Paper-making begins by"cooking"chipped pulpwood to produce pulp. The cell walls of wood fiber consists of several layers composed primarily of cellulose, hemicellulose and lignin. Polymeric constituents of wood are cellulose (40-45 wt% wood), hemicellulose (20-30 wt%), and lignin (20-30 wt%). Pulping is a procedure that disintegrates these fibers by mechanical and chemical means. Ultimately, this pulp will be used for papermaking. The objective of pulping is to separate the cellulose fibers and remove as much lignin and hemicellulose as required by the end use, while preserving the fiber strength.

Kraft pulping is a specific pulping process that uses a wide variety of wood sources to produce quality pulps for manufacture of particularly strong paper. Kraft pulping is a highly alkaline method of wood chip digestion characterized by the use of sodium hydroxide and sodium sulfide in the delignification phase. The kraft process currently is the most widely used pulping process. Lignin is degraded during the cooking process into chromophoric groups which account for approximately 90% of the dark color associated with unbleached kraft pulp (Casey, J. P., Pulp and Paper: Chemistry and Technology. Vol. 1, 3rd ed., John Wiley & Sons, Inc. New York (1980)). Kraft pulp is commonly used for the production of grocery bag stock and linerboard for corrugated containers, but dark pulp must be bleached to eliminate the dark color so bright, high-value paper can be produced.

The bulk of the colored compounds ("chromophores") created during the pulping process diffuse out of the pulp and are removed in the spent liquors or in the washing stages of the kraft pulping. However,

some chromophores remain in the pulp where they are either trapped within the fibers or are chemically bound to the hemicellulosic and cellulosic moieties.

Conventional pulp bleaching is a sequence of treatments using chlorine dioxide and chlorine, among other chemicals (Fengel and Wegner, Wood Chemistry Ultrastructure Reaction. Walter Degruyter. Berlin (1984)). Effluent from chlorine bleaching contains toxic chlorinated compounds. Moreover, the bleaching process damages the pulp fibers by reducing the degree of polymerization of the cellulose, and residual acid left in the paper causes the fibers to degrade slowly over time. Newer bleaching processes employ hydrogen peroxide or, less commonly, oxygen/alkali (02 and NaOH) ; however, the bleaching activity of these chemicals is insufficient, particularly for high yield pulps. Concern about toxic pulp-bleaching effluent has motivated investigation of xylanase catalyzed processes to reduce the dark color of pulp, which consequently would decrease the chlorine required for bleaching and thus the toxic bleaching effluent.

The earliest report of xylanase treatment of pulp described preparation of rayon and cellulase acetate (Paice and Jurasek, 1984).

Viikari, et. al. first reported xylanase delignification of wood pulp (Viikari, et. al., 1986). Since those reports numerous investigators have reported xylanase delignification of wood pulp (see the study by Clark, et. al. Appita, 4 (6), 389-404 (1991), and the paper by Senior, et. al., 1992).

Based on a survey of mill trials and laboratory investigations, xylanase delignification reduced chlorine required for hardwood pulp by 20 to 25% and chlorine required for softwood pulp by 10 to 15% (Dunlop-Jones and Gronberg. Pulp & Paper Canada, 96 (10), 20-24 (1995)).

Xylanase-catalyzed hydrolysis of lignin-carbohydrate complexes was proposed as a reaction mechanism responsible for xylanase delignification of pulp (Yang and Eriksson, Tappi J., 75,95-101 (1992)).

Xylanase modification of pulp-surfaces to enable leaching of lignin from pulp was also proposed to explain xylanase delignification of pulp

(Buchert, et al. Holzforschung, 47 (6), 473-478 (1993)). It seems plausible that either or both of these two mechanisms are responsible for xylanase delignification of pulp; however, neither mechanism explains why xylanase removes only a fraction of total lignin from wood pulp.

It is therefore an object of the present invention to provide an improved means for removing color from wood pulps.

It is a further object of the present invention to provide enhanced xylanase catalyzed removal of chromophores in processing wood pulps.

It is another object of the present invention to reduce or eliminate the amount of bleaching necessary in the processing of wood pulps.

Summary of the Invention A method of removing chromophores from wood pulp is disclosed, using the combination of a pore-enlarging agent, under conditions not substantially degrading the cellulose, and a xylanase, under conditions wherein the lignan is removed from the cellulose. The method includes the steps of first preparing a wood pulp, adjusting the wood pulp to between 1% and 15% consistency (weight %), adjusting the pulp to a pH of between pH 3.0 to 7.0 to maximize cellulase activity, treating the wood pulp with a cellulase to alter the pulp-fiber-pore-structure, adjusting the pulp to between a pH between pH 7.0 and 10.0 to inhibit cellulase activity and maximize xylanase activity, treating the pulp with a xylanase (wherein the xylanase is capable of releasing chromophores from the pulp), and then extracting the pulp to remove chromophores. In a particularly preferred embodiment, the wood pulp is a kraft pulp, the cellulase is obtained from Aspergillus niger, and the xylanase is ICI Ecopulp X-200 (ca 20,000 kDa).

