Bacillus velezensis strain with copper tollerance and laccase production ability and method for decomposing synthetic dtes using the same

Abstract

The present invention provides a strain of Bacillus velezensis that has copper resistance and the ability to produce laccase. The present invention also provides a method for degrading a synthetic dye, comprising the step of introducing said strain into an aqueous solution containing a synthetic dye.

Description

Technology Field

[0001] The present invention relates to a Bacillus velezensis strain having copper resistance and laccase production ability, and more specifically, to a technology for obtaining a strain with further increased copper resistance and laccase production ability through radiation irradiation and using the same. Background Technology

[0002] Most printed materials, textiles, and other products are colored using dyes, and when recycling or disposing of these products, or when treating wastewater from factories, it is necessary to decompose the colored dyes.

[0003] Meanwhile, laccase, one of the representative degrading enzymes, exhibits high degradation ability against azo synthetic dyes and is mainly produced through wood-rotting fungi and recombinant E. coli. However, wood-rotting fungi and recombinant E. coli have low activity and survival rates, making it difficult to apply them directly to environments where dye wastewater is generated.

[0004] Accordingly, a method was studied to immobilize laccase enzymes on a carrier for dye degradation, but there was a problem of inefficiency as it required separate processes such as enzyme separation, purification, and carrier immobilization.

[0005] Meanwhile, Gram-positive bacteria such as Bacillus velezensis are frequently used as microbial factories for the production of related industrial proteins due to their excellent fermentation characteristics and high yields. However, in the case of whole-cell biocatalysts such as bacterial strains, laccase productivity and resistance to copper are closely related, and in particular, conventional technology for producing laccase using bacteria has faced difficulties due to the lack of resistance that said bacteria have to copper toxicity.

[0006] Accordingly, it is expected that if a Bacillus strain with enhanced laccase activity is provided through improved resistance to copper ions, it can be widely applied in related fields such as the environmental industry. Prior art literature

[65535] Japanese Registered Patent No. 6600782 The problem to be solved

[0007] One aspect of the present invention is to solve the problems of the prior art by providing a new strain of the genus Bacillus having copper resistance and laccase production ability.

[0008] Another aspect of the present invention is to provide a method for removing synthetic dyes using the strain of the present invention. means of solving the problem

[0009] According to one embodiment of the present invention, a strain of Bacillus velezensis is provided, which has copper resistance and the ability to produce laccase.

[0010] According to another embodiment of the present invention, a method for degrading a synthetic dye is provided, comprising the step of introducing the strain into an aqueous solution containing a synthetic dye. Effects of the invention

[0011] According to the present invention, a novel strain of the genus Bacillus, Bacillus velezensis, which possesses copper resistance and the ability to produce laccase, can be obtained by utilizing a mutation breeding method through radiation irradiation. When using the strain, it is expected that synthetic dyes contained in wastewater containing dyes can be decomposed more efficiently based on high laccase activity and the survival rate of the strain. Brief explanation of the drawing

