JP3068750B2 - Enzymatic printing method and azo dye decolorizing enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzymatic printing method and an enzyme for decolorizing azo dyes, and more particularly to a substrate dyed with an azo dye or with an azo dye and a non-azo dye. A method in which a substrate having a pattern drawn with a paste liquid containing at least a specific bacterium and an azo dye is treated with a specific bacterium for printing, and an azo dye decolorization for assimilating the azo dye For enzymes. Here, "printing" means direct printing (printing a pattern on a white background or ground dye fabric), discharge printing (printing a pattern on a ground dye fabric (including white discharge, colored discharge, and semi-discharge).) And anti-dyeing (things that write and dye patterns that inhibit dyeing).

[0002]

2. Description of the Related Art The above-mentioned "discharge printing" is performed by applying a paste containing a reducing agent or an alkali onto a previously dyed cloth in a desired pattern and performing a heat treatment to reduce the carboxylic acid ester of the disperse dye or a disperse dye. `` White discharge '' which decolorizes by saponification reaction and gives a white pattern, so-called `` color discharge '' where another color is dyed at the same time as decolorization, the pattern is a little lighter than the ground color by stopping white discharge in the middle "Semi-discharge" is performed. In the "direct printing", a predetermined pattern is applied (printed or the like) on a substrate using a predetermined color paste, and then dried, steamed, and washed to finish.

[0003]

However, in the above-mentioned printing method (particularly in the discharge printing method), since a reducing agent or an alkali is used for decolorization, the first non-color-dyed dye (so-called ground dye) is a dye which is easily decolorized. It is necessary to select a dye, and there is a restriction that the dye to be printed at the time of decolorization (a color dye) is limited to a dye having resistance to a reduction reaction or alkali. Further, there are drawbacks such as the fabric being damaged by the reducing agent and halation (bleeding at the color boundary portion) easily occurring.

[0004] The present invention overcomes the above-mentioned drawbacks, has less restrictions on the dye, has less pain on the base material, and has less bleeding at the color boundary portion, so that fine and complex patterns can be formed and vivid. Coloring discharge printing with various colors is possible, and blurring patterns can be easily created.
It is another object of the present invention to provide an azo dye decolorizing enzyme which enables the above-mentioned useful printing method by acting on an azo dye.

[0005]

Means for Solving the Problems The present inventors focused on the decolorization of azo dyes by enzymes produced by microorganisms and conducted a search to find enzymes that decolorize azo dyes from dyeing wastewater and other samples. As a result, strain Baci belonging to the genus Bacillus
Azoreductase (diaphora) produced by llusOY1-2
They found that an enzyme belonging to se) decolorized azo dyes, and completed the present invention.

[0006] In the method for printing with an enzyme of the first invention, the bacterium Baci of a bacterium belonging to the genus Bacillus is applied to a azo-based dye, a non-azo-based dye or a bacterial species on a dyed substrate dyed with an azo-based dye.
lulusOY1-2 (Accession number; FERM BP-52
61 ) The enzyme Azoreductase (di) produced by
(enzyme belonging to aphorase), a liquid or a paste containing at least the enzyme is applied in a desired shape, and then the enzyme is acted on to perform printing by eliminating or reducing the coloring by the azo dye. And

The method of printing with an enzyme according to the second aspect of the present invention relates to a method of printing an azo dye, a non-azo dye and a bacterium belonging to the genus Bacillus on a composite dyeing substrate dyed with an azo dye and a non-azo dye. Body Bacillus OY1-2 (accession number;
AZRM produced by FERM BP-5261 )
ductase (enzyme belonging to diaphorase)
And applying a liquid or paste containing at least the enzyme into a desired shape, and then performing the enzyme to perform printing by eliminating or reducing the coloring by the azo dye.

The enzymatic printing method according to the third aspect of the present invention is a method for printing an azo dye, a non-azo dye and a bacterium belonging to the genus Bacillus on an undyed substrate or a dyed substrate dyed with a non-azo dye. Bacillus OY1-2
(Accession number: FERM BP-5261 ) At least the azo dye of the enzyme Azoreductase (enzyme belonging to diaphorase) and a liquid or paste containing the enzyme are applied in a desired shape, and then
It is characterized in that printing is carried out by causing the enzyme to act to eliminate or reduce the coloring by the azo dye. Book 4
The printing method using an enzyme according to the invention is a method according to the first to third inventions, wherein the temperature at which the coloring by the azo dye is eliminated or reduced is set to 20 to 70 ° C.

In the printing method using an enzyme according to the fifth aspect of the present invention, a pattern heated to a temperature of 70 ° C. or more is drawn on a part of a desired shape formed by applying a liquid or paste containing the enzyme. It is a method of performing at least one of the steps of drawing a pattern with an alkaline liquid or paste having a pH of 10.5 or more, or an acidic liquid or paste having a pH of 4 or less. It is. In other words, printing is performed by drawing a pattern using a liquid or paste that inhibits the action of the enzyme (inhibits the reaction), thereby preventing decoloration of this portion and maintaining coloring with an azo dye. is there.

