Method for lignin depolymerisation

Abstract

The present invention is in the field of delignifying and/or bleaching of pulp, more in particular wood pulp. Such a process is useful in paper production. More in particular, the method relates to the use of an enzyme for delignification and/or bleaching, more in particular a bacterial laccase. Even more in particular, it provides a method for delignifying and/or bleaching of a pulp, comprising an enzymatic treatment step wherein lignin-containing pulp and a laccase are reacted at alkaline pH, wherein the laccase has an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1.

Claims

1. A method for lignin depolymerization, the method comprising: contacting a solution or suspension containing lignin with a laccase at alkaline pH, the laccase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1; and the lignin is depolymerized.

2. The method according to claim 1, wherein the solution or suspension containing lignin comprises a pulp.

3. The method according to claim 2, wherein the pulp is a mechanical pulp or a chemical pulp.

4. The method according to claim 2, further comprising bleaching the pulp.

5. The method according to claim 4, wherein bleaching the pulp comprises: the pulp with a bleaching chemical selected from the group consisting of peroxide, hydrogen peroxide, oxygen, ozone, chlorine dioxide and a mixture of chlorine dioxide and chlorine gas.

6. The method according to claim 1, wherein the solution or suspension containing lignin comprises at least 40% lignin as a fraction of the dry matter of the solution or suspension.

7. The method according to claim 1, wherein contacting the solution or suspension is carried out at a temperature between 10 and 90 degrees Celsius.

8. The method according to claim 7, wherein the temperature is above 40 degrees.

9. The method according to claim 7, wherein the temperature is below 50 degrees.

10. The method according to claim 1, wherein the pH of the solution or suspension is from 8 up to and including 12.

11. The method according to claim 10, wherein the pH is above 9.

12. The method according to claim 10, wherein the pH is below 12.

13. The method according to claim 1, wherein the laccase is produced in E. coli.

14. The method according to claim 13, wherein the laccase is a Bacillus wakoensis laccase.

15. The method according to claim 1, wherein the solution or suspension does not contain an electron mediator selected from the group consisting of 1-hydroxybenzotriazole (HBT), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), acetosyringone, phenol, and violuric acid.

16. The method according to claim 2, wherein the pulp is wood pulp.

Description

LEGEND TO THE FIGURES

(1) FIG. 1: Diagram showing residual relative activities (% of initial activity) of different laccases in solution after pre-incubation at:

(2) 70 degrees C., at pH 11 for 1-4 hours (FIG. 1A).

(3) 70 degrees C., at pH 9 for 1-4 hours (FIG. 1B).

(4) 40 degrees C., at pH 11 for 1-4 hours (FIG. 1C).

(5) 40 degrees C., at pH 9 for 1-4 hours (FIG. 1D).

(6) Experimental details are provided in Examples 2 and 3.

(7) FIG. 2: Graph showing dissolved oxygen measurements as described in example 4 for delignification of pulp at 70 degrees Celsius and pH 11.

(8) FIG. 3: Diagram showing the stability of different laccases in pulp under different conditions as follows:

(9) 70 degrees C., at pH 11 (FIG. 3A).

(10) 70 degrees C., at pH 9 (FIG. 3B).

(11) 40 degrees C., at pH 11 (FIG. 3C).

(12) 40 degrees C., at pH 9 (FIG. 3D).

(13) Experimental details are provided in Example 4.

(14) FIG. 4: Diagram showing the stability of different laccases in purified high molecular weight lignin, under different conditions as follows:

(15) 70 degrees C., at pH 11 (FIG. 4A).

(16) 70 degrees C., at pH 9 (FIG. 4B).

(17) 40 degrees C., at pH 11 (FIG. 4C).

(18) 40 degrees C., at pH 9 (FIG. 4D).

(19) Experimental details are provided in Example 6

(20) FIG. 5: Diagram showing the decrease in Kappa numbers (K(start)K (end)) of the pulps obtained in delignification experiments with different laccases as described in Example 4. The decrease in Kappa number is a measure of delignification of the pulp.

