Lignin based coating

A method is provided for producing a coated article comprising: (a) preparing a solution of lignin; (b) mixing the lignin solution with a phenol oxidizing enzyme; (c) incubating the mixture from said step (b) under conditions and for a time sufficient to form a solution of a desired viscosity; (d) contacting or spreading the mixture from the step (c) on an article to be coated; and (e) allowing the coating to set onto the article by subjecting the article to conditions and for a time sufficient to form a coating on the surface of the article.

BACKGROUND OF THE INVENTION
 The present invention relates to methods for preparing coatings through
 enzymatic reactions with components of wood. More particularly, the
 present invention relates to the enzymatic polymerization of lignin based
 compositions with phenol oxidizing enzymes.
 It has been estimated that nearly 20% of the chemical pulp and dissolving
 pulp provided worldwide is produced by the sulfite process. Moreover, the
 significance of this process has grown with the large-scale employment of
 modifiable magnesium bisulfite pulping. Nonetheless, although there exist
 a number of uses for lignin sulfonates produced by these processes, it has
 been difficult to find commercially feasible and desirable means of
 disposing of this large waste stream for useful purposes. As described in
 U.S. Pat. No. 4,432,921, the use of spent sulfite liquor as an adhesive
 for paper, wood and other lignocellulosic materials is well known in the
 art, and is facilitated by enzymatic activation of the lignin using a
 phenol oxidizing enzyme. U.S. Pat. No. 5,505,772 describes a method of
 preparing particle boards by conglutinating wood fragments having middle
 lamella lignin with a phenol oxidizing enzyme, the process having the
 advantage that it is necessary to add no additional binder. Further, it
 has been shown that laccase and other oxidoreductase enzymes, such as
 peroxidases, can be used as polymerization or curing catalysts for lignin
 (PCT Publication No. WO/98/31761; EP 648 242).
 PCT Publication No. 93/23477 discloses the production of a solution or gel
 of lignin at high concentrations free of solids by treating the lignin at
 a high pH and then lowering the pH to a desired value. A binder can be
 made by subsequently treating with a phenolic oxidase.
 PCT Publication No. 95/07604 discloses a method of producing fibreboard,
 the method comprising the sequential steps of (a) providing an aqueous
 slurry or suspension of lignin-containing wood fiber material; (b) adding
 a phenol oxidizing system to the fiber slurry; (c) forming the fiber
 slurry into a mat of the wood fiber material; and (d) pressing the formed
 mat by applying heat and pressure to produce the fiberboard.
 PCT Publication Nos. 98/31761, 98/31762, 98/31763 and 98/31764 further
 disclose lignin based adhesives and methods of producing such adhesives
 for the production of fiberboards.
 PCT Publication No. 98/31728 discloses intermediates for the production of
 polymers from lignin derivatives from the pulp industry, produced by
 processing lignin derivatives with phenol oxidizing enzymes in the
 presence of oxidation agents. The invention is characterized in that the
 lignin derivatives are (a) subjected to enzyme treatment for more than 3
 hours in the presence of air; (b) subjected to enzyme treatment for more
 than 10 minutes while air or oxygen is passed through them; or (c) are
 oxidized by treatment with chemical oxidation agents. The intermediate is
 used for the production of polymers of lignin derivatives from the pulp
 industry, of fiber reinforced duroplastic composite materials from plant
 fibers, of water proof papers and cardboards, as well as duroplastics from
 lignin derivatives.
 Thus, there has been much activity in the field of using phenol oxidizing
 enzymes to produce adhesives from lignin. However, other uses of lignin
 and sulfite spent liquors are necessary to economically dispose of the
 large quantity of waste material produced by the pulp and paper industry.
 SUMMARY OF THE INVENTION
 It is an object of the present invention to provide for a method of
 producing a coating for use in the protection, beautifying or otherwise
 treating an object.
 It is a further object of the invention to provide for a method of
 producing such a coating from the waste liquors produced by the pulp and
 paper industry.