In the above, heating to 70 ° C. or more is performed by
This is because the reaction activity of the present enzyme is inhibited.
This heating means is not particularly limited. Further, the types of the respective alkali components and acid components contained in the above-mentioned alkaline solution and the like and the acidic solution and the like are not limited. The predetermined temperature (may be 70 ° C. or higher or lower), pH (10.5 or higher, 4 or lower, or other values) and processing time may be variously selected. The action of the enzyme may be blocked almost completely or incompletely.
In the latter case, a blur pattern (a different color pattern) can be used, and a more complicated and more beautiful pattern can be created by combining the two.

The azo dye decolorizing enzyme according to the sixth aspect of the present invention provides a bacterium B belonging to the genus Bacillus which acts on the azo dye to eliminate or reduce the coloring by the azo dye.
acillus OY1-2 (Accession number; FERM BP
-5261 ) produced enzyme Azoreductase
(Enzyme belongs to the diaphorase) Tona is, following
And wherein the Rukoto that have a nature. (1) an enzyme involved in the oxidation-reduction reaction; (2) an enzyme produced by the microbial cells; (3) production time of the enzyme is produced in stationary phase year; (4) an azo group 2 reduced to the number of amine groups; (5) as a coenzyme for reaction with the enzyme, NADP
H (nicotinamide adenine dinucleotide-phosphorus
Acid), NADH (nicotinamide adenine dinucleotide)
De - hydride) is necessary; (6) is stable at 20~70 ℃; (7) pH4.0~10 in a stable; (8) ethanol, is stable in acetone, ethanol
Can be extracted with acetone.

The "azo dye decolorizing enzyme" of the present invention was isolated by the following method. Sampling place Osaka Dyeing Co., Ltd. Sludge for dye wastewater treatment Sampling date September 22, 1990 The acclimatization treatment method of this enzyme is as follows. That is,
Three platinum loops of the sample were added to a test tube containing 2 ml of the enrichment medium, the cells were shake-cultured at 30 ° C., and the faded or decolorized test tube was inherited as needed. Then, even if the plant was subcultured twice, the test tube, which was still decolorized and faded, was mixed with the following agar medium for general bacteria (adding 0.02% of dye), and the dye was decolorized with flat plate agar. The bacteria that escaped were separated into strains (this was referred to as "Bacillus OY1-2 strain of the genus Bacillus" as shown below).
It was named. ). As this dye, an azo dye `` Roccelline NS conc 120% (CIAc
id Red88) ".

The composition of the enrichment medium is as follows. _{0.1% NaCl K 2 HPO 4 0.1} % MgSO 4 · 7H 2 O 0.05% FeSO 4 · 7H 2 O 0.001% ZnSO 4 · 7H 2 O 0.0001% CuSO 4 · 7H 2 O 0 _{.0001% MnSO 4 · 7H 2 O} 0.0001% (NH 4) 2 SO 4 0.5% dye (Roccelline NS conc 120%) 0.02 % dilution water using distilled water.

The above "Bacillus O of the genus Bacillus"
The "Y1-2 strain" has been deposited with the Institute of Industrial Science and Technology, and has a deposit number of FERM BP-5261 .
The scientific properties of this bacterium are as follows. The morphology is rod-shaped with a size of 0.6-0.8 × 3.0-5.0 μm, which is motile, has flagella and forms spores. Gram staining is positive. It grows in air and grows under anaerobic conditions. Grow at temperatures between 20 ° C and 50 ° C. 38
It grows well at ℃. It grows at a pH of 5-7. Salt does not grow above 2%, but grows well below 2%.

The scientific properties and properties of the bacteria are shown below. Gram reaction + shape spores Size 0.6-0.8 × 3.0-5.0 μm Spore shape Oval position of spore Neutral, semi-vertical, eddy Bacterial expansion-Mobility + in air Growth + growth under anaerobic conditions + catalase + oxidase DOF test-growth at -5C-growth at -10C-growth at 42C + growth at 50C + growth at 55C-growth at pH 5 and 7 + NaCl Growth at 2% + Growth at 5% NaCl-Reduction of nitrate + VP reaction + Indole production-Decomposition of urea + Utilization of citrate + Hydrolysis of starch + Hydrolysis of casein + Hydrolysis of gelatin-Carbohydrate: acid Glucose + mannitol + arabinose-xylose-gas (glucose) production-

The present inventors have described this strain as "Bacillus OY1".
-2 ". The azo dyes decomposed by the bacterium are usually used as a mixture of various isomers, but the bacterium can utilize all of them. Further, since the present bacterium has an activity even at a relatively high temperature of 40 to 50 ° C., it is possible to reduce the time required for the printing process and to prevent the germ from being mixed by killing the germ.

The enzyme of the present invention is produced as follows.
Purified. That is, first, the bacteria of the above Bacillus OY1-2 were inoculated and cultured under predetermined conditions to produce the enzyme,
This enzyme was purified and separated by centrifugation and various types of chromatography. The properties of this enzyme are as follows according to the results obtained in the following examples. [Properties of enzyme] (1) An enzyme involved in a redox reaction. (2) An enzyme produced in the cells. (3) The enzyme is produced in the early stationary phase. (4) The azo group is reduced to two amine groups. (5) NADP is used as a coenzyme in the reaction by this enzyme.
H (nicotinamide adenine dinucleotide-phosphate) and NADH (nicotinamide adenine dinucleotide-hydride) are required. (6) The enzyme reacts at an optimum pH of 7.5. (7) The optimum temperature is 70 ° C. 20-70 ° C is stable. (8) As for pH stability, pH 4.0 to 10 is stable. (9) Since it is stable up to 70 ° C. with respect to temperature, it has heat resistance. Since it has stability at this temperature, it is resistant to storage and decay. Sterilization is possible at 65 ° C. (10) Stable in ethanol and acetone. And
Since the enzyme can be extracted with ethanol and acetone, it is advantageous for industrialization. (12) Since it is an intracellular enzyme, it can be subjected to ultrasonic treatment and lysozyme treatment in order to destroy the cell membrane and elute (extract) the enzyme.

