Laccase-producing serratia marcescens and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to serratia marcescens for producing laccase and application of the serratia marcescens. Specifically, the invention provides a strain of Serratia marcescens ZH-5 capable of realizing high yield of laccase, and a preparation method of the Serratia marcescens ZH-5 capable of realizing high yield of laccase. Experiments prove that the laccase activity of the obtained serratia marcescens in an LB culture medium added with glycerol is remarkably improved and reaches 16822U / L, meanwhile, the strain shows good salt resistance, acid and alkali resistance and heavy metal resistance, and the produced laccase still shows certain enzyme activity at 80 DEG C and shows good enzyme activity in a strong acid environment, so that the laccase has good application prospects. Therefore, the method has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a laccase-producing Serratia marcescens and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Laccase is a type of copper-containing polyphenol oxidase in nature and is regarded as a "green catalyst" by scholars in the field of biotechnology. Currently, laccases have been found in fungi, bacteria, plants and even insects. Laccases are widely present in various biota, participating in the synthesis of lignin in plants, the degradation of lignin in fungi, the biosynthesis of bacterial melanin and copper detoxification. In insects, they can be used for the biosynthesis of hardening and cuticle. The only byproduct of the laccase-catalyzed reaction is water, and the reaction process is carried out with the assistance of four copper ions in its own structure.

[0004] It has been reported that most bacterial laccase molecules have four copper ions, but the copper ions in a small number of bacterial laccase molecules are replaced by iron ions and zinc ions. In essence, laccase can oxidize any substrate with similar properties to diphenol. Laccase is used in many fields. In the food industry, laccase is used because it can promote homopolymerization and heteropolymerization. It is mostly used in beer and wine stabilization, baking, juice processing, etc. In the textile field, it can be used for dyeing and washing. In biofuels, it can synthesize lignin, the raw material for producing fuel ethanol, and as a biocatalyst for organic synthesis. In the cosmetics field, it is also used for the manufacture of personal care products. In nanobiotechnology and biomedicine, people are committed to studying biofuel cells and biosensors. In addition, laccase is also used for enzyme bioremediation. It can be seen that laccase has a wide range of application prospects in biotechnology-related fields such as food, textiles, nanobiotechnology, biofuels, medicine and cosmetics. Therefore, it is of great significance to find new bacterial strains and microorganisms that can produce laccase, thereby broadening the source of laccase. Summary of the invention

[0005] Based on the above-mentioned deficiencies in the prior art, the present invention provides a strain of Serratia marcescens producing laccase and its application. Specifically, the present invention provides a strain of Serratia marcescens ZH-5 with high laccase production. It has been verified by experiments that the laccase activity of the Serratia marcescens obtained by the present invention is significantly improved in the LB medium with glycerol added, reaching 16822U / L. At the same time, the strain shows good salt resistance and acid-base resistance, and the laccase produced still shows a certain enzyme activity at 80°C, and it shows good enzyme activity in a strongly acidic environment. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the present invention relates to the following technical solutions:

[0007] The first aspect of the present invention provides a strain of Serratia marcescens ZH-5, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​June 3, 2024, and its biological deposit number is CGMCC No. 30840.

[0008] The second aspect of the present invention provides a fermentation production method of the above-mentioned Serratia marcescens ZH-5, which comprises: inoculating the Serratia marcescens ZH-5 into a fermentation medium for fermentation culture.

[0009] The third aspect of the present invention provides a microbial agent containing the Serratia marcescens ZH-5 or its fermentation product or metabolite.

[0010] A fourth aspect of the present invention is the use of the above-mentioned Serratia marcescens ZH-5 or microbial agent in the preparation of laccase.

[0011] A fifth aspect of the present invention provides a method for preparing laccase, the method comprising: fermenting and culturing the above-mentioned Serratia marcescens ZH-5 or microbial agent, and obtaining the laccase.

[0012] A sixth aspect of the present invention provides the use of the above-mentioned Serratia marcescens ZH-5, microbial agent or preparation method in the treatment of industrial printing and dyeing wastewater.