In another preferred embodiment, the xylanase is a mixture of xyla- nases in a crude enzyme preparation from a culture of Bacillus stearothermophilus bacteria (ATCC 55696). In another preferred embodiment, the xylanase is a substantially purified preparation of a xylanase obtained from Bacillus stearothermophilus bacteria (ATCC

55696). In another preferred embodiment, the extraction is an alkali/hydrogen peroxide extraction.

The method also includes a method of removing color from a wood pulp which include the steps of first preparing a wood pulp, adjusting the wood pulp to between 1% and 15% consistency (weight %), adding an organic solvent to the pulp (to between 0.01 wt % and 20 wt%), treating the pulp/solvent mixture with a xylanase (wherein the xylanase is capable of releasing chromophores from the pulp), extracting the pulp to remove chromophores, and then heating the pulp/solvent mixture to remove the organic solvent from the pulp. In a particularly preferred embodiment, the wood pulp is a kraft pulp, the solvent is methylamine, the methylamine concentration in the pulp/methylamine mixture is between 1 wt% and 4 wt%, and the xylanase is ICI Ecopulp X-200 (ca 20,000 kDa).

A method of removing color from a wood pulp wherein the wood pulp contains secondary fiber. This method contains the steps of preparing a wood pulp, treating the wood pulp with xylanase (wherein the xylanase is capable of releasing chromophores from the pulp) and extracting the pulp to remove chromophores.

The method of removing chromophores from wood pulp containing the steps of preparing a wood pulp, treating the wood pulp with xylanase, wherein the xylanase is capable of releasing chromophores from the pulp.

Chromophores are released from the pulp and the pulp is then extracted with an alkaline solution. The pulp is then subjected to bleaching, preferably oxygen bleaching.

Another method of removing color from wood pulps begins with the screening of xylanases to obtain a xylanase capable of releasing chro- mophores from wood pulp. Wood pulp is prepared and treated with the xylanase. The pulp is then extracted to remove the chromophores.

Detailed Description of the Invention Xylanases are used to delignify lignocellulosic materials, such as wood pulp, by cleaving the bond between the lignin and the cellulose.

While conventional xylanase treatments are able to achieve some level of delignification, conventional xylanase treatments have been unable to produce delignified wood pulp with high brightness levels (ISO brightness numbers exceeding 85%) without subjecting the pulp to further bleaching steps.

The methods described herein utilize pre-treatment with either cellulose or low molecular weight amines to improve the degree of bleaching obtained with xylanases.

Materials Pulp Pulp is prepared using standard methodologies, from either softwoods, hardwoods, or blends thereof. In preferred embodiments, the pulp is prepared as a slurry having a consistency of between one and fifteen percent by weight. The pH will be adjusted to the optimum of the enzyme to be used, typically a relatively low pH for the cellulase, and a higher pH for the xylanase. A preferred pulp is kraft pulp. In a preferred embodiment, the extraction is an alkali/hydrogen peroxide extraction.

The methods disclosed herein are applicable to the cleavage of lignin-cellulose bonds from virtually any lignocellulosic material.

Preferred lignocellulosic materials are softwood or hardwood pulps, derived from any species. However, other lignocellulosic materials, such as bagasse, can be delignified using the methods disclosed herein.

Preferred wood pulps are softwood pulps. The pulp can be virgin pulp, or can already have been subjected to the Kraft process or other preliminary digestion steps. It is preferred that the pulp has already been subjected to Kraft processing before treatment with cellulase or amines and xylanase.

U. S. Patent No. 5,503,709 to Barton is an example of pulping technology.

Regardless of whether the pulp is virgin or has already been subjected to a preliminary digestion process, the Kappa number of the pulp will be significantly lowered following treatment with cellulase or amines and xylanase as described herein.

The lignocellulosic material is dispersed in a solution, preferably an aqueous solution, to produce a dispersion containing between about 1 and 15% lignocellulosic material by weight of the solution. The solution can also contain water-miscible organic solvents, such as ethylene glycol, which increase the boiling point of the solution.

The lignocellulosic material is treated at a temperature between about 50 and 100 °C. Preferably, the temperature is between 60 and 85 °C, and, more preferably, between 65 and 80 °C. The lignocellulosic material is"cooked"for a period of time between about 1 and 24 hours, preferably between about 2 and 8 hours, and, more preferably, between about 3 and 6 hours, although extended times are not harmful.

