Background of the Invention

Kraft Pulp

The primary chemical method for making pulp from wood involves the digestion of lignin in the wood with sodium sulfide and sodium hydroxide. This is termed the sulfate or kraft process.

Wood pulp produced in the kraft process gene¬ rally contains 5-8% by weight of residual, modified lignin which gives the pulp a characteristic brown color. To obtain a pulp of very high brightness and brightness stability, the lignin must be removed by certain oxidizing agents commonly referred to as bleaching chemicals. Many bleaching processes exist but almost all begin with the chlorination. extrac¬ tion (C-E) stage. There is a loss of cellulosic- fibers during the C-E stage. The C-E effluents resulting from treated pulp contain a very large number of organic compounds having a bound chlorine content of 2.5-3.5 kg/ton pulp. Some of these compounds, primarily the chlorinated phenolics, are known to have toxic, irmtagenic and carcinogenic effects. (Alberti, B.N. and Klibanov, A.M. (1981) Biotechnology and Bioengineering Symp. 11:373-379). These effluents are highly unsuited for recycling within the pulping system due to their high level of corrosive chlorides. Alternatives to chlorine bleaching have, therefore, long been sought by industry.

Hydrogen peroxide has been shown to deligninify sulfite pulps satisfactorily, but on its own it is a relatively ineffective means of- bleaching kraft pulp. When used in sequences with chlorine-con- taining bleaching agents, however, peroxide con¬ tributes significantly to deligninification, pulp brightness and brightness stability.

Oxygen and ozone have been extensively studied for incorporation into the bleaching processes. The major disadvantage of these compounds is their non-specific oxidative attack on- σellulosic fibers. Lower pulp yields•tend to result and the pulp properties are generally inferior to those obtained with chlorine bleaching sequencing. Research sponsored by the U.S. Department of Agriculture's (USDA) Forest Products Laboratory has demonstrated- that 50-75% of the residual lignin was removed by fungal cultures of Phanerochaete chrysos- poriu in 6 to 8 days. Longer incubation resulted in greater lignin reductions, but the data were not quantified. During incubation, the pulp became substantially lighter in color (Kirk, T.K. and Chang, H. (1981) Enzyme Microb. Technol. 3:189-196). Bleaching is impractically slow using whole fungal cultures. It was found that lignin removal (i.e., kappa number decrease) from kraft pulp followed a triphasic pattern: 1) no lignin removal during establishment of the fungus in the pulp over the first two days, 2) rapid deligninification during the following two days, and 3) slower de¬ ligninification thereafter. The initial two-day

lag is due to the secondary metabolic importance of lignin degradation to fungal growth.

Another disadvantage of fungal bleaching is that these organisms contain enzymes which degrade both cellulose and hemicellulose. In any effective bleaching scheme, the degradation of cellulosic fibers must be completely suppressed, since the cellulosic fibers are particularly vulnerable after kraft pulping. Cellulase-less mutants have to some extent overcome this problem, but they are difficult to manage and some are less efficient in degrading lignin than normal fungal cultures. A final dis¬ advantage of using fungal cells is that they can only operate optimally in an environment where temperature ^■ and microbial contamination are care¬ fully controlled:

Mechanical Pulps ■

The objective of mechanical pulping is to produce high-yielded pulps. Several years ago mechanical pulping was limited to a single process, the grinding of round-wood 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 65:95-96. A drawback to the current methods used is that they produce pulp with poor bonding strength and poor brightness stability.

Thermomechanical pulp (TMP) , chemithermo- mechanical 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 bright¬ ening could be enhanced, i.e., prevent brightness

10 reversion. Brightness reversion of commerical 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

15 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

20 being elucidated in several laboratories. It has been postulated that <* -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

25 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.

Coarse TMP can be produced with relatively low

30. energy 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., Neu an, 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 require¬ ments 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 considerably 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.