Further, the action and substrate specificity of the present enzyme will be described with reference to, for example, each of the azo dyes shown in FIGS. 1 and 2, by using the action of the present enzyme by utilizing the action of the coenzyme NADPH. The azo group of the dye is reduced to two amine groups. As a result, the coloring based on the azo group of the azo dye disappears.

The “substrate” in the present invention may be any one that can be dyed with an azo dye or the like.
It is a woven cloth (regardless of the fiber material type and weaving method), but is not limited thereto, and may be a nonwoven fabric, paper, a resin sheet, or the like.

The above-mentioned "azo dyes" include benzene azo dyes,
It can be applied to all of naphthalene azo dyes and heterocyclic azo dyes. In addition, this azo-based dye, regardless of the type of direct dye, acid dye, reactive dye, disperse dye, etc.
The present invention can be applied to all azo dyes classified according to the target materials and the functional groups shown in the above.

[0021]

[Table 1]

In the present invention, a liquid containing a predetermined enzyme (regardless of the presence or absence of a dye; if it contains a dye, it becomes a colored liquid), a paste containing a predetermined enzyme and a paste component (containing a dye) There is no particular limitation on the method of applying the dye on a predetermined base material, regardless of the presence or absence of the dye or the form of a paste or the like.
A desired coating method such as a pattern printing method, an ink jet method, a handwriting method, a roller printing method, and a screen printing method can be used. This paste is usually used for direct printing, and the paste components contained therein include starches (such as wheat starch), processed starches (such as British gum), processed natural gums (such as locust bean gum),
Sodium alginate, sodium carboxymethyl cellulose, synthetic paste (polyvinyl alcohol, polyvinyl acetate, etc.) and the like can be used.

[0023]

The present invention will be described below in detail with reference to examples. A. Production and properties of enzyme Azoreductase (enzyme belonging to diaphorase) (1) Production and purification of enzyme First, dry (normal) broth (Nissui Pharmaceutical Co., Ltd.) 200 m
azo dye (“Roccelline NS conc. 120%”, manufactured by Sumitomo Chemical Co., Ltd.) 0.02% was added to 1 l, and the strain Bacillus OY1-2 was inoculated into a 500 ml Sakaguchi flask.
After culturing with shaking for 8 hours, the culture was centrifuged (3000X).
g, 20 minutes), collect the cells, wash with 0.1 N phosphate buffer (pH 7.0, 1.0 mM DTT), and centrifuge (5000 × g, 10 minutes) to suspend the precipitated cells. Create a suspension. Further, the ultrasonic crusher is used for this.
W, irradiated with ultrasonic waves for 15 minutes (30 second intervals) to break down the cell membrane with the enzyme lysozyme, and then centrifuged (12000 × g, 60 minutes) to obtain an enzyme solution as a supernatant. Was. Furthermore, an ammonium sulfate solution (ammonium sulfate concentration 40%) was added to form a suspension,
The dialysate was purified by successively performing the following chromatographic treatments, and used as an enzyme solution for property evaluation and discharge. DEAE-sepharose CL-6B chromatography (eluent; 0 to
0.5N NaCl) Blue cellulofine affinity chromatography (eluent; 10 mM NADH) Red sepharose CL-6B affinity chromatography (eluent; 10 mM NADH) Gel chromatography (Hiload 16/60 Superdex 200pg)
[(Eluent: 0.02 M phosphto Buffer pH 7.0 (0.1 M NaC
l)]

(2) Optimum pH and stable pH range The optimum pH was determined by conducting an enzyme reaction experiment at each pH shown in Table 2 below. In addition, pH 4.0-5.5 uses an acetate buffer, pH 6.0-8.0 uses a phosphate buffer, and p
H8.5-9.5 used a Tris buffer, and pH10 used a borax buffer. Table 2 shows the results.

Table 2 --------------------------------------------------------------------------------------------------------------------------- 4.0 pH 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.0----------------------enzyme activity ( %) 5.2 8.6 23.1 39.2 57.3 68.5 87.9 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 7.5 pH 8.0 8.5 8.5 9.0 9.5 10.0 10.0 −−−−−− −−−−−−−−−−−−−−−−−−−−−−−−− Enzyme activity (%) 100 83.2 59.7 33.4 11.2 2.5 −−−−−−−−−−−−− From these results, the optimum pH of this enzyme is 7.5, and the pH range of the enzyme reaction is 5.5 to 9.5. It is.