After the lignocellulosic material has been delignified, the solution is transformed into a black liquor which contains, in part, lignin that has been removed from the lignocellulosic material. The black liquor can be separated from the pulp, for example, via centrifugation and washing. If desired, the low molecular weight amines can be removed from the liquor, for example, by distillation, and reused in the next batch of lignocellulosic material to be delignified.

It is often possible, using conventional methods, to recover lignin from the black liquor. The lignin can be used, for example, as a resin binder, a wood rehardening agent, or a glue.

Cellulases A wide variety of cellulases which have optimal activity at different pHs, typically obtained from bacterial or fungal sources, can be used. Cellulases are typically selected based on their pH optimum. A particularly preferred cellulase is obtained from Aspergillus niger, which is used at low pH and inactivated by elevation of the pH. See articles reporting various cellulases, such as Trichoderma reesei cellulase in the bleaching of kraft pulp, by Buchert et al., Applied Microbiology and Biotechnology 40 (6): 941-945 (1994);"Effect of Alkali Pretreatment on Degradation of some Cellulosic Wastes by Aspergillus-sydowii", Ghareib, et al. Zentralblatt Fuer Mikrobiologie 147 (8): 551-556 (1992); and

"Formation of Acetic Acid from Cellulosic substrates by Fusarium- oxysporum"Kumar, et al., Applied Microbiology and Biotechnology 34 (5): 570-572 (1991).

In a particularly preferred embodiment, the wood pulp is a kraft pulp, the cellulase is obtained from Aspergillus niger, and the xylanase is ICI Ecopulp X-200 (ca 20,000 kDa).

Pore-enlarging Organic Solvents The effectiveness of xylanase treatments can be significantly enhanced using the methods disclosed herein by using a low molecular weight amine prior to, or simultaneously with, xylanase treatment. It is believed that the amines expand the pore size in the pulp and allow the xylanase to effectively penetrate the pulp and have access to more of the lignin-cellulose bonds, relative to when xylanase alone is used.

Lignocellulosic materials are delignified with low molecular weight amines and xylanases by contacting the materials with an effective amount of the amines and xylanases, sequentially or simultaneously, at an effective temperature and pressure, for an effective amount of time, to cleave a significant percentage of the lignin-cellulose bonds. As demonstrated in Examples 5, the degree of delignification can be significantly increased when pulp is treated with xylanase and a low molecular weight amine such as methylamine, simultaneously or sequentially, relative to when xylanase alone is used.

Any low molecular weight amine can be used which is able to increase the pore size of the pulp to be treated, does not reduce the activity of the xylanase by more than 50% in the solution in which it is used, and which can be readily removed from the pulp by extraction in an aqueous solution. The amines also assist in cleaving the lignin-cellulose bonds present in the pulp.

Suitable amines include methylamine, monoethanolamine, dimethylamine, ethylenediamine, propylene diamine, hexamethylene diamine, methylaniline, ethylamine and mixtures thereof. The preferred amine is methylamine. To determine whether a particular amine is

compatible with a particular xylanase, it is useful to analyze the xylanase activity in the presence and absence of the amine. As an example, the effect of methylamine on xylanase activity was assessed by comparing xylanase activity in 0 (control) and 10% methylamine solutions. Xylanase activity as assayed by the DNS procedure of Miller (Miller, Anal. Chem., 31: 426-428 (1959)) in 10% methylamine was about 61% of the control value.

The concentration of low molecular weight amines in the dispersion of lignocellulosic material to be treated is between about 1 and 10% by volume of the solution. Preferably, when methylamine is used, the concentration is between about 1 and 4 percent by volume, although concentrations as high as 10% by volume can be used. In a preferred embodiment, lignocellulosic material is first delignified with a low molecular weight amine, such as methylamine, the lignin-containing solution is removed, and then the material is delignified with xylanase.

Xylanases Many different microbial xylanase preparations have been reported in the literature. Most are derived from various fungal sources; a few are from Streptomyces and yeasts. See published PCT applications WO 91/02840, WO 91/02839, U. S. Pat. No. 4,966,850 and published European application 0 383 999. Additional sources are described in U. S. Patent No. 3,909,345 to Parker, et al., 5,369,024 to Jeffries, et al., 5,457,045 to Anker, et al. and 5,405,769 to Campbell, et al.,"Xylanolytic enzyme production by an Aspergillus niger isolate"by Costa-Ferreira, et al., Applied Biochemistry and Biotechnology 44 (3), 231-242 (1994); "Xylanase production by Trichoderma-Longibrachiatum", Royer, et al., Enzyme and Microbial Technology 11 (7): 405-410 (1989);"A cellulase- free xylanase from alkali-tolerant Aspergillus fischeri Fxnl"by Raj and Chandra Biotechnology Letters 17 (3): 309-314 (1995);"Application of xylanase from alkaliphilic thermophilic Bacillus sp. NCIM 59 in biobleaching of bagasse pulp"Kulkarni and Rao Journal of Biotechnology 51 (2): 167-173 (1996);"Biobleaching effect of Streptomyces

