BACKGROUND OF THE INVENTION Lignin is a naturally occurring polymeric material found in the cell walls of vascular plant material such as wood. High molecular weight lignin components present in pulp formed during chemical pulping of wood to produce paper tend to discolor the resulting paper products. It is preferred to remove lignin during the paper production process. Established techniques for removing lignin contaminants during paper production include chemical bleaching of the wood pulp. Chemical treatments can produce dioxin-like compounds that are environmentally unfriendly.

Accordingly, there exists a need for alternative substances and methods for removing lignin during the bleaching process.

SUMMARY OF THE INVENTION This invention features lignin depolymerase enzymes that are effective for depolymerizing lignin in vitro and in vivo.

In one aspect, the invention features a lignin depolymerase containing a lignin depolymerase polypeptide. The lignin depolymerase may have an apparent molecular weight of about 35,000 daltons. It does not require a hydrogen peroxide substrate. In some embodiments, the lignin depolymerase contains a polypeptide having a pI ranging from about 4 to about 6, a pI of about 4.9, a pI of about 5.8, or any combination thereof. The lignin depolymerase may include a polypeptide isolated from a white-rot fungus Trametes cingulata. The lignin depolymerase may be isolated from natural sources or expressed in vitro using recombinant DNA technologies.

In other embodiments, the lignin depolymerase polypeptide is effective for depolymerizing lignin. The depolymerization can take place in vitro, for example in an industrial paper production process. In particular, the lignin depolymerase may be effective for depolymerizing softwood kraft pulp brownstock.

In another aspect, the invention features an article of manufacture containing a lignin depolymerase polypeptide contained within a packaging material wherein the polypeptide is effective for depolymerizing lignin. In some embodiments, the lignin is kraft pulp such as softwood kraft pulp brownstock. Further, the article of manufacture can be used in conjunction with any of the lignin depolymerase polypeptides or combinations of peptides described herein.

In another aspect, the invention features a method for isolating a polymerized lignin derivative by 1) culturing a white-rot fungus in the presence of lignin so as to produce polymerized lignin; and then 2) isolating the polymerized lignin from the culture. In certain embodiments, the culture is grown through or past a primary growth phase. The method may include the step of ultrafiltering the polymerized lignin.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph having a series of curves depicting the elution profiles for a Sephadex (TM) G100 column loaded with degradation products of high molecular weight polymerized kraft lignin samples catalyzed by a lignin depolymerase having a pI of approximately 4.9.

FIG. 2 is a graph having a series of curves depicting the elution profiles for a Sephadex (TM) G100 column loaded with degradation products of high molecular weight polymerized kraft lignin samples catalyzed by a lignin depolymerase having a pI of approximately 5.8.

FIG. 3 is a graph having a series of curves depicting the elution profiles for a Sephadex (TM) G100 column loaded with high molecular weight polymerized kraft lignin samples incubated in a buffer lacking a lignin depolymerase.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention provides a lignin depolymerase source that is useful for depolymerizing lignin. The lignin depolymerases may be one or more individual lignin depolymerases derived from any source and include natural and/or recombinant lignin depolymerases. A lignin depolymerase composition can include, for example,

a single isolated lignin depolymerase or one or more lignin depolymerases that are combined to form a mixture of lignin depolymerases. Natural lignin depolymerases are lignin depolymerases that are isolated from cells or cultures that naturally produce lignin depolymerase. Recombinant lignin depolymerases are lignin depolymerases produced using recombinant DNA technologies. Recombinant DNA technologies include known prokaryotic and eukaryotic expression systems including expression systems useful for expressing exogenous proteins in E. coli, fungi, yeast, insect cells, and other similar systems. Recombinant DNA technologies can be used to express natural lignin depolymerases and genetically engineered lignin depolymerases.

Recombinant lignin depolymerases can include polynucleotide coding sequences of lignin depolymerases operably linked to a regulatory sequence and/or polynucleotide coding sequences of genetically engineered lignin depolymerases operably linked to a regulatory sequence. A genetically engineered lignin depolymerase is a lignin depolymerase containing one or more amino acids not found in a naturally occurring lignin depolymerase.

Useful lignin depolymerases include, in particular, lignin depolymerases isolated from the white rot fungus Trametes cingulata (T. cingulata). Lignin depolymerases having a pI ranging from about 4 to about 6 can be used. Preferred lignin depolymerases have a pI of about 4.9 or about 5.8. Lignin depolymerases, including lignin depolymerases having a pI ranging from about 4 to about 6, may be used alone or in combination.

Lignin depolymerases can be isolated from T. cingulata by culturing the white rot fungus under known conditions that lead to growth of the fungus. The fungus can be allowed to grow through or past the primary growth phase, which is often accompanied by the polymerization of the lignin derivative present in the culture solution. At a point where the polymerization of the lignin derivative slows or stops and the T. cingulata culture exits the primary growth phase, protease inhibitors can be added. Useful protease inhibitors include phenylmethanesulfonyl fluoride (PMSF) and Pepstatin A. The protease inhibitors can be added to a final concentration of about 0.05 mM or about 1 mM or any other effective concentration. After adding the protease inhibitor, the culture solution can be filtered and concentrated.

