BACKGROUND OF THE INVENTION Natural urushi has been used as a lacquer or paint for five thousand years. Natural urushi not only has excellent functional properties such as paint strength, durability or water-resistance but also an excellent gloss and tactility. Its unlimited potentials as a lacquer or paint can be well explained from the reports of some artifacts excavated in China. These artifacts were found with urushi paint on its surface, and have almost kept their original state even after two thousand years.

Natural urushi is in a state of an unstable suspension of oily components and water.

Thus, if natural urushi is directly painted on articles without any pretreatment process, phase separation problems will arise which, in turn, hinders formation and establishment of paint layer and also causes non-uniformity of the paint layer.

Therefore, if natural urushi is effectively used as a paint or lacquer it must be pretreated. The pretreatment process has been called"refinement". The "refinement"used herein means a process of making natural urushi substantially paintable by altering the state of oily components and water contained in natural urushi to a homogeneous suspension state and by providing the necessary amount of

oxygen for enzymatic polymerization reaction.

Up to the present, in the Republic of Korea, China, Japan etc., the refinement has been typically conducted by mixing natural urushi for 2 to 24 hours (or 2 to 15 hours) while maintaining a temperature of 30 to 40°C by using an electrical or infrared heater to vaporize the water component of natural urushi. Depending on the final usage, additives such as an animal or vegetable drying oil, a pigment, a natural or synthetic extender, etc. may be added.

However, such a conventional refinement treatment cannot completely remove bark, impurities, etc. included in the urushi sap. Furthermore, since the drying condition (e. g., temperature, humidity, kind and amount of diluent etc.) is absolutely dependent on the experience of the painter, the product quality is greatly dependent on the painter. It is practically not possible to continuously maintain the operator's skill at a similar or identical level. In addition, such a conventional refinement treatment has problems of the final product quality being greatly dependent on the quality of the source urushi.

Further, if articles are painted with the refined urushi according to the conventional refinement treatment, there may be problems such as undesired crinkles or paintbrush marks of the urushi paint layer. There also may be thermal discoloration or photo- decomposition problems. In addition, the transparency of the urushi paint layer may apparently be lowered. Further, since the drying process of the paint layer proceeds too slowly, the thickness of the layer cannot be controlled.

Due to the problems described above, conventional refinement methods are limited in their industrial application.

Natural urushi is a substance which may be secreted by the urushi tree when the urushi tree bark's is injured on its bark. In general, natural urushi comprises of urushiol, various enzymes including laccase (associated with urushiol hardening reaction), monosaccharides, water-soluble gums (a complex of oligosaccharides and poly saccharides), water-insoluble glycoproteins, moisture, dust etc. It was reported

that when moisture, dust, water-soluble gums, or water-insoluble glycoproteins exist in an excessive amount, it negatively affects functional properties of the paint layer such as paint strength, durability or water-resistance [Polymer (Japan), August 1999, pp. 586-597].

To remove the components that negatively affect the functional properties of the urushi paint layer, methods using concurrent homogenization and dehydration processes or using wiper-type depressurized thin layer evaporation apparatus or 3-axis roll mill were suggested so that moisture content and the diameter of water-soluble gums are decreased [Polymer (Japan), August 1999, pp. 586-597]. However, these methods did not exhibit good results as water-soluble gums and water-insoluble glycoproteins still exist in excess.

Therefore, there is a continuing need for a novel urushi lacquer or paint exhibiting the improved functional properties of the urushi paint layer.

The objective of the present invention is to provide a recombinant urushi lacquer having improved functional properties (e. g., gloss, color, transparency, drying property etc.), operability and economics.

Another objective of the present invention is to provide a process for preparing the recombinant urushi lacquer, which comprises artificially separating natural urushi into its components, and removing some components such as moisture, dust, etc. that negatively affect the functional properties of urushi lacquer and/or artificially controlling some of the components such as water-soluble gums and water-insoluble glycoproteins that are excessively contained in the natural urushi.

Another objective of the present invention is to provide a process for enzymatically polymerizing urushiol which is contained in natural urushi, to keep the excellent functional properties of natural urushi.

Another objective of the present invention is to provide a recombinant urushi lacquer, a process for preparing the same, and the use of the same, said recombinant urushi

lacquer having improved functional properties, these are attained by adding water- soluble enzymes, water-soluble gums or water-soluble glycoproteins to a urushi polymer which is prepared by enzymatic polymerization.

SUMMARY OF THE INVENTION According to the present invention, raw material (urushi) obtained from the urushi tree is separated into its components such as urushiol, water-insoluble glycoproteins, enzymes including laccase, water-soluble gums and other components by using an organic solvent such as acetonitrile etc., for the subsequent recombination of the components. After the recombination process, it can be subjected to enzymatic polymerization within an air injection reactor where laccase and other enzymes are uniformly dispersed or dissolved into urushiol solution by using an organic solvent such as 2-propanol and surfactant. If necessary, the degree of polymerization is further controlled by thermal polymerization or condensation. Also, at least one of the glycoproteins, water-soluble gums, enzymes or natural urushi source material may be added to further improve the functional properties of urushi lacquer.

BRTFF DFSrRIPTTnN HF THE nRAWTNf : <s Fig. 1 represents the molecular structure of urushiol dimer produced by an enzymatic polymerization reaction.

Fig. 2 represents the molecular structure of urushiol dimer produced by thermal polymerization or chemical polymerization.

Fig. 3 represents the changes of electrical conductance of mixture (urushiol + water) according to mole fraction of 2-propanol.

Fig. 4 is a schematic diagram for bubble column reactor.

Fig. 5A is a graph illustrating changes in the degree of polymerization when urushiol polymerization is conducted with 2-propanol and the enzyme solution which is

separated from natural urushi by using acetonitrile according to the present invention.

Fig. 5B is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with 2-propanol and laccase which is obtained from the lower layer of enzyme aqueous solution according to the present invention.

Fig. 6 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with 2-propanol and the enzyme solution obtained by using n-hexane according to the present invention.

Fig. 7 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted only with the enzyme solution obtained according to the present invention.

Fig. 8 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with cetyltrimethylammonium brimide (CTAB) and the enzyme solution obtained according to the present invention.

Fig. 9 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with sodium 1, 4-bis (2-ethylhexyl) sulfosucci nate (AOT) and the enzyme solution obtained according to the present invention.