thermoviolaceus xylanase preparations on birchwood kraft pulp"by Garg, et al., Enzyme and Microbial Technology 18 (4): 261-267 (1996); "Cellulase-Poor Xylanases produced by Trichoderma-Reesei rut C-30 on Hemicellulose substrates"by Gamerith, et al., Applied Microbiology and Biotechnology 38 (3): 315-322 (1992);"Effect of pH on production of xylanase by Trichoderma reesei on xylan-and cellulose-based media" Bailey, et al. Applied Microbiology and Biotechnology 40 (2-3): 224-229 (1993); and"Enhanced stability of cellulase-free xylanase from Chainia sp.

(NCL 82.5.1).

Bandivadekar, et al. Biotechnology Letters 16 (2): 179-182 (1994).

In a preferred embodiment, the xylanase is a mixture of xylanases in a crude enzyme preparation from a culture of Bacillus stearothermophilus bacteria (ATCC 55696). In another preferred embodiment, the xylanase is a substantially purified preparation of a xylanase obtained from Bacillus stearothermophilus bacteria (ATCC 55696).

Xylanase delignification of pulp is a function of xylanase molecular size and pulp pore structure. Xylanase molecular size can limit pulp delignification if pulp pore structure selectively admits xylanases based on molecular size into pulp pores. This relationship was demonstrated by the examples. The examples demonstrate that xylanase molecular size and pulp pore structure determine the amount of lignin that xylanase can remove from pulp. Using softwood pulp, xylanases with molecular weights of 20,000 Da, 39,000 Da, and 67,000 Da removed 48%, 39%, and 30% of lignin respectively. Mild cellulase pre-treatment of softwood pulp increased the apparent median pore size from 37 Angstroms, to 67 Angstroms, which enabled xylanase to reduce chlorine requirement for softwood pulp bleaching by 31%. The same process applied to hardwood pulp eliminated 41.8% of chlorine required for bleaching.

Methods A method of removing chromophores from wood pulp is disclosed, using the combination of a pore-enlarging agent, under conditions not

substantially degrading the cellulose, and a xylanase, under conditions wherein the lignan is removed from the cellulose. Several variations of the method have been developed.

Treatment with Cellulase followed by Xylanase In a first embodiment, the method includes the steps of first preparing a wood pulp, adjusting the wood pulp to between 1% and 15% consistency (weight %), adjusting the pulp to a pH of between pH 3.0 to 7.0 to maximize cellulase activity, treating the wood pulp with a cellulase to alter the pulp-fiber-pore-structure, adjusting the pulp to between a pH between pH 7.0 and 10.0 to inhibit cellulase activity and maximize xylanase activity, treating the pulp with a xylanase (wherein the xylanase is capable of releasing chromophores from the pulp), and then extracting the pulp to remove chromophores. In a particularly preferred embodiment, the wood pulp is a kraft pulp, the cellulase is obtained from Aspergillus niger, and the xylanase is ICI Ecopulp X-200 (ca 20,000 kDa).

Treatment with an Amine followed by Xylanase Alternatively, the color is removed from a wood pulp using the steps of first preparing a wood pulp, adjusting the wood pulp to between 1% and 15% consistency (weight %), adding an organic solvent to the pulp (to between 0.01 wt % and 20 wt%), treating the pulp/solvent mixture with a xylanase (wherein the xylanase is capable of releasing chromophores from the pulp), extracting the pulp to remove chromophores, and then heating the pulp/solvent mixture to remove the organic solvent from the pulp. In a particularly preferred embodiment, the wood pulp is a kraft pulp, the solvent is methylamine, the methylamine concentration in the pulp/methylamine mixture is between 1 wt% and 4 wt%, and the xylanase is ICI Ecopulp X-200 (ca 20,000 kDa).

Pre-treatments Color can be removed from a wood pulp wherein the wood pulp contains secondary fiber. This method contains the steps of preparing a wood pulp, treating the wood pulp with xylanase (wherein the xylanase is capable of releasing chromophores from the pulp) and extracting the pulp

to remove chromophores. The method of removing chromophores from wood pulp containing the steps of preparing a wood pulp, treating the wood pulp with xylanase, wherein the xylanase is capable of releasing chromophores from the pulp. Chromophores are released from the pulp and the pulp is then extracted with an alkaline solution. The pulp is then subjected to bleaching, preferably oxygen bleaching.

Reduction in % Lignin The amount of lignin remaining after treatment can be analyzed using the method described in Llyama and Wallis, Wood Sci. Tech., 22 (3): 271-272 (1988).

The present invention will be further understood by reference to the following non-limiting examples.