The lignin depolymerases can be isolated from the concentrated culture solution using any method including isoelectric focusing, gel chromatography, affinity purification and any other known method for protein purification.

Any polymeric lignin can be depolymerized. Useful lignin sources for depolymerization include wood pulp lignin, kraft lignin, softwood kraft pulp brownstock, and lignin polymerized by cultures of the white-rot fungus T. cingulata.

Isolated lignin depolymerases can be packaged and sold as an article of manufacture. Methods for manufacturing articles of manufacturing are known. The lignin depolymerases can be packaged in the articles of manufacture in any form including in solution, frozen, and/or lyophilized. Such an article of manufacture is useful for depolymerizing kraft pulp.

The invention also features a method of doing business that includes the steps of offering for sale a lignin depolymerase and advertising or communicating to an appropriate audience that the depolymerase is effective for depolymerizing lignin in paper production facilities.

The invention will now be more fully described by way of the following examples.

Example 1. Purification of Lignin Depolymerase A kraft lignin-containing culture of white-rot fungus T. cingulata was constituted in homogeneous aqueous solution as described in Phytochemistry, 49, 1203-1212 (1998), which is hereby incorporated by reference in its entirety. The T. cingulata culture underwent a primary vegetative growth phase. During the primary growth phase, the kraft lignin underwent polymerization. After the primary growth phase, degradation of the polymerized kraft lignin in the T. cingulata culture was observed. Therefore, after the T. cingulata culture exited the primary growth phase and when significant kraft lignin depolymerization had begun, the culture solution was filtered through Mira-cloth (TM). Protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and Pepstatin A were added to the filtrate. The PMSF and Pepstatin A had apparent final concentrations of 1.0 mM and 0.05 mM, respectively. The filtrate was stored for 16-24 h and then any excess solid PMSF and/or Pepstatin A was filtered off leaving a solution. The solution was concentrated using an Amicon PM-10 (TM) ultrafiltration membrane available from Millipore Corporation. After

the initial concentration, the filtrate was equilibrated with 10 to 15 volumes of 0.020 M acetate buffer (pH 4.5) using the PM-10 membrane.

The equilibrated T. cingulata culture filtrate was concentrated from 30-to 80- fold using a PM-10 ultrafiltration membrane and loaded on the anode side of a pH 4.0 -6.5 ampholine polyacrylamide isoelectric focusing gel available from Amersham Pharmacia Biotech. After isoelectric focusing was complete, two proteins with apparent pI's of about 4.9 and about 5.8 were identified in the isoelectric focusing gel and separately electro-eluted from the corresponding ampholine gel segments using an Amicon Centrilutor micro-electroeluter (TM) available from Millipore Corporation according to known methods.

To increase the amount of protein recovered, several gel slices of the appropriate protein, i. e., pI 4.9 or pI 5.8, were placed vertically into a suitable number of 0.5 ml microcentrifuge tubes. Each microcentrifuge tube had narrow circular holes bored at the top and bottom of the tube. Each microcentrifuge tube was then placed in an Amicon Centricon-10 concentrator (TM) fitted with a YM-10 ultrafiltration membrane at a position above the YM-10 membrane. The Centricon-10 concentrator unit was pre-treated with an aqueous 5% polyethylene glycol solution for 12-16 hours and washed with (distilled) water before the electroelution. The entire centricon-10 and microcentrifuge tube assembly was then positioned in the micro-electroeluter between the upper and lower chambers, both of which were filled with aqueous 30 mM Tris-phosphate buffer at a neutral pH, e. g., pH ranging from about 6.5 to about 7.5. Useful pH values used include 7.0 and 7.2. The pH of the electroelution solution was monitored during electroelution to ensure that the pH remained stable. Also, any bubbles appearing beneath the YM-10 membranes in the concentrator units were removed as they formed.

Lignin depolymerase enzymes with apparent pI's of approximately 4.9 and approximately 5.8 purified as described above were run on an SDS PAGE gel to estimate their respective molecular weights. The apparent molecular weights of both proteins were 35,000 daltons when run on the 10% polyacrylamide SDS gel.

Example 2. Isolation of Polymerized Kraft Lignin A white-rot fungus T. cingulata culture was prepared according to the method described in Example 1 and cultured through the primary growth phase. Before

depolymerization of the kraft lignin started, the filtered extracellular culture solution was ultrafiltered with 10 volumes of aqueous 0.020 M acetate at pH 4.5 through an Amicon PM-10 ultrafiltration membrane available from Millipore Corporation. The pH of the retentate was adjusted to pH 13 with 0.50 M NaOH. The retentate was diluted and underwent additional ultrafiltration using an Amicon YM-100 membrane (available from Millipore Corporation) in a 0.10 M NaOH solution until the filtrate exiting the filtration device was colorless. The solution pH was then adjusted to approximately 7.5 by exchanging the NaOH solution in the ultrafiltration device using double distilled water. The polymerized lignin solution was then centrifuged for 20 min at 47,900 x g to remove any particulate material. Aliquots of the polymerized lignin were frozen and then freeze-dried.

