Serratia marcescens producing laccase and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a laccase-producing Serratia marcescens and application thereof. Specifically, the application provides a high-yield laccase-producing Serratia marcescens ZH-5. It is verified through experiments that the Serratia marcescens obtained in the application has significantly improved laccase activity, reaching 16822 U / L, in a LB culture medium added with glycerol. Meanwhile, the strain has good salt tolerance and acid and alkali tolerance, heavy metal tolerance, and the laccase produced by the strain still has certain enzyme activity at 80 DEG C, and has good enzyme activity in a strong acidic environment, thus having good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a laccase-producing Serratia marcescens and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any jurisdiction.

[0003] Laccase is a kind of copper-containing polyphenol oxidase in nature, and is regarded as a "green catalyst" by scholars in the field of biotechnology. At present, laccase has been found in fungi, bacteria, plants and even insects. Laccase widely exists in various biological groups, participates in the synthesis of lignin in plants, the degradation of lignin in fungi, the biosynthesis of bacterial melanin and the detoxification of copper, and can be used for the biosynthesis of sclerotization and cuticle in insects. The only byproduct of laccase catalytic reaction is water, and the reaction process is carried out under the assistance of four copper ions in its own structure.

[0004] It is reported that most of the laccase molecules of bacteria have four copper ions, but a small part of the laccase molecules produced by bacteria are replaced by iron ions and zinc ions. In essence, laccase can oxidize any substrate with similar characteristics to diphenol. Laccase has applications in many fields. In the food industry, laccase is used because it can promote homopolymerization and heteropolymerization, and is mainly used for beer and wine stabilization, baking, fruit juice processing and the like, and in the textile field, it can be used for dyeing and washing, in the field of biofuels, it can be used for the synthesis of raw materials lignin for producing fuel ethanol and as a biological catalyst for organic synthesis, in the field of cosmetics, it is also used for the manufacture of personal care products, in the field of nanobiotechnology and biomedicine, people are committed to the research of biofuel cells and biosensors, in addition, laccase is also used for enzyme bioremediation. As can be seen, laccase has a very wide application prospect in the fields of food, textiles, nanobiotechnology, biofuels, medicine and cosmetics related to biotechnology. Therefore, it is of great significance to find new laccase-producing bacterial strains and expand the sources of laccase. SUMMARY

[0005] Based on the deficiencies of the prior art, the present application provides a laccase-producing Serratia marcescens and its application. Specifically, the present application provides a high-yield laccase-producing Serratia marcescens ZH-5. Through experimental verification, the Serratia marcescens obtained in the present application has significantly improved laccase activity in the LB medium added with glycerol, reaching 16822 U / L. Meanwhile, the strain exhibits good salt tolerance and acid and alkali resistance, and the produced laccase still exhibits certain enzyme activity at 80℃, and exhibits good enzyme activity in a strong acidic environment. Based on the above research results, the present application is completed.

[0006] To achieve the above technical purposes, the present application relates to the following technical solutions:

[0007] In a first aspect of the present application, a Serratia marcescens ZH-5 strain is provided, which has been deposited with the China General Microbiological Culture Collection Center (address: No. 1, Beichen West Road, No. 3, Chaoyang District, Beijing) on June 3, 2024, and has a biological preservation number of CGMCC No. 30840.

[0008] In a second aspect of the present application, a fermentation production method of the above-mentioned Serratia marcescens ZH-5 is provided, which comprises inoculating the Serratia marcescens ZH-5 into a fermentation medium for fermentation culture to obtain the same.

[0009] In a third aspect of the present application, a microbial inoculant containing the Serratia marcescens ZH-5 or its fermentation product or its metabolite is provided.

[0010] In a fourth aspect of the present application, the above-mentioned Serratia marcescens ZH-5 or microbial inoculant is used for preparing laccase.

[0011] In a fifth aspect of the present application, a preparation method of laccase is provided, which comprises fermenting and culturing the above-mentioned Serratia marcescens ZH-5 or microbial inoculant, and obtaining the laccase.

[0012] In a sixth aspect of the present application, the above-mentioned Serratia marcescens ZH-5, microbial inoculant or preparation method is used for industrial printing and dyeing wastewater treatment.

