Microbial in-situ dyeing method of indigo pigment

Abstract

The invention discloses a microbial in-situ dyeing method of indigo pigments. The method specifically comprises the following steps: (1) constructing recombinant bacteria: expressing alcohol dehydrogenase and flavin-containing monooxygenase in host bacteria to obtain the recombinant bacteria; (2) preparing a whole-cell catalytic reaction solution, namely collecting thalli obtained by fermenting recombinant bacteria, resuspending to obtain a thalli suspension, and adding an amino phenethyl alcohol compound and NAD < + > to obtain the whole-cell catalytic reaction solution; and (3) biological in-situ dyeing: adding the fabric into the whole-cell catalytic reaction solution for in-situ dyeing to obtain the indigo pigment dyed fabric. According to the method, indigo pigment microorganism in-situ dyeing of various fabrics is achieved for the first time, the optimal dyeing condition is optimized, and technical support is provided for application of indigo pigment in-situ dyeing; the rubbing fastness, soaping fastness and light fastness of the obtained dyed fabric are all above level 3, and meet the fabric dyeing standard.

Description

Technical Field

[0001] The invention relates to the technical field of textile dyeing and finishing, and relates to a microbial in-situ dyeing method of indigo pigments. Background Art

[0002] Indigo and its derivatives are non-azo colorants. Indigo is one of the oldest pigments known to mankind and is widely used in food, medicine and printing and dyeing industries. Indigo derivatives have wide application research value in the fields of dyes, semiconductor materials, etc.

[0003] At present, the dyeing method of indigo pigments is mainly to reduce the indigo pigments by adding reducing agents such as sodium hydroxide and hydrosulfite, thereby enhancing their solubility and dissolution, and dyeing the indigo pigments. However, the reduction dyeing method leads to a large amount of dyeing wastewater, which causes serious pollution to the environment and requires expensive post-treatment, greatly increasing the production cost and pollution to the environment. Therefore, the development of a biological in-situ dyeing method for indigo pigments has broad prospects and great application value. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for indigo pigment in situ microbial dyeing in view of the shortcomings of the prior art.

[0005] In order to solve the above technical problems, the present invention discloses a method for indigo pigment in situ dyeing by microorganisms, and tests the color fastness of the dyed fabric. The specific technical scheme is as follows:

[0006] A method for indigo pigment in-situ microbial dyeing comprises the following steps:

[0007] (1) constructing a recombinant bacterium: expressing alcohol dehydrogenase and flavin-containing monooxygenase in a host bacterium to obtain a recombinant bacterium;

[0008] (2) Preparing a whole-cell catalytic reaction solution: collecting the bacterial cells obtained by fermentation of the recombinant bacteria in step (1) and resuspending them to obtain a bacterial suspension, adding aminophenylethanol compounds and NAD + , and obtain the whole-cell catalytic reaction solution;

[0009] (3) Biological in situ dyeing: The fabric is added to the whole-cell catalytic reaction solution prepared in step (2) for in situ dyeing to obtain indigo pigment-dyed fabric.

[0010] Wherein, in step (1), the alcohol dehydrogenase is derived from horse liver, preferably, the UniProt protein sequence number is P00327; the flavin-containing monooxygenase is derived from Methylophaga aminisulfidivorans, preferably, the UniProt protein sequence number is Q83XK4. The host bacteria include Escherichia coli or yeast, preferably Escherichia coli, and more preferably Escherichia coli BL21.

[0011] Wherein, in step (1), the specific construction method of the recombinant bacteria is as follows: amplifying the coding gene of the alcohol dehydrogenase (HLADH, the nucleotide sequence is shown in SEQ ID No.1) and the coding gene of the flavin-containing monooxygenase (mFMO, the nucleotide sequence is shown in SEQ ID No.2), cloning them into a co-expression vector to obtain a recombinant vector, and introducing it into a host bacterium, wherein the host bacterium is preferably Escherichia coli.

[0012] Wherein, the co-expression vector is pETDuet-1. Preferably, the HindIII and XhoI sites of pETDuet-1 are digested; the HLADH and mFMO fragment genes containing homology arms are obtained by PCR; and the recombinant vector pETDuet-1-HLADH-mFMO is constructed by one-step cloning.