[0012] Figure 1 shows the results of confirming the growth inhibition rate (Inhibitory effect, %) of Bacillus velezensis by various copper sulfate concentrations. Figure 2 shows the results of comparing the growth curves of the wild-type (WT) strain (Control) and the mutant strain Cu-RE1 with the addition of copper sulfate at concentrations of 5 mM or 10 mM. More specifically, Figure 2(a) shows the results of recording the growth curves by measuring the absorbance at 600 nm while culturing the wild-type (WT) strain and the mutant strain Cu-RE1, respectively, in TSB medium with copper sulfate added at a concentration of 5 mM, and Figure 2(b) shows the results of recording the growth curves by measuring the absorbance at 600 nm while culturing the wild-type (WT) strain and the mutant strain Cu-RE1, respectively, in TSB medium with copper sulfate added at a concentration of 10 mM. Figure 3 shows the results of comparing the difference in laccase activity between the wild-type (WT) strain (Control) and the mutant strain Cu-RE1 following the addition of copper ions (copper sulfate). Figure 4 shows the results of comparing the relative mRNA expression levels of genes related to copper resistance in the wild-type (WT) strain of Bacillus velezensis and the mutant strain Cu-RE1. More specifically, Figure 4(a) shows the results of comparing the expression levels of the Multicopper oxidase gene of each strain. Figure 4(b) shows the results of comparing the expression levels of the CopC / CopD family protein gene of each strain. Figure 4(c) shows the results of comparing the expression levels of the chaperone protein gene of each strain. Figure 5 shows the results of measuring the survival rate of the strains according to the concentration of hydrogen peroxide or cumene hydroperoxide to confirm the resistance of the wild-type (WT) strain and the mutant strain Cu-RE1 to oxidative stress. More specifically, Figure 5(a) shows the results of measuring the survival rate of each strain according to the concentration of hydrogen peroxide. Figure 5(b) shows the results of measuring the survival rate of each strain according to the concentration of cumene hydroperoxide. Figure 6 shows the results of comparing the decolorization rates of the wild-type (WT) strain and the mutant strain Cu-RE1 with respect to synthetic dyes. More specifically, Figure 6(a) shows the results of measuring the decolorization rate of each strain for Congo red. Figure 6(b) shows the results of measuring the decolorization rate of each strain for Crystal violet. Figure 6(c) shows the results of measuring the decolorization rate of each strain for Malachite green. Figure 6(d) shows the results of measuring the decolorization rate of each strain for Methylene blue. Figure 6(e) shows the results of measuring the decolorization rate of each strain for Indigo carmine. Specific details for implementing the invention

[0013] Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0014] According to one embodiment of the present invention, a strain of Bacillus velezensis is provided, which has copper resistance and the ability to produce laccase.

[0015] The activity of the laccase produced by the above-mentioned Bacillus velezensis strain may be 15 to 30 U / L at a copper concentration of 5 mM, preferably 20 to 30 U / L, and more preferably 25 to 30 U / L, when the amount of substrate converted per minute at pH 7 and 25°C is defined as 1 Unit of enzyme activity (U).

[0016] In addition, the above-mentioned Bacillus velezensis strain may have the ability to degrade at least one dye selected from the group consisting of Congo red, Crystal violet, Malachite green, Methylene blue, and Indigo carmine, and preferably has the ability to degrade all of the above dyes.

[0017] Meanwhile, the above-mentioned Bacillus velezensis strain may be a mutant strain obtained by irradiating a Bacillus velezensis MBLB0692 strain obtained from pickles through a serial dilution method.

[0018] For example, the above Bacillus velezensis mutant strain comprises the step of irradiating a Bacillus velezensis strain with radiation; and copper (Cu 2+ It can be prepared by performing a selection step of selecting growing strains by culturing them for 12 to 24 hours in a medium containing ions at the Minimum Inhibitory Concentration (MIC) required to inhibit the growth of wild-type (WT) Bacillus velezensis strains.

[0019] The number of times the screening step is performed is not specifically limited, but, for example, with respect to copper, the minimum inhibition concentration may be 10 mM, and the screening may be to select strains that survive at this concentration. Meanwhile, the screening step may be performed two or more times.

[0020] The basic medium that can be used at this time is any medium known to be suitable for the growth of Bacillus velezensis strains.

[0021] The radiation may be X-rays, electron beams, or gamma rays, and preferably may be gamma rays.

[0022] The above gamma rays may be emitted from any one radioactive isotope selected from the group consisting of cobalt (Co)-60, krypton (Kr)-85, strontium (Sr)-90, and cesium (Cs)-137, and more specifically, the gamma rays may be cobalt (Co)-60, but are not limited thereto.

