequently oxidized using galactose oxidase.
`This preferred aspect of the invention enables the making of
`novel compositions comprising high concentrations of
`reduced molecular weight galactomannans which have been
`enzymatically oxidized by galactose oxidase.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Seed galactomannans, because of their viscous
`properties, have long found use as thickening agents and
`binding or colloidal holding agents in a number of fields,
`including as food additives, commercial
`lubricants, and
`paper additives. However, the use of these inherently vis-
`cous materials has always been subject to intrinsic limita-
`tions because the viscosity of the native galactomannans is
`too high to permit use of the compounds in any but dilute
`concentrations. Further, it has traditionally been difficult and
`or expensive and thus commercially impractical to chemi-
`cally modify the properties of these compounds because of
`the need to effectively carry out
`these reactions at
`low
`concentrations. The present invention addresses this need by
`providing a commercially efficient means to produce high
`concentrations of chemically modified galactomannans;
`most particularly, highly concentrated solutions of low
`molecular weight oxidizcd guar are provided which exhibit
`excellent properties of temporary wet strength in paper-
`making applications.
`
`[0004] Oxidation of galactomannans, particularly when
`achieved enzymatically using galactose oxidase, is known to
`introduce aldehyde groups on the galactose residues within
`the galactomannans.
`It
`is known further that aldehyde-
`containing galactomannans,
`in aqueous solution,
`tend to
`form crosslinks. Frollini et al, Carbohydrate Polymers 27
`(1995) pp. 129-135, and C. Burke (ed.) Carbohydrate Bio-
`technology Protocols, (1999), Humana Press, (N.J p. 79.
`Galactomannan compositions are known to be useful in the
`papermaking industry. For example, see U.S. Pat. Nos.
`5,633,300; 5,502,091; 5,338,407 and 5,318,669.
`
`[0005] Using galactose oxidase to oxidize the galactoman-
`nan gums, especially guar gum has been reported. U.S. Pat.
`No. 3,297,604 (Germino 1967) discloses galactose-contain-
`ing polysaccharides which are oxidized chemically or enzy-
`matically with galactose oxidase. U.S. Pat. No. 5,554,745
`(Chiu 1996) and U.S. Pat. No. 5,700,917 (Chiu 1997)
`describe an enzymatic oxidation process using a dual-
`enzyme system (galactose oxidase and catalase) to convert
`a cationic guar gum to an aldehyde derivative at the C6
`position of the galactose side chain in the guar at 1% solids
`concentration. The guar gum was not enzymatically
`
`degraded prior to the enzymatic oxidation. In fact, efforts
`were made to preserve the molecular weight of such an
`oxidized cationic gum. Frollini et al (supra) and C. Burke
`(supra) reported similar enzymatic oxidation of guar gum.
`However, none of these disclosures report the oxidation of
`gum hydrolyzates or a solution of such oxidized gum
`hydrolyzates at a solids concentration higher than about 1%.
`
`[0006] Use of mannanase to hydrolyze galactomannan
`gums such as guar gum has been practiced for at least five
`decades. Whistler described in 1950 (Whistler et al, J. of
`Chemical Society 72 (1950) 4938-4939) enzyme prepara-
`tions from germinated guar seeds that caused rapid decrease
`in viscosity of a guar gum solution. McCleary (Carbohy-
`drate Research, 71 (1979) 205-230) used mannanase to
`hydrolyze guar gum in order to analyze the fine structure of
`the gum. Japanese patent Hei 10 [1998]-36403 describes
`cationized decomposed galactomannans useful in the cos-
`metic industry.
`Japanese patent Sho 55 [1980]-27797
`describes a method for producing low viscosity guar using
`mannanase. EPAO 557627 A1 (1992) discloses a method of
`hydolyzing guar with mannanase to produce a food grade
`gum, and U.S. Pat. No. 4,693,982 (Carter 1987) discloses a
`method of treating solid guar gum particles with hydrolytic
`enzymes to reduce molecular weight and thereby improve
`solubility.