[0013] Beneficial technical effects of one or more of the above technical solutions:

[0014] The present invention screens and obtains a strain of Serratia marcescens with high laccase production. Experimental verification shows that the activity of laccase prepared by the Serratia marcescens in an LB medium supplemented with glycerol is significantly improved to 16822U / L. At the same time, the strain exhibits good salt resistance, acid and alkali resistance, and heavy metal resistance. The laccase produced still exhibits certain enzyme activity at 80°C, and exhibits good enzyme activity in a strongly acidic environment, so the strain has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0016] Figure 1 The present invention shows the colony morphology of the Serratia marcescens ZH-5 strain on a nutrient agar solid culture medium to which guaiacol has been added.

[0017] Figure 2 This is the effect of copper ion concentration on laccase activity in the present invention.

[0018] Figure 3 This is the effect of temperature on laccase activity in the present invention.

[0019] Figure 4 It is the temperature stability of the laccase of the present invention.

[0020] Figure 5 is the buffer pH of the laccase of the present invention. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0023] In a typical embodiment of the present invention, a strain of Serratia marcescens ZH-5 is provided, which has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​June 3, 2024, and its biological deposit number is CGMCC No. 30840.

[0024] In another specific embodiment of the present invention, a fermentation production method of the above-mentioned Serratia marcescens ZH-5 is provided, and the fermentation production method comprises: inoculating the Serratia marcescens ZH-5 into a fermentation medium for fermentation culture.

[0025] In the present invention, the fermentation production method is not particularly limited, and any conventional bacterial fermentation culture method can be used for culture.

[0026] Wherein, the fermentation medium can be a common bacterial culture medium, such as LB or NA culture medium in a specific embodiment of the present invention. Further, studies have shown that the ZH-5 strain of Serratia marcescens has a high laccase production performance. Analysis of adding different concentrations of copper ions shows that 0.01% copper ion concentration is the optimal copper ion concentration of laccase produced by Serratia marcescens ZH-5. Experiments at different temperatures show that 30°C is the optimal action temperature of the laccase of Serratia marcescens ZH-5. Analysis of the pH of the buffer solution shows that pH 3.0 is the optimal action pH of Serratia marcescens ZH-5.

[0027] In another specific embodiment of the present invention, a microbial agent is provided, which contains the Serratia marcescens ZH-5 or its fermentation product or metabolite.

[0028] In the present invention, the term "fermentation" is used to refer to a fermentation product. The corresponding fermentation may be a liquid obtained from the process of fermenting and culturing Serratia marcescens ZH-5, and therefore, may also be referred to as a fermentation liquid; the liquid may contain fungi (bacteria), but does not necessarily need to contain fungi. The liquid preferably contains metabolites produced by the Serratia marcescens ZH-5 of the present invention.

[0029] And, in an embodiment of the present invention, the fermentation liquid or culture liquid containing the bacteria is separated from the liquid by centrifugation, filtration, sedimentation or other means known in the art, and the liquid remaining after removing the bacterial cells is the "supernatant", and in the present invention, the supernatant contains the extracellular metabolites of Serratia marcescens ZH-5. In an embodiment of the present invention, the bacterial agent may also include the supernatant.

[0030] And, in an embodiment of the present invention, the fermentation liquid or culture liquid containing the bacteria is centrifuged, filtered, sedimented or other means known in the art to separate the bacterial cells grown in the fermentation liquid or culture liquid from the liquid to obtain the bacteria, and the bacteria can be crushed to obtain the bacterial crushed material, and the crushing method can be ultrasound (such as ice bath ultrasonic cell crushing) or other means known in the art, or, further, the bacterial crushed material is centrifuged to collect the supernatant, and the supernatant is recorded as a cell-free extract, and in the present invention, the bacterial crushed material or the cell-free extract contains the intracellular metabolites of Serratia marcescens ZH-5. In an embodiment of the present invention, the bacterial agent may also include the bacterial crushed material or the cell-free extract.

[0031] And, in the embodiments of the present invention, in order to facilitate storage and transportation, improve the survival rate of the strain, etc., the bacterial agent can also be solid, such as a freeze-dried powder. That is, the above-mentioned Serratia marcescens ZH-5 or its fermentation product or its metabolite is further freeze-dried to obtain the freeze-drying technology (including vacuum freeze-drying technology) can be carried out by conventional methods, which will not be repeated here.