 According to the present invention, a method is provided for producing a
 coated article comprising: (a) preparing a solution of lignin; (b) mixing
 the lignin solution with a phenol oxidizing enzyme; (c) incubating the
 mixture from said step (b) under conditions and for a time sufficient to
 form a solution of a desired viscosity; (d) contacting or spreading the
 mixture from the step (c) on an article to be coated; and (e) allowing the
 film to set onto the article by subjecting the article to conditions and
 for a time sufficient to form a film on the surface of the article. In a
 preferred embodiment, the phenol oxidizing enzyme is a catechol oxidase,
 laccase, bilirubin oxidase, monophenol monooxygenase or peroxidase. Also
 preferably, the laccase is derived from Aspergillus, Bacillus, Neurospora,
 Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes,
 Rhizoctonia, Coprinus, Psatyrella, Myceliophthora, Schytalidium, Phlebia
 or Coriolus. Further preferably, the solution of lignin comprises lignin
 sulfonate. Also preferably, the lignin solution further comprises a
 copolymerization agent, for example, acrylic acid.
 In another embodiment of the invention, a coated article is provided,
 wherein the coated article comprises a film comprising lignin and a phenol
 oxidizing enzyme.
 In yet another embodiment of the invention, the coated article is not a
 lignocellulosic, wood or paper based product.
 An advantage of the present invention is that it is possible to prepare a
 useful item, i.e., a coated article, from the waste product of the pulp
 and paper industry.
 Yet another advantage of the present invention is that it is possible to
 produce a coated object using an environmentally safe process comprising
 enzymes and lignin.
 Yet another advantage of the present invention is that it is possible to
 produce a coated object from lignin, wherein the coated object is not a
 lignocellulosic material or derivative there of itself.
 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 "Phenol Oxidase" means an enzyme system which is capable of using hydrogen
 peroxide or oxygen to oxidize oxygen containing phenolic groups. If the
 phenol oxidizing enzyme requires a source of hydrogen peroxide, the source
 may be hydrogen peroxide or hydrogen peroxide precursor for in situ
 production of hydrogen peroxide, e.g., percarbonate or perborate, or a
 hydrogen peroxide generating enzyme system, e.g., an oxidase and a
 substrate for the oxidase, or an amino acid oxidase and a suitable amino
 acid, or a peroxycarboxylic acid or a salt thereof Hydrogen peroxide may
 be added at the beginning of or during the process. If the phenol
 oxidizing enzyme requires oxygen, atmospheric oxygen is usually
 sufficient, or alternatively, oxygen or air can be added to the system by
 percolating oxygen gas through the mixture. Phenol oxidizing enzymes can
 be catechol oxidases (EC 1.10.3.1), laccases (EC1.10.3.2), bilirubin
 oxidases (EC 1.3.3.5), monophenol monooxygenases (EC 1.14.18.1) or
 peroxidases (EC 1.11.1.7). Laccases are especially preferred and are known
 from microbial and plant origins. Preferred microbial laccases are from
 fungi or bacteria and include those from Aspergillus, Bacillus,
 Neurospora, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus,
 Trametes, Rhizoctonia, Coprinus, Psatyrella, Myceliophthora, Schytalidium,
 Phlebia and Coriolus. In a most preferred embodiment, the laccase is
 derived from Trametes, Coprinus, Pleurotus, Stachybotrys or Coriolus.
 "Lignin solutions" means an aqueous solution of lignin. Generally, it is
 possible to obtain suitable lignin solutions from the effluent produced
 during the process of pulping wood fiber and the production of papers,
 particularly the spent sulfite liquors. Examples of suitable lignin
 solutions include lignin sulfonates (also known as lignin sulfonates and
 sulfite lignins) which are the product of sulfite pulping, kraft lignins
 (also called sulfate lignins) which are obtained via the kraft pulping
 process, and lignins obtained from other technologies using organic
 solvents or high pressure steam treatments to remove lignins from plants.