`
`SUMMARY OF THE INVENTION
`
`invention provides galactomannan
`[0007] The present
`compositions having a reduced molecular weight wherein
`the galactomannans are enzymatically oxidized by galactose
`oxidase.
`
`[0008] The preferred galactomannans include guar, locust
`bean and tara gum, with guar being most preferred. The
`preferred reduced molecular weight of the guar will range
`from about 1,000 to about 500,000, while more preferred
`ranges are from about 10,000 to 400,000, from about 50,000
`to about 350,000 and from about 70,000 to about 350,000,
`and from about 70,000 to about 150,000 daltons.
`
`[0009] The molecular weight of the galactomannans of the
`invention can be reduced in a number of ways, including
`with acid treatment, enzymatic treatment and treatment with
`hydrogen peroxide at high temperature being three preferred
`methods. One of the most preferred means of reducing the
`molecular weight of the galactomannan is enzymatically,
`with mannanase being the most preferred enzyme.
`
`[0010] The reduced molecular weight galactomannans of
`the invention are enzymatically oxidized by galactose oxi-
`dase, which acts to oxidize the C6 carbon of the galactose
`residues of the galactomannan to yield an aldehyde group.
`The preferred reduced molecular weight, enzymatically oxi-
`dized galactomannan is guar, having a preferred range of
`oxidation of from about 5% up to about 100% of the C6
`carbon atoms of the galactose residues being oxidized. More
`preferred ranges are wherein the galactose oxidase oxidizes
`from about 15% to about 70% of the galactose C6 carbon
`atoms, from about 15% to about 60% of the galactose C6
`carbon atoms while the most preferred range of oxidation is
`from about 30% to about 45% of the C6 galactose carbon
`atoms being oxidized. Optionally, the enzymatic oxidation
`using galactose oxidase can be carried out in the presence of
`one or more additional enzyme activities, with catalase
`activity and peroxidase activity being most preferred.
`
`

`
`US 2002/0076769 A1
`
`Jun. 20, 2002
`
`[0011] The reduced molecular weight galactomannans of
`the invention can be in derivatized form, with cationic
`derivative groups being most preferred. Derivatization of the
`galactomannan can take place prior to molecular weight
`reduction or after.
`
`In a preferred aspect of the invention, the reduced
`[0012]
`molecular weight, enzymatically oxidized galactomannan is
`made by a process comprising enzymatic molecular weight
`reduction using mannanase, wherein the process comprises
`adding the galactomannan to a prepared solution of man-
`nanase with stirring, and subsequently or simultaneously
`providing galactose oxidase to oxidize the reduced molecu-
`lar weight galactomannan. In this method of the invention it
`is possible to achieve novel compositions comprising enzy-
`matically oxidized galactomannans of reduced molecular
`weight at high concentrations.
`
`In this process of the invention the preferred galac-
`[0013]
`tomannan is guar, which can be made in oxidized form to a
`preferred concentration range of from about 1.5% to about
`80%. More preferred ranges include from about 1.5% to
`about 20%, with the most preferred range being from about
`2% to about 10%. The preferred reduced molecular weight
`range of the guar in this process of the invention is about
`1,000 to about 500,000, with more preferred ranges include
`about 10,000 to 400,000, from about 50,000 to about 350,
`000, from about 70,000 to about 350,000, and from about
`70,000 to about 150,000 daltons.
`
`[0014] The process of the invention yields a preferred
`range of oxidation of guar including from about 5% up to
`about 100% of the C6 carbon atoms of the galactose residues
`being oxidized, with a more preferred range of about 15% to
`about 70%, with an even more preferred range of about 15 %
`to about 60%, while the most preferred range is from about
`30% to about 45%. Additionally,
`in this process of the
`invention the galactomannan can be in derivatized form.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0015] The present invention provides new compositions
`comprising low molecular weight galactomannans, particu-
`larly guar, that have been oxidized by galactose oxidase. The
`invention further provides a preferred method of making the
`oxidized reduced molecular weight galactomannans using
`mannanase, wherein the method is capable of producing
`novel compositions comprising oxidized galactomannans at
`commercially desirable concentrations exceeding 1.5%.