[0032] Furthermore, the fermentation product or its metabolite contains laccase.

[0033] In another specific embodiment of the present invention, the microbial agent may further contain excipients acceptable to the agent.

[0034] In another specific embodiment of the present invention, the auxiliary material is selected from one or more of a dispersant, a wetting agent, a disintegrant, a binder, a defoamer, an antifreeze agent, a thickener, a filler and a solvent. The present invention has no special restrictions on the source of the auxiliary materials acceptable to the bacterial agent, and generally commercially available products can be used.

[0035] In another specific embodiment of the present invention, there is provided use of the above-mentioned Serratia marcescens ZH-5 or microbial agent in the preparation of laccase.

[0036] In another specific embodiment of the present invention, a method for preparing laccase is provided, the method comprising: fermenting and culturing the above-mentioned Serratia marcescens ZH-5 or microbial agent, and obtaining the laccase.

[0037] The fermentation culture comprises: adding Cu 2+ LB medium is added with glycerol and / or glucose for fermentation culture, thereby improving the activity of laccase.

[0038] Furthermore, the Cu 2+ The concentration of is 0.1-2 mM, more preferably 1 mM, and the added concentration of glycerol and / or glucose is 5-30 g / L, further preferably 20 g / L.

[0039] Furthermore, the fermentation culture conditions also include culturing at 30° C., 200 rpm for 6 days, and the fermentation pH is acidic, and further the pH is 3.

[0040] In another specific embodiment of the present invention, there is provided application of the above-mentioned Serratia marcescens ZH-5, microbial agent or preparation method in the treatment of industrial printing and dyeing wastewater.

[0041] The industrial printing and dyeing wastewater may be printing and dyeing wastewater containing any one or more of indigo, indigo red and crystal violet. The printing and dyeing wastewater may contain heavy metals, and the heavy metal is Cd.

[0042] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0043] Example 1 Screening of Laccase-producing Microorganisms

[0044] 1. Materials and Methods

[0045] 1.1 Experimental materials and reagents

[0046] 1.1.1 Experimental Materials

[0047] Plant root soil samples, guaiacol, 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) (ABTS), copper sulfate, acetic acid, and sodium hydroxide.

[0048] 1.1.2 Experimental culture medium

[0049] PDA medium (g / L): potato 20, glucose 20, magnesium sulfate 1.5, agar 20, peptone 5, potassium dihydrogen phosphate 3, distilled water 1000mL;

[0050] NA medium (g / L): glucose 2.5, beef extract powder 3, peptone 5, agar powder 15, distilled water 1000mL;

[0051] LB liquid medium (g / L): 10% tryptone, 10% sodium chloride, 5% yeast extract, and 1000 mL distilled water.

[0052] 1.1.3 Experimental reagents

[0053] pH 4.0 acetic acid-sodium acetate buffer: 0.3 mol / L acetic acid and 0.2 mol / L sodium acetate were mixed in a volume ratio of 4.6:1.

[0054] 1.2 Experimental instruments and equipment

[0055] Electronic balance; high pressure steam sterilizer; constant temperature incubator; ultracentrifuge; clean bench; constant temperature water bath; refrigerated centrifuge; ultrasonic disruptor; PCR instrument; UV-visible spectrophotometer; pH meter; agarose gel electrophoresis instrument; constant temperature magnetic stirrer; microwave oven; shaking incubator; spectrophotometer; gel chromatography; rotor oscillator; measuring cylinder; beaker; conical flask; medicine spoon; glass rod.

[0056] 1.3 Experiments and Methods

[0057] 1.3.1 Screening

[0058] 1. Prepare PDA solid medium and NA solid medium

[0059] Prepare 100 mL of PDA and NA culture medium containing 1 mmol / L CuSO4 respectively, sterilize at 121°C for 20 min. After sterilization, add 0.04% guaiacol at a working concentration after the temperature drops to about 50°C, and pour into plates for later use.

[0060] 2. Dilute the soil

[0061] Place 1 g of the soil sample in a tube containing 9 mL of sterile water, shake it with a rotor oscillator, and let it stand for 30 seconds to 1 minute, which is recorded as 10. -1 Serial dilutions were continued until 10 -3 with 10 -4 .

