US006087135A United States Patent (19) 11 Patent Number: 6,087,135 Kierulff (45) Date of Patent: Jul. 11, 2000 54 MODIFICATION OF POLYSACCHARIDES WO 91/O5839 5/1991 WIPO. BY MEANS OF A PHENOLOXIDIZING WO 93/10158 5/1993 WIPO. ENZYME WO95/O1426 1/1995 WIPO. Wo 96/03440 2/1996 WIPO. 75 Inventor: Jesper Vallentin Kierulff, Roskilde, WO 96/18770 6/1996 WIPO. Denmark WO 97/25468 7/1997 WIPO. OTHER PUBLICATIONS 73 Assignee: Novo Nordisk A/S, Bagsvaerd, Denmark Dialog Info. Services, File 34, SciSearch, Accession No. 03742053 (1994). Hebeish et al., (1994) Cellulose Chem. And Tech. 21 Appl. No.: 09/221,009 28:409-418. 22 Filed: Dec. 23, 1998 Varma et al., (1995) Polymer Degradation and Stability 49:245-25O. Related U.S. Application Data Abdel-Hafiz (1997) Polymer Degradation and Stability 55:9-16. 63 Continuation of application No. PCT/DK98/00563, Dec. 18, SantaceSaria et al., (1994) Carbohydrate Polymers 1998. 23:35-46. 60 Provisional application No. 60/068,647, Dec. 23, 1997. Primary Examiner Francisco Prats 30 Foreign Application Priority Data Attorney, Agent, or Firm-Steve T. Zelson, Esq.; Reza Dec. 19, 1997 DK Denmark ........................... 1997 O1491 Green, Esq. 51 Int. Cl." ............................ C12P 19/04; C12P 19/00; 57 ABSTRACT C12P 7/44; C12N 9/04 The present invention deals with a process for oxidation of 52 U.S. Cl. ............................ 435/101; 435/72; 435/136; a hydroxy group of C and/or C and/or C and/or C and/or 435/142; 435/189; 435/190 Cs and/or C of a Sugar monomer of an oligo- or a polysac 58 Field of Search .............................. 435/101, 72, 136, charide comprising contacting, in an aqueous medium, the 435/142, 190, 189; 536/105 oligo- or the polysaccharide with a phenol oxidizing enzyme and an enhancing agent, whereby an oligo- or a polysac 56) References Cited charide with altered characteristics compared to the native FOREIGN PATENT DOCUMENTS oligo- or polysaccharide is created. 2164394 6/1996 Canada. 6 Claims, 2 Drawing Sheets U.S. Patent Jul. 11, 2000 Sheet 1 of 2 6,087,135 o O O, OH O CH O OH HOH CH CH O O O CH H MN - HO N OH OH O O C. o. O C o or OH" o A B C D E F G U.S. Patent Jul. 11, 2000 Sheet 2 of 2 6,087,135 3.5 3 Tempo 2.5 1714 intreated 1734 2 15 1. 1800 1750 1700 1650 1600 1550 1500 Wavenumbers (cm) Fig. 2 6,087,135 1 2 MODIFICATION OF POLYSACCHARDES various qualities of CMC can thus be produced using BY MEANS OF A PHENOLOXIDIZING cellulose with varying degrees of oxidation. ENZYME There is thus an increasing interest in and need for methods capable of introducing oxidative changes of various CROSS-REFERENCE TO RELATED kinds in oligo- and polysaccharides. APPLICATIONS This application is a continuation of application Ser. No. SUMMARY OF THE INVENTION PCT/DK98/00563 filed on Dec. 18, 1998 and claims priority The present invention relates to a process for Oxidation of under 35 U.S.C. 119 of Danish application serial no. PA a hydroxy group of C and/or C and/or C and/or C and/or 1997 01491 filed Dec. 19, 1997 and U.S. provisional appli Cs and/or C of a Sugar monomer of an oligo- or a polysac cation serial No. 60/068,647 filed on Dec. 23, 1997, the charide comprising contacting, in an aqueous medium, the contents of which are fully incorporated herein by reference. oligo- or the polysaccharide with a phenol oxidizing enzyme, together with a hydrogen peroxide Source when the FIELD OF INVENTION phenol oxidizing enzyme is a peroxidase, and an enhancing 15 The present invention relates to a process for enzymatic agent, whereby an oligo- or a polysaccharide with altered oxidation of Soluble as well as insoluble oligo- or polysac characteristics compared to the native oligo- or polysaccha charides Such as Starch and cellulose wherein the oligo- or ride is created. polysaccharide is oxidized introducing carbonyl groups and/ BRIEF DESCRIPTION OF THE DRAWINGS or carboxylate groups giving products with improved func tional properties. The present invention is further illustrated by reference to the accompanying drawings, in which: BACKGROUND ART FIG. 1 illustrates an idealised model for the progressive Oxidation of polysaccharides by various chemicals are oxidation of cotton cellulose. known in the art: 25 FIG.2 shows FT-IR/PAS spectra of untreated white cotton Oxidation of cellulose, for example, causes changes in the as well as white cotton treated with Tempo/laccase covering Structure and crystallinity of the resulting molecule, which the range 1800–1500 cm as described in Example 1. affects its chemical and physical properties. Varma and DETAILED DESCRIPTION OF THE Chavan Varma, A. J., and Chavan, V. B. (1995), Polymer INVENTION Degradation and Stability, 49, pp. 245-250 found a propor tionality between the decrease in degree of crystallinity and It is the purpose of the present invention to create an increase in degree of