`
`[0016] There are several distinct advantages of the com-
`positions of the inventions. (1) First, these compositions can
`be made at higher concentrations than can be achieved with
`galactomannans at their native molecular weight. For guar
`gum and its derivatives, for example, it is normally difficult
`to make solutions at much higher than 1% because of high
`viscosity. Commercially useful gums in solution form can be
`shipped more conveniently and less expensively at higher
`concentrations, and are ready to use in solution form. (2)
`Second, a higher level of enzymatic oxidation at the C6
`carbon of galactose can be attained using reduced molecular
`weight galactomannans because the enzyme oxidation is less
`inhibited by gel formation. For neutral guar gum at 1%, for
`example, oxidation beyond 20% is difficult because gel
`formation limits the oxidation.
`In contrast,
`a reduced
`molecular weight guar at 1% (and higher) can be enzymati-
`
`cally oxidized up to a level of more than 40%. (3) A third
`advantage is that a liquid gum product with good enzymatic
`oxidation can be achieved at high concentrations. For
`example, native guar (molecular weight approximately 2
`million) at 1% concentration and 20% oxidation of galactose
`C6 is a gel, whereas guar at 70,000 molecular weight and 5%
`concentration is a flowable liquid at up to 35-40% oxidation.
`(4) And finally, the lower molecular weight of the compo-
`sitions of the invention provides better wet strength decay in
`temporary wet strength applications in paper. High initial
`wet strengths can be obtained with the low molecular weight
`compositions, but a particular advantage of the low molecu-
`lar weight composition is that the wet strength is lost more
`quickly and to a greater extent on contact with water than for
`the corresponding high molecular weight oxidized gums.
`
`[0017] These compositions can be especially useful, there-
`fore, in a variety of temporary wet strength applications in
`paper, such as in tissue and towel. For bathroom tissue, for
`example, good wet strength decay prevents pipes from
`getting clogged. Other paper uses include situations where it
`is advantageous to achieve improved dry strength, z-direc-
`tion tensile, Scott bond, Mullen burst, ring crush, STFI,
`tensile energy absorption (TEA), fracture toughness, and
`possibly sizing enhancement. This would include uses in
`paper coating, liquid packaging board, virgin and recycled
`linerboard, lightweight coated paper, fine paper, and news-
`print. Application of these compositions can be at the wet
`end or after the wet end, such as at the size press or in a spray
`application. Other possible uses include cosmetics, oilfield
`recovery, construction, adhesives, tablet coating, paint, tex-
`tiles, toys, and removable adhesives.
`
`[0018] The galactomannans of the invention are well-
`known polysaccharide materials generally derived from
`seed gums. The commercially important galactomannans are
`locust bean gum, guar gum and tara gum. Galactomannans
`are structurally linear polysaccharides based on a backbone
`of [3(1-4)-linked D-mannose residues. Single ot-D-galactose
`residues are linked to the mannose chain by C1 via a
`glycosidic bond to C6 of mannose. The degree of galactose
`substitution on the mannose backbone varies depending on
`the botanical source of galactomannan. In locust bean gum,
`the average galactose to mannose ratio is 1:4; in tara gum the
`ratio is approximately 1:3; and for guar, the most preferred
`galactomannan of the invention, the ratio of galactose to
`mannose is approximately 1:2.
`
`[0019] Within the context of the present disclosure Appli-
`cants intend to include various derivatized forms of galac-
`tomannans within the scope of the invention. Derivatives of
`the galactomannans are very well known in this art, and
`many are commercially available. Roy L. Whistler and
`James N. Bemiller, ed., Industrial Gums; Polysaccharides
`and Their Derivatives (Third Edition), Academic Press, New
`York, 1993. For example, the most common commercially
`available guar derivatives
`include hydroxypropyl, car-
`boxymethyl,
`carboxymethyl-hydroxypropyl,
`and
`2-hy-
`droxy-3-(trimethylammonium chloride) propyl. Other com-
`mon derivatives include hydroxyethyl, ethyl, guar gum
`phosphates, and mixed derivatives including mixed cationic
`and anionic (amphoteric).