CONCLUDING REMARKS

(21) In conclusion, the invention may be described in the following terms: 1. Method for delignifying and/or bleaching of a pulp, comprising an enzymatic treatment step wherein lignin-containing pulp and a laccase are reacted at alkaline pH, wherein the laccase has an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1. 2. Method as described above, characterized in that the bleaching of the pulp comprises a step of contacting the pulp with a bleaching chemical selected from the group consisting of peroxide, hydrogen peroxide, oxygen, ozone, chlorine dioxide and a mixture of chlorine dioxide and chlorine gas. 3. Method as described above, characterized in that the enzymatic treatment step is carried out at a temperature between 10 and 90 degrees Celsius. 4. Method as described above wherein the temperature is between 40 and 80 degrees Celsius. 5. Method as described above wherein the temperature is between 60 and 80 degrees Celsius. 6. Method as described above, characterized in that the pH value of the pulp during the enzymatic treatment is from 7 up to and including 12. 7. Method as described above, characterized in that the pH value of the pulp during the enzymatic treatment is from 8 up to and including 11. 8. Method as described above, characterized in that the pH value of the pulp during the enzymatic treatment is from 9 up to and including 11. 9. Method as described above wherein the laccase is a bacterial laccase. 10. Method as described above wherein the laccase is obtainable from Bacillus wakoensis. 11. Method as described above wherein a laccase mediator is present before or during the enzymatic treatment. 12. Method as described above wherein the laccase mediator is syringaldehyde. 13. Method as described above wherein the pulp is wood pulp. 14. Method as described above wherein the pulp is mechanical pulp. 15. Method as described above wherein the pulp is chemical pulp.

EXAMPLES

Example 1: Preparation of Polypeptides According to SEQ ID NO:s 1-4

(22) The DNA sequences according to SEQ ID NO:s 5-8, encoding the polypeptides according to SEQ ID NO:s 1-4 were commercially synthesized and cloned into a standard plasmid vector pET26a+ under the control of T7-RNA-polymerase promoter for expression in Escherichia coli BL21(DE3).

(23) Protein production was carried out in E. coli BL21(DE3) strain according to the plasmid manufacturer protocol available at internet site richsingiser.com/4402/Novagen%20pET%20system%20manual.pdf. The incubation temperature for protein production was 30 degrees C., which was found optimal for maximum yield of the active protein. Cells were lysed using laccase lysis buffer (20 mM Sodium Citrate pH7.4, 1% Triton X100, 1 mM CuCl2) and heated at 60 degrees C. for 20 min. Coagulated cell debris was removed by centrifugation. The recombinant laccases were detected in the soluble fraction only, consistent with the notion that they are thermostable enzymes.

Example 2: Measuring Laccase Activity in Solution by DMP Oxidation

(24) The term laccase activity is used herein to mean the capability to act as a laccase enzyme, which may be expressed as the maximal initial rate of the specific oxidation reaction. In some experiments relative activity was measured by oxidation of DMP (2,6-Dimethoxyphenol). Reaction course was monitored by change in absorbance at 468 nm (extinction coefficient of oxidized DMP at 468 nm is 14800 M1 cm-1). The appropriate reaction time was determined to provide initial rates of oxidation when color development is linear in time. DMP concentration in the reaction mixture was 1 mM to provide maximum initial rates (substrate saturation conditions).

(25) Typically, reactions were carried out in 200 ul in 96-well plates. 180 l of enzyme dilution in Britton and Robinson buffer (0.04 M H.sub.3BO.sub.3, 0.04 M H.sub.3PO.sub.4 and 0.04 M CH.sub.3COOH that has been titrated to pH 8.0 with 0.2 M NaOH) was prepared in the assay plate and equilibrated to the desired temperature (70 degrees C.), then 20 uL of 10 mM DMP solution was added to start the reaction. The reaction was incubated at 70 degrees C. for 10-20 min. After that optical density at 468 nm was measured using microtiter plate reader. Sample containing no enzyme (only buffer and substrate) was used for background correction, OD reading from this sample was subtracted from all OD values.

