A shrimp shell biomass charcoal prepared by using a composite preparation, and a preparation method and application thereof

Abstract

The present application provides a kind of shrimp shell biomass charcoal prepared by composite preparation and its preparation method and application, the present application is first prepared and obtained the composite preparation containing chitinase, bacillus licheniformis and bacillus subtilis, the chitinase is the chitinase derived from hartz wood enzyme is mutated, constructs mutant library, with trichoderma reesei as expression host, by screening, the excellent mutant Chi M1 obtained, its chitinase activity is significantly improved, is 2 times of original gene. By spraying the fermentation broth of the recombinant bacteria of the present application to obtain enzyme spray powder and the compounding of bacillus licheniformis powder and bacillus subtilis powder, the degradation efficiency of procambarus clarkia shrimp shell can be effectively improved. The composite preparation containing ChiM1 enzyme spray powder has good application prospect in the fields of aquaculture, feed additive and biomass resource utilization.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal biological agents and application technology, specifically relating to a shrimp shell biochar prepared using a compound agent, its preparation method, and its application. Background Technology

[0002] Chitin, also known as chitin, is a high-molecular-weight carbohydrate formed by N-acetyl-D-glucosamine linked by β-1,4-glycosidic bonds. These compounds cross-link with some proteins, phenols, lipids, or other carbohydrates, such as β-glucan, to form a highly ordered polymeric scaffold, constituting the shells of shrimp, crabs, insects, and fungi. When the degree of deacetylation exceeds 40%, it can be converted into chitosan, which is the most abundant biomass resource in the ocean and the second largest biomass resource in nature after cellulose. With the rapid development of aquaculture, the seafood processing industry generates a large amount of waste every year, especially the shells of crustaceans. This waste is usually dumped into landfills or the ocean, causing serious environmental pollution and resource waste.

[0003] The degradation products of chitin and its analogues, namely amino oligosaccharides, possess superior physiological functions and biological activities, and have wide applications in agriculture, medicine, and food. The main processing methods for chitin and other shrimp shells include chemical, physical, and enzymatic methods. Among these, enzymatic methods have become a research hotspot due to their advantages such as mild reaction conditions, single product, and environmental friendliness.

[0004] Chitinases (EC 3.2.1.14) are a class of glycosyl hydrolases that hydrolyze the β-1,4-glycosidic bonds of chitin to produce chitin oligosaccharides or monosaccharides. Most chitinases belong to the glycosyl hydrolases families 18 and 19, with a few belonging to families 20, 23, and 48. Based on their origin, chitinases can be classified into animal, plant, and microbial sources. Microbial chitinases, in particular, possess a wide operating pH range and high catalytic activity, making them more suitable for industrial applications. However, naturally occurring chitinases generally exhibit low enzyme activity; therefore, developing a chitinase with high activity is an urgent problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a shrimp shell biochar prepared using a composite formulation, its preparation method, and its application. Specifically, this invention first mutates chitinase derived from *Trichoderma harzianum*, using *Trichoderma reesei* as the expression host, and obtains a chitinase mutant ChiM1 with enhanced enzyme activity through screening. Recombinant bacteria are then used for fermentation. Furthermore, a composite formulation consisting of enzyme-sprayed dry powder from the fermentation broth of mutant ChiM1, *Bacillus licheniformis* powder, and *Bacillus subtilis* powder is provided. This formulation can effectively improve the degradation efficiency of shrimp shell powder and other wastes, and can be used to degrade drug residues in water.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] This invention provides a composite formulation for preparing shrimp shell biochar, the composite formulation comprising chitinase, Bacillus licheniformis and Bacillus subtilis, wherein the chitinase is a chitinase mutant ChiM1, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0008] This invention provides a chitinase mutant ChiM1, whose amino acid sequence is shown in SEQ ID NO: 3. ChiM1 is obtained by changing the amino acid at position 152 (lysine to glutamic acid), position 171 (glutamic acid to alanine), position 183 (isoleucine to valine), position 261 (asparagine to aspartic acid), and position 390 (leucine to tyrosine) of the chitinase with the amino acid sequence SEQ ID NO: 1.