`
`[0020] Also within the context of the present disclosure
`Applicants intend to include within the scope of the inven-
`tion galactomannans which have been treated with various
`
`

`
`US 2002/0076769 A1
`
`Jun. 20, 2002
`
`wetting and solubility agents. Many such agents are known
`in this art. For example, galactomannan products can be
`mixed with glyoxal or borax to reversibly crosslink the
`surface of the particles and retard hydration. Glyoxalated
`guar requires pH of 7 or above to hydrate, while borated guar
`requires pH below 8 for hydration. These and other treated
`and coated forms of the basic galactomannans are consid-
`ered to be within the scope of Applicants’ invention.
`
`[0021] For purposes of clarity in describing the present
`invention, Applicants use the term ‘reduced molecular
`weight’ of the various galactomannans to refer to a galac-
`tomannan which exists in a form having an average molecu-
`lar weight which is a fraction of its native molecular weight.
`For example, for the preferred galactomannans of the inven-
`tion; guar,
`locust bean and tara,
`the reduced molecular
`weight refers to a value which is approximately one half or
`less than the native molecular weight. Locust bcan (carob)
`gum has a native molecular weight generally reported to be
`in the range of about 300,000 to 360,000 daltons. The
`preferred reduced molecular weight range of the composi-
`tions of the invention for locust is about 1,000 up to about
`150,000 daltons. The most preferred galactomannan of the
`invention, guar, is known to have a native molecular weight
`of approximately 2,000,000 daltons. The preferred reduced
`molecular weight range of the compositions of the invention
`for guar is from about 1,000 to about 500,000 daltons. More
`preferred molecular weight ranges for guar include about
`10,000 to about 400,000 and from about 50,000 to about
`350,000 and from about 70,000 to about 350,000, and from
`about 70,000 to about 150,000 daltons. Another preferred
`galactomannan of the invention is tara gum. Definitive
`molecular weight ranges for native tara gum have not been
`reported, but it is believed that the native molecular weight
`is between the value for native guar and native locust bean.
`Thus, with respect to tara gum in the instant invention, the
`term reduced molecular weight would refer generally to a
`range of about 1000 daltons up to a value representing about
`one half of tara gum’s native molecular weight.
`
`[0022] Within the context of describing molecular weight
`of galactomannans for
`the present disclosure,
`the term
`molecular weight refers to the weight average. More par-
`ticularly, the weight average molecular weight refers to a
`value which is measured by size exclusion chromatography
`analysis (SEC) using a calibration derived from narrow
`distribution polyethylene oxide (PEO) and polyethylene
`glycol (PEG) molecular weight standards.
`
`[0023] Also, it is within the scope of Applicants’ invention
`for an individual galactomannan composition to comprise
`more than a single botanical species of galactomannan. In
`some commercial applications for these gums, final charac-
`teristics of the compositions can be improved and or tailored
`to specific purposes by using a mixture of one of more
`galactomannan. Further,
`the oxidized reduced molecular
`weight galactomannan compositions of the invention may
`comprise additional ingredients, as appropriate and advan-
`tageous for the intended purpose of the material. Many
`additives useful in galactomannan compositions are well
`known in these arts, including, for example bentonite, alum,
`starch, cationic polymers, sizing agents, wet strength addi-
`tives, debonder, defoamers and biocides, any of which might
`be used to impart additional characteristics for a particular
`intended purpose of a composition of the invention.
`
`[0024] The molecular weight of the galactomannans can
`be reduced by a variety of methods, including acid treat-
`ment, enzyme treatment, and heating with hydrogen perox-
`ide. These methods are well known in this art. For example,
`acid treatment and heating with hydrogen peroxide are
`methods known to reduce the molecular weight of the
`galactomannans; Roy L. Whistler and James N. Bemiller,
`ed., Industrial Gums; Polysaccharides and Their Derivatives
`(Third Addition), Academic Press, New York, 1993; and
`U.S. Pat. No. 5,480,984. Commercial preparations of
`reduced molecular weight galactomannans that are made by
`these methods are also available; for example, Galactosol®
`30M1F (Hercules, Inc., Wilmington, Del.).