[0013] The above-mentioned one or more technical solutions have the following beneficial technical effects:

[0014] The present application screens and obtains a Serratia marcescans strain with high yield of laccase, and the laccase activity of the Serratia marcescans strain is significantly improved to 16822 U / L in a LB culture medium added with glycerol, and the strain has good salt tolerance and acid and alkali tolerance and heavy metal tolerance, and the laccase produced by the strain still has certain enzyme activity at 80 DEG C and good enzyme activity in a strong acidic environment, and therefore has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0015] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of these drawings are set to explain the application, and do not constitute an improper limitation to the application.

[0016] Figure 1 The colony morphology of the Serratia marcescans ZH-5 strain of the present application on a solid culture medium of nutrient agar added with guaiacol.

[0017] Figure 2 The influence of copper ion concentration on laccase activity.

[0018] Figure 3 The influence of temperature on laccase activity.

[0019] Figure 4 The temperature stability of laccase.

[0020] Figure 5 The buffer pH of laccase. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed description is merely exemplary and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0022] It should be noted that the terms used herein are merely for the purpose of describing the specific embodiments, and are not intended to limit the exemplary embodiments according to the application. As used herein, the singular form is intended to include the plural form, unless the context clearly indicates otherwise, and it should be understood that, when the terms "comprise" and / or "include" are used in the specification, there is a presence of the 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. This strain was deposited on June 3, 2024, at the China General Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the biological accession number CGMCC No. 30840.

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

[0025] In this invention, no specific limitation is made to the fermentation production method; any conventional bacterial fermentation culture method can be used for cultivation.

[0026] The fermentation medium can be any common bacterial culture medium, such as LB or NA medium in one specific embodiment of the invention. Further research shows that *Serratia marcescens* strain ZH-5 has high laccase production performance. Analysis with different concentrations of copper ions shows that 0.01% copper ion concentration is the optimal copper ion concentration for laccase produced by *Serratia marcescens* ZH-5. Experiments at different temperatures show that 30℃ is the optimal operating temperature for laccase from *Serratia marcescens* ZH-5. Analysis of the buffer pH shows that pH 3.0 is the optimal operating pH for *Serratia marcescens* ZH-5.

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

[0028] In this invention, the term "fermentation product" is used to refer to fermentation products. The corresponding fermentation product can be a liquid obtained from the fermentation culture of *Serratia marcescens* ZH-5, and therefore can also be called fermentation broth; the liquid may contain fungi (cells), but is not necessarily required to contain fungi. Preferably, the liquid contains metabolites produced by *Serratia marcescens* ZH-5 of this invention.

[0029] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is called the "supernatant," and in the present invention, the supernatant contains extracellular metabolites of Serratia marcescens ZH-5. In embodiments of the present invention, the bacterial agent may also contain this supernatant.

[0030] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is centrifuged, filtered, settled, or otherwise known in the art to separate the bacterial cells grown in the fermentation broth or culture medium from the liquid to obtain bacterial cells. The bacterial cells can be broken up to obtain bacterial fragments. The breaking method can be ultrasound (e.g., ice bath ultrasound to break up cells) or other methods known in the art. Alternatively, the bacterial fragments can be centrifuged to collect the supernatant, which is designated as the cell-free extract. In the present invention, the bacterial fragments or cell-free extract contain intracellular metabolites of Serratia marcescens ZH-5. In embodiments of the present invention, the bacterial agent may also contain the bacterial fragments or cell-free extract.

[0031] Furthermore, in embodiments of the present invention, for ease of storage and transportation, and to improve the survival rate of the bacterial strain, the bacterial agent can also be a solid, such as a lyophilized powder. That is, it is obtained by further freeze-drying the aforementioned *Serratia marcescens* ZH-5 or its fermentation products or their metabolites. The freeze-drying technology (including vacuum freeze-drying technology) can be carried out using conventional methods, and will not be elaborated further here.

[0032] Furthermore, the fermentation product or its metabolites contain laccase.

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

[0034] In another specific embodiment of the present invention, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the sources of acceptable excipients for the bacterial agent; commercially available products are generally sufficient.

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

[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 a microbial inoculum, and obtaining the laccase.

[0037] The fermentation culture includes: adding Cu 2+ In LB medium, glycerol and / or glucose are added for fermentation culture to improve laccase activity.

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

[0039] Furthermore, the fermentation culture conditions also include culturing at 30°C and 200 rpm for 6 days, with an acidic fermentation pH, and even more specifically, a pH of 3.

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

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

[0042] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0043] Example 1: Screening of Laccase-Producing Microorganisms

[0044] I. Materials and Methods

[0045] 1.1 Experimental Materials and Reagents

[0046] 1.1.1 Experimental Materials

[0047] Soil samples from plant roots contained 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): tryptone 10, sodium chloride 10, yeast extract 5, distilled water 1000mL.