[0023] The radiation dose for inducing mutations may be 0.1 to 10 kGy, for example, 1 to 8 kGy. If the radiation dose is less than 0.1 kGy, mutation induction may be insufficient, and if the radiation dose exceeds 10 kGy, the mortality rate of the strain may be higher than the mutation rate, making it difficult for the strain to survive.

[0024] More specifically, a Bacillus velezensis mutant strain can be obtained by diluting a liquid-cultured wild-type (WT) strain to an optical density of 0.2 at a wavelength of 600 nm, irradiating the diluted culture with gamma rays of 1, 2, 4, or 8 kGy, and then culturing it on a solid agar medium containing 10 mM copper sulfate until colonies appear.

[0025] The exemplary mutant strain obtained in this invention was named Bacillus velezensis strain Cu-RE1, and said mutant strain was deposited at the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Biotechnology and Bioengineering and assigned accession number KCTC15701BP.

[0026] Meanwhile, a Bacillus velezensis strain according to one embodiment of the present invention may include the nucleotide sequence of SEQ ID NO. 1.

[0027] According to another embodiment of the present invention, a method for degrading a synthetic dye is provided, comprising the step of introducing a Bacillus velezensis strain according to one embodiment of the present invention into an aqueous solution containing a synthetic dye.

[0028] The above synthetic dye may be one or more selected from the group consisting of Congo red, crystal violet, malachite green, methylene blue, and indigo carmine, and may, for example, include all of the above dyes.

[0029] The above-mentioned aqueous solution may be, for example, dye wastewater, but is not limited thereto. In this case, dye wastewater may be, for example, wastewater discharged from a manufacturing facility, such as a factory, where a dyeing process using dyes as described above is carried out.

[0030] In addition, the above aqueous solution contains 1 to 5 mM of Cu 2+ It may contain ions, more specifically 2 to 4 mM of Cu 2+ It may contain ions. The above Cu 2+ The ions may be derived from copper sulfate, but are not limited thereto.

[0031] The above Cu 2+If the ion content exceeds 5 mM, the strain may die due to the excessive copper ion concentration, and consequently, the dye degradation efficiency may decrease.

[0032] The pH of the above aqueous solution may be 5 to 9, and more specifically 6 to 8. If the pH of the above aqueous solution is less than 5 or greater than 9, it falls outside the pH range in which the laccase can be active, and the dye degradation efficiency may decrease.

[0033] In addition, the synthetic dye degradation method described above may be performed under conditions of a temperature of 20 to 40°C, more specifically 30 to 35°C. If the temperature condition under which the synthetic dye degradation method is performed is less than 20°C, the activity of the strain and laccase may be low, and the dye degradation efficiency may decrease. If the temperature condition under which the synthetic dye degradation method is performed exceeds 40°C, the strain may die or the laccase may denature and lose enzyme activity.

[0034] According to the Bacillus velezensis strain of the present invention and the method for degrading synthetic dyes using said strain, there is an advantage in that synthetic dyes contained in dye wastewater, etc. can be degraded more efficiently based on high laccase activity and the survival rate of the strain.

[0035] The present invention will be explained in more detail below through specific embodiments. The following embodiments are merely examples to aid in understanding the present invention and do not limit the scope of the present invention.

[0036] Examples

[0037] 1. Culture of Bacillus velezensis and Confirmation of Copper Resistance

[0038] Cultured the Bacillus velezensis MBLB0692 strain, one of the laccase-producing strains, and copper ions (Cu 2+Resistance to ) was confirmed. At this time, the above Bacillus velezensis was the MBLB0692 strain isolated from pickles, which was isolated from pickles through serial dilution and used.

[0039] More specifically, the above-mentioned wild-type Bacillus velezensis strain is prepared by homogenizing 1 g of pickle in 10 mL of sterile PBS buffer, and then 10 -8 It was diluted using the serial dilution method up to.

