Background of the Invention

The invention disclosed herein is useful in - several processes used by the pulp and paper in¬ dustry. During pulping processes, cellulosic fibers must be liberated from their encasing lignin matrix so that they can associate with one another, yielding strength in the final product. This polymer separa- tion can be accomplished by removal of lignin as in chemical pulps, or by maintaining the lignin as in high yield mechanical pulps. During the bleaching process, lignin is removed and the resulting pulp is brightened. The secondary cell wall of wood, composed of cellulose fibrils, hemicellulose and lignin, imparts physical strength and rigidity to woody plants. The cellulose fibrils are densely packed and surround the cell in regular parallel arrays, or in criss- cross layers. These fibrils are held together by a matrix of hemicellulose and lignin.

Cellulose is the most abundant component of woody tissue, comprising 35-45% of the dry weight. Cellulose is an ordered linear polymer of glucose monomers couples by £-1,4 bonds. The hemicelluloses are branched polymers composed of pentose (5-carbon) monomers, normally xylose and arabinose; and hexose (6-carbon) monomers, consisting of glucose, galac- tose, mannose and substituted uronic acid. Lignin is an extremely complex polymer formed by the free radical polymerization of substituted

cinn.am.yl alcohol procursors. Lignin constitutes 15-35% of dry wood weight.

Lignin is highly resistant to biological attack; not a surprising finding considering the complexity and stability of lignin structure. No - organism has been demonstrated to grow on lignin as the sole carbon source. The complex lignin polymer, however, is completely degraded by pure cultures of various higher order fungi. For reviews see Higuchi (1982) Experientia 38: 159-166, and Janshekar, H. and Feichter, A. (1983) "Advances in Biochemical Engineering/Biotechnology," A. Fiechter and T. . Jeffries, Eds., Vol. 27, pp. 119-178, Springer, Berlin; Kirk, T.K. (1984) in "Biochemistry of Microbial Degradation," D.P. Gibson, Ed., pp.

399-437, Marcel Dekker, N.Y. The major degraders of "fully lignified" tissues (lignin >20%) are the basidiomyσetes that cause the white-rot type of wood decay. The most extensive physiological investiga- tions of lignin biodegradation by white-rot fungi have been conducted with a single member of the family Corticeaceae, Phanerochaete chrysosporium Burds.

Although . chrysosporium is capable of com- pletely degrading lignin, purified lignin will not support its growth. Purified cellulose, however, is a growth nutrient for these fungi. Lignin degrada¬ tion allows these fungi to expose the cellulose food source contained within the lignin matrix. Under defined laboratory conditions, fungal lignin degra¬ dation is not observed during the approximately

first 3 days of culture. Subsequently, the culture becomes starved for carbon or nitrogen. Lignin degradation is first observed one or two days later and is maximal at 6 days. The induction of lignin degradation in response to carbon and nitrogen starvation indicates that fungal lignin metabolism is a secondary metabolic event (Keyser, P., Kirk, T.K. and Zeikus, J.G. (1978) J. Bacteriol. 135:790- 797.) Fungal lignin degradation is commercially impractical for several reasons. The rate of lignin degradation is unacceptably slow since ligninolytic activity must be induced by starvation. Further¬ more, fungi metabolize cellulosic fibers as their primary .food source, resulting in reduced pulp yield and an inferior pulp product.

With regard to the major C-C and C-O-C inter- subunit linkages found in lignin, it is important to note that approximately 80% of intersubunit bonds involve linkages to the C__ or C^ carbons.

