Use of rLDM.TM. 1-6 and Other Ligninolytic Enzymes in the Treatment of Mechanical Pulps

The objective of mechanical pulping is to produce high-yield pulps. Several years ago mechanical pulping was limited to a single process, the grinding of roundwood against a pulpstone, but since then mechanical pulping has expanded into an array of processes that use chemical, thermal and compression technologies (Casey, J. P. [1983] Tappi Journal 66:95-96). A drawback to the current methods used is that they produce pulp with poor bonding strength and poor brightness stability.

Thermomechanical pulp (TMP), chemithermomechanical pulp (CTMP) and chemimechanical pulp (CMP) processes have evolved to improve mechanical pulp quality, expanding its utility in end product applications. Thermomechanical pulping is the dominant alternative high-yield pulping process. Its major limitation is the requirement for high electrical energy input, most of which ends up as low grade heat.

The utilization of thermomechanical pulps would be greatly facilitated if there was an increase of strength properties and if the stability of brightening could be enhanced, i.e., prevent brightness reversion. Brightness reversion of commercial pulps can be related to the presence of oxidized groups. These groups are principally derived from the residual breakdown products of lignin. It is postulated that the introduction of aldehyde and ketone groups into cellulose upon bleaching also contributes to brightness reversion, although to a lesser extent (Springer, E. L. [1983] Tappi Journal 66:93-96.). Breakdown products of lignin cause brightness reversion by mechanisms that are now being elucidated in several laboratories. It has been postulated that .alpha.-carboxyl groups adjacent to aromatic rings in residual lignin absorb daylight and transfer this energy to oxygen which in turn reacts with the phenolic groups of the lignin leading to formation of colored (yellow) quinones (Rapson, W. H. [1969] Appita 23:102-114). This reaction can occur only on "exposed" lignin rings which contain a free hydroxyl group.

Foremost in preventing brightness reversion is the necessity to modify lignin by oxidative cleavage of the exposed aromatic rings so that they cannot form quinones. This should drastically reduce brightness reversion properties.

Coarse TMP can be produced with relatively low inergy input. Subsequent secondary refining, however, requires substantial energy for development of pulp properties (Higuchi, T. [1982] Experientia 38:159-166). Experiments have demonstrated (Pilon, L., Desrochers, M., Jurasek, L., Neuman, P. J. [1982] Tappi Journal 65:93-96) that treatment of coarse TMP with P. chrysosporium cultures for 14 days can substantially reduce the energy requirement for secondary refining without a loss in pulp quality. Preliminary studies showed that the energy requirements to develop a given freeness in fungal-treated pulp was reduced by 25-30% as compared to untreated pulps Furthermore, pulp properties, as measured by the burst index, were also improved considerably. Because the refining of mechanical pulps after swelling in alkali can ccnsiderably improve strength properties, both the fungus-treated and untreated pulps were subjected to refining after swelling in alkali. The fungus-treated pulp then required 50% less refining energy than did the untreated pulp without any loss in strength properties.

In related experiments (Alberti, B. N. and Klibanov, A. M. [1981] Biotech. and Bioeng. Symp. 11:373-379), the water retention value (WRV) of fungal-treated pulps was tested in order to evaluate the effects of biological treatment on mechanical pulps. The WRV is a measure of the swelling and flexibility of the fibers. Increased swelling indicates greater contact between fibers during papermaking, this increasing the strength. An 88% gain in WRV was obtained after pretreatment of pulp with Schizophyllum commune, another white rot fungus, over untreated pulps.

The technical problems in applying organisms to industrial mechanical pulps, including TMP processing, are threefold: (a) in scaling-up with the required careful control of humidity, aeration and temperature; (b) in preventing contamination by unwanted organisms; and (c) in the impractical slowness of lignin degradation.

The subject invention concerns the enhancement of the strength properties of mechanical pulps, including TMP, CTMP, and CMP, by treating them with rLDM.TM. and other ligninolytic enzymes present in the extracellular growth medium from a fermentation of Phanerochaete chrysosporium. The rLDM.TM. selectively degrade only the chemical moieties formed in lignin and will not degrade cellulose or hemicellulose.

rLDM.TM. can enhance the strength properties of these pulps and they are immediately active. Thus, there is no lag in activity as with fungal cultures. Since the rLDM.TM. are biological molecules, they are, advantageously, not corrosive, do not cause pollution, and do not present an environmental hazard when released.

The lignin-degrading enzymes of the invention, referred to as rLDM.TM., are referred to as Pulpases.TM. in co-pending application Ser. No. 755,244.

The rLDM.TM. which can be used in the subject invention process were isolated from a novel stable mutant strain of the white-rot fungus Phanerochaete chrysosporium. 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 upon the grant of a patent disclosing it. The deposit also is available as required by foreign patent laws in countries wherein counterparts 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 derogation of patent rights granted by governmental action.

Novel mutant SC26 was obtained by UV mutagenesis of the wild type Phanerochaete chrysosporium, 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 determining the rate of oxidation of veratryl alcohol to veratrylaldehyde.

Isolation and purification of the ligninases from the extracellular fluid in the fermentation was accomplished by ultrafiltration and fast protein liquid chromatography (FPLC) using an anion exchange column.