[0040] The above diluted dilutions were each plated onto agar media containing basic media (R2A, TSB), etc., and cultured at 30°C for 3 days. Then, they were distinguished according to morphological characteristics such as colony size, shape, and color appearing on the media, and after subculturing into each medium, were pure isolated.

[0041] The isolated strain was identified through 16S rRNA analysis. The wild-type strain of Bacillus velezensis obtained through the above process was named Bacillus velezensis MBLB0692. The Bacillus velezensis MBLB0692 strain was deposited with the Korean Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology on November 27, 2023, and was assigned accession number KCTC15709BP (hereinafter referred to as the "Wild-type (WT) strain").

[0042] Tryptic Soy Broth (TSB), sterilized at 121°C for 20 minutes, was used as the medium for culturing the above-mentioned wild-type (WT) strain of Bacillus velezensis, and the culture was carried out at a temperature of 30°C and under dark conditions while stirring at 200 rpm.

[0043] Next, a Minimum Inhibitory Concentration (MIC) experiment was conducted to confirm the existing resistance to copper of the cultured wild-type (WT) strain of Bacillus velezensis.

[0044] After culturing wild-type (WT) Bacillus velezensis strains for one day in TSB containing 0.8, 0.15, 0.3, 0.6, 1.2, 2.5, 5, or 10 mM copper sulfate, the growth inhibitory effect (i.e., inhibition rate) according to copper sulfate concentration was confirmed by measuring the absorbance at a wavelength of 600 nm.

[0045] The inhibition rate is defined as (absorbance of control group - absorbance of experimental group) / (absorbance of control group) * 100 (%), where the control group refers to a 0 mM copper sulfate condition. When the inhibition rate was 90% or higher, visible growth inhibition was observed, and the copper sulfate concentration at this point was determined to be the minimum inhibition concentration.

[0046] Figure 1 shows the experimental results confirming the Minimum Inhibitory Concentration (MIC) conditions of Bacillus velezensis by various copper (Cu) concentrations.

[0047] Referring to Figure 1, it can be seen that the growth inhibitory effect, or inhibition rate, of wild-type Bacillus velezensis under 10 mM copper sulfate conditions reaches 95%. In other words, it can be seen that the Minimum Inhibitory Concentration (MIC) of copper sulfate for wild-type (WT) Bacillus velezensis strains is 10 mM.

[0048] 2. Induction of Mutation via Radiation and Selection of Copper-Resistant Bacillus velezensis Strains

[0049] Through the following method, a Bacillus velezensis strain capable of growing in a medium containing copper ions at the Minimum Inhibitory Concentration (MIC) required to inhibit the growth of a wild-type (WT) Bacillus velezensis strain was obtained.

[0050] To select strains through irradiation, liquid-cultured wild-type (WT) Bacillus velezensis strains were diluted to an optical density of 0.2 at a wavelength of 600 nm.

[0051] After irradiating the diluted culture with gamma rays of 1, 2, 4, or 8 kGy, it was incubated on a solid agar medium containing 10 mM copper sulfate until colonies appeared.

[0052] An exemplary Bacillus velezensis mutant strain obtained from the colony shown above was named Bacillus velezensis strain Cu-RE1, and the mutant strain was deposited with the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Biotechnology and Bioengineering on November 17, 2023, and was assigned accession number KCTC15701BP.

[0053] The above-mentioned Bacillus velezensis mutant strain Cu-RE1 (hereinafter referred to as "Cu-RE1") was cultured in a medium containing copper sulfate to compare the growth difference with that of the wild-type (WT) strain.

[0054] Figure 2 shows the results of comparing the growth curves of wild-type strains and Cu-RE1 with the addition of copper sulfate at concentrations of 5 mM or 10 mM.

[0055] More specifically, Figure 2(a) shows the results of recording growth curves by measuring absorbance at 600 nm while culturing wild-type (WT) strains and Cu-RE1, respectively, in TSB medium supplemented with 5 mM copper sulfate.