Example 1: A priori estimation of xylanase delignification Methods and Materials The relationship between xylanase-accessible-lignin moieties and xylanase-molecular-size was assumed to map identically to the log-normal distribution of cumulative-pore-volume versus pore-size for spruce kraft pulp (Allan, et. al., 1991; Scallan, 1978; Stone and Scallan, 1968).

Xylanase molecular size was assumed to be twice the radius of gyration obtained from the general relationship between protein molecular weight and radius of gyration (Tyn and Gusek, 1990). The molecular size thus obtained was confirmed with molecular mechanics using xylanase x-ray structure (1BCX ; mw=23, 359 Da) from the Brookhaven Protein Data Base (PDB) (Brookhaven Protein Data Base. October 1995 release. Brookhaven National Laboratory, Upton, NY 11973-5000) and SYBYL 6.0 (Tripos Associates, 1996) running on a Silicon Graphics 4D/310 GTX.

Experimental xylanase-only delignification Ecopulp X-200 xylanase (ICI) (mw=20, 000 Da) was a kind gift of ICI Canada Inc., Ontario. Irgazyme 40 xylanase (Ciba) (mw=67, 000 Da) was a kind gift of Ciba-Geigy Corp., Greensboro, NC. UGA xylanase (mw=39, 000 Da) (UGA) was produced according to the method of Maiti (Maiti, 1996). Pulp was a kind gift of the Pulp and Paper Research

Center, Georgia Institute of Technology, Atlanta, GA. In separate experiments, 50 units of xylanase were added to a mixture of 2 grams (oven dry basis) of softwood pulp (yield 45%) in 100 ml of 50 mM phosphate buffer. Mixture pH values were adjusted to pH=5.5, pH=7.0, and pH=9.0 for ICI, Ciba, and UGA, xylanases respectively. The mixtures were gently agitated and incubated for 2 hours at 55°C, 60°C, 70°C for ICI, Ciba, and UGA xylanases, respectively. Temperature and pH values were optimum for each xylanase's activity. After incubation, each pulp mixture was filtered, and washed with 100 ml each of deionized (D. I.) water, 0.5 N NaOH, D. I. water, 0.5 N HCI, and D. I. water. Pulp was then assayed for residual lignin (Iiyama and Wallis, Wood Sci. Technol., 22 (3), 271-272 (1988)). Control pulp samples were treated identically to the experimental samples except no xylanase was added.

Xylanase delignification of cellulase treated pulp Cellulase, 1 mg, (0.3 units) (Sigma C-1184) was added to a mixture of 2 grams (oven dry basis) of softwood pulp in 100 ml of 50 mM phosphate buffer (pH=5.5). The mixture was gently agitated and incubated for 10 minutes at 35°C. Temperature and pH values were optimum for cellulase activity. After 10 minutes, mixture pH and temperature were rapidly changed to the optimum pH and temperature values described earlier for ICI and Ciba xylanases. The xylanase delignification procedure described earlier was then followed for ICI and Ciba xylanases. A separate control was used where no xylanase was added to the mixture after cellulase pre-treatment. In this control, the pH and temperature were changed to optimum values for ICI and Ciba xylanases, respectively. Residual lignin was determined as before.

Experiments using cellulase-treated pulp followed by ICI xylanase along with the cellulase-only control were repeated and scaled-up to use 80 grams (oven-dry basis) of softwood and hardwood pulp samples.

Residual lignin was determined as before. The pulp-product from each scaled-up experiment was evaluated at the Pulp and Paper Research Center (Georgia Institute of Technology, Atlanta, GA) to determine Kappa

number (TAPPI a). Pulp product from each experiment was also bleached using the C+DEoDD bleaching sequence to determine viscosity (TAPPI b), brightness (TAPPI c) and chlorine reduction (C+D=chlorine + chlorine dioxide, Eo=NaOH extraction reinforced with 0,, D=chlorine dioxide).

Results A priori estimation of xylanase-only delignification Xylanase from the PDB had maximum dimension of 40 Angstroms.

Based on this result, estimates of the maximum dimension values for ICI, UGA, and Ciba xylanases were 41 Angstroms, 48 Angstroms, and 56 Angstroms, respectively. Using these maximum dimension values and the cumulative pore volume distribution, estimates for maximum lignin removal were 50%, 40%, and 32% of original pulp lignin.

Experimental xylanase-only delignification Lignin removed by ICI, UGA and Ciba was 48%, 39%, and 30% respectively. Fitting these data to the log-normal distribution of cumulative pore volume versus pore-size yielded a median pore size of 40.5 Angstroms, with standard deviation of 2.1 Angstroms (Allan et. al., 1991). Lignin removal from control pulp samples was non-detectable.