 However, one of ordinary skill in the art would recognize lignins which
 are suitable.
 According to the process of the invention, the lignin solution is contacted
 with the phenol oxidizing enzyme system and contacted with the article to
 be coated. In practice, it is appropriate to incubate the lignin solution
 with the phenol oxidizing enzyme system for a time and under conditions
 sufficient to facilitate some polymerization of the lignin and develop a
 viscosity which is appropriate for the coating of the article. For
 example, it may be preferred in the event that a thick, textured coating
 is desired to allow the mixture to incubate for a longer time than if a
 thin coating is desired, prior to coating the article with the combined
 lignin and phenol oxidizing enzyme system solution. As suggested above, if
 a thick textured coating is desired, the viscosity of the solution should
 be permitted to become relatively greater such that in applying the
 solution to the article, the texture of, for example, the application
 brush, is permanently set into the coating. On the other hand, thin
 coatings would preferably start from a relatively less viscous solution of
 lignin and enzyme. Routine experimentation may be necessary to develop the
 precise timing and conditions to obtain the appropriate appearance of the
 coated article, however, such experimentation is well within the ability
 of one of ordinary skill in the art.
 In any event, the incubation time will be dependent on the conditions under
 which the reaction takes place, including consideration of temperature,
 pH, concentration of lignin, concentration of phenol oxidizing enzyme and
 the presence or not of accelerator compounds which promote the activity of
 the phenol oxidizing enzyme system.
 The concentration of lignin should be such as to facilitate polymerization
 of the lignin in a suitable amount to produce the coating and will be
 dependent to some extent on the concentration of enzyme used. For example,
 in a lower concentration of lignin, it may still be possible to produce a
 suitable coating by adding a relatively higher amount of phenol oxidizing
 enzyme system. Likewise, in a higher concentration of enzyme, it may be
 possible to obtain sufficient coating production with the addition of less
 phenol oxidizing enzyme system. Preferably, the concentration of lignin in
 solution is 10 g/l to 600 g/l, more preferably from 25 g/l to 400 g/l and
 most preferably from 50 g/l to 200 g/l.
 In the process of the invention, the concentration of oxygen or hydrogen
 peroxide can be determined using routine methods as an appropriate
 concentration or amount to facilitate the polymerization of the lignin.
 The concentration of phenol oxidizing enzyme is that amount which is
 sufficient to form the coating as provided herein, and will depend on, for
 example, the activity of the enzyme and its performance characteristics,
 the types of lignin and other components in the mixture. Nonetheless, the
 concentration of the enzyme can be determined routinely by the worker of
 ordinary skill.
 Generally, the temperature and pH will be dependent on the optimal
 conditions for the phenol oxidizing enzyme system. For example, where the
 phenol oxidizing enzyme system is from Trametes versicolor, the optimal pH
 is between 4.5 and 6.0 and the optimal temperature is from 15.degree. C.
 to 40.degree. C. Alternatively, where the phenol oxidizing enzyme system
 is a laccase from Stachybotrys chartorum, the optimal pH is between 6.5
 and 8.0 and the optimal temperature is from 15.degree. C. to 40.degree. C.
 The time required for the coating to set will depend on whether conditions
 are optimal or not, but will generally be at least 1 minute, and will
 usually range depending on conditions from between 10 minutes and 10
 hours.
 Other components may be added to the mixture prior to the application of
 the coating to the article to accelerate the polymerization of the lignin.
 For example, mediators may be added to the mixture such as ABTS
 (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or HOBT
 (1-hydroxybenzotriazole). Similarly, known mediators for use accelerating
 the activity of phenol oxidizing enzyme systems, for example in bleach
 compositions, may be used, see e.g., PCT Publication Nos. WO 96/12846 and
 WO 95/01426.