Fig. 10 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with Triton-X-100 and the enzyme solution obtained according to the present invention.

Fig. 11 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with 1,4-dioxan and the enzyme solution obtained according to the present invention.

Fig. 12 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with ethanol and the enzyme solution obtained according to the present invention.

Fig. 13 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with acetonitrile and the enzyme solution obtained according to the present invention.

Fig. 14 is a graph illustrating changes in the degree of polymerization when the urushiol polymerization is conducted with tetrahydrofuran (THF) and the enzyme solution obtained according to the present invention.

Fig. 15 is a comparative illustration of the surface shape changes of the urushi lacquer of the present invention and the conventional urushi lacquer when the lacquer surface is irradiated by IR for 450 hours.

Fig. 16 is a comparative illustration of the surface shape changes of the urushi lacquer of the present invention and the conventional urushi lacquer when the lacquer surface is treated with acid and base.

Fig. 17 is a comparative illustration of the surface shape changes of the urushi lacquer of the present invention and the conventional urushi lacquer when the lacquer surface is treated with organic solvent.

Fig. 18A is a photograph illustrating the suspension state of the urushi prepared according to the conventional refinement process.

Fig. 18B is a photograph illustrating suspension state of the recombinant urushi prepared according to the present invention. nFTATI, Fn nF. ('RTPTTON OF THF. TNVFNTION The first step of the present invention is to separate natural urushi into its components.

A method which has been reported to be used as the separation method is one that uses acetone or ethanol [Tsukumanotami, Progress in Organic Coatings, 26 (1995); pp.

163-195]. By this method, natural urushi is separated into the upper layer

comprising urushiol and the lower layer comprising the other components. This conventional method requires additional steps in order to separate natural urushi into urushiol, water-insoluble glycoproteins, water soluble gums, and enzymes as in the present invention (described in detailed below). Thus, a costly multi-step process cannot be avoided. Further, even with such a multi step process, the purity of the separation product is not that high.

The present inventors have conducted intensive studies to develop a more effective separation process. We have found a novel process where natural urushi can be separated into urushiol, enzymes, glycoproteins, gums, moisture, and the other components in just a single-step (or two steps at most) process by using specific organic solvents. This novel process is advantageous in terms of high purity and low process cost.

The following Table 1 shows representative examples of organic solvents that may be used in the present invention for the separation of natural urushi. However, it should be understood that the kinds of organic solvents to be employed in the present invention is not limited to those shown in Table 1.

Table 1 Non-polar organic solvent Biphenyl,perchloro Ethane, pentachloro Naphthalene 1,3-Butadiene Ethane, 1,1,2,2-tetrachloro Nonane 1,3-Butadiene, 2-methyl Ethanethiol Octane Butane Ethane, 1,1,2-trichloro Octane Pentane, Butanic acid, nitrile, sulfur dioxide Ethane trichloro-trifluoro pentans, 1-bromo Carbontetrachloride Ethene Pentane, 1-bromo Chloroform Ethene, tetrachloro Pentanoic acid nitrile Cyclohexane Ethene, trichloro Cyclohexane, methyl Heptene Pentene-l Cyclohexane, perfluoro Heptene, perfluoro Phenanthrene Propane Cyclopentane Hexane Propane Decalin# Hexene-1 Propane, 1-bromo Cenane Malonic acid, dinitrile Propane, 2,2-dimethyl Dimethylsulfide Methane Propane, 2-nitro Ethane Methane, bromo Propane, 2-methyl Ethane, bromo Methane, dichloro Ethane chloro Methane, dichloro difluoro Propanoic acid nitrile Ethane, chloro Methane, dichloro difluoro Propanoic acid nitrile Ethane,1,2-dibromo Methane, dichloro monofluoro Styrene Ethane,1,1-dichloro Methane, nitro Hydrogenated Terpenil Ethane, difluoro tetrachioro Methane, tetrachloro difluoro W@ Ethane,nitro Methane, trichloro monofluoro m-Xylene Polar organic solvent Acetic acid 1-Dodecanol Acetic acid amide, N-ethyl Ethene,chloro Pentane, 1-emino Acetic acid, butyl ester Ethanol Pentane, I-Iodo Acetic acid, dichloro Ethanol, 2-chloro 1,3-Pentanediol, 2-methyl Acetic acid, ethyl ester Ether, 1,1,-dichloroethyl 1-Pentanol Acetic acid, anhydride Ether, diethyl 2-Pentanol Acetic acid, methyl ester Ether, dimethyl 2-Pentanoate Acetic acid, pentyl ester Ether, dipropyl Pentanone-2,4-hydroxy,4-methyl Acetic acid, propyl ester Ethylene glycol Pentanone-2,4-methyl Acetic acid amide, N,N-diethyl Ethylene, monobutyl ether Phosphoric acid, triphenyl ester Acetic acid amide, N,N-dimethyl Ethylene, monoethyl ether Phosphoric acid, tri-2-tolyl ester Acetic acid, buthyl ether Ethylene, monomethyl ether Phthalic acid, dibytyl ester Acetic acid, ethyl ether Formic acid Phthalic acid, diethyl lester Acetic acid, methyl ether Formic acid amide, N-ethyl Phthalic acid, dihexyl ester Acrylic acid Formic acid amide, N-methyl Phthalic acid, dimethyl ester Adipic acid, dioctyl ester Formic acid amide, N,N-diethyl Phthalic acid, di-2-metyyl ester Amine, diethyl Formic acid amide, N,N-dimethyl Phthalic acid, dipentyl ester Amine, ethyl Formic acid, ethyl ester Phthalic acid, dipropyl ester Amine, methyl Formic acid, methyl ester Piperidine Ammonia Formic acid, 2-methyl butyl ester 2-Piperidione Aniline Formic acid, propyl ester propane, 1,2-epoxy Aniline, N,N-dimethyl Furan 1,2-Propanediol Benzene, 1-methyoxy-4-propenyl Furan, tetrahydro 1-Propanol Benzoic acid, ethyl ester Furfural 2-Propanol Benzoic acid, methyl ester Glycerol 1-Propanol, 2-methyl Butanal 2-Heptanone 2-Propanol, 2-methyl Butane, 1,3-diol 2,3-Hexane diol 2-Propenol Butane, 1,4-diol 1,3 Hexane diol, 2-ethyl Propionic acid Butane, 2,3-diol Hexanoic acid, 6-aminolactam Propionic acid anhydride Butane, 1-Iodo Hexanic acid, 6-hydroxylactone Propionic acid, ethyl ester Butanoic acid, 4-hydroxlactone 1-hexanol Propinioc acid, methyl ester 1-Butanol 1-hexanol, 2-ethyl 1,2-Propanediol 2-Butanol Isophorone Pyrdin 1-Butanol, 2-ethyl Latic acid, butyl ester 4-Piroan 1-Butanol, 2-methyl Lactic acid, ethy ester 2-Pyrolidone 2-Butanone Maleic acid, anhydride 2-Pyrrolidone, 1-methyl Butyric acid Methacrylic acid Quinoline Carbonic acid, diethyl ester Methacrylic acid amide, N-methyl Sebaci acid, dioctyl ester Carbonic acid, dimethyl ester Methacrylic acid, butyl ester Sehaci acid, dibutyl ester Cyclohexanol Methacrylic acid, ethyl ester Stearic acid, butyl ester Cyclohexanone Methacrylic acid, methyl ester Succinic acid Cyclopetanone Methanol Sulfone, diethyl 2-Decanone Methanol, 2-furyl sulfone, dimethyl Diethylene glycol 1-Nonanol Sulfone, dipropyl Diethylene glycol, monobutyl ether Oxalic acid, diethyl ester Tetraethylene glycol Diethylene glycol,monoethyl ether Oxalic acid, ethyl ester Toluene, 3-hydroxy Dimethyl sulfuxide Oxirane 1,4-Dioxane