[0052] 1.1.3 Experimental Reagents

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

[0054] 1.2 Experimental Instruments and Equipment

[0055] Electronic balance; autoclave; incubator; ultracentrifuge; clean bench; water bath; refrigerated centrifuge; ultrasonic disruptor; PCR instrument; UV-Vis spectrophotometer; pH meter; agarose gel electrophoresis apparatus; magnetic stirrer; microwave oven; shaking incubator; spectrophotometer; gel chromatograph; rotor shaker; graduated cylinder; beaker; conical flask; spatula; glass rod.

[0056] 1.3 Experiments and Methods

[0057] 1.3.1 Screening for bacteria

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

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

[0060] 2. Dilute the soil

[0061] Place 1g of the collected soil sample into a tube containing 9mL of sterile water. After shaking with a rotor shaker, let it stand for 30s to 1min, and record this as 10. -1 Continue with a series of dilutions to 10. -3 With 10 -4 .

[0062] 3. Apply to flat plate

[0063] Take a dilution of 10 -3 With 10 -4 The diluted solutions were plated on PDA and NA solid media in a clean bench. One plate was prepared for each dilution on each medium, and the plates were clearly labeled.

[0064] 4. Cultivation

[0065] The labeled solid culture medium was placed in an incubator at 28°C and inverted for five to seven days, with observations every two days. The morphology of the colonies was observed, and single colonies with good growth and a reddish-brown oxidation zone were selected for isolation and purification. The labeled colonies were then streaked onto the same type of culture medium and incubated inverted at 28°C until single colonies appeared.

[0066] 1.3.2 Identification

[0067] 1. Rapid method for extracting genomes

[0068] Use a pipette to transfer 500 μL of 20 mmol / L NaOH into a 1.5 mL centrifuge tube. Pick a small amount of the test bacteria into the tube, incubate in boiling water for 10 min to break the cells, cool slightly, and then centrifuge at 10000 r / min for 5 min. Collect the supernatant to obtain the template DNA.

[0069] 2. Confirmatory PCR

[0070] For each bacterial species, the full-length 16S rDNA was amplified using universal primers for bacteria (27F / 1492R) and the full-length 18S rDNA was amplified using universal primers for fungi (ITS-4 / ITS-5). Each sample was processed in a 15 μL PCR reaction system.

[0071] Table 1 PCR reaction system

[0072]

[0073] 3. Electrophoresis

[0074] (1) Rubber preparation

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

[0076] (2) Spotting

[0077] Vertically add 5 μL of sample and 5 μL of marker to the well of the gel. If the sample does not contain loading buffer, mix the loading buffer with the sample before loading.

[0078] (3) Electrophoresis

[0079] Electrophoresis should be performed immediately after all samples and markers have been loaded. Electrophoresis can be stopped when the electrophoresis indicator has moved to half of the gel plate, and the bands can be observed and compared under the UV light of a gel imaging system.

[0080] 4. Sequencing PCR

[0081] Based on the validated PCR results, each bacterium was sequenced using a 100 μL PCR system with the bacterial 16S rDNA primers. KMM-101 could be replaced with KMM-201, which has better fidelity. After verifying the band size by electrophoresis, the PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0082] 5. Strain identification

[0083] The gene sequences obtained from sequencing were analyzed using NCBI's BLAST homology comparison to confirm the species of the isolated bacteria.

[0084] 1.3.3 Determination of laccase activity

[0085] 1. Seed culture medium

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

[0087] 2. Cultivating bacterial cells

[0088] Take 1 mL of seed culture in a clean bench and inoculate it into 50 mL of LB liquid medium for culture. Culture at 150 r / min and 25 ℃ for 3 days.

[0089] 3. Cell disruption

[0090] Pour 25 mL of the cultured bacterial solution into a centrifuge tube and place it in a refrigerated centrifuge. Set the temperature to 4℃ and centrifuge at 9000×g for 10 min. Discard the supernatant and rehydrate the centrifuged bacterial cells with 25 mL of distilled water. Disrupt the bacterial cells using an ultrasonic homogenizer for 30 min, with 3-second intervals between each disruption, at 45% power.