Tien and Kirk have disclosed a preparation capable of oxidatively cleaving C _Λ - Z bonds in lignin model compounds (Tien, M. and Kirk, T.K. (1984) Proc. Natl. Acad. Sci. 81:2280-2284). This preparation displays on an SDS-polyacrylamide gel predominantly one protein with an apparent molecular weight of 42 kilodaltons and several minor bands. Thus the preparation is a mixture of proteins without any means suggested for isolating the dominant protein from the minor bands. Thus the preparation is a mixture of proteins without any means suggested for isolating the dominant protein

from the minor band. Subsequent to the publication of this paper, several scientific papers were published disclosing an inability to isolate the major protein from the mixture. These articles are as follows: Huynh, V-B and Crawford, R.L. (1985) - FEMS Microbiology Letters 28:119-123; Leisola, M. et al. (1985) Lignin Biodegradation Workshop; and Gold, M.H. e_t al . (1985) Lignin Biodegradation Workshop. These protein isolations have been done by either ion-exchange chromatography or size exclu¬ sion-ion exchange column chromatography. The fractions containing the indicated component have been analyzed by isoelectric focusing or SDS-poly¬ acrylamide gel electrophoresis, and have shown multiple proteins. The scientists who performed this work are at the forefront of the lignin enzyme field, as evidenced by their participation in the Lignin Biodegradation Workshop held in Vancouver, BC, in 1985. These failures suggest a seemingly insurmountable problem in resolving the prior art mixtures.

Isolation of the active component in the Tien and Kirk mixture is a highly desirable objective because, inter alia, the character of such mixtures has been found to substantially impede their prac¬ tical use. Specifically, Tien and Kirk and similar mixtures have been found to be highly unstable at practical storage temperatures and at those tempera¬ tures suitable for effecting their practical activi- ties. In particular, the useful activities of the Tien and Kirk mixture are substantially degraded in about 2 days time at room temperature and such

mixtures have been found to contain destructive or protein degrading native proteases as indicated by the well-known azocoll test.

In addition, there is a clear need to isolate and identify other enzymes which can be used to catalyze the degradation and modification of lignin.

Brief Summary of the Invention

The invention disclosed herein has successfully solved the problem by producing a substantially pure enzyme preparation, herein designated rLDM TM 6, which is substantially free of destabilizing pro- teases. Advantageously, the rLDM TM 6 preparation of the present invention possesses desirable properties for practical usage which the Tien and Kirk prepara- tion did not have.

Also, by working up the extracellular fluid from a culture of a novel strain of Phanerochaete chrysosporium in the manner hereinafter described, we have isolated not only rLDM TM 6, the apparent major protein in the Tien and Kirk mixture discussed above, but a series of other distinct lignin-de- grading enzymes which are substantially free of destabilizing native proteases. These enzymes have stability suitable for practical use, are imme- diately active and require no metabolic induction. The enzymes provided by the invention not only degrade lignin and effect various reactions as indicated on lignin model compounds but also will not attack cellulose or hemicellulose, and are

further indicated, as of particular significance, as useful in the selective degradation and modification of lignin in wood pulp, and hence, are indicated for use in operations in the pulp and paper industry in which such a result is sought. The various pro¬ perties of the enzyme preparation of the invention including their substantial purity which frees the enzymes from activity degrading native proteins is of distinct advantage of such usage. Hence, the subject invention concerns novel lignin-degrading enzymes which are called rLDM TM 1,

TM TM TM TM TM rLDM ^*1 2, rLDM 3, rLDM 4, rLD 5, and rLDM

6, each substantially free of native protein which naturally degrades these enzymes. These novel compounds, advantageously, possess- the properties of (1) reducing the amount of lignin in kraft pulp, (2) enhancing the strength properties of thermo echanical pulp (TMP) and (3) decolorizing

TM kraft lignin. The rLDM of the subject invention are characterized herein by the critical property of being able to catalyze the oxidation of veratryl alcohol to veratrylaldehyde, and the following physical parameters:

(1) molecular weight as determined by SDS- PAGE;

(2) a ino acid composition;

(3) heme content;

(4) homology of antibody reactivity;

(5) specificity of activity against lignin model substrates; and

(6) elution from a FPLC column at specified sodium acetate molarities.