[0013] Further preferably, the recombinant bacteria is preferably recombinant Escherichia coli E. coli pETDuet-1-HLADH-mFMO, named recombinant Escherichia coli M1.

[0014] Wherein, in step (2), the fermentation is carried out as follows: the recombinant bacteria are inoculated into the fermentation medium, and cultured at 30-40°C until the OD 600 When the concentration of the inducer is 0.6 to 1.0, an inducer with a concentration of 0.05 to 0.5 mM is added to continue inducing fermentation for 12 to 36 hours; wherein the inducer is IPTG. Preferably, the induction fermentation temperature after adding the inducer is 25°C. Further preferably, the recombinant bacteria is preferably recombinant Escherichia coli, and the fermentation medium is preferably LB medium. More preferably, the recombinant Escherichia coli is inoculated into the LB medium and cultured at 37°C until the OD 600 When the pH value was between 0.6 and 1.0, an inducer with a concentration of 0.5 mM was added to continue inducing fermentation for 12 h.

[0015] Wherein, in step (2), the resuspending is performed by using a phosphate buffer with a pH of 7 to 10 to resuspend the bacteria; the OD of the bacterial suspension is 600 Preferably, the bacterial cells are resuspended in a phosphate buffer solution with a pH of 8; the OD of the bacterial cell suspension is 600The phosphate buffer is preferably a potassium phosphate buffer.

[0016] Wherein, in step (2), the aminophenethanol compound is a 2-aminophenethanol compound, preferably 4-bromo-2-aminophenethanol or 2-aminophenethanol.

[0017] Wherein, in step (2), in the whole cell catalytic reaction solution, the concentration of aminophenylethanol compound is 1-10 mM, NAD + The concentration of the aminophenylethanol compound is 0.05-2 mM. Preferably, the concentration of the aminophenylethanol compound is 5 mM, NAD + The concentration is 0.1 mM.

[0018] Wherein, in step (3), the dyeing temperature is 20-35° C. and the dyeing time is 2-24 h; the fabric comprises any one of nylon, wool, acrylic, polyester, acetate fiber or cotton fabric, preferably any one of nylon fabric, wool fabric or cotton fabric; the structural formula of the indigo pigment is as shown in Formula I:

[0019]

[0020] Wherein, R1, R2, R3 or R4 are independently selected from H, F, Cl, Br, I, Me, OMe, NO 2 、COOH、CN、NH 2 Or any one of OH.

[0021] Preferably, the indigo pigment includes Tyrian violet or indigo. In the Tyrian violet, R1, R2 and R4 are all H, and R3 is Br; in the indigo, R1, R2, R3 and R4 are all H. Further preferably, the indigo pigment includes Tyrian violet.

[0022] Preferably, the dyeing time and temperature are 30° C. and 12 h, respectively.

[0023] Beneficial effects:

[0024] The present invention provides for the first time a method for indigo pigment in-situ dyeing of a variety of fabrics with microorganisms. The in-situ dyeing method of the present invention realizes for the first time a scheme of synthesizing and dyeing indigo pigments, overcomes the drawbacks of indigo pigment dyeing in the prior art, and provides technical support for further application of indigo pigment microbial in-situ dyeing. The dyed fabrics obtained by the present invention have rubbing fastness, soaping fastness, and light fastness of all above level 3, which meets the fabric dyeing standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0026] Figure 1 The following are samples of different fabrics dyed with Tyrian purple. Column a is the biological in-situ dyeing method, column b is the one-step enzyme method, and column c is the two-step enzyme method. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are all repeated three times, and the results are averaged.

[0029] In the following examples, the HLADH is an alcohol dehydrogenase derived from horse liver, and the UniProt protein sequence number is P00327; the mFMO is a flavin-containing monooxygenase derived from Methylophaga aminisulfidivorans, and the UniProt protein sequence number is Q83XK4. The HLADH and mFMO are synthesized by a commissioned company, wherein the gene sequence encoding HLADH is shown in SEQ ID No. 1, and the gene sequence encoding mFMO is shown in SEQ ID No. 2.