[0009] The present invention provides the encoding gene of the chitinase mutant ChiM1, the nucleotide sequence of which is shown in SEQ ID NO: 4.

[0010] The present invention also provides an expression vector containing the chitinase mutant ChiM1 encoding gene, wherein the vector is pCDH, pCBH, or pPIC9K.

[0011] The present invention provides a genetically engineered bacterium containing the chitinase mutant ChiM1 encoding gene, wherein the genetically engineered bacterium is Bacillus subtilis, Trichoderma reesei, or Pichia pastoris.

[0012] This invention also provides a method for preparing shrimp shell biochar using the aforementioned composite formulation, the method comprising the following steps:

[0013] (1) Shrimp shell cleaning and drying: The shrimp shells are washed multiple times with clean water, detergent and deionized water to remove impurities. The washed shrimp shells are dried and cooled to room temperature.

[0014] (2) Crushing: The dried shrimp shells are crushed using a crusher and then passed through a 40-80 mesh sieve;

[0015] (3) Add the compound preparation to the shrimp shell powder at a mass ratio of 0.1%-2%, add water and mix thoroughly, ferment, and after fermentation, dissolve and filter.

[0016] (4) The solid obtained in step (3) is placed in a quartz boat and heated in a tube furnace at 700°C for 2 hours under nitrogen protection. After cooling to room temperature, the shrimp shell biochar is obtained by sieving.

[0017] Furthermore, the compound preparation contains ChiM1 enzyme spray dry powder, Bacillus licheniformis powder, and Bacillus subtilis powder in a mass ratio of 5:3:2. The ChiM1 enzyme spray dry powder has an enzyme activity greater than 2000 U / g, the Bacillus licheniformis powder has a bacterial content greater than 10 billion CFU / g, and the Bacillus subtilis powder has a bacterial content greater than 10 billion CFU / g.

[0018] Furthermore, the fermentation medium formula for the ChiM1 mutant recombinant bacteria is as follows: soybean meal 1-3%, neutral protease 1000U / g, glucose 1-3%, ammonium sulfate 0.1-0.5%, magnesium sulfate 0.01-0.1%, calcium chloride 0.01-0.1%, potassium dihydrogen phosphate 0.2-0.8%, Tween 80 0.02-0.05%, and corn steep liquor 3-10%.

[0019] This invention also provides the application of the shrimp shell biochar in removing drug residues from aquaculture water.

[0020] Furthermore, the drugs include florfenicol, enrofloxacin, doxycycline hydrochloride powder, and sulfadiazine.

[0021] Compared with existing technologies, the advantages and technical effects of this invention are as follows: This invention mutates and improves chitinase derived from harzianum enzyme, obtaining a mutant ChiM1 with significantly enhanced enzyme activity through screening. This mutant is twice the size of the original gene, with mutation sites K152E / E171A / I183V / N261D / L390Y. The recombinant ChiM1 bacteria are fermented to obtain a fermentation broth, which is then spray-dried to obtain ChiM1 enzyme spray-dried powder. This ChiM1 enzyme spray-dried powder, along with Bacillus licheniformis powder and Bacillus subtilis powder, are compounded at a mass ratio of 5:3:2 to obtain a composite preparation. This preparation can effectively improve the degradation efficiency of waste such as shrimp shell powder and has promising application prospects in aquaculture, feed additives, and biomass resource utilization. Attached Figure Description

[0022] Figure 1 This is the result of the amplification of the chitinase gene;

[0023] Figure 2 The activity of the chitinase mutant is described.

[0024] Figure 3 The chitinase mutant was fermented in 30L using Trichoderma reesei as the host.

[0025] Figure 4 SDS-PAGE results of concentrated fermentation broth of ChiM1 chitinase;

[0026] Figure 5The effect of chitinase mutant fermentation broth enzyme spray dry powder, combined with Bacillus licheniformis powder and Bacillus subtilis powder, on the degradation of shrimp shell powder.

[0027] Figure 6 This is an electron microscope image of the shrimp shell biochar prepared by the present invention;

[0028] Figure 7 This is the adsorption result of enrofloxacin and doxycycline hydrochloride on the shrimp shell biochar prepared by the present invention.