Example 3. Degradation of Polymerized Kraft Lignin in Homogeneous Solution Aliquots of polymerized kraft lignin prepared according to the method of Example 2 were diluted in 0.020 M acetate at pH 4.5. Protein samples of depolymerases prepared according to the method of Example 1 were added to the diluted kraft lignin solution. The aliquots were incubated at ambient temperature for 0 to 21 days.

The extent of kraft lignin depolymerization was measured using size-exclusion chromatography. Samples of the depolymerase reactions were loaded on a Sephadex (TM) G-100 column equilibrated with 0.10 M NaOH. Elution of each sample was monitored at 280 nm using a UV/visible detector attached to the G-100 column.

Absorbance was monitored as a function of eluent volume exiting the G-100 column, which is a measure of the molecular weight of the compounds exiting the column.

As shown in FIG. 1, the lignin depolymerase having a pI of 4.9 depolymerized high molecular weight polymerized kraft lignin for at least 21 days. In FIG. 1, samples were run at 0.025 days, 5 days and 21 days.

As shown in FIG. 2, the lignin depolymerase having a pI of 5.8 depolymerized high molecular weight polymerized kraft lignin for at least 10 days. In FIG. 2, samples were run at 0.025 days, 5 days and 10 days.

As shown in FIG. 3, incubating high molecular weight polymerized kraft lignin without a lignin depolymerase did not result in depolymerization of the kraft lignin for at least 10 days. In FIG. 3, samples were run at 1 day and 10 days.

As such, both polypeptides isolated according to the methods described herein degraded high molecular weight kraft lignin components that had been previously polymerized by the white-rot fungus.

Example 4. Delignification of Kraft Pulp Lignin depolymerase isoenzymes are believed to be the active catalytic agents in the delignification of industrial softwood kraft pulp brownstock by T. cingulata culture solution. See N. Nutsubidze and S. Sarkanen, Proc. 9th Int'l Symp. Wood Pulp. Chem., G6,1-5 (1997), which is hereby incorporated by reference. A T. cingulata culture solution as described in Example 1 was prepared and allowed to develop for 30-35 days. After 30-35 days of incubation, the culture was depolymerizing the kraft lignin substrate in the culture medium. T. cingulata cultures were pooled, filtered through glass wool, centrifuged at 36,000 x g for 30 minutes and filter-sterilized by passing the supernatant through a 0.2 micron serum acrodisc (TM) (Gelman No. 4525). PMSF (final concentration 0.13 g/L) was added to the cell-free filter-sterilized solution. After 16 hours, the protease inhibitor containing solution was filtered to remove the remaining solid inhibitor and then subjected to ultrafiltration using a 10,000 nominal molecular with cutoff membrane (PM-10 available from Amicon Corporation) using twenty volumes of 0.02 M acetate buffer (pH 4.5). The filtered supernatant was concentrated 3-fold and then removed from the ultrafiltration cell.

Unbleached industrial softwood kraft pulp, primarily from Jack pine (Pinus banksiana), was washed with distilled water and then autoclaved at 121°C for 30 min. to achieve a sterilized pulp. The pulp underwent a second wash with distilled water.

The resulting brownstock was then extracted with aqueous 0.40 M NaOH for 5 min at room temperature and thoroughly washed with distilled water. Samples of the softwood kraft pulp (2.5 g, 42% consistency) were apportioned into individual 500- mL conical flasks and autoclaved for a second time at 121°C.

These 2.5 g samples of the sterilized softwood kraft pulp brownstock (42% consistency) were immersed in 50 ml aliquots of the 3-fold concentrated cell-free T.

cingulata culture solution to which flavin adenine dinucleotide (FAD) had been added bringing the final FAD concentration to 1.6 x 10 M. The FAD had been sterilized by passing a stock solution through a 0.2 micron serum acrodisc available from Gelman Sciences (No. 4525). Although FAD was not required and its use is not meant to indicate a particular mode of action, FAD was added to facilitate enzyme turnover.

The resulting mixtures were incubated without agitation at ambient temperature in 500 ml conical flasks. The mouth of each flask was equipped with a neoprene stopper fitted with a glass tube, into which a glass wool plug had been loosely inserted to allow air-exchange. Kappa numbers for measuring pulp delignification were then determined according to TAPPI Test Method T236 cm-85, which is hereby incorporated by reference in its entirety, after thorough washing of the pulp with distilled water before and after extraction for 5 min using 30 ml of a 0.10 M NaOH solution at room temperature. The kappa number of the softwood kraft pulp brownstock was reduced by 50% after a single treatment of the pulp samples in this way with the cell-free T. cingulata culture solution.