(26) One unit of laccase activity is defined as the enzyme amount oxidizing 1 micro mole of substrate per minute, one microkatal is the amount of enzyme oxidizing 1 micromol of substrate per second, and hence 10 millikatal equals 600,000 units. Absolute value of enzyme activity as measured in units or katals depends on the conditions under which this activity was determined and on the substrate used for activity measurement.

Example 3: Stability of Laccase Enzymes in Solution

(27) Enzyme solutions at a concentration of 0.1 mg per ml in Britton and Robinson buffer were adjusted to pH 9.0 or 11.0 before pre-incubation. Pre-incubation was carried out at a temperature of 40 degrees Celsius or 70 degrees Celsius for 0 h, 1 h, 2 h, 3 h or 4 h.

(28) After that, the residual activity was determined spectrophotometrically as described in Example 2. Initial activity (0 h) was taken as 100% for each enzyme (FIGS. 1A-1D).

Example 4: Pulp Delignification by Laccase

(29) Pulp used in these experiments was high lignin spruce Kraft pulp (kappa number 56) collected after oxygen delignification stage before bleaching stage. The pulp was washed thoroughly with hot tap water prior to the experiments. The delignification process mediated by laccase was analyzed by measuring the consumption of dissolved oxygen in samples during laccase treatment and eventually by measuring of kappa number of the resulting pulp.

(30) The dissolved oxygen measurements were made with a SensorLink PCM800 meter using a Clark oxygen electrode.

(31) The reactions were run in a 1 L stirring reactor with automatic pH and temperature control at 0.5% pulp dry weight content at 40 and 70 degrees C. The pH in the reactor was adjusted with NaOH solution to pH 9 or pH 11 and maintained constant. The laccase was dosed at 1 ukat/g of the pulp dry weight as measured by DMP oxidation in solution at pH 8, 70 degrees C. with photometric detection as described in Example 2.

(32) Prior to the reaction, the pulp suspension was equilibrated to the desired temperature and pH and air-saturated by stirring at 500 rpm in the reactor so that dissolved oxygen level was stable. This level was set as 100%. After that, the enzyme was added to the pulp and mixing continued for 1 min to distribute the enzyme and then stopped for 15 min. Substrate oxidation by the enzyme was followed by the gradual drop of dissolved oxygen as monitored by the oxygen probe. This oxygen drop is attributed to the fact that laccase is using oxygen as electron acceptor in the oxidation reaction and converts it to water, thus oxygen consumption can be directly linked to the enzymatic activity. The oxygen decrease remained essentially linear for at least 15 min. The slope value of oxygen decrease was taken as a measure of laccase enzyme activity (FIG. 2).

(33) After 15 minutes of incubation, the mixing was resumed and oxygen levels stabilized again typically at a slightly lower level than 100% due to continuous oxygen consumption by the enzyme. After 45 min, stirring was stopped for 15 min again and residual laccase activity was measured again by following the oxygen depletion rate. The experiment was continued for 4 hours and 15 min in the same manner, stopping mixing in the beginning of every hour for 15 min, and finally at the end of the 4 hour incubation period. After the experiment, pulp was washed with 2% NaOH and subjected to Kappa number measurement.

Example 5: Measurement of Kappa Number

(34) The Kappa number estimates the amount of chemicals required during bleaching of wood pulp to obtain a pulp with a given degree of whiteness. Since the amount of bleach needed is related to the lignin content of the pulp, the Kappa number can be used to monitor the effectiveness of the lignin-extraction phase of the pulping process. It is approximately proportional to the residual lignin content of the pulp.

(35) The kappa number or lignin content can be calculated using the following formula: Kc*l, wherein K: Kappa number; c: constant6.57 (dependent on process and wood); l: lignin content in percent.

(36) The Kappa number for bleachable pulps are in the range of 25-30, sack paper pulps in the range 45-55 and pulps for corrugated fiberboard are in the range 60-90.

(37) Kappa number was determined according to standard protocol Kappa Standard: ISO 302:2015 PulpsDetermination of kappa number; available at internet site: www.iso.org/iso/home/store/catalogue_ics/catalogue_detail_ics.-htm?csnumber=66 533.