[0029] Figure 8 This is a comparison of the removal rates of commonly used antibiotics by the shrimp shell biochar prepared in this invention. Detailed Implementation

[0030] To facilitate understanding of the present invention, the invention will be described more fully and in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0031] Molecular biology experimental methods not specifically described in the following examples can be performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions. Unless otherwise specified, the reagents and biological materials used in the specific examples are commercially available.

[0032] 1. Strains and vectors

[0033] Trichoderma reesei QP4 (uracil-deficient), plasmid pCBH, plasmid pET-21a(+), Escherichia coli BL21, etc., were purchased from Invitrogen.

[0034] 2. Reagents and Culture Media

[0035] Plasmid extraction kit, fragment purification and recovery kit, restriction endonucleases, etc. were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; ampicillin, kanamycin, uracil, etc. were purchased from Sangon Biotech (Shanghai) Co., Ltd.; protein marker: Blue Plus II Protein Marker (14-120 kDa) was purchased from Beijing TransGen Biotech Co., Ltd.

[0036] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl;

[0037] Trichoderma reesei transformation medium (g / L):

[0038] Upper layer of conversion: 10g glucose, 1g MgSO4·7H2O, 10g KH2PO4, 6g (NH4)2SO4, 3g sodium citrate, 0.005g FeSO4·7H2O, 0.0016g MnSO4·H2O, 0.0014g ZnSO4·7H2O, 0.002g CoCl2·2H2O, 182.18g sorbitol, 6g agarose. Sterilize at 115℃ for 30 min.

[0039] Lower layer of conversion: 10g glucose, 1g MgSO4·7H2O, 10g KH2PO4, 6g (NH4)2SO4, 3g sodium citrate, 0.005g FeSO4·7H2O, 0.0016g MnSO4·H2O, 0.0014g ZnSO4·7H2O, 0.002g CoCl2·2H2O, 12g agar powder, sterilized at 115℃ for 30 min.

[0040] All of the above culture media require the addition of 10 mM uracil when culturing uracil-deficient genotypes.

[0041] PDA medium: 20% potato extract, 2% glucose.

[0042] MM medium: Glucose 20g, MgSO4·7H2O 0.6g, CaC12 0.6g, KH2PO4 15g, (NH4)2SO45g, Peptone 2g, FeSO4·7H2O 0.005g, MnSO4·H2O 0.0016g, ZnSO4·7H2O 0.0014g, CoC12·2H2O 0.002g.

[0043] Transformation-related reagents:

[0044] Solution 1 (200mL): sorbitol 43.72g, potassium dihydrogen phosphate 2.72g, pH adjusted to 5.5 with sodium hydroxide solution.

[0045] Solution 2 (100 mL): 18.22 g sorbitol, 0.735 g calcium chloride dihydrate, 10 mM Tris-HCl, adjust pH to 7.48.

[0046] Solution 3 (100mL): PEG6000 25g, calcium chloride dihydrate 0.735g, 10mM Tris-HCl adjusted to pH 7.5.

[0047] Fermentation medium: soybean meal 1-3%, neutral protease 1000U / g, glucose 1-3%, ammonium sulfate 0.1-0.5%, magnesium sulfate 0.01-0.1%, calcium chloride 0.01-0.1%, potassium dihydrogen phosphate 0.2-0.8%, Tween 80 0.02-0.05%, corn steep liquor 3-10%.

[0048] 3. Chitinase activity was determined by spectrophotometry, referring to GB / T34799-2017 "Chitinase Activity Determination Method".

[0049] 4. Transformation methods of Trichoderma reesei

[0050] (1) Experimental preparation: PDA plates, sterile physiological saline (0.9% sodium chloride, 0.05% Tween-20), funnel with four layers of lens paper or filter paper (sterilized), cellophane. Cut the cellophane to the size of the petri dish, place a filter paper in the middle of each layer, place it in the petri dish, and sterilize.

[0051] (2) Culture of the fungus: Take QP4 spores on a PDA (containing uracil) plate and culture at 30°C until conidia appear; take fresh spores, add 5 ml of sterile physiological saline, wash off the spores to obtain a spore suspension; add a layer of cellophane on the PDA (containing uracil) plate and spread it evenly with a spreading stick, add 100 μl of spore suspension on the cellophane, spread it evenly, and culture at 30°C until a lot of hyphae grow.