The lignin-degrading enzymes of the invention referred to as rLDM TM, hheeirein, have previously been referred to as PuplasesT ^' M

Detailed Description of the Invention The isolation of the novel rLDM TM of the subject invention was facilitated by use of a novel stable mutant strain of Phanerochaete chrysosporium, which elaborates high amounts of ligninolytic enzymes into the fermentation medium. The novel mutant strain, designated SC26, has been deposited in the permanent collection of a public culture repository, to be maintained for at least 30 years. The culture repository is the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois 61604, USA. The accession number is NRRL 15978, and the deposit date is July 3, 1985. This deposited culture is available to the public as required by patent laws in countries wherein counter¬ parts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in dero¬ gation of patent rights granted by governmental action. Novel mutant SC26 was obtained by UV muta- genesis of the wild type Phanerochaete chrysos¬ porium, ATCC 24725.

Novel mutant SC26 was grown on a nitrogen- limited trace element medium supplemented with glucose and buffered at pH 4.5.

Ligninase activity in the fermentation medium was measured periodically by standard means deter¬ mining the rate of oxidation of veratryl alcohol to r veratrylaldehyde. Isolation and purification of the novel rLDM TM- of the subject invention from the extracellular fluid in the fermentation was accomplished by ultrafiltration and FPLC using an anion exchange column. Following are examples which illustrate the novel enzymes and procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percen¬ tages are by weight and all solvent ^' mixture pro- portions are by volume unless otherwise noted.

Example 1—Growth of Mutant SC26 (NRRL 15978) to Produce Fermentation Medium Containing Novel Ligninases

Inoculum was prepared by homogenizing 50 ml of 1.5-day cultures of mutant SC26 grown in 1 liter flasks containing the following medium, designated nitrogen-limited Bill/glucose medium:

The Bill medium contains 1.08 x 10 M ammonium tartrate, 1.47 x 10 -2 M KH-PO., 2.03 x 10-3 M MgS0 ₄ • 7H-0, 6.8 x 10~ ⁴ M CaCl- • 2H-0, 2.96 x 10 M thiamine»HCl and 10 ml»L~ of a trace element solution. The trace element solution contains 7.8 x 10 M nitriloacetic acid, 1.2 x 10~ ² M MgS0 ₄ • 7H-0, 1.7 x 10 ^~2 M NaCl, 3.59 x 10~ ⁴ M FeS0 ₄ • 7H ₂ 0, 7.75 x lθ ^"4 M CoCl ₂ , 9.0 x

10 ^"4 M CaCl-,, 3.48 x lθ ^"4 M ZnS0 ₄ • 7H-0, 4 x

10 ^"5 , M CuS0 ₄ • 5H ₂ 0, 2.1 x 10~ ⁵ M A1K(S0 ₄ ) ₂ «

12H20, 1.6 x 10 ^"4 M_ H3.B03-,' 4.1 x lθ ^"5 M NaMoO4.'

2H2_0 and 2.9 x 10 M MnSO4. • H2„0. The medium was supplemented with 10% (by,* wt/liter) of glucose.

The medium was buffered with 10 mM trans- aconitic acid, pH 4.5

Flasks (125 ml, containing 10 ml sterile medium having the above-described medium) were each inoculated with 0.5 ml of the above homogenate and kept stationary at 39°C. The flasks were flushed on days 0, 3, and 6 with water-saturated O-,. Alternatively, a rotating biological contractor (RBC) was used to grow the fungus. 2.5 liters of the above-described medium was inoculated with 100 ml of the above homogenate and grown at 39°C with the RBC rotating at 1 rpm with continuous oxygenation. Ligninase activity was measured periodically by determining the rate of oxidation of veratryl alcohol to veratrylaldehyde. Reaction mixtures contained 275 1 of extracellular fluid (from flasks or the RBC) , 2 mM veratryl alcohol, 0.4 mM H-O- and 0.1 mM sodium tartrate, pH 2.5 in a final volume of 0.5 ml. The reactions were started by H-0-, addition immediately after buffer was added and were monitored at 310 n . Protein was determined according to Bradford (Bradford, M.M. (1976) Anal. Biochem. 72:248-254) using bovine serum albumin (Sigma Chemical, St. Louis, MO) as standard.