As shown in Table 1, the organic solvents to be employed in the present invention are classified into polar organic solvents and non-polar organic solvents. The preferred solvents for the present invention are acetonitrile, benzene, toluene, chloroform, hexane, heptane or octane.

Separation process using polar organic solvents Natural urushi may be separated with a polar organic solvent, such as acetonitrile.

Natural urushi solution and acetonitrile is mixed in a volume ratio of about 1.0: 0.5 to 1.0: 3.0. The mixture is cetrifuged at 3,000 to 10,000 rpm. Depending on the kind or origin of natural urushi, water may also be added. After centrifugation, the natural urushi is separated into the following three layers: (1) an upper layer of thick dark brown solution comprising urushiol; (2) a middle layer of thin brown solid comprising water-insoluble glycoproteins and water-soluble gums; and (3) a lower layer of azure solution comprising water-soluble enzymes such as laccase, peroxidase, and small amount of water-soluble gums.

As stated above, in the conventional separation method using acetone or ethanol, natural urushi is separated into two layers, the upper layer comprising urushiol and the lower layer comprising the other components [Tsukumanotami, Progress in Organic Coatings, 26 (1995); 163-195]. However, according to the present invention, natural urushi is separated into three layers. When acetone or ethanol is used, the lower layer of the centrifugation product is very viscous and sticky such that it attracts solid parts which otherwise may be in the middle layer separately. In comparison, when acetonitrile is used, the viscosity is not high and thus a separate middle solid layer may be formed on the lower solution layer.

After the phase separation is completed, the lower layer comprising water-soluble enzymes can be separated from the three layers by using any method well known in the art. As the lower layer contains highly concentrated water-soluble enzymes, it may be used as an enzyme source (so called"concentrated enzyme solution") in the enzymatic urushiol polymerization process that will be explained in detail below. In consideration of the fact that the cost incurred for the enzyme purification step is almost all (for example 60 to 90%) of the total cost incurred for the whole enzyme reaction process, it is greatly advantageous to directly use the lower layer as an enzyme source.

If necessary, the lower layer can be further separated into water-soluble gums and pure enzyme by using any method well known in the art, such as the precipitation or column method. Thus, the obtained pure enzyme can also be used as an enzyme source.

Any method well known in the art, such as the filtration method may be employed to separate the upper layer from the middle layer, as the upper layer is a solution comprising urushiol and acetonitrile and the middle layer is solid.

Separation process using non-poiar organic solvents Natural urushi may also be separated using a non-polar organic solvents, such as benzene, toluene, chloroform, hexane, heptane, or octane etc, as set forth below.

Water and a non-polar organic solvent in a desired ratio are added to natural urushi.

The natural urushi is then separated into the following three layers: (1) an upper layer of thick dark brown solution comprising urushiol; (2) a middle layer of thin brown solid comprising water-insoluble glycoproteins and water-soluble gums; and (3) a lower layer of azure solution comprising water-soluble enzymes such as laccase, peroxidase, and water-soluble gums.

Depending on the separation condition, the interface between the middle layer and the upper layer may not be clear (i. e., the two layers form a mixed layer). However, subsequent centrifugation with 3,000 to 10,000 rpm makes the mixed layer separated into an upper layer (thick dark brown solution comprising urushiol) and a middle layer (thin brown solid comprising water-insoluble glycoproteins and water-soluble gums). Once such a phase separation is completed, each layer can be separated from the three layers by using any well known method in the art.

As described above, since the lower layer contains highly concentrated water-soluble enzymes, it may be used as an enzyme source (so called"concentrated enzyme solution") in the enzymatic urushiol polymerization process, which will be discussed later in detail.

If necessary, pure enzyme can be separated from the lower layer by using any method well known in the art, such as the precipitation or column method. Thus obtained pure enzyme can also be used as an enzyme source.

The above separation methods according to the present invention have an advantage in that the natural urushi can be separated into urushiol, enzymes, water-soluble gums and water-insoluble substances in just a single step operation. In addition, as moisture is collected in the lower layer in these methods, the problem associated with the negative effect of the moisture component on the functional properties of the urushi lacquer may be solved by simply removing the lower layer. Further, organic solvents such as aceitonitrile, etc. are so highly volatile that they may be simply recovered from the urushiol solution and be re-used, thus the separation method according to the present invention is economical in terms of the process cost.

Preparation of recombinant urushi A process for preparing a recombinant urushi by separating natural urushi into three layers by using the separation method described above, and subsequently recombining the components of each layer, will be described in the below.

In order to prepare a recombinant urushi, polymerization of the urushiol in the upper layer should be performed first. Enzymatic polymerization, thermal polymerization or chemical polymerization can be employed.