Experimental xylanase delignification of cellulase treated pulp Softwood pulp. Lignin removed from softwood pulp by ICI and Ciba xylanases was 67.7% and 40.4% respectively. These data were fitted to the log-normal distribution of cumulative pore volume versus pore-size, which yielded a median pore size of 66.7 Angstroms (standard deviation of 2.1 Angstroms) for cellulase-treated pulp.

In the scaled-up experiments, softwood pulp had a Kappa number of 26.7 and a viscosity of 22.72 cp prior to any enzyme treatment. Pulp brightness, viscosity, and Kappa number after the bleaching sequence were 85.5%, 9.12 cp, and 19.8, respectively. Softwood pulp treated with enzyme before bleaching required 31% less chlorine than the control.

Lignin removal from control softwood pulp samples: pulp treated with no enzyme, and, pulp treated with cellulase and no xylanase, was non- detectable.

Hardwood pulp. Hardwood pulp had a Kappa number of 12.7 and viscosity of 27.5 cp prior to any enzyme treatment. Pulp brightness, viscosity, and Kappa number after the bleaching sequence were 89.0%, 13.75 cp, and 6.4, respectively. Hardwood pulp treated with enzyme before bleaching required 41.8% less chlorine than the control.

As before, lignin removal from controls: pulp treated with no enzyme, and, pulp treated with cellulase and no xylanase, was non- detectable.

Conclusions The purpose of this study was to explore putative relationships between xylanase molecular size, pulp pore structure, and xylanase pulp delignification. Except for the assumption that xylanase must be transported within pulp pores to effect lignin removal, no catalytic mechanism of xylanase delignification was considered. Literature reports of pulp pore-size distribution allowed estimation of lignin removal for xylanases of any size. These estimates indicate the molecular size of xylanases used commercially in pulp mills are within the range of softwood pulp pore-sizes. Based on this finding we conclude that pulp- pore structure along with xylanase molecular size can possibly affect xylanase delignification of kraft pulp.

Xylanases with molecular weights of 20,000 Da, 39,000 Da, and 67,000 Da removed 48%, 39%, and 30% of lignin respectively from softwood kraft pulp. These data, where initial pulp pore-size remained constant and xylanase size varied, confirm the finding that lignin removal from typical softwood kraft pulp is a function of xylanase molecular size.

The goal of cellulase treatment of pulp was to increase accessibility of lignin moieties to xylanase. Xylanases were used as probes to provide a measure of accessibility based on lignin removal. Cellulase treatment increased accessibility presumably by increasing the median pulp pore diameter. This result, where pore-size was apparently increased and xylanase size remained unchanged, provides additional support for our proposition that the pulp pore-size, xylanase-molecular-size relationship

determines lignin removal. Decreases in Kappa number values for xylanase delignification of cellulase-treated softwood and hardwood pulp, confirm lignin-removal results obtained earlier. Based on this study, it is estimated that using typical bleaching sequences, chlorine could be reduced by 31% for softwood pulp and could possibly be eliminated from hardwood bleaching with no deleterious effects on pulp physical properties (Hsieh and Dhasmana, Pulp and Paper Research Center, Georgia Institute of Technology, Atlanta, GA., 1996).

Example 2: Isolation of Xylanase-producing Organisms Materials and Methods Organism Isolation Soil samples were suspended in sterile water, transferred to agar plates (yeast extract 1 gram/liter, polypeptone 1 gram/liter, agar 15 grams/liter, phosphate buffer 50 mM, pH=7) and incubated at 56°C for 48 hours. Colonies were transferred to xylan agar plates (xylan 10 gram/liter, yeast extract 1 gram/liter, polypeptone 1 gram/liter, agar 15 gram/liter in phosphate buffer 50 mM, pH 9) and incubated at 56°C. Sterile xylan agar was hazy so a clear zone surrounding a colony was presumptive for xylanase secretion. A single zone-clearing colony was transferred to fresh xylan agar, incubated, and stored at-85°C on glycerol slants. A single colony from this incubation was submitted to ATCC for identification and storage.

Growth parameters Stock culture was prepared by transferring colonies from storage to xylan media (yeast extract 1 gram/liter, xylan 5 gram/liter, polypeptone 1 gram/liter in 50 mM phosphate buffer, pH=7) and incubated in shake flasks (Innova 3000, New Brunswick Scientific Co. Inc., Edison, NJ., USA) at 56°C 120 rpm for 28 hours. Xylan media (100 ml) was added to each of 24 shake flasks. Seven groups with three flasks each were adjusted to pH 5,6,7,8,9,10, and 11 respectively, and inoculated with ten ml of stock culture; three flasks were sterile controls. The flasks were incubated in a shaker at 56°C, and 120 rpm for 30 hours. Media from each

flask was centrifuged (Dupont Sorvall RC 28S, Newton, CT, USA) at 9000 x g for 15 minutes. The pellet from each culture was dried at 75°C.