`
`[0025] A particularly preferred method of reducing the
`molecular weight of galactomannan is accomplished using
`the enzyme mannanase. Mannanase, which has been well
`characterized in the art, is known to hydrolyze mannans
`(mannan endo-1, 4-[3-mannosidase E.C. 3.2.1.78) wherein
`the endo-mannanase randomly cleaves 1,4-[3-D mannosidic
`linkage in mannans. See, for example, European Patent
`Application 0 557 627A1 or McCleary, Carbohydrate
`Research 71 (1979), pp. 205-230. Mannanase activity can be
`provided in the form of purified mannanase enzyme, or
`alternatively, mannanase activity can be provided by using
`one of the commercial preparations of hemicellulases or
`cellulases which are known to contain mannanase activity.
`Examples of such commercially available preparations
`include Hemicellulase GMTM sold by Amar1o; EnzeboTM
`cellulase CRX sold by Enzyme Development Corp. of N.Y.;
`and Gamanase 1.0L sold by Novo Nordisk.
`
`[0026] The compositions of Applicants’ invention com-
`prise galactomannan having reduced molecular weight
`wherein the galactomannan is enzymatically oxidized by
`galactose oxidase. In a well-characterized reaction mecha-
`nism, galactose oxidase is known to specifically oxidize the
`C6 carbon atom of the galactose residues, wherein the
`alcohol OH group is oxidized to an aldehyde C=O group.
`Mazur, A. W. ACS Symposium Series, 466 (1991) 99; U.S.
`Pat. No. 3,297,604 (Germino); and Knowles, P. F. and Ito,N.,
`Perspectives in BioOrganic Chem., Vol.2,207-244, JAI Press
`LTD (1993). Galactose oxidase may be produced by the
`fungus Dactylium dendroides, recently renamed Fusarium
`sp., and has been given the E. C. Number 1. 1.3.9. Within the
`context of the compositions of Applicants’ invention, oxi-
`dation of the C6 of galactose by galactose oxidase is
`accomplished on about 5% up to about 100% of the C6
`carbon atoms on the galactose residues. More preferred
`ranges of oxidation are about 15% up to about 70%; about
`15% up to about 60%; with a range of about 30% up to about
`45% being most preferred.
`
`[0027] The oxidation process can be accomplished using
`an effective amount of the single enzyme galactose oxidase,
`however, in a preferred aspect the oxidation reaction can be
`improved by incorporating a catalase activity and or a
`peroxidase activity in the galactose oxidase reaction mix-
`ture. The presence of either or both of these additional
`activities can improve the effectiveness of the oxidation
`reaction, and enables effective oxidation more efficiently and
`less expensively when commercial quantities of oxidized
`galactomannan are desired. The increased catalytic activity
`of galactose oxidase in the presence of a peroxidase and
`catalase has been shown by Radin, et al., in The Use of
`Galactose Oxidase_in Lipid Labeling, J . Lipid Res., Vol.
`
`

`
`US 2002/0076769 A1
`
`Jun. 20, 2002
`
`22:536-541, (1981). Applicant has discovered, with respect
`to the oxidation of galactomannans, that the activity level of
`galactose oxidase can be increased, i.e., it can be continu-
`ously activated, by carrying out the reaction in the presence
`of a one-electron oxidant such as peroxidase or laccase,
`together with a hydrogen peroxide remover such as catalase.
`Galactose oxidase in combination with catalase has been
`
`reported in the oxidation of galactomannans, but Applicants
`have improved this reaction by the addition of a peroxidase
`activity, wherein surprisingly,
`the oxidation reaction
`becomes more economically efficient for large scale com-
`mercial applications, even when the three-enzyme system is
`used.
`
`[0028] This separate invention regarding improving the
`activity level of galactose oxidase by the addition of a
`one-electron oxidant to continually activate the galactose
`oxidase, in the presence additionally of a hydrogen peroxide
`remover to decompose the hydrogen peroxide which is
`formed as a coproduct in the oxidation of alcohols, is the
`subject of a separate commonly-owned and concurrently-
`filed patent application.