In enzymatic polymerization, the phenol oxidase is usually employed. A phenol oxidase comprises laccase, peroxydase, or catalase etc. For example, laccase has a function wherein it provides reactivity to a phenyl group by altering the phenol group of urushiol to quinone to form a radical. As a result, urishiol can form various types of dimers, as illustrated by Fig. 1. Such dimers formed by enzyme reaction are highly reactive since they have alkyl groups having a double bond on the outer position.

On the other hand, as for thermal or chemical polymerization, the reactivity of double bonds on the alkyl groups are relatively higher than that of double bonds on phenyl groups, as shown in Fig. 2. Thus, it is difficult to obtain the desired properties of natural urushi from thermal or chemical polymerization, in view of the chemical structure, variety and component ratio.

Thus, in order to obtain the functional properties expected by natural urushi, it is essential to prepare dimers having low steric hindrance and high reactivity via enzyme reaction at the initial stage of the polymerization. The degree of urushiol polymerization can be further increased by preparing polymers to some extent by enzyme polymerization at an initial stage of the process and, then, if required, by thermal or chemical polymerization.

Enzymatic polymerization of urushiol Enzymatic polymerization of the urushiol existing in the upper layer separated by using the separation method according to the present invention is carried out as follows.

First, the urushiol solution of the upper layer is concentrated by using any method of the conventional processes well known in the art, such as depressurized concentration process. Then, the lower aqueous layer separated by using the separation method according to the present invention is diluted with water to about 1/100 to 1/10 (v/v).

The diluted enzyme solution thus obtained is added to the concentrated urushiol solution in a volume ratio of about 1/10, 000 to 1/10. Consequently, laccase contained in the lower aqueous layer catalyzes the polymerization of the concentrated urushiol.

For the polymerization of urushiol, a pure enzyme solution separated from the lower aqueous layer may be used instead of the diluted solution as above. A commercially available laccase may also be used. However, it is preferable to use the lower aqueous solution or pure enzyme obtained according to the present invention, since

the price of the enzyme is very expensive.

In order to further improve the functional properties of urushi solution, glycoproteins or gums may be added in a volume ratio of about 1/100 to 1/10. The middle layer separated according to the present invention as described above may be used as the source of glycoproteins or gums. For instance, a solution obtained by diluting the middle layer to a volume ratio of about 1/100 to 1/10 may be used.

The components to be added and the component ratio described above can be properly adjusted considering the desired properties of the recombinant urushi lacquer layer and/or painting conditions which are required for specific use.

In general, the reaction rate of an enzyme reaction increases as the contact area between the enzyme and the substrate increases. Thus, the reaction rate of the present invention can further increase by increasing the contact area between laccase and urushiol, the substrate of laccase.

Specifically, laccase as an aqueous enzyme exists in water drops in the urushiol solution, so that the reaction rate of laccase is determined by the degree of dispersion of water drops in the urushiol solution and the diameter of water drops. As the dispersion degree of water drops increases and the size of water drops decreases, the contact area between laccase and urushiol increases, which leads to the increase in the reaction rate of laccase.

Any method known to increase the contact area between an enzyme and a substrate may be employed in the present invention. For example, surfactant addition, organic solvent addition, vigorous stirring, etc. may be preferably used.

The surfactants which can be used to increase the contact area include cationic surfactants (such as cetyltrimethylammonium bromide), anionic surfactants (such as sodium 1,4-bis (2-ethylhexyl) sulfosuccinate), nonionic surfactants (such as Triton-X- 100), or zwitterionic surfactants (such as n-dodecyl-N-betaine). However, any type of surfactant may be used if it provides the function of increasing the contact area.

Table 2 shows a list of other examples of the surfactants.

Table 2 Anionic Detergent Zwitterionic Detergent Aerosol 22 Aerosol'-OT CHAPS Salt of : Alginic acid CHAPSO Carprylic acid N-Decyl-N, N'-dimethyl-3-ammonio-1- Cholic acid Propanesulfonate 1-Decanesulfonic acid N-Dodecyl-N, N-dimethyl-3-ammonio-1- Dehydrocholic acid Proapnesulfonate Deoxycholic acid N-Hexadecyl-N, N-dimethyl-3-ammonio-1- Dioctyl sulfosuccinate Propanesulfonate 1-Dodecanesulfonic acid N-Octadecyl-N, N-dimethyl-3-ammonio-1- 1-Heptanesulfonic acid Propanesulfonate 1-Hexanesulfonic acid N-Otcyl-N, N-dimethyl-3-ammonio-1- I-Nonanesulfonic acid Propanesulfonate I-Octanesulfonic acid Phosphatidylcholine I-Petanesulfonic acid N-Tetradecyl-N, N-dimethyl-3-ammonio-1- N-Laurylsarcosine Propanesulfonate Lauryl sulfate (dodecyl sulfate) Cationic Detergent Nonionic Detergent BIGCHAP Decanoyl-N-methylglucamide n-Decyl-D-glucopyranoside Alkyltrimethylammonium bromide n-Decyl-D-glucopyranoside Bezalkonium chloride n-Decyl-D-maltoside Benzethonium chloride Heptanoyl-N-methylglucamide Benzyldimethyldodecylammonium bromide n-Heptyl-D-glucopyranoside Benzyldimethylhexadecylammonium chloride N-Heptyl-D-thioglucopyranoside Benzyldimethyltetradecylammonium chloride n-Nonyl-D-glucopyranoside Benzyltrimethylammonium methoxide octanoyl-N-methylglucamide Cetyldimethylethylammonium bromide n-Octyl-D-glucopyranoside Cetylpyridinium n-Octyl-D-glucopyranoside Decamethonium bromide Octyl-D-thiogalactopyranoside Dimethyldioctadecylammonium bromide Octyl-D-thioglucopyranoside Methylbenzenthornium chloride Polyoxyethylene ester Methyl mixed trialkylammonium chloride Polyoxyethlene ether Methyltrioctylammonium chloride Polyoxyethylenesorbitan ester N. N', N'-polyoxyethylene (10)-N-tallow-1, 3- Sortitan ester diaminopropane Tergitol n-Tertadecyl-D-maltoside Triton Tyloxapol n-Undecyl-D-glucopyranoside

There are inherent problems with surfactant in that it is difficult to be separate them after the polymerization reaction. Particularly, in cases where a surfactant is used in a large amount, the control of the amount of the surfactant is very important because the surfactant could easily be scattered all over the urushi paint layer and deteriorate the functional properties of the urushi paint layer. Thus, the amount of surfactant should be properly controlled depending on the type of surfactant and the subject of urushi paint.