[0030] Example 1 Tyrian purple in-situ dyeing of nylon fabric

[0031] 1. Preparation of recombinant Escherichia coli M1 for indigo pigment production

[0032] (1) Construction of recombinant plasmid

[0033] The HindIII and XhoI sites of pETDuet-1 were digested and gel-recovered to obtain the purified pETDuet-1 linearized vector;

[0034] Then, PCR technology was used to amplify the HLADH and mFMO fragment genes containing homology arms using HLADH-F / HLADH-R and mFMO-F / mFMO-R as PCR primers, and the HLADH coding gene (sequence shown in SEQ ID No. 1) and mFMO coding gene (sequence shown in SEQ ID No. 2) synthesized by the company as templates; wherein the primer sequences are as follows:

[0035] HLADH-F: ctgcaggtcgacaagcttATGACTGGTGGACAGCAAAT (SEQ ID No. 3),

[0036] HLADH-R: ttaagcattatgcggccgcTCAAAAGGTCAGAAT (SEQ ID No. 4),

[0037] mFMO-F: cgtcggtaccctcgagATGGCAACCCGCATTG (SEQ ID No. 5),

[0038] mFMO-R: gcggtttctttaccagaTTAGGCTTCTTTGGCAA (SEQ ID No. 6).

[0039] The pETDuet-1 linearized vector, HLADH fragment gene and mFMO fragment gene prepared above were cloned in one step to construct the recombinant plasmid pETDuet-1-HLADH-mFMO.

[0040] (2) Construction of recombinant strains

[0041] The pETDuet-1-HLADH-mFMO recombinant plasmid was introduced into E. coli BL21 (DE3) competent cells by chemical transformation to obtain recombinant E. coli M1 (E. coli pETDuet-1-HLADH-mFMO). 2. Preparation of E. coli whole cell catalytic reaction solution

[0042] The preparation of the whole-cell catalytic reaction solution includes the following steps:

[0043] (1) The recombinant E. coli M1 prepared above was inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, shaken at 37°C and 200 rpm overnight for 12 h, and then inoculated into LB liquid medium containing 50 μg / mL kanamycin at a volume ratio of 1% inoculum, and cultured at 37°C until OD 600When the concentration of 50 μg / mL was 0.6-1.0, IPTG was added at a final concentration of 0.05-0.5 mmol / L (0.5 mmol / L in this case), and the expression was induced at 25°C and 200 rpm for 12-36 h (12 h in this case), and then centrifuged at 4°C and 8000 rpm for 5 min to collect the precipitated bacteria (i.e., the recombinant cells), and the collected bacteria were resuspended in phosphate buffer (50 mmol / L, pH 7.0) to obtain a bacterial suspension, and the OD of the bacterial suspension was ensured to be 600 is 15.

[0044] (2) Add 1-10 mmol / L (5 mmol / L in this example) of 4-bromo-2-aminophenylethanol and 0.05-2 mmol / L (0.1 mmol / L in this example) of NAD to the bacterial suspension prepared in step (1). + , and obtain the Escherichia coli whole cell catalytic reaction liquid. Wherein, the structural formula of 4-bromo-2-aminophenylethanol is shown in Formula II:

[0045]

[0046] 3. Biological in situ dyeing of nylon fabric

[0047] A 6cm*6cm standard nylon fabric was added to the prepared E. coli whole cell catalytic reaction solution to fully infiltrate the fabric in the reaction solution. The fabric was dyed at 30°C and 200rpm for 12h to obtain a dyed fabric. The NMR information of the biosynthetic product was as follows: 1 H NMR (400 MHz, D 2 O)δ7.42(d,J=11.3Hz,4H),7.02(d,J=7.8Hz,2H), it was determined that the synthesized product was Tyrian purple, and the biological in-situ dyeing of nylon fabric was achieved. The structural formula of Tyrian purple is shown in formula III:

[0048]

[0049] 4. Enzyme-catalyzed dyeing

[0050] This example uses the method of preparing pure enzyme in the prior art to perform enzyme catalysis dyeing as a control group, specifically including two-step enzyme method and one-step enzyme method:

[0051] HLADH pure enzyme and mFMO pure enzyme were prepared by referring to the preparation method of pure enzyme described in Examples 1 and 2 of patent CN116676354A.