The enzyme activity (titer) was measured as follows. The same applies to the following test examples. [Measurement of enzyme activity (titer)] The substrate is a dye of 40 μM Rocc.
10 ml β-N in 1.0 ml ellin NS 120% solution
The ADPH solution was adjusted to 100 µl, the volume of the solution was adjusted to 2.1 ml with an enzyme solution and 0.1 M phosphate buffer, and 505 n at 37 ° C.
This was done by following the decrease in absorbance of m. The enzyme unit was the amount of enzyme capable of reducing 1 μM substrate per minute at 37 ° C. The composition of the enzyme activity (titer) measurement solution is shown below.

(3) Stable pH range The pH stability experiment was carried out at 37 ° C. for 1 hour at a predetermined pH shown in Table 3, and then 1M phosphate buffer (pH 7.0).
After adding 5) and cooling with ice, the enzyme activity was measured. Table 3 shows the results.

Table 3 ------------------------------------------------------------------------ 4.0 pH 4.5 5.0 5.5 6.0 6.5 7.0 7.0----------------------enzyme activity ( %) 48.5 58.9 76.9 85.9 92.0 96.3 101 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 7.5 pH 8.0 8.5 9.0 9.0 9.5 10.0 −−−−−−−−−−− −−−−−−−−−−−−−−−−−−−−−− Enzyme activity (%) 100 101 96.3 86.1 80.0 62.1 −−−−−−−−−−−−−−−−−−−− −−−−−−−−−−−− From this result, the stable pH range is a wide range of pH 5.0 to 9.5. Even at pH 4.0 and pH 10, only about 50% is inactivated in about one hour.

(4) Range of optimum temperature for the experiment The experiment for the optimum temperature is performed by setting the enzyme reaction to each temperature shown in Table 4. Table 4 shows the results. Table 4--------------------------------Temperature (° C) 20 30 37 40 50 60 65 65 70 75 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Enzyme activity (U) 65 139 207 207 266 438 603 603 695 715 596 − −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Temperature (° C ) 80 85 −−−−−−−−−− Enzyme activity (U) 470 452 −−−−−−−−−−−−−− From these results, the optimum temperature is 70 ° C.

(5) Temperature Stability of Enzyme In the temperature stability experiment of the enzyme, the enzyme solution was adjusted to pH with a phosphate buffer.
Adjusted to 7.0, reacted at each temperature shown in Table 5 for 1 hour,
Immediately on ice and the enzyme activity was measured. Table 5------------------------------Temperature (° C) 20 30 37 40 50 55 60 65 70 70 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Enzyme activity (U) 426 449 426 426 454 423 456 425 426 − −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Temperature (° C ) 75 80 −−−−−−−−−−− Enzyme activity (U) 51 12 −−−−−−−−−−−−−−

(6) Conditions for deactivation by pH, temperature, etc. Deactivation by pH is, as inferred from the above-mentioned stability of pH, 50% or more between pH 4.5 and 10.0. Enzyme-stable, but not more than pH 4.0, pH 10.5
The above seems to be deactivated. Inactivation by temperature is stable up to 70 ° C. from the above-mentioned temperature stability, but above that temperature, the enzyme is inactivated.

(7) Crystal structure Although the entire amino acid sequence of the protein of this enzyme (Azoreductase) has not been elucidated yet, the sequence is partially analyzed from the N-terminus by analysis. The following is the N-terminal methionine (the details are shown in Table 6). That is, MKLLVINGGPRK
Twenty FGRTRVAs have been determined. Table 7 shows the types and abbreviations of the amino acids constituting the protein.

Table 6: Call First Second Third ------------------------------------ ------------------------------------------ 1 MGS2K PN3LEW4VYM5VTSS6ICD7NWF8GY S9TQF10PHC11RPC12KRA13FSE14GLI15RPC16TIV17RILI18VAL19VCW20AGS- −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

[0034]

[Table 7]

B. Examples of Use of the Discharge Enzyme Solution of the Present Invention Hereinafter, use examples of the enzyme solution of the present invention will be described with reference to specific examples and comparative examples. (1) White discharge dyeing Example 1 3% by weight azo direct dye “Direct Brilliant Blu”
When a cloth made of 100% rayon taffeta was dyed by a conventional method using "eRW" (manufactured by Mitsubishi Kasei Hoechst Co., Ltd.), the cloth was dyed blue. A rubber plate with a polka dot pattern cut out was placed on this blue-dyed cloth, and the main discharge dyeing enzyme solution shown above was poured into the cut-out pattern, and after removing the rubber plate, the cloth was wetted. 60 ℃
Was placed in a constant temperature bath for 1 hour to perform white discharge treatment. As a result, the azo dye dyeing the cloth was completely decolorized, and a white polka dot pattern was obtained on a blue background. In addition, a clear pattern was shown without bleeding of the color boundary portion.

Example 2 In this example, nylon 1 was used as the material of the cloth used.
As a dye for dyeing this cloth, a 3% by weight azo acid dye "SuminolMilling B" was used.
rilliant Red BS "(manufactured by Sumitomo Chemical Co., Ltd.) (ground color: red), a triangular cut-out rubber plate for injecting an enzyme solution was used, and the discharge treatment conditions were 50 ° C., 2
Except for the time, white discharge printing was performed in the same manner as in Example 1. As a result, a white triangular pattern was obtained on a red ground. Also in this case, a clear pattern was shown.