Further, the reaction rate of the enzyme polymerization can be improved by adding an organic solvent. Organic solvent facilitates urushiol being easily dispersed or dissolved, which leads to the increase in the reaction rate. In other words, depending on the amount of organic solvent added, the enzyme solution is dispersed in a small size, or the enzyme dissolved in water drops forms a single-phase with urushiol.

Thus, reactivity of laccase with urushiol may increase. However, when performing an enzyme reaction with organic solvents, the reactivity may decrease, depending on the properties of the organic solvent, by lowering the enzyme stability. In some cases, the reaction process cannot be easily controlled because of generation of bubbles during the reaction. Thus, the organic solvent should be appropriately selected depending on the purpose of use or the subject to be painted.

Representative examples among usable solvents are listed in Table 1.

In order to increase the contact area, a suspension or an emulsion state, where the water drops containing laccase are dispersed in urushiol, may be employed.

Further, a microemulsion state, where the diameter of the dispersed particle is about 10 to 200 nm, may be employed.

In a microemulsion, surfactant forms thermodynamically stable minute cells. Thus, total phase separation does not occur even though it is in a dispersed state. At this time, the minute cells theoretically have a remarkably broad surface area of about 100m2 in 1 ml.

In the present invention, enzyme reactivity was further improved by the use of a microemulsion system having such properties.

It has been known that 2-propanol can serve as a surfactant to form microemulsion [Y.

L. Kmelnizski, A. V. Revashov, N. L. Kliachiko, K. Martinek, Enzyme Microb. Tech., 10,710 (1988); G. Lund, S. L. Holt, JACOS, August, 264 (1980)]. With the knowledge that a microemulsion can be formed between urushiol and laccase without employing a surfactant if 2-propanol is used, the present inventors succeeded in effectively providing a recombinant urushi.

In view of the fact that a microemulsion can be established without using a surfactant (detergent), such a system is particularly referred to as"DMS (Detergentless Microemulsion System)". Since 2-propanol has high volatility and a low boiling point, it can be completely removed by mild heating, and the problems caused by adding a surfactant of low volatility do not occur. In addition, since the microemulsion system forms a single-phase stable medium, substrate transfer hindrance which usually occurs in 2-phase or 3-phase separation process can be avoided.

According to the previous report [Y. L. Kmelnizski, A. V. Revashov, N. L. Kliachiko, K. Martinek, Enzyme Microb. Tech., 10,710 (1988); G. Lund, S. L. Holt, JACOS, August, 264 (1980)], phase change occurs when a microemulsion is generated, and the electrical conductivity abruptly changes near the point of the phase change.

Fig. 3 shows the measurement of electrical conductivity during the addition of 2- propanol to a mixture of water and urushiol (1: 9 v/v). As can be shown in Fig. 3, electrical conductivity is changed remarkably between the 2-propanol mole fraction of about 0.4 to 0.45. This phenomenon occurs with an abrupt increase in the degree of dispersion of water drops and means that a microemulsion occurs in the range of such mole fraction. However, when the amount of 2-propanol added exceeds a certain

mole fraction (in case of Fig. 3, molar fraction of 0.45), the microemulsion breaks down and changes to a dissolved or coagulated state (Fig. 3,2-propanol mole fraction range of 0.45 to 0.7). Fig. 3 shows that electrical conductivity decreases again when the mole fraction of 2-propanol exceeds about 0.7. The reason for such decrease in conductivity is because the system itself has a relatively low conductivity, as the amount of 2-propanol increases. With a different volume ratio of water and urushiol, the mole fraction of 2-propanol where microemulsion occurs would then be altered.

Since laccase requires oxygen in its reaction, oxygen should be effectively supplied to the reaction system in order to successfully perform an enzyme polymerization using laccase. The reaction system should be well mixed, and affordable if performed in a large-scale for industrial purposes.

An example of the reaction system satisfying such requirements is bubble column reactor. The reactor is not restricted to this type, and any type of reactor may be used as long as air can be provided to the reactor. A person having ordinary skill in the art can properly select or alter the type, shape and operation mode of the reactor.

The enzyme reaction process according to the present invention is advantageous in that it is performed under ambient temperature and pressure conditions without producing any contaminants or by-products, and it is environmentally friendly since almost the total amount of the used solvent can be recovered and be re-used.

The recombinant urushi thus prepared according to the separation process and enzyme polymerization process according to the present invention basically has a far higher degree of polymerization than the urushi obtained according to conventional processes. Thus, the recombinant urushi prepared according to the present invention can be successfully used as paint material for crafts or as an industrial paint.

In some practical cases of painting, a specific degree of polymerization and a curing rate are required depending upon the type of surface to be painted or the painting

environment. In this case, the degree of polymerization of the recombinant urushi and the curing rate of the coated layer can be increased by adding glycoproteins or gums in a volume ratio of about 1/100 to 1/10. The middle layer containing glycoproteins and gums, which was separated according to the separation process of the present invention, may be employed as the source of glycoproteins or gums.

The degree of polymerization of the recombinant urushi solution can be additionally increased by thermal polymerization with heating.

In order to further improve the functional properties of the urushi paint layer, pure urushi may be added in a volume ratio of about 1/20 to 1/4.

In addition, a commercially available laccase may be added, if required, in a volume ratio of 1/100 to 1/10. It is preferable to use the enzyme solution itself which was separated according to the separation process of the present invention, a diluted solution of said enzyme solution [dilution ratio of 1/10,000 to 1/10 (v/v) with water], or pure enzymes obtained from said enzyme solution, instead of commercially available laccase.

The components to be added and the component ratio described above can be properly adjusted depending upon the desired properties of the urushi coated layer and/or painting condition which are required for specific use.

The present invention will now be described in detail by means of Examples.

These Examples are described only to illustrate the present invention, so that it should be noted that the scope of the present invention is not restricted or limited by the Examples by any means.