oxidation. Further, Varma and Chavan enzymatic oxidative modification of oligo- or polysaccha from cellulose created sodium 2,3-dicarboxy cellulose by rides whereby new functional groups are produced. oxidizing cellulose to 2,3-dialdehyde cellulose by periodate Accordingly, we have created a process for oxidation of oxidation followed by oxidation of the 2,3-di-aldehyde 35 a hydroxy group of C and/or C and/or C and/or C and/or cellulose to 2,3-dicarboxy cellulose by sodium chlorite. The Cs and/or C of a Sugar monomer of an oligo- or a polysac 98% sodium 2,3-dicarboxy cellulose was found to be water charide comprising contacting, in an aqueous medium, the Soluble. oligo- or the polysaccharide with a phenol oxidizing enzyme, together with a hydrogen peroxide Source when the Further, oxidized polyglucosides have interesting proper 40 ties as calcium Sequestrants and are useful as tripolyphoS phenol oxidizing enzyme is a peroxidase, and an enhancing phate Substitutes in detergent formulation SantaceSaria, E., agent, whereby an oligo- or a polysaccharide with altered Trulli, F., Brussani, G. F., Gelosa, D., and Di Serio, M. characteristics compared to the native oligo- or polysaccha (1994), Carbohydrate Polymers, 23, pp 35-46). ride is created. Further, various kinds of polysaccharides, in particular 45 In particular we have created a proceSS for oxidation of a Starch, are of utmost importance as a sizing agent in the hydroxy group of C and/or C and/or C of a Sugar textile industry, in addition to application as a processing aid monomer of an oligo- or a polysaccharide comprising in printing and finishing. However, the properties of the contacting, in an aqueous medium, the oligo- or the polysac native Starch is not always optimal compared to the prop charide with a phenol oxidizing enzyme, together with a erties required for the particular application. One of the 50 hydrogen peroxide Source when the phenol oxidizing problems of native Starch is the very large molecular size, enzyme is a peroxidase, and an enhancing agent, whereby an the insolubility, the instability of viscous solutions under oligo- or a polysaccharide with altered characteristics com varying temperature, and its Susceptibility to microbial pared to the native oligo- or polysaccharide is created. degradation. Consequently, chemical modification of Especially, we have created a process for Oxidation of a Starches has become an important tool to overcome the 55 hydroxy group of C of a Sugar monomer of an oligo- or a problems and create Starches having altered characteristics polysaccharide comprising contacting, in an aqueous compared to the native Starch. Common treatments involve medium, the oligo- or the polysaccharide with a phenol acid treatment, oxidation, etherification, esterification, oxidizing enzyme, together with a hydrogen peroxide Source grafting, and preparation of poly (vinyl)-Starch composites when the phenol oxidizing enzyme is a peroxidase, and an Abdel-Hafiz, S. A. (1997), Polymer Degradation and 60 enhancing agent, whereby an oligo- or a polysaccharide with Stability, 55, pp 9-16). altered characteristics compared to the native oligo- or In addition, Hebeish et al. Hebeish, A., El-Kashouti, M. polysaccharide is created. A., Abdel-Thalouth, I., Haggag, K., and El-Halwagi, A. According to the present invention an oligosaccharide (1994), Cellulose Chemistry and Technology, 28, pp contains at least 20 monomers, preferably at least 50 409–418 have shown, that the degree of substitution when 65 monomers, in particular at least 100 monomers. Carboxy Methyl Cellulose (CMC) is produced depends on The Oxidation according to the invention may result in the degree of oxidation of the cellulose base-material, and creation of carbonyl groups (ketones and aldehydes) and/or 6,087,135 3 4 carboxylate groups in the oligo- or polysaccharide resulting Oxidation of cellulose initially results in the formation of in an oligo- or a polysaccharide with altered characteristics carbonyl groups, with further oxidation resulting in the compared to the native oligo- or polysaccharide. formation of carboxylic acids or carboxylate anions depend An example of an idealised model for the progressive ing on the pH. oxidation of cotton cellulose is illustrated in FIG. 1. This Staining methods for the determination of carboxylate and model demonstrates Some of the types of changes to the carbonyl groups have been extensively used to indicate the cotton cellulose which might be expected with progressive extent of oxidation in cotton fabrics. The application of the oxidation. The number of carbonyl groupS/carboxylate Tollen's reagent (Ag(NH))OH) to oxidized cotton results groups formed will increase with Oxidation: in black Staining of all aldehyde groups due to precipitation “A” illustrates unmodified glucose, of silver metal Skoog, D.