Embodiment 3 In this embodiment, wool 10 was used as the material of the cloth used.
0% muslin was used, and 1% by weight azo acid dye "KayanolMilling Y" was used as a dye for dyeing this cloth.
ellow 0 "(manufactured by Nippon Kayaku Co., Ltd.) and 1% by weight azo acid dye" Kayanol Milling Red BW "(manufactured by Nippon Kayaku Co., Ltd.) (ground color: orange), star pattern A white discharge treatment was carried out in the same manner as in Example 1 except that a rubber plate for injecting an enzyme solution was used, and the conditions for discharge treatment were 40 ° C. and 3 hours. As a result, two types of azo dyes dyeing the cloth were assimilated simultaneously, and a white star pattern was obtained on an orange background.
The same result can be obtained even when the discharge printing conditions are set at 20 ° C. for 8 hours. The azo acid dye “Kayanol Mill”
"ing Red BW" is not used for ordinary discharge printing because of its poor dischargeability with a reducing agent. Therefore, according to this example, it was possible to reliably perform discharge printing even when using an azo acid dye that cannot be normally used for discharge printing.

(2) Color Discharge Dyeing Example 4 In this example, 100% cotton was used as the material of the cloth used.
The use of GABA, 1
Weight percent anthraquinone-based reactive dye "Mikasion Bri
lliant Blue RS "(Nippon Kayaku Co., Ltd.) and 1% by weight
Concentration of an azo reactive dye “Remazol Red B 150” (manufactured by Mitsubishi Kasei Hoechst Co., Ltd.) (ground color: purple),
Using a brush on the dyed cloth, the pattern was drawn using the enzyme solution for discharge used in Example 1, and the conditions for discharge treatment were 70
A color discharge treatment was carried out in the same manner as in Example 1 except that the temperature was 1 ° C. and 1 hour. As a result, as for the treated cotton cloth, the azo dye dyeing the cloth was assimilated, and the non-azo dye dyeing the cloth remained without being decolorized, and was drawn with a discharge enzyme solution on a purple background. The pattern was obtained as a blue color (colored by the anthraquinone dye). In addition, a clear pattern was shown without bleeding of the color boundary portion.

Embodiment 5 In this embodiment, wool 10 was used as the material of the cloth used.
The use of 0% muslin and the use of 1% by weight anthraquinone acid dye "Alizar
ine Rubinol 3G 115% "(manufactured by Yamada Chemical Industry Co., Ltd.) and 1% by weight azo acid dye" Kayanol Navy Blue "
R "(manufactured by Nippon Kayaku Co., Ltd.) (ground color: purple), a rubber plate for injecting an enzyme solution with a polka dot cut out, and a discharge treatment condition of 50 ° C. for 3 hours Except for the above, the color discharge treatment was performed in the same manner as in Example 1. As a result, in the treated 100% wool cloth, the azo dye dyeing the cloth is successfully decolorized in a short time at 50 ° C., and the non-azo dye dyeing the cloth remains without decoloring. As a result, a red polka dot pattern was obtained on a purple ground. Also in this case, a clear pattern was shown.

Example 6 In this example, diacetate 10 was used as the cloth used.
A 0% plain woven fabric was used, and a 3% by weight azo disperse dye "KayalonFast Yell" was used as a dye for dyeing the fabric.
ow G ”(manufactured by Nippon Kayaku Co., Ltd.), 3% by weight azo disperse dye“ Kayalon Fast Rubine B ”(manufactured by Nippon Kayaku Co., Ltd.) and 3% by weight anthraquinone disperse dye“ Kayalon Fast Blue FN ” (Manufactured by Nippon Kayaku Co., Ltd.) (ground color: black), a triangular cutout rubber plate for injecting an enzyme solution was used, and the discharge treatment conditions were 60
A color discharge treatment was performed in the same manner as in Example 1 except that the temperature was 2 hours at ℃. As a result, the two types of azo dyes that dye the cloth are simultaneously assimilated, and the non-azo dye that dyes the cloth remains without being decolorized, and a blue triangle pattern is obtained on a black background. Was. Also in this case, a clear pattern was shown.

Example 7 In this example, the enzyme solution used in Example 1 was used as the enzyme solution, and the azo acid dye "Kayanol Milling Yellow" was used.
3GW "(manufactured by Nippon Kayaku Co., Ltd.), 4% by weight was used, wool was used as the material of the cloth, and an anthraquinone acid dye" Alizarine Rubinol 3G 115 "was used as a dye for dyeing the cloth. % ”(Yamada Chemical Industry Co., Ltd.) and the azo acid dye“ Kayanol Navy Blue R ”
(Manufactured by Nippon Kayaku Co., Ltd.) (ground color: purple), followed by discharging, followed by steam treatment at 100 ° C. for 60 minutes at normal pressure, followed by washing with water and hot water. In the same manner as in Example 1, colored discharge printing was performed. As a result, both the azo dye on the enzyme solution side and the azo dye that dyes the cloth are substantially decolorized, and the non-azo dye that dyes the cloth remains without decolorization and remains substantially red on a purple background. Was obtained.