FXAMPIFS Example 1 : Separation and purification oLthe components of satural urushi by using acetonitrile 50 ml of natural urushi crude solution and 50 ml of acetonitrile were mixed and stirred and left for 30 minutes. This mixture was filled into two 50 ml volume corn type tubes and subjected to centrifuge for 10 minutes at 4,000 rpm by using bench-top swing bucket centrifuge.

As result of the centrifuge, the urushi solution was separated into three layers; namely, an upper layer comprising urushiol (hereinafter,"separated urushiol"), a middle layer comprising water-insoluble glycoproteins and water-soluble gums, and a lower layer comprising water-soluble enzymes. Each layer was separated and recovered by a conventional method. If conducting such a process on a large scale, a large volume centrifuge or continuous outflow centrifuge may be used.

Before conducting said process, the mixture of natural urushi lacquer crude solution and acetonitrile may be primarily filtered, or left for at least 24 hours to induce phase separation by gravity. A supernatant containing a small amount of solids can then be separated, which leads to a great improvement in work efficiency of the solid precipitate separation.

ExampleExample 2: Separation and purification of the components of natural urushi by using n-hexane 50 ml of natural urushi crude solution, 50 ml of n-hexane and 25 ml of water were mixed, stirred and left for 30 minutes. The solution was separated into an upper layer comprising urushiol, glycoproteins and gums and a lower layer comprising water-soluble enzymes. The lower layer was separated by a conventional method and the upper layer was subjected to centrifuge for 10 minutes at 4, 000 rpm by using

bench-top swing bucket centrifuge.

As result of the centrifuge, the upper layer was again separated into two layers; namely an upper layer comprising urushiol and a lower layer comprising glycoproteins and gums. Each layer was separated and recovered by conventional methods.

Example 3: Polymerization nf separateLurushioLbFusing enzyme solution layer (Acetonitrile) 200g of natural urushi crude solution was separated and purified by the acetonitrile method according to Example 1. About 20ml of the lower aqueous solution layer thus obtained was diluted 20 times with water and used as an enzyme solution (hereinafter,"separated enzyme solution"). 10 ml of the separated enzyme solution and 45 ml of 2-propanol were added to 90 ml of the separated urushiol.

Polymerization was then carried out in a 200 ml volume bubble column reactor (Fig.

4) with an air dispersion device, a reaction column and an air pump.

During polymerization, air injection speed into the reactor was maintained at 1.5L per minute at a temperature of 30°C. Samples were taken at 3 to 6 hour intervals and viscosity was measured. The viscosity was determined by the method described in Rotational Rheometry (I) [Polymer Science and Technologies No. 5 (3), 1994; pps.

275-283]. The result of viscosity measurement is shown in Fig. 5 A. The Y-axis in Fig 5A represents viscosity, and viscosity is an index which directly represents the degree of polymerization [R. J. Young, P. A. Lobel, Introduction to Polymers, 2nd ed., Chapman & Hall, London, 1991]. As shown in Fig. 5A, by using the separated enzyme solution, polymerization of the separated urushiol was successfully carried out. The same result was obtained in a 2L reactor.

When conducting such a process on a large a scale, an air compressor and a large volume reaction column made by material that is not corroded by urushiol and

involved solvents, such as stainless steel, glass, polypropylene and high density polyethylene, can be used. High density polyethylene tube with a depth of at least 0.05 mm can be used as a handy reaction column. Preferably, the length of the reaction column should not exceed 15 times the diameter of the reaction column, and the reaction solution should be less than 70% of the column volume.

Example 4: Polymerization of separatedLuiushiol by using enzyme solution layer (n-Hexane) 200g of natural urushi crude solution was separated in a manner similar to Example 2, except that n-hexane was used instead of acetonitrile. 100 ml of the resultant lower aqueous solution layer was diluted 20 times with water and used as an enzyme solution in this Example. 10 ml of the separated enzyme solution and 45 ml of 2- propanol were added to 90 ml of the separated urushiol. Polymerization was then carried out in a 200 ml volume bubble column reactor (Fig. 4) with an air dispersion device, a reaction column and an air pump.

During polymerization, air injection speed into the reactor was maintained at 1.5L per minute at a temperature of 30°C. Samples were taken by 3 to 6 hour intervals and viscosity was measured. As shown in Fig. 6, by using the separated enzyme solution, polymerization of the separated urushiol was successfully carried out.

Example 5: Polymerization of separated urushiol by using laccae A separated enzyme solution was obtained according to Example 3. From this solution, laccase was separated using the chromatography method. Primary purification was conducted via cationic chromatography, which fills Sephadex C-50 within an 8 x 25cm filling column. Inasmuch as the enzyme contains copper, it is easily recognized by its blue color. Secondary purification was then carried out based on anionic chromatography, which fills diethylaminoethyl (DEAE) A-50 within a 1.5 x 10cm column. The third purification was carried out by cationic

chromatography, which fills Sephadex C-50 within a 5 x 5cm column. The purified laccase thus obtained was diluted with 400 ml of water (pH 6.0). 10 ml of pure laccase and 45 ml of 2-propanol were added to 90 ml of the separated urushiol.

Polymerization was then carried out in a 200 ml volume bubble column reactor (Fig.

4) with an air dispersion device, a reaction column and an air pump.

During polymerization, air injection speed into the reactor was maintained at 1.5L per minute at a temperature of 30°C. Samples were taken by 3 to 6 hour intervals and viscosity was measured. As shown in Fig. 5B, by using pure laccase, polymerization of the separated urushiol was successfully carried out.

Example 6: Polymerization of separated urushiol by using separated enzyme solution only (ComparativeExample) Polymerization was carried out in the same manner shown in Example 3, with the exception that 10 ml of separated enzyme solution was used with 90 ml of separated urushiol. The viscosity measurement result is given in Fig. 7. Polymerization of the separated urushiol was successfully carried out even without prior addition of 2- propanol.

Example Z : Polymerizatioln of separated urushiol by using cetyltrimethylammonium bromide Polymerization was carried out in the same manner as shown in Example 3, with the exception that 10 ml of separated enzyme and 2 ml of cetyltrimethylammonium bromide were added to 88 ml of separated urushiol. The viscosity measurement result is given in Fig. 8. Polymerization of separated urushiol was successfully carried out.