[0052] (3) Protoplast preparation and transformation: Take a clean petri dish, add 0.1 g of lysin, then add 20 mL of solution 1 and mix well; wash the cultured hyphae into a plate containing lysin and lyse at 30 °C for 90 minutes; after lysis, filter the hyphae with a funnel containing filter paper (lens paper), and collect the protoplasts by centrifugation; resuspend in 4 mL of solution 2, centrifuge at 2500 rpm at 4 °C for 10 min, and discard the supernatant; after resuspending in solution 2, dispense into 200 μL tubes, add 15 μL of transformation plasmid to each tube, and incubate on ice for 5 minutes; then add 2 mL of solution 3 and 4 mL of solution 2 respectively, add 30 mL of transformation supernatant, and transfer to a plate; incubate at 30 °C until transformants grow.

[0053] Example 1: Construction of chitinase mutant gene and verification of its expression in Trichoderma reesei

[0054] After preliminary screening and verification, a harzianum enzyme with high chitinase activity was obtained. The sequence was translated into the corresponding nucleotide sequence and then optimized by gene sequence before being artificially synthesized. Its amino acid sequence is shown in SEQ ID NO: 1 and its corresponding nucleotide sequence is shown in SEQ ID NO: 2.

[0055] The primers are designed as follows: an Nco I restriction site is designed at the 5' end, and a Sac I restriction site is designed at the 3' end.

[0056] Using the GeneMorph II random mutagenesis PCR kit, a synthetically produced gene was randomly mutated. The primer sequences used are as follows, and the PCR amplification results are shown below. Figure 1 As shown, the Nco I and Sac I sites of plasmid pCBH were cloned, and the recombinant plasmid was transformed into *Trichoderma reesei* QP4 according to the above genetic transformation method to obtain recombinant bacteria. The fermentation medium consisted of: soybean meal 1-3%, neutral protease 1000 U / g, glucose 1-3%, ammonium sulfate 0.1-0.5%, magnesium sulfate 0.01-0.1%, calcium chloride 0.01-0.1%, potassium dihydrogen phosphate 0.2-0.8%, Tween 80 0.02-0.05%, and corn steep liquor 3-10%. Fermentation was carried out in shake flasks at 30℃ for 4-5 days, and the culture medium was centrifuged to obtain the supernatant for later use.

[0057] Chi-F1: CATGCCATGGATGCTCAGCTTCCTCGGCAA (SEQ ID NO: 5)

[0058] Chi-R1: CGAGCTCCTAGTTGAGGCCGTTCTTAATG (SEQ ID NO: 6).

[0059] Following the above-described method, the supernatant after centrifugation of the fermentation broth was taken, and the enzyme activity in the supernatant of each mutant was measured. After repeated verification, five mutants with enzyme activities higher than the control group were screened out. Figure 2 As shown, the recombinant bacteria with the highest enzyme activity were selected for gene amplification and sequencing analysis. After sequencing, the chitinase mutant ChiM1 was obtained, and its amino acid sequence is shown in SEQ ID NO: 3, and its corresponding nucleotide sequence is shown in SEQ ID NO: 4. The enzyme activity of ChiM1 is approximately twice that of the original gene.

[0060] SEQ ID NO: 3

[0061] MLSFLGKSVALLAALQATLSSPKPGHRRASVEKRANGYANSVYFTNWGIYDRNFQPADLV ASDVTHVIYSFMNLQADGTVISGDTYADYEKHYADDSWNDVGTNAYGCVKQLFKVKKANR GLKVLLSIGGWTWSTNFPSAASTDANRKNFAETAITFMKDWGFDGIDIDWAYPADATQAS NMVLLLKEVRSQLDAYAAQYAPGYHFLLTIAAPAGKDNYSKLRLADLGQVLDYINLMAYD YAGSFSPLTGHDANLFNNPSDPNATPFNTDSAVKDYINGGVPANKIVLGMPIYGRSFQNT AGIGQTYNGVGSGSWEAGIWDYKALPKAGATVQYDSVAKGYYSYNSATKELISFDTPDMI NTKVAYLKSLGLGGSMFWEASADKKGADSYIGTSHRALGGLDTTQNLLSYPNSKYDNIKNGLN