[0056] Figure 2(b) shows the results of recording growth curves by measuring absorbance at 600 nm while culturing wild-type (WT) strains and Cu-RE1, respectively, in TSB medium supplemented with 10 mM copper sulfate.

[0057] Referring to Figure 2(a), it can be seen that Cu-RE1 cultured in TSB medium with 5 mM copper sulfate added has a significantly higher absorbance at 600 nm compared to the wild-type strain as culture time progresses.

[0058] From the above results, it can be confirmed that Cu-RE1 has a higher growth rate in TSB medium with 5 mM copper sulfate added compared to wild-type strains.

[0059] In addition, referring to Figure 2(b), the absorbance of the wild-type strain cultured in TSB medium with 10 mM copper sulfate was measured to be lower at a wavelength of 600 nm compared to the start of culture, and from the above results, it can be confirmed that the wild-type strain did not grow in TSB medium with 10 mM copper sulfate. On the other hand, in the case of Cu-RE1, it can be confirmed that growth occurred even in TSB medium with 10 mM copper sulfate.

[0060] From the above experimental results, it can be confirmed that Cu-RE1 has high resistance to copper ions and, accordingly, exhibits a higher growth rate compared to the wild type, which is inhibited in a medium containing copper sulfate.

[0061] 3. Confirmation of Laccase Activity of Bacillus velezensis Wild-Type Strain and Cu-RE1 Following Copper Sulfate Addition

[0062] Differences in laccase activity were compared between the wild-type (WT) Bacillus velezensis strain and Cu-RE1 with the addition of copper ions (copper sulfate).

[0063] Laccase activity was measured by the ABTS assay. The procedure was as follows. First, each strain was cultured for 24 hours in TSB medium supplemented with various concentrations of copper sulfate. After culture, the supernatant was collected and reacted with 0.5 mM ABTS in a 0.1 M sodium acetate buffer solution at pH 4.5. The reaction was carried out under dark conditions at 25°C. After 5 minutes of reaction, the absorbance was measured at 420 nm to calculate the laccase activity. Laccase activity (U / L) was calculated as (difference in absorbance × total reaction volume) / (reaction time × extinction coefficient × optical depth × volume of supernatant).

[0064] The results of comparing the difference in laccase activity according to the addition of copper ions (copper sulfate) are shown in Figure 3.

[0065] Referring to Figure 3, Cu-RE1 has higher laccase activity compared to the wild-type (WT) strain in the presence of copper sulfate, and in particular, the difference in laccase activity of Cu-RE1 compared to the wild-type (WT) strain is greatest in the presence of copper sulfate at a concentration of 5 mM.

[0066] 4. Analysis of Metabolisms of Bacillus velezensis Wild-Type Strain and Cu-RE1

[0067] To confirm functional changes related to copper tolerance and laccase activity between Bacillus velezensis wild-type (WT) strains and Cu-RE1, changes in the relative mRNA amounts of genes related to copper tolerance were compared.

[0068] After culturing the above wild-type (WT) strain and Cu-RE1 for 8 hours in TSB liquid medium containing 0, 2.5, or 5 mM copper sulfate, respectively, RNA was extracted from each strain, and cDNA was synthesized from the extracted RNA.

[0069] Real-time polymerase chain reaction (real-time-PCR) was performed using the above cDNA to confirm the differences in the relative expression levels of genes related to copper tolerance and laccase activity following the addition of copper sulfate, based on the expression levels of the wild-type (WT) strain measured under 0 mM copper sulfate conditions.

[0070] Figure 4 shows the results of comparing the relative mRNA expression levels of genes related to copper resistance in Bacillus velezensis wild-type (WT) strains and Cu-RE1.