[0062] 3. Coating the flat plate

[0063] Take the dilution of 10 -3 with 10 -4 The dilutions were plated on PDA and NA solid culture media in a clean bench. Each dilution was plated on each culture medium and marked.

[0064] 4. Cultivation

[0065] Place the marked solid culture medium in an incubator at 28°C and invert it for five to seven days, and observe it every two days. Observe the morphology of the colonies, select and mark the single colonies with good growth and reddish-brown oxidation zones around them, and separate and purify them. Continue to streak the marked colonies on the same type of culture medium and invert them in a 28°C incubator until a single colony appears.

[0066] 1.3.2 Identification

[0067] 1. Rapid method for genome extraction

[0068] Use a pipette to draw 500 μL of 20 mmol / L NaOH into a 1.5 mL centrifuge tube, pick a small amount of the bacteria to be tested into the tube, boil it in a boiling water bath for 10 minutes to break the cells, cool it down slightly and centrifuge it at 10,000 r / min for 5 minutes to take the supernatant, and you will get the template DNA.

[0069] 2. Confirmatory PCR

[0070] For each bacterial species, the universal primers for bacteria (27F / 1492R) were used to amplify the full-length 16S rDNA of the species, and the universal primers for fungi (ITS-4 / ITS-5) were used to amplify the full-length 18S rDNA of the species. A 15 μL PCR reaction system was prepared for each sample.

[0071] Table 1 PCR reaction system

[0072]

[0073] 3. Electrophoresis

[0074] (1) Glue preparation

[0075] Weigh 0.25 g of agarose using an electronic balance and place it in a conical glass bottle that has been added with 25 mL of 1×TAE. Heat it in a microwave oven until all the agarose melts. After cooling until it is not hot to the touch, add 1.5 μL of dye (APE BIO) and mix well. Prepare gel and wait for about 30 minutes for the gel to solidify before use.

[0076] (2) Sample

[0077] Add 5 μL of sample and marker vertically to the sample well of the gel. If the sample does not contain Loading Buffer, you need to mix the Loading Buffer with the sample before spotting.

[0078] (3) Electrophoresis

[0079] After all samples and markers are spotted, electrophoresis is performed immediately. When the electrophoresis indicator moves to half of the gel plate, electrophoresis can be stopped and the bands can be observed and compared under the ultraviolet light of a gel imager.

[0080] 4. Sequencing PCR

[0081] According to the verified PCR results, 100 μL PCR system with 16S rDNA primers of each bacterium is used for sequencing. KMM-101 can be replaced with KMM-201 with better fidelity. After electrophoresis to verify that the band size is correct, the PCR product is sent to Shanghai Bioengineering Company for sequencing.

[0082] 5. Identification of bacterial species

[0083] The gene sequences obtained by sequencing were compared and analyzed by NCBI's BLAST homology analysis to confirm the species of the isolated bacteria.

[0084] 1.3.3 Determination of laccase activity

[0085] 1. Cultivate seed solution

[0086] Take the purified strains and inoculate them into 50 mL of LB liquid culture medium in the clean bench. Set the shaker temperature to 25°C. After marking the flasks, culture them on the shaker for three days. The resulting bacterial solution is the seed solution.

[0087] 2. Cultivation of bacteria

[0088] Take 1 mL of seed solution in a clean bench and inoculate it into 50 mL of LB liquid culture medium for culture. Shake the flask at 150 r / min and 25°C for 3 days.

[0089] 3. Cell disruption

[0090] Take 25 mL of the cultured bacterial solution and pour it into a centrifuge tube. Place it in a refrigerated centrifuge, set the temperature to 4°C, and centrifuge it at 9,000×g for 10 minutes. Discard the supernatant and re-dissolve the centrifuged bacteria with 25 mL of distilled water. Use an ultrasonic pulverizer to crush the bacteria. The crushing time is 30 minutes, 3 seconds each time, 3 seconds interval between each time, and the power is 45%.

[0091] 4. Collect crude enzyme solution

[0092] Place the cell disruption solution in a refrigerated centrifuge, set the temperature to 4°C, and centrifuge at 12,000×g for 10 minutes. Take the supernatant, which is the crude enzyme solution, and store it at 4°C for later use.