[0062] SEQ ID NO:4

[0063] ATGCTCAGCTTCCTCGGCAAGAGCGTCGCCCTCCTCGCCGCCCTCCAGGCCACCCTCAGC

[0064] AGCCCGAAGCCGGGCCACCGCCGCGCCAGCGTCGAGAAGCGCGCCAACGGCTACGCCAAC

[0065] AGCGTCTACTTCACCAACTGGGGCATTTACGACCGCAACTTCCAGCCGGCCGACCTCGTC

[0066] GCCAGCGACGTCACCCACGTCATTTACAGCTTCATGAACCTCCAGGCCGACGGCACCGTC

[0067] ATTAGCGGCGACACCTACGCCGACTACGAGAAGCACTACGCCGACGACAGCTGGAACGAC

[0068] GTCGGCACCAACGCCTACGGCACTGTCAAGCAGCTCTTCAAGGTCAAGAAGGCCAACCGC

[0069] GGCCTCAAGGTCCTCCTCAGCATTGGCGGCTGGACCTGGAGCACCAACTTCCCGAGCGCC

[0070] GCCAGCACCGACGCCAACCGCAAGAACTTCGCCGAGACCGCCATTACCTTCATGAAGGAC

[0071] TGGGGCTTCGACGGCATTGACATTGACTGGGCCTACCCGGCCGACGCCACCCAGGCCAGC

[0072] AACATGGTCCTCCTCCTCAAGGAGGTCCGCAGCCAGCTCGACGCCTACGCCGCCCAGTAC

[0073] GCCCCGGGCTACCACTTCCTCCTCACCATTGCCGCCCCGGCCGGCAAGGACAACTACAGC

[0074] AAGCTCCGCCTCGCCGACCTCGGCCAGGTCCTCGACTACATTAACCTCATGGCCTACGAC

[0075] TACGCCGGCAGCTTCAGCCCGCTCACCGGCCACGACGCCAACCTCTTCAACAACCCGAGC

[0076] GACCCGAACGCCACCCCGTTCAACACCGACAGCGCCGTCAAGGACTACATTAACGGCGGC

[0077] GTCCCGGCCAACAAGATTGTCCTCGGCATGCCGATTTACGGCCGCAGCTTCCAGAACACC

[0078] GCCGGCATTGGCCAGACCTACAACGGCGTCGGCAGCGGCAGCTGGGAGGCCGGCATTTGG

[0079] GACTACAAGGCCCTCCCGAAGGCCGGCGCCACCGTCCAGTACGACAGCGTCGCCAAGGGC

[0080] TACTACAGCTACAACAGCGCCACCAAGGAGCTCATTAGCTTCGACACCCCGGACATGATT

[0081] AACACCAAGGTCGCCTACCTCAAGAGCCTCGGCCTCGGCGGCAGCATGTTCTGGGAGGCC

[0082] AGCGCCGACAAGAAGGGCGCCGACAGCTACATTGGCACCAGCCACCGCGCCCTCGGCGGC

[0083] CTCGACACCACCCAGAACCTCCTCAGCTACCCGAACAGCAAGTACGACAACATTAAGAAC

[0084] GGCCTCAACTAG

[0085] Example 2: Fermentation and preparation of ChiM1 recombinant strain in a 30L fermenter

[0086] The genetically engineered ChiM1 recombinant bacteria expressed in the above examples were streaked onto PDA plates and cultured at 30°C until spores appeared. After approximately 7 days of culture, the spores were washed off with sterile water for single-spore isolation. This involved diluting the spores and spreading them onto MM (containing Triton-X100) medium until colonies appeared but then fell off. The culture was then repeated to produce more spores. This process was repeated three times to obtain a pure culture. The spores from the pure culture were washed off with 100 mL of sterile water and used as seed culture for inoculating fermenters.