[0071] Referring to Figure 4(a), it can be seen that the expression level of Multicopper oxidase, an enzyme possessed by Bacillus velezensis, was directly increased in Cu-RE1. Furthermore, unlike the wild-type (WT) strain, which showed little change in Multicopper oxidase expression levels depending on copper sulfate concentration, it can be seen that the Multicopper oxidase expression level in Cu-RE1 increased most significantly under the 5 mM copper sulfate condition. Multicopper oxidase has the function of oxidizing substrates using copper ions as a cofactor, and laccase is a type of Multicopper oxidase. Therefore, the increase in Multicopper oxidase expression is directly related to the increase in laccase activity.

[0072] Referring to Figure 4(b), it can be seen that as the concentration of copper sulfate added increases, the expression of CopC / CopD family proteins, which are proteins related to copper ion resistance, increases. In addition, the expression of CopC / CopD family proteins in Cu-RE1 increased more rapidly compared to the wild-type (WT) strain, and it can be seen that the difference between the expression of CopC / CopD family proteins in the wild-type (WT) strain and the expression of CopC / CopD family proteins in Cu-RE1 is greatest under the copper sulfate 5 mM condition.

[0073] Referring to Figure 4(c), it can be seen that as the concentration of copper sulfate added increases, the expression level of chaperones related to protein folding also increases. The chaperone expression level of Cu-RE1 increased more rapidly compared to the wild-type (WT) strain, and the difference between the chaperone expression level of the wild-type (WT) strain and the chaperone expression level of Cu-RE1 was found to be greatest under the 5 mM copper sulfate condition.

[0074] Through this, it can be confirmed that the above Cu-RE1 has improved resistance to copper through an increase in the expression of proteins related to copper metabolism.

[0075] 5. Detailed variant analysis of Cu-RE1 through genome sequencing

[0076] To confirm the degree of genetic mutation of the above Cu-RE1, genetic mutation information was obtained through NGS sequencing and compared with the gene locations of the existing MBLB0692 reference strain, and the results are as shown in Tables 1 to 3 below.

[0077] Total mutations gene rRNA tRNA Coding 143 70 10 3 Non-coding 213 - - - gun 356 - - -

[0078] Insertion Deletion Substitution Coding 17 15 111 Non-coding 60 34 119 gun 77 49 230

[0079] More specifically, referring to Table 1, the genomic analysis results identified a total of 356 mutations in the Cu-RE1 genome, and among them, 143 mutations were found in 70 genes within the functional gene group. Additionally, regarding the types of mutations occurring in Cu-RE1, referring to Table 2, it was found that mutations involving base insertion and deletion accounted for approximately 20% and 12% of the total mutations, respectively, while cases involving substitution accounted for approximately 67%. In particular, among genes with confirmed functions, cases involving substitution accounted for 77%. Notably, multiple mutations occurred in some genes, and genes in Cu-RE1 that had mutations involving the addition, removal, or substitution of three or more bases were analyzed, with the results presented in Table 3 below.

[0080] Gene number Gene function Mutation frequency O7R04_01250 ABC transporter permease 7 O7R04_01260 ABC transporter permease 6 O7R04_01180 class I SAM-dependent methyltransferase 5 O7R04_01265 ABC transporter permease 5 O7R04_01275 DUF4879 domain-containing protein 5 O7R04_17135 hypothetical protein 5 O7R04_18700 PLP-dependent aminotransferase family protein 5 O7R04_01245 ABC transporter ATP-binding protein 4 O7R04_03925 ABC transporter ATP-binding protein 4 O7R04_14615 hypothetical protein 4 O7R04_18695 3-hydroxybutyrate dehydrogenase 4 O7R04_01165 protein kinase 3 O7R04_01170 lanthioninesynthetaseC family protein 3 O7R04_01215 hypothetical protein 3 O7R04_08655 hypothetical protein 3 O7R04_10390 glutamic-type intramembrane protease PrsW 3 O7R04_17140 stage II sporulation protein M 3 O7R04_18720 rhodanese-like domain-containing protein 3

[0081] Referring to Table 3 above, it can be seen that among the 70 genes in which mutations occurred in Cu-RE1, 18 genes had mutations in which three or more bases were added, removed, or substituted within a single gene. However, it can be seen that among the genes in which mutations occurred intensively as described above, there are no genes related to laccase expression or copper ion resistance, such as CopC / CopD, which are well-known.