[0093] 5. Determination of enzyme activity

[0094] Add 200 μL ABTS, 1.8 mL pH 4 acetic acid-sodium acetate buffer, and 1 mL crude enzyme solution to the reaction tube, a total of 3 mL reaction system, and measure the absorbance change at 420 nm for 5 minutes. Use the mixed solution containing the inactivated enzyme solution as a control (heat 1 mL crude enzyme solution in a boiling water bath for 5 minutes). Measure the absorbance change of bacteria No. 5 and calculate the enzyme activity. One unit of enzyme activity is defined as the amount of enzyme required to hydrolyze 1 umol ABTS per minute.

[0095] Note: Enzyme activity (U / mL) = 10 3 / ε×Vtotal / Venzyme×△OD / △t, where Vtotal and Venzyme represent the total volume of the reaction system and the volume of the enzyme solution, respectively, ε is the absorption coefficient, and the molar extinction coefficient of ABTS is ε420=36mL / (μmol·cm). If dilution is performed, it is also necessary to multiply by the dilution factor.

[0096] 1.3.4 Analysis of factors affecting laccase activity

[0097] 1. Laccase activity is affected by copper ions

[0098] Add 200 μL ABTS, 1.7 mL pH 4 acetic acid-sodium acetate as buffer and 1 mL crude enzyme solution into the reaction tube, and add 100 μL of copper sulfate solution with a concentration increased by 2 times from 0.0025% to 0.16% respectively, for a total of 3 mL reaction system. Measure the absorbance change at 420 nm in a UV-visible spectrophotometer and calculate the enzyme activity.

[0099] 2. Effect of temperature on enzyme activity

[0100] The prepared ABTS solution, pH 4 acetic acid-sodium acetate buffer and crude enzyme solution were placed in a constant temperature water bath at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C respectively for insulation. 200 μL ABTS, 1.8 mL pH 4 acetic acid-sodium acetate as buffer and 1 mL crude enzyme solution were added to the reaction tube, totaling 3 mL reaction system, and the absorbance change was measured at 420 nm by UV-Vis spectrophotometer and the enzyme activity was calculated.

[0101] 3. Analysis of enzyme activity and thermal stability

[0102] The crude enzyme solution was placed at 50℃, 60℃, 70℃, and 80℃ for one hour. 200μLABTS, 1.8mL pH4 acetic acid-sodium acetate as buffer and 1mL of the crude enzyme solution that had been treated in advance were added to the reaction tube, totaling 3mL of reaction system. The absorbance change was measured at 420nm in a UV-visible spectrophotometer and the enzyme activity was calculated.

[0103] 4. Effect of pH on enzyme activity

[0104] Add 200 μL ABTS solution, 1.8 mL acetic acid-sodium acetate buffer and 1 mL crude enzyme solution to the reaction tube, control the pH value of the buffer to 2.5, 3.0, 4.0, 5.0, and 6.0, for a total of 3 mL of reaction system, measure the absorbance change at 420 nm using a UV-visible spectrophotometer and calculate the enzyme activity.

[0105] 2. Results and Analysis

[0106] 2.1 Appearance and identification of bacterial strains

[0107] The inventor obtained 3 strains with laccase activity after preliminary screening, among which strain ZH-5 was white and opaque in appearance in the solid medium of nutrient agar with guaiacol, with obvious reddish-brown oxidation zone in the center, round or oval, regular edges, and smooth and moist surface. According to the colorimetric analysis of the plate with guaiacol, the laccase activity of strain ZH-5 was relatively the largest. Therefore, this strain was selected for further study. The 16S rDNA gene sequence was obtained by amplifying and sequencing the 16S rDNA gene sequence of the strain, and the sequence was compared using BLAST, and the physiological and biochemical analysis was performed to determine that strain ZH-5 was Serratia marcescens.

[0108] 2.2 Determination of enzyme activity of Serratia marcescens

[0109] The Serratia marcescens with the most obvious color development on the plate was selected and cultured in LB liquid medium for 3 days. The laccase activity was measured and the enzyme activity of the Serratia marcescens was measured to be 1483.3 U / L.

