Effects of Manganese Peroxidase on Residual Lignin of Softwood Kraft Pulp

Abstract

Manganese peroxidase treatment lowered the kappa number of kraft pulp and increased the alkali extractability of the residual lignin but did not directly solubilize it. This indicates that MnP partially oxidizes the lignin in the pulp but does not degrade it to soluble fragments.

Manganese peroxidase (MnP) has been implicated as an important enzyme in delignification of kraft pulps by white rot fungi (6, 8, 12, 15). Treating kraft pulps with MnP can lower their kappa numbers and increase their bleachability (10, 13, 14). In the presence of the surfactant Tween 80, the enzyme can increase the brightness of hardwood kraft pulp (9). This enzyme also releases methanol from methoxyl groups on rings with free phenolic hydroxyls (15). The bleaching effect is thought to be caused by residual lignin oxidation by chelated Mn³⁺, generated by enzymatic oxidation of Mn²⁺, but the reacting sites within the lignin and the products of delignification have not yet been identified. To better understand pulp bleaching by MnP, we have investigated its action on softwood kraft pulp (SWKP) containing ¹⁴C-labelled residual lignin. Delignification of this pulp by the white rot fungus Trametes versicolor has been described previously (16). The fungus extensively depolymerized and solubilized the residual lignin in the pulp and mineralized 22% of it to CO₂.

We show here that the decrease in kappa number caused by MnP treatment is not associated with removal of lignin carbon from the pulp.

Pulps.

SWKP labelled with ¹⁴C in its residual lignin (SWKP*2) was prepared by laboratory pulping of lignin-labelled white spruce as previously described (16). The labelled pulp was washed with 0.1 M NaOH and rinsed with water just before use to remove radioactive materials that had leached from the pulp during storage. Unlabelled pulp was prepared from white spruce in the Paprican pilot plant.

Enzymes.

Glucose oxidase (Gox) was purchased from Sigma. The activity of the Gox was assayed at pH 4.5, with peroxidase to detect the production of H₂O₂ from oxidation of glucose (1). MnP was produced from Trametes versicolor 52J as previously described (13) and assayed by spectrophotometric detection of Mn(III) malonate formation (15). Crude cellulase was purchased from Iogen Corp., Ottawa, Ontario, Canada.

Pulp treatments.

SWKP (0.5 g) was suspended in 50 ml of 5 mM malonate buffer, pH 4.5, containing 50 U of MnP, 0.2 mM MnSO₄, and 10 mM glucose, in a 125-ml Erlenmeyer flask. In experiments using labelled pulp, 1 × 10⁵ dpm of SWKP*2 was added to each flask. Gox (1.76 U) was added to start the reaction, and the flasks were incubated on a gyrotory shaker at 250 rpm for 24 h at 30°C. Control flasks lacking either MnP or Gox were incubated in parallel with the treatment flasks. After incubation, the flask contents were fractionated into soluble, alkali-extractable, and cellulase lignin fractions as previously described (16). Alkali extraction was carried out at 10% consistency and 60°C for 60 min with 1.6% NaOH. Cellulase lignin was the fraction soluble in 0.1 M NaOH at 37°C after digestion of the pulp polysaccharides with crude cellulase.

Analytical methods.

Kappa numbers were determined by the micro-kappa method (3). Klason lignin was measured by the method of Effland (4), with estimation of acid-soluble lignin from the A₂₀₅ of the hydrolysis filtrates (11).

Effect of MnP treatment on distribution of ¹⁴C from lignin-labelled pulp.

Gox was used in these experiments to supply H₂O₂ continuously at low concentration; MnP is inactivated by H₂O₂ at millimolar concentrations (5). Treatment of the labelled pulp with the complete system of MnP plus Gox did not release significant amounts of ¹⁴CO₂, nor did it solubilize the labelled lignin; the level of soluble radioactivity after the treatment of the pulp with both MnP and Gox was low, intermediate between those of the two controls lacking one of the enzymes (Table 1). Treatment of the pulp with Gox alone (no MnP control) consistently decreased the amount of soluble radioactivity; the basis for this effect was not determined. The enzyme treatment did increase the amount of radioactivity extracted by alkali from ca. 15% to ca. 25% of the total and decreased the amount of label in the cellulase lignin fraction.