`
`[0029] A preferred embodiment of Applicants’ invention
`is a process for making a composition comprising galacto-
`mannan at a concentration of at least about 1.5%, wherein
`the galactomannan is enzymatically hydrolyzed by mannan-
`ase and oxidized by galactose oxidase to yield an aldehyde
`group on at least about 5% up to about 100% of the C6
`carbon atoms of the galactose residues. The process com-
`prises preparing a solution of an effective concentration of
`mannanase, and slowly adding to that solution, while stirring
`or otherwise agitating the solution, galactomannan to a
`concentration of at least about 1.5% up to about 80%. Then,
`with continued stirring or agitation, an effective amount of
`galactose oxidase and a source of oxygen are added. Option-
`ally, this last step wherein galactose oxidase is added can be
`carried out in the presence of one or more additional activity
`components including a catalase activity and or a peroxidase
`activity.
`
`In another aspect of Applicants’ process, the reduc-
`[0030]
`tion of molecular weight of the galactomannan using man-
`nanase can be carried out simultaneously with the oxidation
`of the galactomannan. In this aspect, the process comprises
`preparing a solution comprising effective amounts of man-
`nanase and galactose oxidase, and a source of oxygen, and
`slowing adding to this solution, with continued stirring or
`other agitation, galactomannan to a concentration of at least
`about 1.5% up to about 80%. In this aspect also, optionally,
`the oxidation using galactose oxidase can be carried out in
`the presence of one or more additional activities including a
`catalase activity and or a peroxidase activity.
`
`[0031] One important consideration in the process of the
`invention will be the form in which the galactomannan is
`added to the mannanase solution. Galactomannans exist in a
`
`number of solid, particulate and slurry forms, well known in
`this art. A preferred technique for the present invention is
`that galactomannan is added to the mannanase solution in
`the form of particles. This method allows for putting the
`galactomannan into solution in a highly concentrated form
`without rapid viscosity increase that will impair the molecu-
`lar weight reduction, and without the production of visible
`“grits” in the final solution product. Galactomannan particle
`size should be selected carefully. For example, using guar, if
`
`the guar particles are too fine, the viscosity of the water-
`soluble gum will increase so rapidly that the dispersion and
`solubilization of the gum at high concentration will be
`virtually impossible and impractical at industrial production
`scale. If the guar particles are too coarse, the final product
`will be heterogeneous and have visible large particles
`(“grits”). The preferred particle size range for a guar gum,
`for example, is between about 40 to about 250 mesh, more
`preferably between about 60 to about 200 mesh, and most
`preferably between about 80 to about 200 mesh (75-180
`ym). Given these parameters, the preferred particle sizes of
`other galactomannans are easily determined empirically,
`depending upon the desired final characteristics of the
`intended solution and the properties of the starting galacto-
`mannan. Coarsely ground gum or the dehulled seeds from
`which the gum is obtained, e.g., guar splits, can also be used,
`but mechanical homogenization may be needed to eliminate
`visible particles in the finished product.
`[0032] Another important aspect of Applicants’ process is
`the step of adding the galactomannan to the mannanase
`solution, instead of adding the mannanase to the galacto-
`mannan, as is traditionally done. By adding the galactoman-
`nan into the enzyme-containing solution, while stirring or
`otherwise agitating,
`the enzyme is able to effectively
`degrade the galactomannan as the galactomannan is added
`while continually lowering its viscosity. This aspect of the
`process allows for hydrolyzing guar, for example, up to a
`high concentration in solution without problematic lumping
`or difficulty of mixing due to the otherwise rapid viscosity
`build-up that is traditionally experienced in the process of
`solubilizing galactomannans. If one tries to disperse the
`galactomannan into solution without controlling the particle
`size and or having the mannanase present in solution prior
`to addition of the galactomannan, it will be very difficult and
`impractical to make even a 1.5% galactomannan solution at
`large scale. Applicants have discovered that if proper con-
`sideration is not given to particle size and manner of addition
`of the galactomannan to the mannanase solution, it will not
`be possible or practical to carry out the enzymatic oxidation
`reaction at high concentrations of galactomannan. One
`skilled in the art could resort to making low molecular
`weight low viscosity gum hydrolyzates at normal solids
`concentration (0.5-1.5%), then using spray-drying or alcohol
`precipitation methods
`to obtain powered hydrolyzates
`before re-dissolving it at higher concentration, but such
`processes are cumbersome and expensive for commercial
`production.