Example 8 In this example, an azo acid dye "Kayanol Navy Blue R" was added to the enzyme solution used in Example 1 as the enzyme solution used.
(Nippon Kayaku Co., Ltd.) and an anthraquinone-based acid dye “Alizarine Rubinol 3G 115%” (Yamada Chemical Co., Ltd.) added at 2% by weight.
The use of wool as the material of the fabric and the azo acid dye "Kayanol Navy Blue R" as a dye for dyeing this fabric
Color discharge printing was performed in the same manner as in Example 7 except that (Nippon Kayaku Co., Ltd.) was used (ground color: blue). As a result, both the azo dye on the enzyme solution side and the azo dye dyeing the cloth are almost decolorized, and the non-azo dye on the enzyme solution side remains without decolorization, and red to magenta on a blue background. Was obtained.

Example 9 In this example, the enzyme solution used in Example 1 was used in combination with the anthraquinone-based acid dye "Suminol Fast".
Blue G ”(manufactured by Sumitomo Chemical Co., Ltd.), 3% by weight of a colored discharge liquid was used, wool was used as a material of the cloth, and an azo acid dye“ Kayanol Milling ”was used as a dye for the cloth. Yellow 0 ”(manufactured by Nippon Kayaku Co., Ltd.) 2% by weight and azo acid dye“ Kayanol Milling Re
Color discharge printing was performed in the same manner as in Example 7 except that 2% by weight of “d RS 125” (manufactured by Nippon Kayaku Co., Ltd.) was used (ground color: orange). As a result, the two types of azo dyes dyeing the cloth were decolorized, and the non-azo dye on the enzyme solution side remained without being decolorized, and a predetermined blue pattern was obtained on an orange background. .

Example 10 In this example, an anthraquinone-based disperse dye “Diacelliton” was added to the enzyme solution used in Example 1 as the enzyme solution used.
"Fast Pink R" (manufactured by Mitsubishi Kasei Hoechst Co., Ltd.), 3% by weight of a colored discharge liquid was used, a 100% diacetate plain woven cloth was used as a cloth, and an azo dye was used as a dye for dyeing the cloth. Disperse dye `` Kayalon Fast Yel
low G ”(Nippon Kayaku Co., Ltd.) 1% by weight, azo-based disperse dye“ Kayalon Fast Rubine B ”(Nippon Kayaku Co., Ltd.) 1% by weight, and anthraquinone-based disperse dye“ Suminol ”
Fast Blue G ”(manufactured by Sumitomo Chemical Co., Ltd.) (ground color: black), steam treatment conditions were 100 ° C., 30
The color discharge treatment was performed in the same manner as in Example 7 except that the color was discharged. As a result, the two types of azo dyes dyeing the cloth are decolorized, and the two dyes on the enzyme solution side and the dyed cloth side are decolorized.
The non-azo dyes remained without being decolorized, and a predetermined light purple pattern was obtained on a black background.

Example 11 In this example, a non-azo reactive dye (anthraquinone reactive dye) "Mikacion Brilliant Blue RS" (manufactured by Nippon Kayaku Co., Ltd.) was used as the enzyme solution used in Example 1 above. A) to which 3% by weight) was added and 0.1% by weight of the dye
Using the added liquid B, using cotton as the material of the cloth, and using the azo-based reactive dye “Remazol Red B150” (manufactured by Mitsubishi Kasei Hoechst Co., Ltd.) as a dye for dyeing the cloth.
2% by weight was used to dye a red background, the prescribed pattern was drawn using the two kinds of liquids A and B, and the discharge printing condition was 70 ° C. for 1 hour.
Example 7 was repeated except that the post-treatment conditions were as follows: immersion in an alkaline solution (90 ° C.) for 15 seconds, washing with water, immersion in a 5% by weight aqueous solution of acetic acid for 15 seconds to neutralize, and then washing with hot water and water.
Color discharge was performed in the same manner as described above. As a result, the non-azo dye on the side of the enzyme solution having a different concentration remained as it was, and two predetermined patterns of dark blue and light blue were obtained on a red background.
The composition of the above-mentioned alkaline liquid (the following “%” represents% by weight) is as follows. Anhydrous sodium sulfate: 10%, sodium carbonate: 15%, potassium carbonate: 5%, sodium hydroxide: 2%, sodium silicate: 1%, water: 67% (total 100%).

Comparative Example 3% by weight azo reactive dye "Remazol / Diamira Bril"
A rubber plate from which a polka dot pattern was cut out was placed on a 100% cotton broad cloth dyed by a conventional method using "l.Red 5B" (manufactured by Mitsubishi Kasei Hoechst Co., Ltd.). Then, the following colored discharge paste is printed on the cut out pattern portion. After drying, heat treatment is performed at 100 ° C. for 10 minutes, then superheated steam treatment is performed at 170 ° C. for 7 minutes, and after washing with water, sodium perborate 5 g / liter, bath ratio 1:40, oxidation treatment is performed at 50 ° C. for 10 minutes. And then washed and dried. [Composition of colored discharge paste] (The following “%” represents% by weight.) Sleek dye “Mikethren Brilliant Blue R” (manufactured by Mitsui Toatsu Dye Co., Ltd.): 3%, Rongalit C: 12%, potassium carbonate: 7%, Nafka crystal gum 35% aqueous solution: 60%, glycerin: 3%, water: 15% (total 10%)
0%). As a result, a blue polka dot pattern was obtained on a red ground. In this case, equipment for steam treatment is required, it is necessary to prepare various types of chemicals, and the process is complicated.