[0087] Fermentation process: Soybean meal 1-3%, neutral protease 1000U / g, glucose 1-3%, ammonium sulfate 0.1-0.5%, magnesium sulfate 0.01-0.1%, calcium chloride 0.01-0.1%, potassium dihydrogen phosphate 0.2-0.8%, Tween 80 0.02-0.05%, corn steep liquor 3-10%. pH is natural, temperature 30℃, stirring speed 600rpm, aeration rate 1.5 (v / v), dissolved oxygen controlled above 20%. Fermentation is carried out at a natural pH. Enzyme activity is measured after 24 hours of fermentation. After fermentation is complete (generally 48 hours), the fermentation broth is processed through a plate and frame filter press to obtain crude enzyme solution, which is then spray-dried into powder for application testing.

[0088] Fermentation process curve as shown Figure 3 As shown: Samples were taken every 8 hours to measure enzyme production levels, and enzyme activity levels reached their peak after 60 hours of fermentation.

[0089] Example 3: SDS-PAGE detection of ChiM1 chitinase expression

[0090] After centrifuging and filtering the fermentation broth from Example 2, the supernatant was collected and then passed through a protein purification column to filter out protein and other molecular components below 10KD. The fermentation broth was further concentrated and stored at -20 degrees Celsius.

[0091] Electrophoresis: 100 μL of the concentrated fermentation enzyme sample was added to 100 μL of Loading Buffer, boiled for 10 min, and 20 μL was loaded onto the sample.

[0092] Electrophoresis conditions: For stacking gel, voltage 80-90 mV and current around 30 mA; for separating gel, voltage 110-120 mV and current around 40 mA. Stop electrophoresis when the sample tip is 1 cm from the edge of the gel. Peel the gel, stain for 2 hours, and destain for 24 hours, changing the destaining solution during this period.

[0093] The test results are as follows Figure 4 As shown, the expression level is basically consistent with the theoretical molecular weight of 46KD, and there is a relatively concentrated protease band, indicating that the expression level of ChiM1 chitinase is high, which is convenient for subsequent testing and application.

[0094] Example 4: Application Experiment of ChiM1 Recombinant Bacterial Fermentation Broth and Compound Formulation of Bacillus licheniformis and Bacillus subtilis

[0095] 1. Compound preparation: After centrifuging the ChiM1 recombinant bacterial fermentation broth, obtain the supernatant and spray dry to obtain ChiM1 enzyme spray dry powder (enzyme activity greater than 2000 U / g). Add 50% ChiM1 enzyme spray dry powder, 30% Bacillus licheniformis (bacterial content greater than 10 billion CFU / g) and 20% Bacillus subtilis powder (bacterial content greater than 10 billion CFU / g) according to the following mass ratios. When using, add at a dosage of 0.1-2% of the shrimp shell powder mass ratio.

[0096] 2. Compound preparation of degraded shrimp shell powder

[0097] Experimental group: 200g of shrimp shell powder (passed through an 80-mesh sieve) was placed in a sealed bag. The compound preparation was added to the shrimp shell powder at a dosage of 1%, i.e., 2g of the compound preparation was weighed, dissolved in 40mL of deionized water, and then mixed into the shrimp shell powder until thoroughly combined. The mixture was then placed in a 37℃ incubator for anaerobic fermentation for 3 days. After fermentation, the solids were dissolved and filtered, and the remaining solids were dried and weighed.

[0098] Control group: Take 200g of shrimp shell powder (passed through an 80-mesh sieve) in a sealed bag, add 40mL of deionized water directly, mix thoroughly. Place in a 37℃ incubator for anaerobic fermentation for 3 days. After fermentation, dissolve and filter, dry the remaining solids, and weigh.

[0099] After repeated verification, the results are as follows: Figure 5As shown, the degradation rate of shrimp shell powder in the experimental group was 45.5%, while the degradation rate in the control group was only 15%. This indicates that adding a compound preparation containing ChiM1 chitinase, Bacillus licheniformis, and Bacillus subtilis to shrimp shell powder can increase the degradation rate by more than 2 times, significantly improving the decomposition of shrimp shells containing a large amount of chitin and promoting the recycling of biomass resources.

[0100] Example 5: Preparation of biochar from the shells of Procambarus clarkii

[0101] 1. The method for preparing shrimp shell biochar by degrading the shell of Procambarus clarkii using the aforementioned composite agent includes the following steps:

[0102] (1) Cleaning and drying of shrimp shells: The shells of the red swamp crayfish are cleaned multiple times with water, detergent and deionized water to remove impurities (residual oil, remaining meat). The cleaned shrimp shells are dried in an oven at 105°C for about 3 hours and then cooled to room temperature.