[0091] 4. Collect the crude enzyme solution

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

[0093] 5. Determine enzyme activity

[0094] Add 200 μL of ABTS, 1.8 mL of pH 4 acetate-sodium acetate buffer, and 1 mL of crude enzyme solution to a reaction tube, for a total reaction system of 3 mL. Measure the absorbance change at 420 nm for 5 minutes. Use a mixture containing the inactivated enzyme solution as a control (1 mL of crude enzyme solution was heated in a boiling water bath for 5 minutes). Measure the absorbance change of bacteria 5 and calculate the enzyme activity. One unit of enzyme activity is defined as the amount of enzyme required to hydrolyze 1 μmol of 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 enzyme solution volume, respectively, and ε is the absorbance coefficient. The molar extinction coefficient of ABTS is ε420=36mL / (μmol·cm). If dilution is required, it needs to be multiplied 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 of ABTS, 1.7 mL of pH 4 acetate-sodium acetate buffer, and 1 mL of crude enzyme solution to the reaction tube. Add 100 μL of copper sulfate solution with a concentration of 0.0025% (increased by 2 times) to each of the two solutions, for a total of 3 mL of reaction system. Measure the absorbance change at 420 nm using a UV-Vis spectrophotometer and calculate the enzyme activity.

[0099] 2. The effect of temperature on enzyme activity

[0100] The prepared ABTS solution, pH 4 acetate-sodium acetate buffer, and crude enzyme solution were incubated in constant temperature water baths at 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, respectively. 200 μL of ABTS, 1.8 mL of pH 4 acetate-sodium acetate buffer, and 1 mL of crude enzyme solution were added to each reaction tube, for a total reaction system of 3 mL. The absorbance change was measured at 420 nm using a UV-Vis spectrophotometer, and the enzyme activity was calculated.

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

[0102] The crude enzyme solution was treated at 50℃, 60℃, 70℃, and 80℃ for one hour, respectively. 200 μL of LABTS, 1.8 mL of pH 4 acetate-sodium acetate buffer, and 1 mL of the pre-treated crude enzyme solution were added to the reaction tube, for a total reaction system of 3 mL. The absorbance change was measured at 420 nm using a UV-Vis spectrophotometer, and the enzyme activity was calculated.

[0103] 4. The effect of pH on enzyme activity

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

[0105] II. Results and Analysis

[0106] 2.1 Appearance and Identification of Fungal Strains

[0107] After initial screening, the inventors obtained three strains with laccase-producing activity. Among them, strain ZH-5 appeared as a white, opaque solid medium supplemented with guaiacol, with a distinct reddish-brown oxidation zone in the center. The strain was round or oval with regular edges and a moist, smooth surface. Colorimetric analysis of guaiacol-supplemented plates showed that strain ZH-5 had the highest laccase activity. Therefore, this strain was selected for further research. After amplification and sequencing of the 16S rDNA gene, the 16S rDNA gene sequence was obtained. BLAST sequence alignment and physiological and biochemical analysis confirmed that strain ZH-5 is *Serratia marcescens*.

[0108] 2.2 Enzyme activity assay of Serratia marcescens

[0109] Serratia marcescens, which showed the most obvious color development on agar plates, was selected and cultured in LB liquid medium for 3 days. Laccase activity was then measured, and the enzyme activity of Serratia marcescens was found to be 1483.3 U / L.

[0110] 2.3 Enzyme Activity Analysis of Serratia marcescens

[0111] Using ABTS as the reaction substrate, the absorbance changes were measured spectrophotometrically by adding different concentrations of copper ions. The results are as follows: Figure 2 As shown in the figure. Analysis of the results shows that when the copper ion concentration is between 0.0025% and 0.01%, the enzyme activity gradually increases with increasing copper ion concentration; when the copper ion concentration is between 0.01% and 0.08%, the enzyme activity gradually decreases with increasing copper ion concentration; and the enzyme activity reaches its maximum value at a copper ion concentration of 0.01%. Therefore, it is determined that 0.01% copper ion concentration is the optimal concentration for Serratia marcescens laccase.

[0112] Using ABTS as the reaction substrate, different reaction temperatures were set, and the absorbance changes were measured spectrophotometrically. The results are as follows: Figure 3 As shown in the figure. Analysis of the results shows that enzyme activity gradually increases with increasing temperature between 25℃ and 30℃; enzyme activity reaches its maximum value at 30℃; and enzyme activity gradually decreases with increasing temperature between 30℃ and 50℃. Therefore, it is determined that the optimal operating temperature for 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. Then, using ABTS as the reaction substrate, the absorbance change was measured spectrophotometrically after the reaction, and the laccase activity was calculated. The results are as follows: Figure 4 As shown, after treatment at 50℃ for 1 hour, the enzyme activity loss was only 57.7%. After treatment at 80℃ for 1 hour, 26.9% of the enzyme activity was still retained. This indicates that the laccase of this bacterium has good stability.