(3) Direct printing Example 12 In this example, 4% by weight of an azo-based direct dye "Kayarus Supra Yellow RL" (manufactured by Nippon Kayaku Co., Ltd.) and copper phthalocyanine were added to the enzyme solution used in Example 1 above. System direct dye `` Sumi
light Supra Turquoise Blue G conc. "(Sumitomo Chemical Co., Ltd.) 4% by weight and British gum 10% by weight
(The rest is water.) The kneaded paste is kneaded and kneaded as a colored discharge paste. On the other hand, when a 3% by weight azo-based direct dye “Kayarus Supra Red 6BL” (manufactured by Nippon Kayaku Co., Ltd.) is used to dye 100% rayon fabric using a conventional method, the fabric is dyed red. Went up. A 100% rayon dyed red dyeing paste is squeezed out of the tube to form a pattern. This cloth was placed in a constant temperature bath at 50 ° C. for 3 hours in a wet state, and then taken out of the constant temperature bath, subjected to steam treatment at 100 ° C. for 40 minutes at normal pressure, and washed with water and hot water. As a result, the size liquid side and the dyed cloth side
The azo dyes of the type are substantially decolorized (the azo dyes on the size liquid remain to some extent), and the non-azo dyes on the size liquid side remain without being decolorized, leaving a blue-green or A green-blue pattern was obtained.

(4) Degree of Discharge Example 13 In this example, the enzyme solution used in Example 1 was used, and two kinds of cloth, rayon or cotton, were used. Azo-based direct dye “Kayarus Su”
pra Yellow RL ”(manufactured by Nippon Kayaku Co., Ltd.) (ground color: yellow).
The test was performed in the same manner as in Example 1 except for the time. As a result, in the case of cotton, the coloring by the azo dye was completely decolorized, but in the case of rayon, the color was lightened but not completely decolorized. When this rayon was treated in the same manner at the same temperature for 5 hours, the color was almost completely decolorized. Therefore, it is understood that the coloring by the azo dye is bleached or reduced depending on the conditions, and the degree of bleaching varies depending on the type of fabric and the bleaching temperature.

Example 14 In this example, the enzyme solution used in Example 1 was added to the enzyme solution used in Example 1 above, and the azo-based direct dye "Kayarus Supra Yellow R" was used.
L "(manufactured by Nippon Kayaku Co., Ltd.), 3% by weight of colored discharge printing liquid was used, rayon was used as the material of the cloth, and 3% by weight of an anthraquinone-based reactive dye" Mikacion Brilliant Blue RS ”(manufactured by Nippon Kayaku Co., Ltd.) was used (ground color: blue), and the semi-colored discharge treatment was performed in the same manner as in Example 1 except that the discharge condition was 50 ° C. for 1 hour. Was. As a result, all of the azo-based direct dye was not decolorized and a part thereof remained, so that a blue-colored greenish pattern appeared on a blue background. As described above, the semi-discharge printing is performed under the conditions of the discharge printing using the rayon base material, so that the blur dyeing becomes possible.

Example 15 In this example, a copper phthalocyanine-based direct dye “Sumiligh” was added to the enzyme solution used in Example 1 as a colored discharge paste solution.
t Supra Turquoise Blue G conc. "(manufactured by Sumitomo Chemical Co., Ltd.), 4% by weight, azo direct dye" Kayarus Supra Yellow "
RL "(manufactured by Nippon Kayaku Co., Ltd.), 4% by weight and 10% by weight of British gum (the remainder is water) and kneaded, and the azo direct dye" Kayaru "
s SupraYellow RL ", a glue B not containing only" Supra Yellow RL ", 100% rayon as a material for the cloth, and the cloth being undyed.
A direct printing process was performed in the same manner as in Example 12, except that a predetermined pattern was drawn using each type of paste (agent A and agent B). As a result, under these discharge conditions, while a part of the azo dye on the paste A side is decolorized, the paste B
Since no azo dye to be decolorized was contained on the side, two kinds of patterns, greenish blue (partly bleached) and blue, were formed on a white background.

(5) Effects of the Examples The various azo dyes (reactive dyes, direct dyes, acid dyes and disperse dyes) used in each of the above examples are decolorized and non-azo dyes ( Anthraquinone-based reactive dye, anthraquinone-based acid dye, anthraquinone-based disperse dye and copper phthalocyanine-based direct dye)
As can be seen from the fact that is not decolorized, the cells show excellent selectivity and a wide range of applications of the decolorizable azo dye.

Also, various azo dyes contained in the enzyme solution and various azo dyes dyed on the cloth are decolorized, and even if various base materials are variously changed, the various azo dyes dyed on the cloth are Although the degree of bleaching may be different, it was similarly bleached. Therefore, the type of dye (whether or not an azo dye or a non-azo dye is included) contained in the enzyme solution or the like and the type of dye dyed on the cloth (whether or not an azo dye or a non-azo dye is used) )) Can be selected in various ways,
By simultaneously performing a plurality of each combination, a colored and / or blurred pattern suitable for the purpose can be freely created.

Further, the degree of discharge can be changed by changing the type of the base material, the decolorization temperature, the decolorization time, and the like (semi-discharge discharge can be performed freely), so that a pattern such as "blur" can be freely formed. . In addition, discharge can be performed even at a relatively high temperature of 50 to 70 ° C, and particularly at a temperature of about 65 ° C, many germs are killed.