The technical problems in applying organisms to industrial mechanical pulps, including TMP proces¬ sing, 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.

Decolorization of El Effluent

The primary chemical method for making pulp from wood involves the digestion of lignin in the wood with sodium sulfide and sodium hydroxide. This is termed the sulfate or kraft process.

Wood pulp produced in the kraft process gene¬ rally contains 5-8% by weight of residual, modified lignin which gives pulp a characteristic brown color. To obtain pulp of very high brightness and brightness stability, the lignin must be removed by certain oxidizing agents commonly referred to as bleaching chemicals. Many bleaching processes exist but almost all begin with the chlorination-extrac- tion (C-E) stage. The spent liquor from the first alkali extraction stage of bleaching following chlorination, commonly referred to as El effluent, contains over 80% of the effluent color emanating from a kraft bleach plant (Kirk, T.K. and Chang, H-M. (1981) Enzyme Microb. Technol. 3:189-196). The effluent must be discharged due to its high content of corrosive chlorides. Polymeric lignin degrada¬ tion products, the main contributors to color of bleach plant effluent, are resistant to the current bacteria-based effluent treatment process. Alter- nate treatment processes such as ultrafiltration, carbon adsorption, and massive lime precipitation are required for effective color removal, but are quite expensive. Economical color removal systems do not presently exist and would be desirable for effluent treatment prior to its discharge to re¬ ceiving waters. .

Fungal decolorization systems have been stu¬ died. In USDA sponsored laboratory experiments (Kirk, T.K. (1983) in The Filamentous Fungi, Vol. 4, Fungal Technology, Smith, J.E., Berry, D.R., Kris- tiansen, B., eds., Edward Arnold Press, London), greater than 80% decolorization of bleaching

effluent prepared by chlorination and alkali treat¬ ment of kraft-cooked synthetic lignins has been achieved in 24 hr using Phanerochaete chrysosporium cultures. There are three problems in using fungal cultures to decolorize bleach plant effluents: (1) fungi require earful culture conditions (i.e., humidity, aeration, temperature and pH) not com¬ patible with industrial processing environments; (2) fungi require long lag times and then only very slowly degrade lignin; a'nd (3) fungi cannot grow on lignin. ^' An additional food source must be added to support fungal growth.

Brief Summary of the Invention The subject invention concerns the bleaching of kraft pulp 'with rLDM TM and other lignmolytic enzymes. rLDM TM are ligninases which are highly specific and which will degrade the hard-to-remove residual lignin polymers in chemical pulps without damaging cellulosic fibers. rLDM TM can bleach kraft pulp 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 aa.s rLDM TM, were previously referred to as Pulpases T ^' M The subject invention also concerns the enhance¬ ment of the strength properties of mechanical pulps,

including TMP, CTMP, and CMP, by treating them with rLDM TM and other lignmolytic enzymes. These other ligninolytic enzymes are present in the extra¬ cellular growth medium from a fermentation of Phanerochaete σhrysosporium. 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 pollu¬ tion and do not present an environmental hazard when released.- Further, the subject invention concerns the decolorization of El effluent by treating the effluent with rLDM TM antl other lignmolytic enzymes present in the extra ^' cellular growth medium from a fermentation of Phanerochaete chrysosporium. rLDM TM are ligninases which are highly specific and which will degrade lignin polymers. rLDM TM do not require precise culture conditions and are immediately active to efficiently decolorize effluents in a non-corrosive and non-polluting manner.

Detailed Description of the Invention The rLDM TM which can be used m 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. Depart¬ ment 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 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 uta- genesis of the wild type Phanerochaete chryso- sporiu ', ATCC 24725.