[0114] Using ABTS as the reaction substrate, the absorbance change was measured spectrophotometrically by controlling the pH of the buffer solution. The results are as follows: Figure 5 As shown in the figure. Analysis of the results shows that enzyme activity gradually increases between pH 2.5 and 3.0; the enzyme activity reaches its maximum at pH 3.0; therefore, it is determined that the optimal pH for the activity of *Serratia marcescens* is 3.0. This further broadens its application areas and scope.

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

[0116] This embodiment relates to the characterization of the acid, alkali, salt, and high-temperature resistance properties of Serratia marcescens ZH-5, and the specific methods are as follows:

[0117] (1) Serratia marcescens ZH-5 bacterial suspension was inoculated into LB liquid medium at an inoculation ratio of 1% (v / v) and cultured at 30℃ and 200 rpm for 24 h. The growth of each strain under different pH conditions (3.0-10.0) 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 values

[0119]

[0120] Where "-" indicates that bacteria basically do not grow (OD of bacterial solution). 600 <0.2), "+" indicates bacterial growth, and the OD of the bacterial solution is... 600 The value is 0.2–0.5, where “++” represents bacterial growth, and OD… 600 The value is 0.5–1.0, with “+++” indicating bacterial growth and the bacterial culture OD value being [missing value]. 600 It is greater than 1.0.

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

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

[0123]

[0124] Where "-" indicates that bacteria basically do not grow (OD of bacterial solution). 600 <0.2), "+" indicates bacterial growth, and the OD of the bacterial solution is...600 The value is 0.2–0.5, where “++” represents bacterial growth, and OD… 600 The value is 0.5–1.0, with “+++” indicating bacterial growth and the bacterial culture OD value being [missing value]. 600 It is greater than 1.0.

[0125] (3) Serratia marcescens ZH-5 bacterial suspension was inoculated into LB liquid medium at an inoculation ratio of 1% (v / v). After incubation at 30℃ and 200rpm for 24h, the bacterial suspensions of each strain were analyzed under different Cd conditions. 2+ Growth status at (CdSO4·8 / 3H2O) concentration. The results are shown in Table 4. The results indicate that ZH-5 has excellent heavy metal tolerance, even when Cd... 2+ It can still grow even when the concentration reaches 350 mg / L.

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

[0127]

[0128] Where "-" indicates that bacteria basically do not grow (OD of bacterial solution). 600 <0.2), "+" indicates bacterial growth, and the OD of the bacterial solution is... 600 The value is 0.2–0.5, where “++” represents bacterial growth, and OD… 600 The value is 0.5–1.0, with “+++” indicating bacterial growth and the bacterial culture OD value being [missing value]. 600 It is greater than 1.0.

[0129] (4) Serratia marcescens ZH-5 bacterial suspension was inoculated into 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℃) was analyzed. The results are shown in Table 5. The strain can grow in the above temperature range.

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

[0131]

[0132] Wherein, "+" represents bacterial growth, and the bacterial solution OD 600 The value is 0.2–0.5, where “++” represents bacterial growth, and OD… 600 The value is 0.5–1.0, with “+++” indicating bacterial growth and the bacterial culture OD value being [missing value]. 600 It is greater than 1.0.

[0133] Example 3: Optimization of Laccase Activity

[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 for determining laccase activity typically uses sodium acetate solution as a buffer solution, with a final concentration in the reaction system usually of 0.02, 0.05, or 0.10 mol / L, and a pH mainly between 3 and 5. A commonly used final concentration of ABTS in the reaction system is 0.5 mmol / L. The reaction is generally carried out at room temperature. After laccase action, ABTS forms ABTS radicals. At 420 nm, the absorbance coefficient of ABTS radicals is much greater than that of the substrate ABTS. As the concentration of ABTS radicals increases, the absorbance value increases; therefore, changes in the OD value are detected at 420 nm.

[0135] Reagents: 0.1 mmol / L acetate-sodium acetate buffer, crude enzyme solution, 1 mmol / L ABTS solution, 100 mmol / 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 solution containing 1 mM Cu 2+ In LB, the fermentation broth is initially OD 600 Laccase activity was measured after culturing at 0.1 g / L, 30°C, and 200 rpm for 6 days as a control group. OD after 6 days... 600 It is 2.23.