The rLDM.TM. used in the subject invention process were prepared as follows :

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 BIII/glucose medium:

The BIII medium contains 1.08.times.10.sup.-3 M ammonium tartrate, 1.47.times.10.sup.-2 M KH.sub.2 PO.sub.4, 2.03.times.10.sup.-3 M MgSO.sub.4.7H.sub.2 O, 6.8.times.10.sup.-4 M CaCl.sub.2 2H.sub.2 O, 2.96.times.10.sup.-6 M thiamine.multidot.HCl and 10 ml.multidot.L.sup.-1 of a trace element solution. The trace element solution contains .7H.sub.2 O, 1.7.times.10.sup.-2 M NaCl, 3.59.times.10.sup.-4 M FeSO.sub.4.7H.sub.2 O, 7.75.times.10.sup.-4 M CoC.sub.2, 9.0.times.10-4M CaCl.sub.2, 3.48.times.10.sup.-4 M ZnSO.sub.4. 7H.sub.2 O, 4.times.10.sup.-5 M CuSO.sub.4.5H.sub.2 O, 2.1.times.10.sup.-5 M AlK(S.sub.4).sub.2 O, 1.6.times.10.sup.-4 M H.sub.3 BO.sub.3, 4.1.times.10.sup.-5 M NaMoO.sub.4.2H.sub.2 O and 2.9.times.10.sup.-3 M MnSO.sub.4.H.sub.2 O.

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.degree. C. The flasks were flushed on days 0, 3, and 6 with water-saturated O.sub.2. 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.degree. 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 .mu.l of extracellular fluid (from flasks or the RBC), 2 mM veratryl alcohol, 0.4 mM H.sub.2 O.sub.2 and 0.1 mM sodium tartrate, pH 2.5 in a final volume of 0.5 ml. The reactions were started by H.sub.2 O.sub.2 addition immediately after buffer was added and were monitored at 310 nm. 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.

The extracellular growth media from cultures grown in flasks, as described above, was harvested by centrifugation at 5000.times.G, 10 min., 4.degree. C. Extracellular growth media was then concentrated by ultrafiltration through a 10K filter. The resulting concentrate is called the Ligninolytic Mixture.TM.. The rLDM.TM. contained in this Ligninolytic 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 1M. rLDM.TM. 1, 2, 3, 4, 5, and 6 elute from the column in a typical preparation at the following sodium acetate molarities, respectively: 0.16, 0.18, 0.34, 0.40, 0.58, and 0.43M to give essentially pure rLDM.TM. 1-6. Each rLDM.TM. is substantially free of other rLDM.TM. and native proteins.

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) amino 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 activity is defined as the production of 1 micromole of veratrylaldehyde aldehyde in the rLDM.TM. catalyzed reaction. The specific activities of typical preparations at about 24.degree. C. are as follows:

Heme and carbohydrate content--rLDM.TM. 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).

This procedure was used to further characterize the rLDM.TM.. It is a standard procedure which is disclosed in Towbin et 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 (1) a primary probe, rabbit anti-rLDM.TM. antibody and (2) a secondary probe, goat anti-rabbit antibody coupled to horseradish peroxidase.

rLDM.TM. 1, 3, 4, 5, and 6 react to polyclonal antibodies made to rLDM.TM. 2 and 6, using the above immunoblot procedure. rLDM.TM. 2, in the same procedure, reacts to polyclonal antibodies made to rLDM.TM. 6.

All the rLDM.TM. disclosed herein have the following unique activities on lignin model substrates:

(1) oxidative cleavage of C.sub..alpha. -C.sub..beta. ;

(2) hydroxylation of benzylic methylene groups

(3) oxidation of benzyl alcohols to aldehydes;

(4) phenol oxidation; and

(5) oxidation of methoxy and ethoxy benzene.

"Lignin model substrates" are chemicals which resemble parts of lignin. The above activities are characteristic of the rLDM.TM. disclosed herein.

Following are Examples which illustrate the best mode for practicing the invention. These Examples should not be construed as limiting. In all Examples herein, percentages are by weight and solvent mixture proportions are by volume unless otherwise noted.

The Ligninolytic Mixture.TM., as described in Preparative Example 2, (0.15-1.5 mg protein total) was added to 10 gm of TMP (dry weight) at 3% consistency in 10 mM trans-aconitic acid, pH 4.5, 400 .mu.M H.sub.2 O.sub.2 and 100 .mu.M MnSO.sub.4. The pulp slurry was flushed with O.sub.2 and incubated with slow shaking at 39.degree. C. for 12 hr, after which time the TMP was washed with water. The tensile, tear and burst indices as well as breaking length of the pulp was measured and found to be of enhanced strength versus an untreated sample. The brightness reversion of the treated sample was less than the untreated sample; therefore, brightness stability was increased with the Ligninolytic Mixture.TM. treatment.

The use of MnSO.sub.4 is optional.

Regarding the above conditions, for each of the parameters there is a range of values which can be used to achieve the desired result. Typical values and acceptable ranges for each parameter are shown in Table 2.

rLDM.TM. 1 through 6, individually, or mixtures thereof, can be used to treat TMP using essentially the same procedures as disclosed in Example 1, including ranges, or obvious modifications thereof. The resulting pulp is of high quality.

Upon substituting the Ligninolytic Mixture.TM. of Example 1 with extracellular growth medium, prepared as disclosed in Preparative Example 1, there is obtained pulp of high quality.

Upon substituting the Ligninolytic Mixture.TM. of Example 1 with a mixture comprising all of the following or any combination thereof: rLDM.TM. 1-6, individually or mixtures thereof; Ligninolytic Mixture.TM. ; and extracellular growth medium there is obtained pulp of high quality.

Upon substituting CTMP or CMP for the TMP in Examples 1-4, there is obtained pulp of high quality.

The rLDM.TM. of the subject invention can be used in the crude form, in a purified form, wherein each rLDM.TM. is substantially free of other rLDM.TM. 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 in accordance with the purity of the rLDM.TM. preparation.

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