 Where it is desired that the coating be waterproof, it is possible to add
 copolymerization agents to the lignin solution prior to the application of
 the coating to facilitate water impermeability of the coating once it is
 set on the article. For example, acrylic acid, cellulose, starch, pulp,
 other carbohydrates, and chemical monomers capable of forming a
 copolymerization product with lignin may be used as a copolymerization
 agent.
 Articles suitable for coating using the invention provided herein include
 commonly coated articles such as wood, glass, plastics, metal, ceramics or
 other solid items capable of supporting the coating. An especially useful
 application of the present invention is with porous items for which it is
 desired to provide a sealing effect. Another especially useful application
 of the invention is due to the potential anti-microbial properties of the
 laccase enzyme in the mixture.
 In one embodiment of the invention, using the present invention it is
 possible that the article to be coated may surprisingly not be comprised
 of a substantial portion of lignocellulosic material, wood or paper.
 In order to further illustrate the present invention and advantages
 thereof, the following specific examples are given with the understanding
 that they are being offered to illustrate the present invention and should
 not be construed in any way as limiting its scope.

EXAMPLES
 Calculation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
 Units
 Reagents:
 50 mM Na acetate (pH 5.0): 1.5 ml
 4.5 mM ABTS in water : 0.2 ml
 Enzyme sample : 0.1 ml
 Dilutions of the enzyme are made in McIlvaine buffer pH 5 (mixture of 50 mM
 citric acid and 100 mM NaH.sub.2 PO.sub.4)
 Conditions:
 Wavelength : 420 nm
 Time : 30 seconds
 Temperature : 30.degree. C.
 Procedure:
 Add 0.2 ml ABTS to 1.5 ml 50 mM Na acetate (pH 5.0) in a cuvette and
 equilibrate to 30.degree. C. in a water bath. Transfer the cuvette to the
 thermostated cell holder of a spectrophotometer. Add the enzyme sample,
 mix the contents of the cuvette thoroughly and start measuring, using
 "auto rate assay". Read for 30 seconds, in 6 second intervals.
 Calculation:
EQU U/ml=(.DELTA.A.sub.420 nm/2) (Dilution factor)
 Reaction mixture A (using Trametes villosa laccase)
 2 g of ligninsulfonic acid (sodium salt), available from Extrasynthese
 (Genay, France), is dissolved in 5 ml McIlvaine buffer pH 5 (mixture of 50
 mM citric acid and 100 mM Na.sub.2 HPO.sub.4 in water). To this solution
 3.5 ml Trametes vilfosa laccase (470 ABTS units), 0.23 ml 1 M. citric acid
 and 0.12 ml water are added, giving a total volume of 10 ml at pH 5.5.
 (Optimum pH for this system)
 Reaction mixture B (with Stachybotrys chartarum laccase)
 2 g of ligninsulfonic acid (sodium salt), available from Extrasynthese
 (Genay, France), is dissolved in a mixture of 3.34 ml water and 5 ml.
 McIlvaine buffer pH 6 (mixture of 50 mM citric acid and 100 mM Na.sub.2
 HPO.sub.4 in water). To this mixture 0.165 ml of Stachybotrys chartarum
 laccase (140 ABTS units), 0.05 ml 1 M citric acid and 0.30 ml water are
 added, giving a total volume of 10 ml at pH 7. (Optimum pH for this
 system)
 Experimental Procedure
 2 ml of either reaction mixture A or B was placed in a glass reaction tube
 (outer diameter 1.6 cm, length 15 cm) The tube was dosed with a screw cap,
 placed in a rotary mixer (type Heidolph REAX 2) and rotated at a speed of
 75 rpm giving end over end rotation of the reaction tube. The reaction
 mixture flowed along the walls of the tubes facilitating contact between
 the liquid film and the oxygen in the air. The reaction tubes were
 incubated in this manner, at 24.degree. C. for 50 minutes the viscosity of
 the reaction mixture increases significantly after approximately 50
 minutes. After the incubation period ended, the walls of the reaction tube
 were covered with a solid coating formed of a layer of polymerised lignin.