[0052] (1) The specific steps of the two-step enzymatic method are as follows:

[0053] Step 1: Add 0.1mM NAD +, 0.2 mg HLADH and 5 mM 4-bromo-2-aminophenylethanol, in 50 mM KPi7.0 buffer, react at 30 °C for 12 h before proceeding to the second step;

[0054] Step 2: Add 0.2 mg mFMO and standard nylon fabric, react at 30°C (dyeing temperature) for 12 h (dyeing time) to obtain dyed nylon fabric.

[0055] (2) The specific steps of the one-step enzymatic method are as follows:

[0056] 0.1mM NAD + , 0.2 mg HLADH, 0.2 mg mFMO, 5 mM 4-bromo-2-aminophenylethanol and standard nylon fabric were reacted in 50 mM KPi 7.0 buffer at 30°C for 12 h.

[0057] Example 2 Dyeing method optimization

[0058] 1. The influence of dyeing temperature on dyeing rate

[0059] A series of biological in-situ dyeing experiments of nylon fabrics were carried out according to the method described in Example 1, wherein the dyeing temperature was 20, 25, 30 or 35°C (wherein the dyeing temperature described in the two-step enzyme method refers to the temperature after the fabric is added in the second step), and the dyeing time was 4h. The K / S value of the dyed fabric was measured using a Hunterlab Ultrascanp computer colorimeter, and the colorimetric light source was a D65 light source with a 10° viewing angle. Each sample was measured 4 times to take an average value, and the results are shown in Table 1.

[0060] Table 1 Effect of different dyeing temperatures on K / S value

[0061]

[0062] It can be seen from Table 1 that when the dyeing temperature reaches 30°C, the biological in situ dyeing method can obtain a higher K / S value (3.37), which is much higher than the one-step enzyme method and the two-step enzyme method. Considering factors such as production efficiency and energy consumption, it is determined that the dyeing temperature of 30°C is more appropriate.

[0063] 2. The influence of dyeing time on dyeing rate

[0064] A series of biological in situ dyeing experiments of nylon fabrics were carried out according to the dyeing method described in Example 1, wherein the dyeing time was 1, 2, 4, 8, 12, and 24 h, respectively (wherein the dyeing time described in the two-step enzyme method refers to the time after the nylon fabric is added in the second step), the dyeing temperature was 30°C, and the dye uptake results are shown in Table 2.

[0065] Table 2 Effect of different dyeing time on dyeing rate

[0066]

[0067] It can be seen from Table 2 that when the dyeing time is 12h, the dyeing rate of the biological in situ dyeing method reaches the highest value of 67.79%, which is much higher than the one-step enzyme method and the two-step enzyme method. Considering factors such as production efficiency and energy consumption, it is determined that the dyeing time of 12h is more appropriate.

[0068] 3. Effect of dye bath pH value on dye uptake

[0069] A series of biological in situ dyeing experiments of nylon fabrics were carried out according to the method described in Example 1, wherein the pH value of the dye bath in the dyeing process was adjusted to 6, 7, 8, 9, 10 and 11 in sequence (i.e., phosphate buffers of different pH values ​​were prepared for preparing the bacterial suspension), the dyeing temperature was 30°C, the dyeing time was 12 h, and the dyeing rate results are shown in Table 3.

[0070] Table 3 Effect of different dye bath pH on dye uptake value

[0071]

[0072] It can be seen from Table 3 that when the pH of the dye bath is 8, the biological in situ dyeing method can obtain a higher dye uptake rate (75.22%), so it is more appropriate to determine the pH value of the dye bath to be pH=8.