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

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: Preparative

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-li ited Bill/glucose medium:

_3 The Bill medium contains 1.08 x 10 M ammonium tartrate, 1.47 x 10~ ² M KH ₂ PO. , ^' 2.03 x 10~ ³ M

MgS0 ₄ • 7H ₂ 0, 6.8 x lθ ^"4 M CaCl ₂ • 2H ₇ 0, 2.96 x 10 M thiamine-HCl and 10 ml _' L ^-1 of a trace element solution. The trace element solution

_3 contains 7.8 x 10 M nitπloacetic acid, 1.2 x

10~ ² M MgS0 ₄ • H ₂ 0, 1.7 x 10~ ² M NaCl, 3.59 x

10~ ⁴ M FeS0 ₄ • 7H ₂ 0, 7.75 x 10~ ⁴ M CoCl ₂ , 9.0 x 10 ^"4 M CaCl ₂ , 3.48 x lθ ^"4 M ZnS0 ₄ • 7H ₂ 0, 4 x

10~ ⁵ , M CuS0 ₄ • 5H ₂ 0, 2.1 x lθ ^"5 M A1K(S0 ₄ > ₂ '

12H ₂ 0, 1.6 x 10~ ⁴ M H ₃ B0 ₃ , 4.1 x 10~ ⁵ M NaMo0 ₄ '

2H20 and 2.9 x 10~ ³ M MnSO4.. H20. The medium was supplemented with 10% (by wt/liter) of glucose.

The medium was buffered with lOmM 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 0 _~ . 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 rp with continuous oxygenation. Ligninase activity was measured periodically by determining the rate of oxidation of veratryl alcohol to veratrylaldehyde. Reaction mixtures

contained 275 μ1 of extracellular fluid (from flasks or the RBC), 2 mM veratryl alcohol, 0.4 mM H 0 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.

Preparative

Example 2—Isolation and Purification of the Novel rLDM TM ⁿ

The extracellular growth media from cultures grown in flasks, as described above, was harvested by centrifugation at 5000 xG, 10 min, 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 TM ^* s or other ligninolytic enzymes in varying proportions. The rLDM TM contained in this Lignmo- 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 in a typical preparation at the following sodium acetate molarities, respectively: 0.16, 0.1818,

0.34, 0.40, 0.58, and 0.43 M to give essentially pure rLDM TM 1-6. Each rLDMTM is substantially free of other rLDM TM and native proteins including substantial freedom from undesirable native de-

structive 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) .

TM Characterization of the Novel rLDM 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 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 veratryl-aldehyde in the rLDM catalyzed reaction.

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

rLDM TM

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 e_t a_l. (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

TM Heme and carbohydrate content—rLDM 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

TM the rLDM . It is a standard procedure which is disclosed in Towbiri e_t a_l. (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

TM antibodies made to rLDM 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. All the rLDM TM disclosed herein have the following unique activities on lignin model sub-

Ta le I ^

Amino Acid Composition of rLDM

Amino Acid r _τ L _n D _M MTM , 1

Ratio

3.0 asp/asn ι l I..-4 A 2 ^• - ^• 0 ^w _{? l} ⁵ _{6 8 1} ⁵ ₉ -.° ₉ 8 o. n0 .0 7.7 16.8 ser ^ ^•3 7 -. 13.9 3 i. i. 3.2 ' ^• - ^»

>P»

6.5 5 ³ - 7 ^/ 2A.0 ^k - _ ^η ,,. _9_ giy

2 2 3.5 . thr ^L - ^L _{2 9} . A.8 1.3

1 1 1.2 ^L ' ^y arg -' _u 13.8 6.7

7 7.9 ^1M - ala ' ^'J , ₀ i.o 0.2

-y- met A 6.5 A.2

1 6 ² - -. ? ^val , n 7 0 3.3 ^3'2 phe ., 3.6

1 0 -■ - -• ~x i i ^le 6.5 6.0 3-- leu s 2.3 ^*• n ς 1.0 '- ⁾ lys 0.5

6

-15-

strates, i.e., veratryl alcohol, 1- (3' ,4'-dimethoxy- phenyl)glycerol-^-guaiacyl ether, phenol, methoxy- lated benzenes such as 1,4-dimethoxybenzene: (1) oxidative cleavage of C _Λ -C__ ; (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 reaction products of

TM the model compounds with rLDM s can have practical utility particularly to but not limited to food, pharmaceutical and chemical industries as chemical feedstocks. The above activities are characteristic

TM of the rLDM 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.