Net growth for each flask was computed by subtracting the dry mass of the sterile control from that of the sample. Xylan media (100 ml, pH=8.7) was added to each of 15 shake flasks. Twelve flasks were inoculated with ten ml each of stock culture. Each group of flasks were separately incubated in a shaker (120 RPM) for 30 hours at 40°C, 60°C, 70°C, and 80°C respectively. Media from each flask was centrifuged (Dupont Sorvall RC 28S, Newton, CT, USA) at 9000 x g for 15 minutes. The pellet from each culture was dried as before. Net growth for each flask was computed as before.

Xylanase Analysis and Preparation Xylanase activity was determined using the dinitrosalicylic acid (DNS) method (Miller Anal. Chem. 31,426-428 (1959); Gruninger and Fiechter Enzyme Microb. Technol., 8 (May), 309-314 (1986)). Assay temperature and pH were 65°C and 8.7 unless otherwise specified. Ten ml of stock culture was transferred to 100 ml xylan media (pH=8.7) in shake flasks. Each hour during incubation (70°C, pH=8.7), samples were drawn from the flasks, centrifuged, and the supernatant assayed for xylanase activity. When maximum activity was achieved, flask contents were centrifuged at 9000 x g for 15 minutes. Xylanase in the supernatant was concentrated via ultrafiltration according to the procedure detailed by Maiti,"Advanced Delignification Technology", Ph. D. Dissertation, University of Georgia, Athens, GA 30602 (1996). Xylanase molecular weight was determined by SDS-PAGE using a PhastSystem (Pharmacia, Uppsala SWEDEN). Protein was determined according to Lowry (Lowry, et. al., J. Biol. Chem. 193,265 (1951)).

The effect of pH on xylanase activity was determined with the DNS assay modified to use 50 mM phosphate buffer at pH values of 6,7, and 8, and 50 mM glycine-NaOH buffer at pH values 9,10,11. The effect of temperature on xylanase activity was determined by using the DNS assay at 40°C, 50°C, 60°C, 70°C, and 80°C at pH 9.

Kinetic parameters were estimated using the DNS procedure (65°C and pH 8.7) to obtain initial velocity values at xylan concentrations from 0.4 mg/ml to 5.0 mg/ml. Softwood pulp was treated with xylanase according to the procedure of Maiti (1996). Lignin was measured using the acetyl bromide procedure of Iiyama and Wallis (1988).

Example 3: Xylanase-only Delignification Materials and Methods Ecopulp X-200 xylanase (ICI) (mw=20, 000 Da) was a kind gift of ICI Canada Inc., Ontario. Irgazyme 40 xylanase (Ciba) (mw=67, 000 Da) was a kind gift of Ciba-Geigy Corp., Greensboro, NC. UGA xylanase (mw=39, 000 Da) (UGA) was produced according to the method of Maiti (Maiti, 1996). Pulp was a kind gift of the Pulp and Paper Research Center, Georgia Institute of Technology, Atlanta, GA.

In separate experiments, 50 units of xylanase was added to a mixture of 2 grams (oven dry basis) of softwood pulp (yield 45%) in 100 ml of 50 mM phospate buffer. Mixture pH values were adjusted to pH=5. 5, pH=7.0, and pH=9.0 for ICI, Ciba, and UGA xylanases respectively. The mixtures were gently agitated and incubated for 2 hours at 55°C, 60°C, 70°C for ICI, Ciba, and UGA xylanases respectively.

Temperature and pH values were optimum for each xylanase's activity.

After incubation, each pulp mixture was filtered, and washed with 100 ml each of D. I. water, 0.5 N NaOH, D. I. water, 0.5 N HCI, and D. I. water.

Pulp was then assayed for residual lignin (Iyama and Wallis, 1988).

Control pulp samples were treated identically to the experimental samples except no xylanase was added.

Example 4: Xylanase Delignification of Cellulase Treated Pulp Materials and Methods Cellulase, 1 mg, (Sigma C-1184) was added to a mixture of 2 grams (oven dry basis) of softwood pulp in 100 ml of 50 mM phospate buffer (pH=5.5). The mixture was gently agitated and incubated for 10 minutes at 35°C. Temperature and pH values were optimum for cellulase activity. After 10 minutes, mixture pH and temperature were rapidly

changed to the optimum pH and temperature values described earlier for ICI and Ciba xylanases. The xylanase delignification procedure described earlier was then followed for ICI and Ciba xylanases. A separate control was used where no xylanase was added to the mixture after cellulase pre- treatment. In this control, the pH and temperature were changed to optimum values for ICI and Ciba xylanases respectively. Residual lignin was determined as before.

Experiments using cellulase-treated pulp followed by ICI xylanase along with the cellulase-only control were repeated and scaled-up to use 80 grams (oven-dry basis) softwood and hardwood pulp samples. Residual lignin was determined as before. Kappa number values were determined for the pulp-product from each scaled-up experiment [TAPPI, 1960].