[0110] 2.3 Analysis of enzyme activity of Serratia marcescens

[0111] Using ABTS as the reaction substrate, by adding different concentrations of copper ions, the absorbance change was measured by spectrophotometry. The results are as follows: Figure 2 As shown in the figure, the results show that when the copper ion concentration is between 0.0025% and 0.01%, the enzyme activity gradually increases with the increase of copper ion concentration; when the copper ion concentration is between 0.01% and 0.08%, the enzyme activity gradually decreases with the increase of copper ion concentration; when the copper ion concentration is 0.01%, the enzyme activity reaches the maximum value. Therefore, 0.01% copper ion is the optimal concentration of Serratia marcescens laccase.

[0112] Using ABTS as the reaction substrate, setting different reaction temperatures, and measuring the absorbance change by spectrophotometry, the results are as follows: Figure 3 As shown in the figure, the results show that the enzyme activity increases with the temperature from 25℃ to 30℃; the enzyme activity reaches the maximum value at 30℃; the enzyme activity decreases with the temperature from 30℃ to 50℃. Therefore, it can be judged that the optimal action temperature of laccase produced by Serratia marcescens is 30℃.

[0113] The reaction tube containing the crude enzyme solution was treated at a specific water bath temperature for one hour, and ABTS was used as the reaction substrate. After the reaction, the absorbance change was measured by spectrophotometry and the enzyme activity of laccase was converted. The results were as follows: Figure 4 As shown in the figure, after treatment at 50℃ for 1 hour, the enzyme activity loss was only 57.7%. After treatment at 80℃ for 1 hour, there was still 26.9% enzyme activity, indicating that the laccase of this fungus has good stability.

[0114] Using ABTS as the reaction substrate, the pH of the buffer solution was controlled and the absorbance change was measured by spectrophotometry. The results are as follows: Figure 5 As shown in the figure, the results show that the enzyme activity gradually increases between pH 2.5 and 3.0, and reaches the maximum at pH 3.0. Therefore, it is judged that the most suitable pH for Serratia marcescens is 3.0. This will further broaden its application field and scope.

[0115] Example 2 Study on the acid and alkali resistance, salt resistance, heavy metal resistance and high (low) temperature resistance of Serratia marcescens ZH-5

[0116] This embodiment relates to the characteristic identification of acid and alkali resistance, salt resistance and high temperature resistance of Serratia marcescens ZH-5, and the specific method is as follows:

[0117] (1) The Serratia marcescens ZH-5 suspension was inoculated into LB liquid culture medium at an inoculation ratio of 1% (v / v), and cultured at 30°C and 200 rpm for 24 hours, and the growth of each strain under different pH (3.0-10.0) conditions was analyzed. The results are shown in Table 2. The strain can grow in the pH range of 3.0-9.0, indicating that it has good acid and alkali resistance.

[0118] Table 2 Growth status of ZH-5 at different pH

[0119]

[0120] Among them, “-” means that the bacteria basically do not grow (bacterial solution OD 600 <0.2), “+” represents bacterial growth, bacterial solution OD 600 The value is 0.2 to 0.5, and “++” indicates bacterial growth. 600 The value is 0.5 to 1.0, and “+++” indicates bacterial growth. 600 is>1.0.

[0121] (2) The Serratia marcescens ZH-5 suspension was inoculated into LB liquid culture medium at an inoculation ratio of 1% (v / v), and cultured at 30°C and 200 rpm for 24 h, and the growth of each strain under different salt concentrations (1-13%) was analyzed. The results are shown in Table 3, and the strain ZH-5 can grow in the range of salt concentrations of 1-12%.

[0122] Table 3 Growth status of ZH-5 at different NaCl concentrations

[0123]

[0124] Among them, “-” means that the bacteria basically do not grow (bacterial solution OD 600 <0.2), “+” represents bacterial growth, bacterial solution OD600 The value is 0.2 to 0.5, and “++” indicates bacterial growth. 600 The value is 0.5 to 1.0, and “+++” indicates bacterial growth. 600 is>1.0.

[0125] (3) The suspension of Serratia marcescens ZH-5 was inoculated into LB liquid medium at a ratio of 1% (v / v). After culturing at 30°C and 200 rpm for 24 h, the activity of each strain at different Cd 2+ The results are shown in Table 4. The results show that ZH-5 has excellent heavy metal tolerance, even when Cd 2+ When it reaches 350mg / L, it can still grow.