TM Example 1—Bleaching of Kraft Pulp with rLDM and

Other Ligninolytic Enzymes

TM The Ligninolytic Mixture , as described in Preparative Example 2, was added to kraft pulp having a characteristic brown color at 3% consis¬ tency in 10 mM trans-aconitic acid, pH 4.5, 400 J M H_0 ₂ and 100 μ M MnSO.. The pulp slurry was flushed with 0_ and incubated with slow shaking at 39°C for

12 hr, after which the kraft pulp solution was decanted, and 1 M NaOH solution was added to the pulp and incubated for 60 min at 65°C. This was then decanted and the kraft pulp was washed in water. The resulting kraft pulp no longer had a dark brown color, but instead had a desired lighter color.

The use of MnSO. 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.

Example 2 rLDM TM 1 through 6, individually, or mixtures thereof, can be used to treat kraft pulp using essentially the same procedures as disclosed in Example 1, including ranges, or obvious modifica¬ tions thereof. The resulting kraft pulp is of the desired lighter color.

Example 3 Upon substituting the Ligninolytic Mixture TM of

Example 1 with extracellular growth medium, prepared as disclosed in Preparative Example 1, there is obtained kraft pulp having a desired lighter brown color.

Example 4 UUppoonn substituting the Ligninolytic Mixture TM of Example 1 with a mixture comprising all of the

following or any combination thereof: rLDMTM 1-6, individually or mixtures thereof; Ligninolytic

TM Mixture ; and extracellular growth medium; there is obtained kraft pulp having a desired lighter brown color.

Example 5—Treatment of TMP with rLDM TM and Other

Ligninolytic Enzymes The Lign olytic 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% con¬ sistency in 10 mM trans-aconitic acid, pH 4.5, 400 μM H-O- and 100_^M MnSO.. The pulp slurry was flushed with 0_ and incubated with slow shaking at 39°C for 12 hr, after which time the TMP was washed , with wajier. 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 that of the untreated sample; therefore, brightness stability was increased with the Lignmolytic Mixture TM treatment.

The use of MnSO. 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 3.

Example 6 rrLLDDMM T ^' M 1 through 6, individually, or mixtures thereof, can be used to treat TMP using essentially

the same procedures as disclosed in Example 5, including ranges, or obvious modifications thereof. The resulting pulp is of high quality.

Example 7

TM Upon substituting the Lignmolytic Mixture of

Example 5 with extracellular growth medium, prepared as disclosed in Preparative Example 1, there is obtained pulp of high quality.

Example 8 UUppoonn substituting the Lignmolytic Mixture TM ^* of

Example 5 with a mixture comprising all of tthhee following or any combination thereof: rLDM T TMM following or any combination thereof: rLDM 1-6, individuuaallly or mixtures thereof; Ligninolytic

T TMM MMiixxttuurree ;; aanndd eexxttrraacceelllluullaarr growth medium; there is obtained .pulp of high quality

Example 9

Upon substituting CTMP or CMP for the TMP in Examples 5-8 there is obtained pulp of high quality.

TM Example 10—Decolorization of Effluent with rLDM and Other Ligninolytic Enzymes The Ligninolytic Mixture TM, as described in

Preparative Example 2, was added to a 0.2% solution of El effluent in 10 mM trans-aconitic acid, pH 4.5,

400 μU H-0 ₂ and 100 μM MnSO.. The solution was flushed with 0_ and incubated with slow shaking at

39°C for 12 hr. The solution was monitored spectro- photometrically in the ultraviolet and visible

regions. El effluent treated as above was no¬ ticeably decolorized and reduced in absorbance at 465 nm. (Note that color is measured by A465 nm wherein an absorbance of 1.0 at 465 nm, pH 7.6 equals 3774 National Council for Air and Stream Improvement color units.)