[0103] (2) Crushing: The dried shrimp shells are crushed using a crusher and then passed through an 80-mesh sieve;

[0104] (3) Take 200g of shrimp shell powder (passed through an 80-mesh sieve) into a sealed bag. Add the compound preparation to the shrimp shell powder according to the addition amount of 1%, that is, weigh 2g of the compound preparation, dissolve it in 40mL of deionized water, and then mix it into the shrimp shell powder. Mix thoroughly. Place it in a 37℃ incubator for anaerobic fermentation for 3 days. After fermentation, dissolve and filter it.

[0105] (4) The solid obtained in step (3) is placed in a quartz boat and heated in a tube furnace at 700°C for 2 hours under nitrogen protection. After cooling to room temperature, it is passed through an 80-mesh sieve to obtain shrimp shell biochar.

[0106] 2. Characterization of shrimp shell biochar

[0107] After the shrimp shell biochar production was completed, the elemental composition of the shrimp shell biochar was measured using X-ray photoelectron spectroscopy (XPS). The specific surface area of ​​the adsorbent was determined using the Brunauer-Emmett-Teller (BET) method. The surface morphology of the samples was scanned using scanning electron microscopy (SEM). SEM images of the shrimp shell biochar are shown below. Figure 6 As shown in Table 1, the surface functional groups of shrimp shell biochar were analyzed by Fourier transform infrared spectroscopy (FT-IR). The elemental composition and specific surface area of ​​shrimp shell biochar are shown in Table 1.

[0108] Table 1. Elemental composition and specific surface area of ​​crayfish shell biochar

[0109]

[0110] Example 6: Application of Procambarus clarkii shell biochar in removing residual drugs from aquaculture water

[0111] In aquaculture, antibiotics are often mixed into feed, but excessive drugs can cause serious water pollution. The shrimp shell biochar prepared in Example 5 is used to improve the adsorption efficiency of residual drugs.

[0112] 1. Adsorption experiments of shrimp shell biochar on enrofloxacin and doxytetracycline hydrochloride:

[0113] Experimental group: 1L of aquaculture water (pH 5.0-8.0) containing 0.5‰ antibiotic (enrofloxacin or doxycycline hydrochloride) was added to shrimp shell biochar treated with compound preparation at an addition rate of 0.2%. The adsorption efficiency was measured after 24 hours of constant temperature stirring at 15°C to 35°C.

[0114] Control group: 1L of aquaculture water (pH 5.0-8.0) containing 0.5‰ antibiotic (enrofloxacin or doxycycline hydrochloride) was added to shrimp shell biochar that had not been treated with compound preparation at an addition rate of 0.2%. The adsorption efficiency was measured after 24 hours of constant temperature stirring at 15°C to 35°C.

[0115] After adsorption, drug residues were detected by high performance liquid chromatography (HPLC), and the antibiotic removal rate was calculated.

[0116] Experimental results: such as Figure 7 As shown, the shrimp shell biochar treated with the compound preparation can significantly improve the adsorption efficiency of residual drugs, which is about 25% higher than that of the control group, and the drug residue removal efficiency reaches more than 80%.

[0117] 2. Comparison of removal rates of commonly used antibiotics by shrimp shell biochar

[0118] Take 1L of aquaculture water (pH 5.0-8.0) containing 0.5‰ of antibiotics (florfenicol powder, enrofloxacin powder, doxycycline hydrochloride powder, and sulfadiazine), stir at a constant temperature of 35 degrees Celsius, and after 24 hours, determine the removal rate of the four antibiotics by the shrimp shell biochar treated with the compound preparation.

[0119] The results are as follows Figure 8 As shown, the removal rates of florfenicol powder, enrofloxacin powder, doxycycline hydrochloride powder, and sulfadiazine by shrimp shell biochar treated with the compound preparation were 72%, 81%, 84%, and 70%, respectively, with the best removal effect on doxycycline hydrochloride powder.