[0126] Table 4ZH-5 in different Cd 2+ Growth status at (mg / L) concentration

[0127]

[0128] Among them, “-” means that the bacteria basically do not grow (bacterial solution OD 600 <0.2), “+” represents bacterial growth, bacterial solution OD 600 The value is 0.2 to 0.5, and “++” indicates bacterial growth. 600 The value is 0.5 to 1.0, and “+++” indicates bacterial growth. 600 is>1.0.

[0129] (4) The Serratia marcescens ZH-5 suspension was inoculated into the LB liquid medium at an inoculation ratio of 1% (v / v), and cultured at 200 rpm for 24 h. The growth of each strain at different temperatures (15-50° C.) was analyzed. The results are shown in Table 5. The strain can grow within the above temperature range.

[0130] Table 5 Growth status of ZH-5 at different temperatures

[0131]

[0132] Among them, "+" represents bacterial growth, and the OD of the bacterial solution is 600 The value is 0.2 to 0.5, and “++” indicates bacterial growth. 600 The value is 0.5 to 1.0, and “+++” indicates bacterial growth. 600 is>1.0.

[0133] Example 3 Laccase enzyme activity optimization

[0134] 2,2'-diazo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) is a commonly used substrate for determining laccase activity. The ABTS method usually uses sodium acetate solution as a buffer solution for determining laccase activity. The final concentration in the reaction system is usually 0.02, 0.05 or 0.10 mol / L, and the pH is mainly between 3 and 5. The commonly used final concentration of ABTS in the reaction system is 0.5 mmol / L. The determination reaction is generally carried out at room temperature. ABTS forms ABTS free radicals after being acted upon by laccase. The absorption coefficient of ABTS free radicals at 420 nm is much greater than that of the substrate ABTS. As the concentration of ABTS free radicals increases, the absorbance value increases, so the change in OD value is detected at 420 nm.

[0135] Reagents: 0.1mmol / L acetic acid-sodium acetate buffer, crude enzyme solution, 1mmol / L ABTS solution, 100mmol / L Cu 2+ Solution (CuSO4).

[0136] Preparation of crude enzyme solution: same as in Example 1.

[0137] Serratia marcescens ZH-5 was inoculated into a medium containing 1 mM Cu 2+ LB was added to make the fermentation broth start at OD 600 The laccase activity was measured after culturing at 30°C, 200 rpm for 6 days as a control group. 600 is 2.23.

[0138] Prepare the reaction system: pH = 3 buffer solution 1.8 mL, crude enzyme solution 1.0 mL, and use the control group to determine the optimal Cu 2+ The results are shown in Tables 6 and 7.

[0139] Table 6 Different Cu 2+ Enzyme activity at concentration

[0140]

[0141] Table 7 Enzyme activity at different ABTS volumes

[0142]

[0143]

[0144] Finally, the experimental group reaction system Cu 2+ The concentration was 1.5 mM and the ABTS volume was 0.40 mL.

[0145] Serratia marcescens ZH-5 was inoculated into a medium containing 1 mM Cu 2+ LB was added to make the fermentation broth start at OD 600The value was 0.1, and 5 g / L, 10 g / L, 15 g / L, and 20 g / L of glucose and glycerol were added as carbon sources, respectively, and the laccase activity was measured after culturing at 30°C and 200 rpm for 6 days. The results are shown in Tables 8 and 9.

[0146] Table 8 Enzyme activity at different glucose concentrations

[0147]

[0148] Table 9 Enzyme activity at different glycerol concentrations

[0149]

[0150] Serratia marcescens ZH-5 was inoculated into a medium containing 1 mM Cu 2+ LB was added to make the fermentation broth start at OD 600 The value was 0.1, 15 g / L glycerol and glucose were added as carbon sources, respectively, and the laccase activity was measured after culturing at 37°C and 200 rpm for 6 days. The results are shown in Table 10.

[0151] Table 10 Enzyme activity under different temperature culture

[0152]

[0153] Experiments show that the efficiency of enzyme production at 37°C is not high. Combined with previous work, 30°C is the most suitable temperature for cultivating ZH-5 bacteria to produce laccase.