[0073] 4. Effect of recombinant E. coli M1 concentration on dye uptake rate

[0074] A series of biological in-situ dyeing experiments of nylon fabrics were carried out according to the dyeing method described in Example 1. The dyeing temperature was 30°C, the dyeing time was 12 h, the dye bath pH was 8, and the concentrations of recombinant Escherichia coli M1 were OD 600 =5, 10, 15, 20, 25 and 30, and the dyeing rate results are shown in Table 4.

[0075] Table 4 Effect of different recombinant E. coli M1 concentrations on the dye uptake value

[0076] <![CDATA[Recombinant Escherichia coli M1 concentration (OD 600 )]]> Dyeing rate (%) 5 40.18 10 61.81 15 75.22 20 79.44 25 69.90 30 60.01

[0077] It can be seen from Table 4 that when the concentration is OD 600 When =20, the biological in situ dyeing method can obtain a higher dyeing rate and the amount of microorganisms used is relatively small, which meets the requirements of low carbon and environmental protection.

[0078] 5. Color fastness test of dyed fabrics

[0079] The dyeing temperature was 30°C, the dyeing time was 12 h, the pH value of the dye bath was 8, and the concentration of recombinant E. coli M1 was OD 600=20 for the biological in situ dyeing experiment of nylon fabric, the dyeing temperature of the one-step enzyme dyeing experiment and the two-step enzyme dyeing experiment were 30°C, the dyeing time was 12h, and the pH value of the dye bath was 8. Then the dyed fabric was subjected to the color fastness experiment, wherein the determination of rubbing fastness was carried out in accordance with the test method of GB / T 3920-2008 "Textiles - Tests for color fastness - Color fastness to rubbing", the determination of washing fastness was carried out in accordance with the test method of GB / T 3921-2008 "Textiles - Tests for color fastness - Color fastness to washing with soap", and the determination of light fastness was carried out in accordance with the test method of GB / T 8427-2008 "Textiles - Tests for color fastness - Color fastness to light". The results are shown in Table 5.

[0080] Table 5 Color fastness test results of biological in situ Tyrian purple dyed nylon fabric

[0081]

[0082] It can be seen from Table 5 that the rubbing fastness, soaping fastness and sunlight fastness of the nylon fabric dyed according to the biological in-situ dyeing method of the present invention are all level 3 or above, which meets the nylon dyeing standard and is significantly higher than the nylon dyeing standard obtained by the enzyme catalysis dyeing method.

[0083] 6. Color calibration test of dyed fabrics

[0084] The dyeing temperature was 30°C, the dyeing time was 12 h, the pH value of the dye bath was 8, and the concentration of recombinant E. coli M1 was OD 600 =20 for the biological in situ dyeing experiment of nylon fabric. The dyeing temperature of the one-step enzymatic dyeing experiment and the two-step enzymatic dyeing experiment was 30°C, the dyeing time was 12h, and the pH value of the dye bath was 8. Then the dyed fabric was subjected to color calibration experiment. The results are shown in Table 6.

[0085] Table 6 Color experimental results of biological in situ Tyrian purple dyed nylon fabric

[0086] Dyeing method <![CDATA[Brightness L * > <![CDATA[Red-green index a * > <![CDATA[Yellow-Blue index b * > In situ staining 44.97 35.02 -13.24 One-step enzymatic method 15.97 12.04 -7.25 Two-step enzymatic method 5.38 5.99 -3.87

[0087] As can be seen from Table 6, according to the color experiment of nylon fabric dyed by the biological in-situ dyeing method of the present invention, the biological in-situ dyeing method achieves the natural color of Tyrian purple dyeing of nylon fabric.

[0088] Example 3 Tyrian purple in-situ dyeing of other fabrics

[0089] The biological in situ staining process is as shown in Example 1, specifically, the staining temperature is 30°C, the staining time is 12h, the pH value of the dye bath is 8, the concentration of recombinant Escherichia coli M1 is OD 600=20 parameters for other fabrics (wool, acrylic, polyester, cotton or acetate) biological in situ dyeing experiments. The dyeing temperature of the one-step enzyme method and the two-step enzyme method dyeing experiments was 30°C, the dyeing time was 12h, the dye bath pH value was 8, and the sample was as shown in the figure. Figure 1 As shown, the dyed fabrics were then subjected to color fastness experiments, where the rubbing fastness was determined in accordance with the test method of GB / T 3920-2008 “Textiles - Tests for color fastness - Color fastness to rubbing”, the washing fastness was determined in accordance with the test method of GB / T 3921-2008 “Textiles - Tests for color fastness - Color fastness to washing with soap”, and the light fastness was determined in accordance with the test method of GB / T 8427-2008 “Textiles - Tests for color fastness - Color fastness to sunlight”. The dyeing results of some fabrics are shown in Table 7.