The MnSO. 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 4.

Example 11

TM rLDM 1 through 6, individually, or mixtures thereof, can be used to treat effluent using essen- tially the same procedures as disclosed in Example

10, including ranges, or obvious modifications thereof. The resulting effluent is decolorized.

Example 12 Upon substituting extracellular growth medium from a Phanerochaete chrysosporium fermentation, obtained as disclosed in Preparative Example 1, for the Lignmolytic Mixture TM of Example 10, there is obtained decolorized El effluent.

Example 13 Upon substituting the Lignmolytic Mixture TM of

Example 10 with a mixture comprising all of the following or any combination thereof: rLDM TM 1-6, individually or mixtures thereof; Ligninolytic

MixtureTM; and extracellular growth medium; there is obtained decolorized El effluent. The rLDM TM of the subject invention can be used in the crude form, in a purified form, wherein each rLDM TM i.s substanti.ally free of other rLDMTM and native proteins, and in mixtures thereof. It is particularly desirable to use the rLDM TMs which are substantially purified and essentially free of degradative proteases. 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. The rLDMTMs may be combined with various diluents, adjuvants and other chemicals including proteins which are non-deleterious to the rLDM TMs and their use, for various purposes such as providing marketable forms an ' enhancing their use.

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

Table 2

Para eter Typical Range

Cons is tency 37. 0 . 01 to 20 *

Concentration of trans ' 10 mM 0 . 0 05 to 0 . 5 M aconitic acid ^, *

pH 4.5 2 to 7

Concentration of H _^ C^ A00 2 μM to 10 mM

Concentration of MnSO, 100 μM 10 to 500 μM

Incubation of pulp slurry 12 hr 2 min to.-48 hr (First incubation)

Temperature of first 39°C 15° to 50°C incubation

Concentration of NaOH*** 1 M 0.01 to 5 M

Incubation of pulp after 60 min 2 min to 48 hr alkaline treatment (Second incubation)

Temperature of second 65°C 5° to 100°C incubation

'- ^' 'Concentrations greater than 7. can be used if the fluid consistency of the medium is maintained.

-'"''Other nontoxic enzyme buffers such as ammonium tartrate can be used.

*** 0H or other alkaline solutions can be used.

Table

Parameter ^{' '} " Typical Range

Consis ency 3% 0.01 to 20%'

Ratio of Ligninolytic 0.08 0.015 to 0.15 Mixture TM to mechanical pulps

₍ mg of protein/g of pulp ⁾

Concentration of 10 mM 0.005 to 0.5 M trans-aconitic acid-*

4.5 2 to 7 pH

Concen _t ra _t ion of H ₂ 0 ₂ 400 μM 2 ^μ M ^t o 10

Concen _t ra _t ion of c _M- n,Sn0 _T OO uM 10 to 500 ^μ M

'A 1UU ^t -

Incubation period 12 hr 2 min to 48 hr

Temperature during 39°C 15 to 50°C

can be used.

Table A

Parameter Typical Range

Concentration of 0.2% 0.01 to 20% effluent

Concentration of 10 mM 0.005 to 0.5 M trans-aconitic acid*

Concentration of 1 VAO/ 0.01 to 30 Units/ml

Ligninolytic Mixture TM Unit/ml**

pH 4.5 2 to 7

Concentration of H-C^ 400 μM 2 μM to 10 mM

Concentration of MnSO, 100 μM 10 to 500 μM

Incubation period 12 hr 2 min to 48 hr

Temperature during 39°C 15° to ^' 50°C incubation

*0ther nontoxic enzyme buffers such as ammonium tartrate can be used. ^Sr" 'VA0/Unit = veratryl alcohol oxidation activity unit