[0120] 3. Application of shrimp shell biochar in removing drug residues from water under different conditions

[0121] (1) Add enrofloxacin to the pond water to a final concentration of 1 mg / L;

[0122] (2) The shrimp shell biochar was used to treat enrofloxacin in water. The experimental temperature was 25℃, the time was 1h, and the amount added was 0.05g / 50ml.

[0123] (3) At pH 7, the average removal rates of enrofloxacin were 85.36%, 92.57% and 84.26% when shrimp shell biochar was added at amounts of 0.05 g / 50 ml, 0.1 g / 50 ml and 0.15 g / 50 ml, respectively, with the 0.1 g / 50 ml addition amount showing the best effect.

[0124] (4) The average removal rates at pH 5, 7 and 9 were 65.25%, 77.62% and 59.65% respectively, with the best adsorption effect under neutral conditions (pH=7).

[0125] 4. The application steps of the present invention using the shrimp shell biochar to remove drug residues in water are as follows: add shrimp shell biochar to the water at an addition rate of 2 g / 1L, and perform adsorption treatment at pH=7.

[0126] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. 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 the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A composite formulation for preparing shrimp shell biochar, characterized in that, The compound preparation contains chitinase, Bacillus licheniformis and Bacillus subtilis, wherein the chitinase is a chitinase mutant ChiM1, and its amino acid sequence is shown in SEQ ID NO:

3.

2. A chitinase mutant ChiM1, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

3. The ChiM1 is obtained by changing the amino acid at position 152 of the chitinase with amino acid sequence SEQ ID NO: 1 from lysine to glutamic acid, the amino acid at position 171 from glutamic acid to alanine, the amino acid at position 183 from isoleucine to valine, the amino acid at position 261 from asparagine to aspartic acid, and the amino acid at position 390 from leucine to tyrosine.

3. The gene encoding the chitinase mutant ChiM1 according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

4.

4. A recombinant expression vector comprising the chitinase mutant ChiM1 encoding gene as described in claim 3, characterized in that, The expression vector is pCBH or pPIC9K.

5. A genetically engineered bacterium comprising the recombinant expression vector of claim 4, characterized in that, The genetically engineered bacteria are Bacillus subtilis, Trichoderma reesei, or Pichia pastoris.

6. A method for preparing shrimp shell biochar using the composite formulation described in claim 1, characterized in that, The preparation method includes the following steps: (1) Shrimp shell cleaning and drying: The shrimp shells are washed multiple times with clean water, detergent and deionized water to remove impurities. The washed shrimp shells are dried and cooled to room temperature. (2) Crushing: The dried shrimp shells are crushed using a crusher and then passed through a 40-80 mesh sieve; (3) Add the compound preparation to the shrimp shell powder at a mass ratio of 0.1%-2%, add water and mix thoroughly, ferment, and after fermentation, dissolve and filter. (4) Place the solid obtained in step (3) into a quartz boat, and under nitrogen protection, use a tube furnace at a temperature of 700℃ for 1.5-3 hours. After cooling to room temperature, sieve to obtain shrimp shell biochar. The compound preparation contains ChiM1 enzyme spray-dried powder, Bacillus licheniformis powder, and Bacillus subtilis powder in a mass ratio of 5:3:

2. The ChiM1 enzyme spray-dried powder has an enzyme activity greater than 2000 U / g, the Bacillus licheniformis content is greater than 10 billion CFU / g, and the Bacillus subtilis powder has a content greater than 10 billion CFU / g. The ChiM1 enzyme spray-dried powder is obtained by spray-drying the fermentation broth of ChiM1 recombinant bacteria. The fermentation culture medium for ChiM1 recombinant bacteria has the following formula: soybean meal 1-3%, neutral protease 1000 U / g, glucose 1-3%, ammonium sulfate 0.1-0.5%, magnesium sulfate 0.01-0.1%, calcium chloride 0.01-0.1%, potassium dihydrogen phosphate 0.2-0.8%, Tween 80 0.02-0.05%, and corn steep liquor 3-10%.

7. The application of the shrimp shell biochar prepared according to claim 6 in removing drug residues from aquaculture water.

8. The application according to claim 7, characterized in that, The drugs include florfenicol, enrofloxacin, doxycycline hydrochloride, and sulfadiazine.

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