Example 2—Isolation and Purification of the Novel rLDM TM

The extracellular growth media from cultures grown in flasks, as described above, was harvested by centrifugation at 5000 xG, 10 in, 4°C. Extra-,- cellular growth media was then concentrated by ultrafiltration through a 10K filter. The resulting concentrate is called the Ligninolytic Mixture TM The lignmolytic Mixture TM can contain one or more of rLDM TMs or other ligninolytic enzymes in varying proportions. The rLDM TM contained in this Lignino- lytic Mixture TM were separated by fast protein liquid chromatography (FPLC) using a Pharmacia Mono

Q column (Pharmacia, Piscataway, NJ) and a gradient of sodium acetate buffer, pH 6, from 10 mM to 1 M. rLDM TM 1, 2, 3, 4, 5, and 6 elute from the column a typical preparation at the following sodium acetate molarities, respectively: 0.16, 0.18, 0.34,

0.40, 0.58, and 0.43 M to give essentially pure

TM TM rLDM 1-6. Each rLDM is substantially free of other rLDM TM and native proteins including sub¬ stantial freedom from undesirable native destructive proteases. There are indications of these proteases in crude mixtures which are difficult to separate (each substantially pure rLDM TM gives a negative result in the Azocoll test) .

Characterization of the Novel rLDM TM The rLDM TM have been characterized by the following criteria;

(1) ability to catalyze the oxidation of veratryl alcohol to veratrylaldehyde;

(2) molecular weight as determined by SDS- PAGE; (3) a ino acid composition;

(4) heme content;

(5) homology by antibody reactivity;

(6) specificity of activity against lignin model substrates; and (7) elution from an FPLC column at specified sodium acetate molarities. All of the rLDM TM catalyze the oxidation of veratryl alcohol to veratrylaldehyde, as monitored spectrophotometrically at 310 nm. A unit of acti- vity is defined as the production of 1 micromole of

TM veratrylaldehyde in the rLDM catalyzed reaction.

The specific activities of typical preparations at about 24°C are as follows:

TM rLDM Specific Activity 2.6 17.1 5.1 9.7 9.4 12.4 Units/MG Minute

Molecular 38 38 42 42 43 42

Weight kD

Amino acid composition—Amino acid composition was determined by a modification of the procedure of Jones et. aL. (Jones, B.N., Paabo, S. and Stein, S. (1981) J. Liquid Chromatography 4:565-586) . . The ratio of amino acids is approximately due to the limitation of technique and quantity of protein used in the determination. See Table 1

Tab le I Λinino Acid ComDOs ition of rLDM TM

Amino Acid rLDM™ 1 rLDM™ 2 rLDM™ 3 rLDM T ^u M TM rLDM ¹ " 6

Ratio Ratio Ratio Ratio Ra io

asp/asn 1.4 2.0 5.4 5.0 3.0 glu/gln 6.0 7.7 16.8 19.9 8.0 ser 'ι.3 4.1 14.0 22.3 6.8 his 4.4 . 1 7.3 15.9 3.2

1 giy 6.5 5.7 24.0 44.7 8.3 r-> t_

1 1

Chr 2.2 3.5 - - 4.9 arg 1.1 1.2 2.9 4.8 1.3 ala 7.3 7.9 14.4 13.8 6.7 tyr 0.2 - 1.0 1.0 0.2 met - - 1.2 - 0.14 val 1.6 2.6 7.4 6.5 4.2 phe 1.1 3.0 7.0 3.3 3.2 ile 1.0 2.2 4.1 3.6 2.4 leu 1.5 2.6 6.5 5.0 3.3 lys 0.5 1.0 2.5 2.3 1.0

Heme and carbohydrate content—rLDMTM 1 , 2, 3 , 4, 5, and 6 each contain a single protoheme IX moiety.

All are glycosylated according to periodic acid staining (PAS) and binding to Con A-Sepharose (Sigma) .