TABLE 1.

Treatment of ¹⁴C-lignin-labelled SWKP with MnP increases the alkali extractability of the label but not its solubility

SWKP	% of total radioactivitya			n
	Soluble	Alkali extractable	Cellulase lignin
MnP treated	3.6a	25.8a	45.9a	11
Control (no MnP)	1.8b	17.0b	56.8ab	4
Control (no Gox)	5.1c	14.2b	65.6b	7
SD	±1.0	±3.8	±8.4

^aData are the means of n (4 to 11) replicates. Within each column, numbers followed by the same letter are not significantly different at the 95% probability level by the Student-Newman-Kuels test. 

Effect of MnP treatment on Klason lignin content of pulp.

Treatment with MnP plus Gox lowered the kappa number of the pulp by about 20%, but it did not change the Klason lignin content (Table 2). There were no significant differences among the controls lacking one or both of the enzymes. When the pulps were extracted with alkali, the kappa numbers of the treated and control pulps all decreased by about six units, but the pulp treated with the complete enzyme mixture lost significantly more Klason lignin (1.5% on pulp) than the control pulps did (0.5% on pulp).

TABLE 2.

Kappa numbers and Klason lignin contents of enzyme-treated pulps before and after alkaline extraction

Enzyme treatment		Before extractiona		After extractiona
MnP	Gox	Kappa number	Klason lignin (%)	Kappa number	Klason lignin (%)
+	+	27.0a	4.9a	22.0a	3.4a
+	−	34.3b	5.0a	27.1b	4.3b
−	+	33.1b	4.8a	27.0b	4.2b
−	−	32.8b	4.6a	27.6b	4.4b
		±0.9	±0.1	±0.9	±0.1

^aData are means of four replicates. Within each column, numbers followed by the same letter are not significantly different at the 95% probability level by the Student-Newman-Kuels test. The last value in each column is the standard error.  

Thus, the Klason lignin content of the pulp reacted to enzyme treatment like the ¹⁴C-labelled lignin: it was not directly solubilized, but its alkali extractability was increased. The agreement between these two measures of lignin content shows that lignin was not removed from the pulp by MnP treatment. However, the capacity of the lignin to reduce permanganate (i.e., kappa number) was lowered, probably because the lignin was partially oxidized by the enzyme. The ratio of kappa number to Klason lignin decreased from 7 in the control pulp to 5.5 in the enzyme-treated pulp. MnP treatment of kraft pulps releases methanol from methoxyl groups on lignin rings bearing free phenolic hydroxyls (15); the likely coproducts are o-quinones. MnP-treated pulps have enhanced susceptibility to peroxide bleaching, which may also result from the presence of o-quinones (13). Alternatively, the enhanced bleachability may be due to formation of α-carbonyls, which are easily degraded by alkaline peroxide (7). MnP is known to oxidize lignin model compounds predominantly to α-carbonyl products (17, 18).

The partial oxidation of the lignin apparently did not cause conversion to fragments small or hydrophilic enough to diffuse out of the fiber walls. However, the enzyme treatment did increase the proportion of the lignin that could be extracted by alkali. It seemed that the lignin extracted by alkali included the partially oxidized lignin, because the ratio of kappa number to Klason lignin in the extracted pulp was 6.5, similar to the ratios in the extracted control pulps. The enhanced extractability of lignin after oxidation with MnP parallels the effect of lignin oxidation by chlorine or chlorine dioxide in conventional bleaching (2). The ability of alkali to solubilize the oxidized lignins is attributed to ionization of acidic groups and to decreased association of the lignin molecules; there are also alkali-promoted chemical reactions that cleave oxidized fragments and liberate ionizable groups in the lignin (2).

The inability of MnP to solubilize the residual lignin from kraft pulp differs from the ability of intact cultures of Trametes versicolor to extensively solubilize this lignin (16). MnP probably functions at an early stage in the degradation of lignin by this fungus, leading to demethoxylation (15) and increased alkali extractability (16). Other enzymes then probably continue the degradation to release soluble lignin fragments. Alternatively, the fungal mycelium might enhance the ability of MnP to degrade lignin by secreting thiols (18) or other redox mediators (12) or by trapping fragments produced transiently.