Example8 : Polymerization of separated urushiol by using sodiuml, Sbis (2- ethylhexyl)sulfousccinate Polymerization was carried out in the same manner as shown in Example 3, with the exception that 10 ml of separated enzyme and 2 ml of sodium 1, 4-bis (2- ethylhexyl) sulfosuccinate were added to 88 ml of separated urushiol. The viscosity measurement result is given in Fig. 9. Polymerization of separated urushiol was successfully carried out.

Example 9 : Polymerization of separated urushiol by using Triton-X-100 Polymerization was carried out in the same manner as shown in Example 3, with the exception that 10 ml of separated enzyme and 2 ml of Triton-X-100 were addied to 88 ml of separated urushiol. The viscosity measurement result is given in Fig. 10.

Polymerization of separated urushiol was successfully carried out.

Example 1Q : Polymerization of separated urushiol by using 1, 4-dioxane Polymerization was carried out in the same manner as shown in Example 3, with the exception that 10 ml of separated enzyme and 20 ml of 1,4-dioxane were added to 90 ml of separated urushiol. The viscosity measurement result is given in Fig. 11.

Polymerization of separated urushiol was successfully carried out.

Example 11: Polymerization feparated-urushiol by usingthanoJ Polymerization was carried out in the same manner as shown in Example 3, with the exception that 10 ml of separated enzyme and 20 ml of ethanol were added to 90 ml of separated urushiol. The viscosity measurement result is given in Fig. 12.

Polymerization of separated urushiol was successfully carried out.

Example 12.: Poxmerization-of senarated nrushiol by usingacetonitrile Polymerization was carried out in the same manner as shown in Example 3, with the exception that 10 ml of separated enzyme and 20 ml of acetonitrile were added to 90 ml of separated urushiol. The viscosity measurement result is given in Fig. 13.

Polymerization of separated urushiol was successfully carried out.

ExampleM : Polymerization of separated urushialby using tetrahydrofuran Polymerization was carried out in the same manner as shown in Example 3, with the exception that 10 ml of separated enzyme and 20 ml of tetrahydrofuran were added to 90 ml of separated urushiol. The viscosity measurement result is given in Fig. 14.

Polymerization of separated urushiol was successfully carried out.

Example 14: Thermal polymerization Enzymatic polymerization proceeded until no more polymerization occurred, even when additional amount of enzyme were applied. It was then stirred in a 150° C oil bath for 2 hours. Consequently, further improvement in viscosity was observed. In this thermal polymerization, if the degree of polymerization excessively increases, a spongy type gel containing air is formed so that the urushi polymer cannot be painted.

In such cases, however the desired degree of polymerization can be controlled by simultaneously conducting decompression and thermal polymerization. At that time, depending on the purpose, sodium hydroxide, oxalic acid, hydrochloric acid or ammonia, and formaldehyde, phenol, furfural, urea, hexamine, linseed oil, castor oil, etc. may be added as a polymerization stimulating agent.

Example 1 ; ChChinnnt urnshi paint lsyer depending on the recoibination ratin For urushiol polymer according to Examples 3 to 14, various recombination ratios

were tested. Table 3 shows various recombination ratios between urushiol polymer, water-insoluble glycoproteins, water-soluble gums and separated enzyme solution, and characteristics of the resulting paint layer.

Table 3 Glycoprotein Additional Surface Drying Surface Adhesive Urushiol polymer Drying Transparency (%) and gums Enzymatic Time Time Quality power (%) (%) Solution Urushiol 99 1 0 4 16 **** **** ***** Polymer 95 5 0 1 5 ***** ***** **** Polymerized By enzyme 85 15 0 1 3. *** *** *** 98 1 1 6 24 97 1 2 4 18 **** **** ***** 96 2 2 3.5 12 ***** ***** ***** 95 3 2 2 8 ***** ***** ***** Urushiol 93 4 3 1.5 6 ***** ***** **** Polymer Polymerized 90 7 3 1 ***** ***** **** by heat 85 10 5 1 **** **** **** 80 15 5 1 *** *** *** 75 20 5 1 ** *** * 75 10 15 1. 5 4 *** *** *** High quality natural Korean Urushi 1 *** *** ** High quality natural Chinese Urushi 4#6 15#30 ** ** * ***** :A(excellent) ; ****: B (very good) ; ***: C (good); **: D (poor); *: F (bad)

As the composition of urushi paint, in general, shows large variation depending on the origin, harvest season, harvest method, storage period etc., the component ratio cannot be uniformly specified. However, roughly calculated, domestic high-grade natural urushi, before passing through purification process, comprises 60-70% of urushiol, 20-35% of water, 5-10% of water-soluble gums, 1-5% of glycoproteins and trace amounts of laccase. Chinese low-grade natural urushi comprises 50% of urushiol, 38% of water, 10% of water-soluble gums, 2% of glycoproteins and laccase.

As can be seen from Table 3, when glycoproteins and gums were combined to an

amount similar to that of natural urushi without the addition of enzyme, overall properties of the urushi paint layer was equal, despite the smaller amount of enzymes compared to natural urushi lacquer. When the amount of gums exceeds 10 to 15%, the amount that exists in natural urushi, deterioration of the paint layer properties was observed.

Urushiol polymer enzymatically polymerized according to the present invention is in a state where polymerization was already highly accomplished. Thus, the curing rate of paint can be accelerated by adding small amounts of natural urushi or by adding a certain amount of glycoproteins and gums which are obtained from the middle layer, as described above.

After having passed through thermal polymerization, separated enzyme solution can be added. At this time, the drying speed of the urushi paint layer increases as the amount of glycoproteins and gums increases. Depending on the purpose, the combination ratio can be optionally regulated. Preferably, desirable properties of the paint layer may be obtained by controlling the addition ratio of glycoproteins and gums within a range of from 1 to 10%. An enzyme solution may be added in an excessive amount, but an increase in the amount of enzyme-containing aqueous solution may cause a negative effect on the characteristics of the paint layer. Thus, adding an excessive amount of enzyme solution is not desirable.

Example 16 : Resistance-toJJV For the recombinant urushi paint according to the present invention, a UV exposure test was conducted according to Korea Standard Formula (KSF) 2274-95. With respect to the urushi paint according to conventional techniques, the gloss degree decreased below 50% upon exposure to UV for 450 hours, yet the paint layer retained the same strength as before exposure, confirming the possibility for use as external paint. This exposure of 450 hours corresponds to exposure to natural light of 5 to 8 years and the corresponding humidity.