[0082] From the above results, it is presumed that Cu-RE1 developed mutations in the 18 genes listed in Table 3 due to radiation irradiation, and acquired copper resistance and high laccase productivity through a new, previously unknown method by being directly or indirectly affected by the mutations.

[0083] 6. Analysis of Oxidation Resistance Characteristics of Cu-RE1

[0084] Since no mutations in the previously known copper transporter and laccase activity-related genes were found in the gene mutation analysis, it was hypothesized that Cu-RE1 would have different characteristics of resistance to copper ions compared to the conventional Bacillus velezensis strain.

[0085] More specifically, since metal ions including copper induce oxidative stress, it was hypothesized that Cu-RE1 would possess increased resistance to its own oxidative stress, allowing it to contain and utilize high concentrations of copper ions. Therefore, to verify the above hypothesis, the following oxidation resistance characteristic analysis experiment was conducted.

[0086] The survival rates of wild-type (WT) strains and Cu-RE1 were determined by treating them with hydrogen peroxide (H2O2), a representative oxidizing agent, at concentrations of 20 to 400 mM, and cumene hydroperoxide (CHP), at concentrations of 10 to 80 mM, respectively. The survival rate of the strains refers to the colony-forming units (CFU) of the experimental group compared to the control group, and was calculated using the formula log(colony-forming units of the experimental group / colony-forming units of the control group). Here, the experimental group refers to cases treated with hydrogen peroxide or cumene hydroperoxide, while the control group refers to culture conditions without any treatment.

[0087] The results of measuring the survival rate of strains according to the concentration of hydrogen peroxide or cumene hydroperoxide are as shown in Figure 5.

[0088] Referring to Figure 5, it can be seen that the Cu-RE1 strain has a higher survival rate under the same oxidative stress than the wild-type (WT) strain.

[0089] More specifically, under conditions of 400 mM hydrogen peroxide concentration, it can be confirmed that the colony-forming unit of Cu-RE1 is 8 times higher than that of the wild-type strain.

[0090] In addition, referring to Figure 5(b), it can be seen that the colony-forming unit of Cu-RE1 under conditions of 80 mM cumene hydroperoxide concentration is 4.5 times higher than that of the wild-type strain.

[0091] From the above results, it is determined that Cu-RE1 improved the utilization capacity of copper ions by enhancing resistance to self-oxidative stress.

[0092] This implies that it is easy to utilize in industries requiring biological oxidation processes in terms of the strain's own resistance to oxidative stress.

[0093] 7. Comparison of Synthetic Dye Degradation Ability of Bacillus velezensis Wild-Type Strain and Cu-RE1

[0094] To determine the difference in synthetic dye degradation ability between Bacillus mutants and wild-types, decolorization experiments on various synthetic dyes were conducted as follows.

[0095] Cu-RE1 and wild-type (WT) strains cultured for one day were inoculated into TSB liquid medium at an OD of 600 nm = 1, respectively. At this time, the pH of the TSB medium was adjusted to 7.

[0096] After adding 0, 2.5, or 5 mM copper sulfate to the culture medium, Congo red, Crystal violet, Malachite green, Methylene blue, and Indigo carmine were inoculated as synthetic dyes at an initial dye concentration of 200 ppm each, and the culture was shaken at 200 rpm and 30°C for 6 hours.

[0097] The concentration of the synthetic dye remaining after culture was measured, and the ratio of the removed dye to the initial dye concentration, i.e., the decolorization rate, was calculated.

[0098] Figure 6 shows the results of comparing the decolorization rates of Cu-RE1 and wild-type strains for synthetic dyes.