[0090] Table 7 Color fastness test results of biological in situ Tyrian purple dyed cotton and wool fabrics

[0091]

[0092] It can be seen from Table 7 that the rubbing fastness, soaping fastness and sunlight fastness of the cotton and wool fabrics dyed according to the biological in situ dyeing method of the present invention are all level 3 or above, which meets the cotton and wool dyeing standards and is significantly higher than the cotton and wool dyeing standards obtained by the enzyme catalytic dyeing method.

[0093] The present invention provides a method and idea for indigo pigment microbial in situ dyeing. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for indigo pigment in situ dyeing by microorganisms, characterized in that: The following steps are involved: (1) constructing a recombinant bacterium: expressing alcohol dehydrogenase and flavin-containing monooxygenase in a host bacterium to obtain a recombinant bacterium; (2) Preparing a whole-cell catalytic reaction solution: collecting the bacterial cells obtained by fermentation of the recombinant bacteria in step (1) and resuspending them to obtain a bacterial suspension, adding aminophenylethanol compounds and NAD + , and obtain the whole-cell catalytic reaction solution; (3) Biological in situ dyeing: The fabric is added to the whole-cell catalytic reaction solution prepared in step (2) for in situ dyeing to obtain indigo pigment-dyed fabric.

2. The microbial in situ staining method according to claim 1, characterized in that: In step (1), the alcohol dehydrogenase is derived from horse liver; and the flavin-containing monooxygenase is derived from Methylophagaaminisulfidivorans.

3. The microbial in situ staining method according to claim 1, characterized in that: In step (1), the host bacteria include Escherichia coli or yeast.

4. The microbial in situ staining method according to claim 1, characterized in that: In step (1), the specific construction method of the recombinant bacteria is as follows: amplify the coding gene of the alcohol dehydrogenase and the coding gene of the flavin-containing monooxygenase, clone them into a co-expression vector to obtain a recombinant vector, and introduce them into the host bacteria.

5. The microbial in situ staining method according to claim 4, characterized in that: The co-expression vector is pETDuet-1.

6. The microbial in situ staining method according to claim 1, characterized in that: In step (2), the fermentation is carried out as follows: the recombinant bacteria are inoculated into a fermentation medium and cultured at 30-40°C until the OD 600 When the pH value is 0.6-1.0, an inducer with a concentration of 0.05-0.5 mM is added to continue inducing fermentation for 12-36 hours; wherein the inducer is IPTG.

7. The microbial in situ staining method according to claim 1, characterized in that: In step (2), the resuspending is performed by using a phosphate buffer having a pH of 7 to 10 to resuspend the bacteria; the OD of the bacterial suspension is 600 It is 10 to 25.

8. The microbial in situ staining method according to claim 1, characterized in that: In step (2), the aminophenethanol compound is a 2-aminophenethanol compound.

9. The microbial in situ staining method according to claim 1, characterized in that: In step (2), in the whole cell catalytic reaction solution, the concentration of the aminophenylethanol compound is 1 to 10 mM, and NAD + The concentration is 0.05~2mM.

10. The microbial in situ staining method according to claim 1, characterized in that: In step (3), the dyeing temperature is 20 to 35° C. and the dyeing time is 2 to 24 h; The fabric includes any one of nylon, wool, acrylic, polyester, acetate or cotton fabric; The structural formula of the indigo pigment is shown in Formula I: Wherein, R1, R2, R3 or R4 are independently selected from any one of H, F, Cl, Br, I, Me, OMe, NO2, COOH, CN, NH2 or OH.

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