`[0033]
`In Applicants’ process any galactomannan can be
`used. Locust bean, tara and guar gums are preferred; with
`guar gum being the most preferred. As discussed earlier, the
`galactomannan can be in native form, or it can be used in
`derivatized form, and or additionally treated to alter the
`wettability and solubility aspects of the gum. If the starting
`galactomannan has been treated or coated to improve its
`wettability or solubility characteristics,
`the form of the
`galactomannan which is added to the mannanase solution
`will be adjusted accordingly, which adjustments are easily
`determined empirically within the parameters of the inven-
`tion. For example, if guar is selected as the galactomannan
`to be reduced and oxidized and the starting guar particles are
`in a form coated with borate, the particle size and rate of
`addition of the guar will be less critical. The rate of addition
`of the galactomannan to the mannanase and the form of
`stirring or agitation of the solution while the galactomannan
`
`

`
`US 2002/0076769 A1
`
`Jun. 20, 2002
`
`is added are also important aspects of this step of the
`process. Typically, effective stirring and rate of galactoman-
`nan addition are adjusted easily within the parameters of the
`invention to prevent lumping during addition of the galac-
`tomannan to the enzyme solution.
`
`In the process of the invention the molecular
`[0034]
`weight, the degree of the polymerization and the viscosity of
`the galactomannan are reduced to desired levels in order to
`accommodate the subsequent or simultaneous enzymatic
`oxidation reaction at high galactomannan concentrations,
`and to meet the performance requirements in the desired
`application. When using guar in the process of the invention,
`for example, the reduced molecular weight of the hydro-
`lyzed guar is preferably about 1,000 to 500,000 daltons,
`more preferably about 10,000 to about 400,000 daltons or
`from about 50,000 to about 350,000 or from about 70,000 to
`about 350,000, or from about 70,000 to about 150,000
`daltons. If other galactomannans are used in the process of
`the invention, their reduced molecular weight after treatment
`with mannanase will be approximately one half or less than
`the starting native molecular weight of the selected galac-
`tomannan.
`
`[0035] The process of the invention carries out molecular
`weight reduction of the starting galactomannan using man-
`nanase mannan
`endo-1,4-[3-mannosidase E.C.3.2.1.78
`hydrolysis. This reaction, which has been well studied in the
`art, is typically carried out at ambient temperature up to 80
`degrees C., and in a pH range of about 3 to about 9. The
`effective concentration range of the mannanase will be
`adjusted dependent upon the desired final molecular weight
`and desired final concentration of the selected galactoman-
`nan. Cost and time are also factors to be considered. A
`
`convenient concentration of mannanase will be approxi-
`mately 1000 units per gram of galactomannan. The man-
`nanase of the process is essentially free of galactose side
`chain cleaving ot-galactosidase and exo-mannanase activity.
`After the molecular weight range of the starting galacto-
`mannan is reduced to the desired molecular weight range,
`the mannanase is deactivated by conventional methods such
`as heat to prevent uncontrolled hydrolytic reaction.
`
`[0036] The next step in the process of the invention,
`(which step can also be carried out simultaneously with the
`mannanase molecular weight reduction in one aspect of the
`invention), is the enzymatic oxidation of the galactomannan
`using galactose oxidase. The term galactose oxidase, for
`purposes of the present invention, means that enzyme clas-
`sified as E.C. No. 1. 1.3.9 and those enzymes which function
`in a substantially similar manner, including, for example,
`glyoxal oxidase, (CAS Registration No. 109301-01-1). Also
`included are all enzymes, including those obtained through
`any form of genetic manipulation, with a catalytic domain
`which is substantially homologous with galactose oxidase or
`glyoxal oxidase. As used herein, the term galactose oxidase
`includes each of the thre