(38) FIG. 5 shows the decrease in kappa numbers (indicative of the delignification of the pulp) after 4.25 hours of incubation as described in Example 4. Some decrease in Kappa number (increase in brightness) is caused by solubilisation of lignin in alkaline conditions at elevated temperature. This is why the control (without enzyme) shows a decrease in kappa number of 5. Oxidative degradation of lignin enhances the process of lignin solubilisation. That oxidative part can be followed by the oxygen consumption (FIG. 2).

Example 6: Lignin Depolymerization by Laccase

(39) Lignin used in these experiments was Cat #471003 Sigma ALDRICH Lignin alkali low sulfonate content.

(40) The lignin depolymerization process mediated by laccase was analyzed by measuring the consumption of dissolved oxygen in samples during laccase treatment (similar to example 4) and eventually by measuring of the molecular weight distribution of the lignin by size exclusion chromatography (SEC).

(41) The dissolved oxygen measurements were made with a SensorLink PCM800 meter using a Clark oxygen electrode.

(42) The reactions were run in a 1 L stirring reactor with automatic pH and temperature control at 2.2 gram per liter lignin at 40 or 70 degrees C. The pH in the reactor was adjusted with NaOH solution to pH 9 or 11 and maintained constant.

(43) The laccase was dosed at 1 ukat/g of the lignin dry weight as measured by DMP oxidation in solution at pH 8 at 70 degrees C. with photometric detection as described in Example 2.

(44) Prior to the reaction, the lignin solution or suspension was equilibrated to the desired temperature and pH and air-saturated by stirring at 500 rpm in the reactor so that dissolved oxygen level was stable. This oxygen level was set as 100%. A control experiment was performed without any enzyme. The enzyme was added to the lignin solution or suspension and mixing continued for 1 min to distribute the enzyme and then stopped for 15 min. Substrate oxidation by the enzyme was followed by the gradual drop of dissolved oxygen as monitored by the oxygen probe. The oxygen decrease remained essentially linear for at least 15 min. The slope value of oxygen decrease was taken as a measure of laccase enzyme activity (FIG. 2).

(45) After 15 minutes of incubation, the mixing was resumed and oxygen levels stabilized again typically at a slightly lower level than 100% due to continuous oxygen consumption. After 45 min, stirring was stopped for 15 min again and residual laccase activity was measured again by following the oxygen depletion rate. The experiment was continued for 4 hours and 15 min in the same manner, stopping mixing in the beginning of every hour for 15 min, and finally at the end of the 4 hour incubation period. After the experiment, the solution or suspension was filtered with a 0.45 micrometer pore size filter and the flow-thrue was subjected to molecular weight distribution measurement by size exclusion chromatography (Example 7).

Example 7: Size Exclusion Chromatography

(46) The molar mass measurements were performed with size exclusion chromatography using alkaline eluent (0.1M NaOH). For the molar mass measurements, the samples were diluted with 0.1M NaOH for the measurement consentration. In all cases the samples were filtered (0.45 m) before the measurement.

(47) The SEC measurements were performed in 0.1 M NaOH eluent (pH 13, 0.5 ml/min, T=25 C.) using PSS MCX 1000 & 100000 ngstrom columns with a precolumn. The elution curves were detected using Waters 2998 Photodiode Array detector at 280 nm. The molar mass distributions (MMD) were calculated against polystyrene sulphonate (8PSS, 3420-148500 g/mol) standards, using Waters Empower 3 software.

(48) We found that in the control samples, the average molecular size was above 2000 Daltons for all temperatures and pH values. The enzyme according to SEQ ID NO: 1 was superior in depolymerizing lignin at all conditions tested (pH 9 or 11 and 40 and 70 degrees Celsius. The depolymerization was most effective at 70 degrees Celsius and pH 11. Under these conditions, over 70% of the lignin molecules obtained with the laccase according to SEQ ID NO: 1 were below 1000 Daltons after 4 hours of incubation, whereas the samples obtained with the enzymes according to SEQ ID NO: 2-4 were more comparable to the control samples.