[0154] Serratia marcescens ZH-5 was inoculated into a 1.5 mM Cu 2+ LB was added to make the fermentation broth start at OD 600 The value was 0.1, and the culture was carried out at 30°C and 200 rpm for 6 days as the control group. The reaction system of enzyme activity was further optimized, and the buffer solution was 1.8 mL, the crude enzyme solution was 1.0 mL to explore the ABTS volume, and the results are shown in Table 11.

[0155] Table 11 Enzyme activity at different ABTS volumes

[0156]

[0157] Serratia marcescens ZH-5 was inoculated into a 1.5 mM Cu 2+ LB was added to make the fermentation broth start at OD 600 The value was 0.1, and 5 g / L, 10 g / L, 15 g / L, and 20 g / L of glucose and glycerol were added as carbon sources, respectively, and cultured at 30°C and 200 rpm for 6 days. The buffer was 2.0 mL, the crude enzyme solution was 0.5 mL, and the Cu 2+ The concentration was 1.5 mM, the volume of ABTS was 0.8 mL, and the results are shown in Tables 12 and 13.

[0158] Table 12 Enzyme activity measured at different glucose concentrations

[0159]

[0160] Table 13 Enzyme activity measured at different glycerol concentrations

[0161]

[0162] After adding 1.5 mM Cu 2+ In the LB medium, after the crude enzyme extraction process was mastered and the reaction system was optimized, the laccase activity reached 8817U / L. After adding carbon source, especially in the medium of LB+20g / L glycerol, the laccase activity was significantly improved to 16822U / L, showing the good application prospect of the laccase produced by this bacterium.

[0163] Example 4 Laccase decolorization of industrial printing and dyeing wastewater

[0164] Indigo, indigo carmine and crystal violet were used as research objects to study the decolorization of dyes by bacterial laccase. The decolorization system was as follows: 3 mL of 50 mg / L indigo, indigo carmine and crystal violet solution, 0.2 mL of crude enzyme solution prepared in Example 1, Cu 2+ The concentration of (CuSO4) was 1.5mM, and the reaction was carried out in a 30℃ water bath for 1 hour for decolorization. The absorbance change rate before and after the reaction was measured at 680nm, 605nm and 590nm wavelengths, and the decolorization rate of the dye was calculated. Among them, the decolorization rate of indigo was 93.2%, the decolorization rate of indigo red was 87.6%, and the decolorization rate of crystal violet was 81.5%. It shows that it has a good ability to decolorize industrial printing and dyeing wastewater.

[0165] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A strain of Serratia marcescens ZH-5, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 3, 2024, and its biological deposit number is CGMCC No.30840.

2. The fermentation production method of Serratia marcescens ZH-5 according to claim 1, characterized in that: The fermentation production method comprises: inoculating Serratia marcescens ZH-5 into a fermentation medium for fermentation culture.

3. A microbial agent, characterized in that: It contains the Serratia marcescens ZH-5 or its fermentation product or metabolite as described in claim 1.

4. Use of the Serratia marcescens ZH-5 according to claim 1 or the microbial agent according to claim 3 in the preparation of laccase.

5. A method for preparing laccase, characterized in that: The preparation method comprises: fermenting and culturing the Serratia marcescens ZH-5 according to claim 1 or the microbial agent according to claim 3, and obtaining the laccase.

6. The preparation method according to claim 5, characterized in that: The fermentation culture comprises: adding Cu 2+ LB medium was added with glycerol and / or glucose for fermentation.

7. The preparation method according to claim 6, characterized in that: The Cu 2+ The concentration is 0.1-2mM.

8. The preparation method according to claim 6, characterized in that: The added concentration of the glycerol and / or glucose is 5-30 g / L.

9. The preparation method according to claim 6, characterized in that: The fermentation culture conditions include culturing at 30° C. and 200 rpm for 6 days, and the fermentation pH is acidic, and further the pH is 3.

10. Use of the Serratia marcescens ZH-5 according to claim 1, the microbial agent according to claim 3, or the preparation method according to any one of claims 5 to 9 in the treatment of industrial printing and dyeing wastewater.

Images