[0099] Referring to Figure 6(a), it can be seen that the decolorization rate of Cu-RE1 against Congo Red was highest at 76% in a medium supplemented with 5 mM copper sulfate. On the other hand, the decolorization rate of the wild-type strain against Congo Red was highest at 48% in a medium supplemented with 2.5 mM copper sulfate, and it can be seen that the decolorization rate actually decreased in a medium supplemented with 5 mM copper sulfate.

[0100] Referring to Figure 6(b), it can be seen that the decolorization rate of Cu-RE1 against crystal violet is close to 100% in a medium with 5 mM copper sulfate added. On the other hand, the maximum decolorization rate of the wild-type strain against crystal violet is 73%, and it can be seen that the decolorization rate in a medium with 5 mM copper sulfate added is lower than in the case without copper sulfate added.

[0101] Referring to Figure 6(c), it can be seen that the decolorization rate of Cu-RE1 against malachite green is close to 100% in a medium supplemented with 5 mM copper sulfate. On the other hand, the decolorization rate of the wild-type strain against malachite green under 0 mM copper sulfate conditions is 59%, it is highest at 63% in a medium supplemented with 2.5 mM copper sulfate, and it can be seen that the decolorization rate actually decreases in a medium supplemented with 5 mM copper sulfate.

[0102] Referring to Figure 6(d), it can be seen that the decolorization rate of Cu-RE1 for methylene blue was highest at 55% in a medium supplemented with 5 mM copper sulfate. The decolorization rate of the wild-type strain for methylene blue was highest at 31% in a medium supplemented with 5 mM copper sulfate, but it can still be seen that it is significantly lower than that of Cu-RE1.

[0103] Referring to Figure 6(e), it can be seen that the decolorization rate of Cu-RE1 against indigo carmine was highest at 34% in a medium supplemented with 5 mM copper sulfate. The decolorization rate of the wild-type strain against indigo carmine was highest at 26% in a medium supplemented with 5 mM copper sulfate, but it can still be seen that it is significantly lower than that of Cu-RE1.

[0104] From the results of Figures 6(a) to 6(e) above, it can be confirmed that the decolorization efficiency of Cu-RE1 was improved by 1.5 to nearly 2 times compared to the wild-type strain with the addition of copper sulfate.

[0105] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resources Center (KCTC) Trustee Number: KCTC15701BP Date of Deposit: 2023-11-17 Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resources Center (KCTC) Trustee Number: KCTC15709BP Date of Deposit: 2023-11-27

Claims

Claim 1 A strain of Bacillus velezensis that is copper-resistant and capable of producing laccase, and is the Bacillus velezensis Cu-RE1 strain deposited under accession number KCTC15701BP. Claim 2 delete Claim 3 In claim 1, the Bacillus velezensis strain is a Bacillus velezensis strain comprising the nucleotide sequence of SEQ ID NO.

1. Claim 4 In claim 1, the Bacillus velezensis strain is a Bacillus velezensis strain having a laccase activity of 15 to 30 U / L at a copper concentration of 5 mM. Claim 5 In claim 1, the Bacillus velezensis strain is a strain having the ability to degrade one or more dyes selected from the group consisting of Congo red, Crystal violet, Malachite green, Methylene blue, and Indigo carmine. Claim 6 A method for degrading synthetic dyes, comprising the step of introducing a strain of any one of claims 1 and 3 to 5 into an aqueous solution containing synthetic dyes. Claim 7 A method for degrading synthetic dyes according to claim 6, wherein the synthetic dye is one or more selected from the group consisting of Congo red, Crystal violet, Malachite green, Methylene blue, and Indigo carmine. Claim 8 In claim 6, the aqueous solution is 1 to 5 mM Cu 2+ A method for decomposing synthetic dyes containing ions. Claim 9 In claim 6, the aqueous solution containing the synthetic dye is dye wastewater, a method for decomposing synthetic dyes.

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