[0138] The reaction system was prepared as follows: pH=3 buffer solution 1.8 mL, crude enzyme solution 1.0 mL. The optimal Cu was determined using a control group. 2+ Concentration and ABTS volume. 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] The final determination of the Cu reaction system in the experimental group 2+ The concentration was 1.5 mM, and the volume of ABTS was 0.40 mL.

[0145] Serratia marcescens ZH-5 was inoculated into a solution containing 1 mM Cu 2+ In LB, the fermentation broth is initially OD 600The value was 0.1. Glucose and glycerol at concentrations of 5 g / L, 10 g / L, 15 g / L, and 20 g / L were added as carbon sources, and the mixture was incubated at 30℃ and 200 rpm for 6 days before laccase activity was measured. 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 solution containing 1 mM Cu 2+ In LB, the fermentation broth is initially OD 600 The value was 0.1. Glycerol and glucose (15 g / L) were added as carbon sources, and the mixture was incubated at 37℃ and 200 rpm for 6 days before laccase activity was measured. The results are shown in Table 10.

[0151] Table 10 Enzyme activity under different culture temperatures

[0152]

[0153] Experiments show that enzyme production efficiency is not high when cultured at 37℃. Based on previous work, 30℃ is the appropriate temperature for cultivating laccase produced by ZH-5 bacteria.

[0154] Serratia marcescens ZH-5 was inoculated into a solution containing 1.5 mM Cu. 2+ In LB, the fermentation broth is initially OD 600 The control group was culturing at 0.1, 30℃, and 200rpm for 6 days. The reaction system for further optimizing enzyme activity was explored using 1.8 mL of buffer solution and 1.0 mL of crude enzyme solution to determine the ABTS volume. 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 solution containing 1.5 mM Cu. 2+ In LB, the fermentation broth is initially OD 600 The value was 0.1. Glucose and glycerol at concentrations of 5 g / L, 10 g / L, 15 g / L, and 20 g / L were added as carbon sources, and the mixture was incubated at 30°C and 200 rpm for 6 days. The buffer solution was 2.0 mL, the crude enzyme solution was 0.5 mL, and Cu... 2+ The concentration was 1.5 mM, and the volume of ABTS was 0.8 mL. The results are shown in Tables 12 and 13.

[0158] Table 12 Enzyme activity at different glucose concentrations

[0159]

[0160] Table 13 Enzyme activity at different glycerol concentrations

[0161]

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

[0163] Example 4: Decolorization treatment of industrial dyeing and printing wastewater using laccase

[0164] Indigo, indigo red, and crystal violet were used as research subjects to study the decolorization of dyes by bacterial laccase. The decolorization system specifically consisted of 3 mL each of 50 mg / L indigo, indigo red, and crystal violet solutions, 0.2 mL of the crude enzyme solution prepared in Example 1, and Cu... 2+ Decolorization was performed by reacting 1.5 mM CuSO4 in a 30°C water bath for 1 hour. The changes in absorbance before and after the reaction were measured at wavelengths of 680 nm, 605 nm, and 590 nm, and the decolorization rate of the dyes was calculated. The decolorization rates were 93.2% for indigo, 87.6% for indigo red, and 81.5% for crystal violet. This indicates that the treatment has good decolorization capabilities for industrial dyeing and printing wastewater.

[0165] Finally, it should be noted that the above description is merely 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A strain of Serratia marcescens ( Serratia marcescens ZH-5, this strain was deposited at the China General Microbiological Culture Collection Center on June 3, 2024, with the biological accession number CGMCC No. 30840.

2. The fermentation production method of Serratia marcescens ZH-5 according to claim 1, characterized in that, The fermentation production method includes: inoculating Serratia marcescens ZH-5 into a fermentation medium and carrying out fermentation culture to obtain the product.

3. A microbial inoculant, characterized in that, It contains Serratia marcescens ZH-5 as described in claim 1.

4. The use of Serratia marcescens ZH-5 as described in claim 1 or the microbial agent as described in claim 3 in the preparation of laccase.

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

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

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

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

9. The preparation method according to claim 6, characterized in that, The fermentation conditions included culturing at 30°C and 200 rpm for 6 days, with an acidic fermentation pH.

10. The preparation method according to claim 9, characterized in that, The fermentation pH is 3.

11. The *Serratia marcescens* strain according to claim 1 (… Serratia marcescens The application of ZH-5, the microbial agent of claim 3, or the preparation method of any one of claims 5-10 in the treatment of industrial dyeing and printing wastewater.

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