Immunoblot Procedure

This procedure was used to further characterize the rLDM TM. It• i.s a standard procedure which is disclosed in Towbin e_t al.. (Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA

76:4350) . The procedure involves separating the proteins by electrophoresis in a gel, transfer of the proteins to a solid matrix, and reacting with

TM (1) a primary probe, rabbit anti-rLDM antibody and (2) a secondary probe, goat anti-rabbit antibody coupled to horseradish peroxidase.

TM rLDM 1, 3, 4, 5, and 6 react to polyclonal antibodies made to rLDM TM 2 and 6, using the above

TM immunoblot procedure. rLDM 2, in the same proce-

TM dure, reacts to polyclonal antibodies made to rLDM

6.

TM All the rLDM disclosed herein have the following unique activities on lignin model sub¬ strates, i.e., veratryl alcohol, 1- (3 ' , 4 '-dimethoxy- phenyl) glycerol- d? -guaiacyl ether, phenol, methoxy- lated benzenes such as 1,4-dimethoxybenzene:

(1) oxidative cleavage of C -C ;

(2) hydroxylation of benzylic methylene groups; (3) oxidation of benzyl alcohols to aldehydes;

(4) phenol oxidation; and

(5) oxidative cleavage of methoxyl groups.

"Lignin model substrates" are chemicals which resemble parts of lignin. The reaction products of the model compounds with rLDM TMs can have practical utility particularly to but not limited to food, pharmaceutical and chemical industries as chemical- feedstocks. The above activities are characteristic of the rLDM TM disclosed herein.

Example 3—Bleaching of Kraft Pulp w th rLDM T-M rLDM TM 1-6, alone, or mixtures thereof, are added to kraft pulp having a characteristic brown color at 3% consistency in 10 mM trans-aconitic acid, pH 4.5, 400 μM H-,0- and 100 μM MnS0 ₄ . The pulp slurry is flushed with 0_ and incubated with slow shaking at 39°C for 12 hr, after which the kraft pulp solution is decanted, and a 1 M NaOH solution is added to the pulp and incubated for 60 min at 65°C. This is then decanted and the kraft pulp is washed in water. The resulting kraft pulp no longer has a dark brown color, but instead has a desired lighter color.

The use of MnSO. is optional.

Example 4—Treatment of Thermomechanical Pulp (TMP) with rLDM TM rLDM TM 1-6, alone, or mixtures thereof, are added to 10 gm of TMP (dry weight) at 3% consistency in 10 mM trans-aconitic acid, pH 4.5, 400 juM H_0_ and 100 ,uH MnSO.. The pulp slurry is flushed with

0_ and incubated with slow shaking at 39°C for 12 hr, after which.time the TMP is washed with water.

The tensile, tear and burst indices as well as breaking length of the pulp measured and found to be of enhanced strength versus an intreated sample. The brightness reversion of the treated sample is less than the untreated sample; therefore, bright-*- ness stability is increased with the rLDM TM treat¬ ment.

The use of MnSO. is optional. The rLDM TM of the subject invention can be used

TM in a purified form, wherein each rLDM is sub-

TM stantially free of other rLDM and native proteins, and in mixtures thereof. It is well within the skill of a person skilled in the art to adjust amounts of rLDM TM used m accordance with the purity

TM TM of the rLDM preparation. The rLDM s may be combined with various diluents, adjuvants and other chemicals including proteins which are non-dele-

TM teπous to the rLDM s and their use, for various purposes such as providing marketable forms and enhancing their use.

"Native proteins" as used herein refers to other proteins present in the extracellular fermen¬ tation medium, as described above.

Example 5—Treatment of Softwood Kraft Pulp with rLDM

One part of softwood kraft pulp is treated with about 10 x 10~ to about 20 x 10~ parts of a rLDM™ in about 40 mM trans-aconitic acid, pH 4.5, at about 39°C for about 1 to about 16 hr. The pulp is- then washed in about 1 M NaOH at about 65°C for about 1

hour, and rinsed in water. This treatment of the softwood kraft pulp results in the removal of about 1/3 of the lignin as evidenced by the reduction of kappa number from about 18 to about 13.