It has been known that the aging of urushi paint due to UV light exposure is attributable to water-insoluble glycoproteins and gums included in the urushi solution [Polymer (Japan), August 1999, page 586]. In the present invention, the amount of glycoproteins and gums within the purified urushi lacquer was controlled to an amount smaller than that of natural urushi lacquer, i. e., 10%. As a result, UV tolerance was observed to be very superior to paint samples according to conventional techniques. In addition, even against LTV exposure of 450 hours, reflection ratio of about 80% was maintained and neither crack nor exfoliation was observed.

On the other hand, to investigate the aging degree of a urushi paint layer due to UV exposure, the strength of paint layer was determined by a pencil hardness measurement apparatus. Fig. 15 reveals that gums were carbonized by UV in the lacquer sample according to conventional techniques. Thus, the light reflection level decreased in all specimens, and the strength was below B grade. However, the urushi lacquer of the present invention retained the degree of strength that it had before exposure, i. e., 2H grade.

Example 17 Iluarability and functional properties To investigate industrial applicability for the various utilities of the recombinant urushi lacquer according to the present invention, suitable durability as well as functional properties were tested. All test items generally applied to paint were covered and the test method was in accordance with the Korea Standard Method (KSM) and the American Standard Test Method (ASTM). Table 4 summarizes the test result. It was confirmed that the recombinant urushi paint of the present invention can be used as an industrial paint for various uses.

Table 4 Test items Test condition Test result Test method 5%,30%,70+%H2SO4, Acid resistance No effect KSM5307* roomtemp., 168hr Decrease of 95%,30%NaOh, Polishment at 30% Base resistance NaOH treatment, no KSM 5307 room temp., 168hr change of surface hardness 95%benzene, toluene, No surface Organic solvent hexane, acetone, expansion, KSM3296 resistance tetrahydrofuran, ethanol No damage Heavy metal, harmful solvent etc. Cu Pb Cd Cr6+ Cr- Hg Environmental Not detected KSD8502 Trichloroethylene Tetrachloroethylene Organic phosphate Temperature No expanison and -20°C/84hr, 80°C/84hr KSM3752 fluctuationtest crack Adhesionstrength kg f/cm2 552 KSM3734 03crack 10ppm, 40°C, 48hr No crack KSM6518 Leak out water Waterpressure No leak KSM7564 At 100 kg f/cm2 Pencil hardness system High temp. Hardness hardness: 7H, room KSM3037 test temp. hardness: 2H Adhesion test At 100 kg f/cm² No detachment ASTM4541** anti-corrosive Sea-water spray, No corrosive damage ASTM6543 performance 35°C, 7 days Water repelling 10xl0cm/5m spray, Excellent, less than ASTM7519 ability residue measurement 135 mg Eleftric Conductance coating Less than 0.001# KSM4531 conductance 1 cm space Insulation coating, Electiricinsulation More than 5 k# KSM4531 1 cm space, 3,000 V

Example 18: Resistance to acid or akali Recombinant urushi lacquer's resistance to acid or alkali was measured. Tests were carried out against the standard conventionally applied to paints, i. e., 5% sulfuric acid or sodium hydroxide (Table 4), and harsher conditions, that is, 30% hydrochloric acid, sulfuric acid, acetic acid, nitric acid, ammonium hydroxide or sodium hydroxide, were also tested. As illustrated in Fig. 16, the recombinant lacquer of the present invention did not show any deterioration or degeneration of the paint layer upon treatment with 30% hydrochloric acid, sulfuric acid, acetic acid, nitric acid or ammonium hydroxide. When sodium hydroxide was treated, some decline in surface gloss occurred, but the physical properties such as paint layer hardness and transparency did not change.

Ex {<T) pte 19 : Resistance to organic solvent Recombinant urushi lacquer's resistance to organic solvents was tested. Tests were conducted using the general test standard for paints, using at least 95% and, under more severe conditions, 99% organic solvent. As shown in Fig. 17, the recombinant lacquer of the present invention did not experience any deterioration or degeneration upon exposure to at least 99% ethanol, benzene, acetone, xylene, toluene or tetrahydrofuran.

Example e 20: Comparison of suspension state between recombinant lacqner of thepresentinvention and that of conventional techniques As illustrated by Fig. 18, the suspension state of conventional lacquer was heterogeneous, that is, large lumps exist due to excessive glycoproteins and gums; whereas the recombinant lacquer of the present invention formed homogeneous suspension, that is, lumps were relatively small in size and uniformly distributed.

In view of above description, a person skilled in the art could easily understand the

essential feature of the present invention and make various modifications within the range of the present invention and apply them to various use and conditions.

TN1) TTSTRIAI APPT ICARlT ITY The present invention provides an innovative process for separating natural urushi into its components. Further, based on such separation techniques, the present invention provides economic recombinant urushi lacquer paint, which is far superior to natural urushi lacquer paint by conventional technique in terms of paint layer characteristics.

The recombinant lacquer of the present invention has high solid component content and low moisture content. Thus, the functional features of the paint layer, such as drying property, are superior. The recombinant lacquer of the present invention is very glossy, has few impurities, and has unique properties such as acid-resistance, alkali-resistance, resistance to chemical, tolerance to weather and bio-decomposition tolerance, and provides additional features, such as UV stability, owing to minimization of glycoproteins and gums.

The urushiol paint of the present invention can be processed by conventional methods into various paints, such as for the hull of ship, rust-proof paint, insulating paint, car paint, airplane paint, chemical-proof paint, lumber paint, industrial paint and musical instrument paint. In addition, conventional painting methods such as brush painting, roller-brush painting, spray coating, airless spray coating can be easily employed.

Moreover, urushiol polymer paint according to the present invention exhibits a high degree of polymerization. Thus, the occurrence of allergy and dermatitis, which occur with conventional lacquer paint, is noticeably low, maximizing the satisfaction of the painter and user.

The present invention has been described with reference to various specific examples.

However, it should be understood that numerous variations and modifications are possible to those skilled in the art without departing from the spirit of the present invention, and all such variations and modifications are intended to be within the scope of the claims which follow.