Results of treatment with cellulase then xylanase 2 grams of softwood pulp was treated with 1 mg solid of cellulase and thereafter with 1 ml xylanase (ICI Ecopulp X-200; 13,000 birch xylan international units/ml, BXIU/ml) (55°C, pH=6. 5). % lignin reduction by AcBr trial 1 90.4 trial2 63. 6 trial 3 71. 8 2 grams of softwood pulp was treated with 1 mg solid of cellulase and thereafter with 1 ml xylanase (CIBA IRGAZYME 40; 1,000 XAU units/ml) (60°C, pH=7.5). % lignin reduction by AcBr trial 1 70. 7 trial 2 40. 7 trial3 40. 1

80 grams of softwood pulp was treated with 40 mg solid of cellulase and thereafter with 5 ml xylanase (ICI Ecopulp X-200; 13,000 birch xylan international units/ml, BXIU/ml) (55°C, pH=6.5). Kappa number reduction = 26.3-19.9 = 6.4; percentage lignin reduction (AcBr) = 83.1%; viscosity reduction= 22.7 cp-14.3 cp = 8.4 cp; final brightness (based on CEDED) = 85.9% (ISO 83% brightness achieved) 80 grams of hardwood pulp was treated with 40 mg solid of cellulase and thereafter with 5 ml xylanase (ICI Ecopulp X-200; 13,000 birch xylan international units/ml, BXIU/ml) (55°C, pH=6.5). Kappa number reduction = 12.8-6.56 = 6.24; chlorine reduction (based on [C+D Eo D1 D2] sequence) = 47.8%; final brightness= 89% (ISO 83% brightness achieved).

80 grams of softwood pulp was treated with 40 mg solid of cellulase and thereafter with 5 ml xylanase (ICI Ecopulp X-200; 13,000 birch xylan international units/ml, BXIU/ml) (55 o C, pH=6.5). Kappa number reduction = 26.3-20.3= 6.0; chlorine reduction (based on [C+D Eo Dl D2] sequence) = 31% ; viscosity reduction (based on complete sequence) = 13.6 (standard is 11 to 12 cP); final brightness= 85.5% (ISO 83% brightness achieved).

Xylanase was prepared according to the method of Maiti (Maiti, 1995). In all cases, 50 mM phosphate buffer (pH=8.7), 2% consistency pulp, and 63°C temperature was used. Xylanase activity was assayed by the DNS procedure of Miller (Miller, 1959). Lignin was assayed by the procedure of Iiyama and Wallis (Iiyama, 1988).

Example 5: Methylamine use in pulp delignification Methods and Materials Xylanase was prepared according to the method of Maiti (Maiti, 1995). In all cases, 50 mM phosphate buffer (pH=8.7), 2% consistency pulp, and 63°C temperature was used. Xylanase activity was assayed by the DNS procedure of Miller (Miller, 1959). Lignin was assayed by the procedure of Iiyama and Wallis (Iiyama, 1988).

Methylamine effect on xylanase. Xylanase was assayed in buffer of 0% and 10% methylamine respectively.

Methylamine effect on xylanase delignification. Xylanase, pulp, and methylamine were combined to yield a final concentration of 0%, 4% or 10% methylamine in a final volume of 100 ml and incubated for 2 hours as indicated in Table 1.

Results Xylanase activity in 10% methylamine was 61% of the control value in 0% methylamine. Control experiments used no xylanase and no methylamine; lignin remaining in pulp after control treatment was considered as 100%.

Table 1 % lignin remaining in pulp Xylanase,Control 4% ml methylamine 0 100. 0 59. 5 1 68. 2 53. 0 2-51. 8 Table 2

% lignin remaining in pulp Xylanase,Control 10% ml methylamine 0 100. 0 67. 6 1 65. 0- 2 47. 4 46. 4 When 4% methylamine was used in the absence of xylanase, about 60% of the lignin remained in the pulp. When 1 mL of a xylanase solution was used, about 68% of the lignin remained in the pulp.

However, when the xylanase and methylamine were used simultaneously, the lignin content dropped to about 53%. Doubling the amount of xylanase dropped the lignin content to about 47%. The combination of the increased xylanase and 4% methylamine was slightly less effective, resulting in 52% remaining lignin.

The experiment was repeated using a 10% concentration of methylamine. Using methylamine alone, the lignin content was reduced to about 68% of the original value. Using 1 mL of the xylanase solution, the remaining lignin was about 65% of the original value. When the amount of xylanase was doubled, the amount of residual lignin was reduced to about 47%. However, when the treatment included 10% methylamine and 2 mL of a xylanase solution, the lignin content was reduced to about 46%.