The present invention is not limited to the specific embodiments described above, but may be variously modified within the scope of the present invention in accordance with the purpose and application. That is,
The combination of the dye contained in the enzyme solution and the dye to be dyed on the base material and the type of the base material can be other than the above examples, and the number of dyes used simultaneously is not particularly limited. The number may be other than the above embodiment. Furthermore, various decolorization (reaction) conditions can be selected as long as the enzyme solution is within a temperature range in which the enzyme solution is active. Further, since the fixing of the dye is carried out with an alkaline solution, the fixing can be carried out not only by immersion in an alkaline solution but also by microencapsulation of the alkaline solution and breaking it by temperature or shearing force.

[0055]

According to the method for printing with an enzyme of the present invention,
Since no reducing agent or alkali is used, there are few restrictions on dyes, there is little pain on the base material, and there is little bleeding at the color boundary portion, so that a fine and complicated pattern can be created. Further, according to the printing method, it is possible to perform discharge printing having a vivid color, and a blurred (semi-discharge) pattern can be freely and easily created by selecting discharge printing conditions. Furthermore, by selecting various combinations of the type of the dye contained in the enzyme solution and the type of the dye dyed on the cloth, a colored and / or blurred pattern suitable for the purpose can be created. In addition, the activity of the enzyme can be easily suppressed by heating or treatment with an alkaline solution having a pH of 10.5 or more, so that the dyeing can be easily and reliably performed. In addition, by using the azo dye decolorizing enzyme of the present invention, coloring by the azo dye can be surely and easily eliminated or reduced.

[Brief description of the drawings]

FIG. 1 shows the use of the enzyme and coenzyme of the present invention to produce an azo dye.
It is explanatory drawing explaining the effect | action which decolorizes Roccellin.

FIG. 2 shows the use of the enzyme and coenzyme of the present invention to produce an azo dye.
It is explanatory drawing explaining the effect | action which decolorizes Methyl red.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/02 C12R 1:07) (72) Inventor Tadashi Yokoyama 1580 Tabata, Samukawa-cho, Takaza-gun, Kanagawa Prefecture Yushiro Chemical Industry Co., Ltd. (58) Investigated field (Int.Cl. ⁷ , DB name) D06P 5/13 C12N 9/00-9/99 C12N 1/00-1/38 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. An azo dye, a non-azo dye, and cells Bacillus OY1-2 of a species belonging to the genus Bacillus (accession number: F) on a dye substrate dyed with an azo dye.
Azored enzyme produced by ERM BP-5261 )
A liquid or paste containing at least one of octase (enzyme belonging to diaphorase) is applied in a desired shape, and then the enzyme is acted on to perform printing by eliminating or reducing the coloring by the azo dye. A printing method using an enzyme, characterized in that: 2. Bacillus OY, a bacterium belonging to the genus Bacillus, comprising an azo dye, a non-azo dye and a bacterial species belonging to the genus Bacillus, on a composite dyeing substrate dyed with an azo dye and a non-azo dye.
1-2 (Accession number; FERM BP-5261 ) produced enzyme Azoreductase (diaphora)
(enzymes belonging to se), a liquid or paste containing at least the enzyme is applied in a desired shape, and then the enzyme is acted to eliminate or reduce the coloring by the azo dye, thereby performing printing. Printing method using an enzyme. 3. An azo dye, a non-azo dye and a bacterial cell Bacill belonging to the genus Bacillus on an unstained base material or a dyed base material dyed with a non-azo dye.
rusOY1-2 (Accession number; FERM BP-526)
Azoreductase (dia) produced by 1 )
azo dye) and a liquid or paste containing the enzyme is applied in a desired shape, and then the enzyme is acted on to eliminate or reduce the coloring by the azo dye, thereby printing. Printing method using an enzyme. 4. The method according to claim 1, wherein the temperature at which the coloring by the azo dye is eliminated or reduced is 20 to 70 ° C. 5. A step of drawing a pattern heated to a temperature of 70 ° C. or more on a part of a desired shape formed by applying a liquid or a paste containing the enzyme, The enzymatic printing method according to any one of claims 1 to 4, wherein at least one of the steps of drawing a pattern with a liquid or paste or an acidic liquid or paste having a pH of 4 or less is performed. 6. An enzyme Azo produced by Bacillus OY1-2 (accession number: FERM BP-5261 ) of a bacterium belonging to the genus Bacillus, which acts on an azo dye to eliminate or reduce coloring by the azo dye.
produced in (2) intracellular; (1) an enzyme involved in the redox reaction: reductase (enzyme belonging to diaphorase) Tona is, azo dyes bleaching enzymes characterized by having the following properties an enzyme; (3) production time enzymes are produced in the stationary phase year; as (5) coenzyme to reaction with the enzyme; that reduction to up to (4) an azo group to two amine groups NADP
H (nicotinamide adenine dinucleotide-phosphorus
Acid), NADH (nicotinamide adenine dinucleotide)
De - hydride) is necessary; (6) is stable at 20~70 ℃; (7) pH4.0~10 in a stable; (8) ethanol, is stable in acetone, ethanol
Can be extracted with acetone.