Shrimp shell biomass charcoal prepared from compound preparation as well as preparation method and application of shrimp shell biomass charcoal

Abstract

The invention provides shrimp shell biomass charcoal prepared by utilizing a compound preparation and a preparation method and application thereof.The preparation method includes the steps that the compound preparation containing chitinase, bacillus licheniformis and bacillus subtilis is prepared, the chitinase is obtained by mutating chitinase from trichoderma harzianum to construct a mutant library, and the mutant library is used for preparing the shrimp shell biomass charcoal; trichoderma reesei is used as an expression host, the chitinase activity of the excellent mutant Chi M1 obtained through screening is remarkably improved and is two times that of an original gene. Enzyme spray-dried powder obtained by spray-drying fermentation liquor of the ChiM1 recombinant bacteria, bacillus licheniformis powder and bacillus subtilis powder are compounded, and the degradation efficiency of the procambarus clarkia shells can be effectively improved. The compound preparation containing the ChiM1 enzyme spray-dried powder has a good application prospect in the fields of aquaculture, feed additives, biomass resource utilization and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal biological preparations and applications, and specifically relates to shrimp shell biomass charcoal prepared by using a composite preparation, and a preparation method and application thereof. Background Art

[0002] Chitin, also known as chitosan, chitosan, chitosan, etc., is a high-molecular carbohydrate formed by N-acetyl-D-glucosamine connected by β-1, 4-glycosidic bonds. These compounds and some proteins, phenols, lipids or other carbohydrates, such as β-glucan, are cross-linked to form a highly ordered high-molecular scaffold, which constitutes shrimp shells, crab shells, insect shells and fungal cell walls. When the degree of deacetylation is above 40%, it can be converted into chitosan. It is the most abundant biomass resource in the ocean and the second largest biomass resource in nature besides cellulose. With the rapid development of aquaculture, the aquatic product processing industry generates a large amount of waste every year, especially the shells of crustaceans. These wastes are usually thrown into landfills or the ocean, causing serious environmental pollution and waste of resources.

[0003] Chitin and its analogs, amino oligosaccharides, have excellent physiological functions and biological activities and are widely used in agriculture, medicine, food and other fields. The main processing technologies for chitin and other shrimp shells are chemical, physical and enzymatic methods. Among them, the enzymatic method has the advantages of mild reaction conditions, single product and green environmental protection, which has become a hot topic in research.

[0004] Chitinase (EC 3.2.1.14) is a type of glycosyl hydrolase that hydrolyzes chitin β-1,4-glycosidic bonds to generate chito-oligosaccharides or monosaccharides. Most chitinases belong to glycosyl hydrolase families 18 and 19, and a few are distributed in families 20, 23, and 48. Chitinase can be divided into animal, plant, and microbial sources according to its source. Among them, chitinase from microorganisms has the characteristics of wide pH range and high catalytic activity, so it is more suitable for industrial application. However, the enzymatic activity of chitinase from natural sources is generally very low. Therefore, the development of a chitinase with high enzyme activity is an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide a shrimp shell biomass charcoal prepared by using a composite preparation, and a preparation method and application thereof. Specifically, the present invention first mutates chitinase derived from Trichoderma reesei, uses Trichoderma reesei as an expression host, obtains a chitinase mutant ChiM1 with improved enzyme activity by screening, and uses recombinant bacteria for fermentation, and further provides a composite preparation composed of enzyme spray-dried powder of the fermentation liquid of the mutant ChiM1, Bacillus licheniformis powder, and Bacillus subtilis powder, which can effectively improve the degradation efficiency of wastes such as shrimp shell powder, and is used to degrade drug residues in water bodies.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The invention provides a composite preparation for preparing shrimp shell biochar. The composite preparation comprises chitinase, Bacillus licheniformis and Bacillus subtilis. The chitinase is a chitinase mutant ChiM1, and its amino acid sequence is shown in SEQ ID NO:3.

[0007] The invention provides a chitinase mutant ChiM1, whose amino acid sequence is shown in SEQ ID NO: 3. The ChiM1 is obtained by changing the 152nd amino acid of the chitinase with the amino acid sequence of SEQ ID NO: 1 from lysine to glutamic acid, the 171st amino acid from glutamic acid to alanine, the 183rd amino acid from isoleucine to valine, the 261st amino acid from asparagine to aspartic acid, and the 390th amino acid from leucine to tyrosine.

[0008] The present invention provides a coding gene for the chitinase mutant ChiM1, and the nucleotide sequence thereof is shown in SEQ ID NO:4.

[0009] The present invention also provides an expression vector comprising the chitinase mutant ChiM1 encoding gene, wherein the vector is pCDH or pCBH or pPIC9K.

[0010] The present invention provides a genetically engineered bacterium comprising the chitinase mutant ChiM1 coding gene, wherein the genetically engineered bacterium is Bacillus subtilis, Trichoderma reesei or Pichia pastoris.

[0011] The present invention also provides a method for preparing shrimp shell biomass charcoal using the composite preparation, and the preparation method comprises the following steps: (1) Washing and drying shrimp shells: The shrimp shells are washed with clean water, detergent and deionized water for multiple times to remove impurities. The washed shrimp shells are dried and cooled to room temperature. (2) Crushing: The dried shrimp shells are crushed with a crusher and then passed through a 40-80 mesh sieve; (3) adding the composite preparation to shrimp shell powder at a mass ratio of 0.1% to 2%, adding water to mix thoroughly, and fermenting. After the fermentation is completed, dissolving and filtering; (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 solid is sieved to obtain shrimp shell biochar.

[0012] Furthermore: the composite preparation contains ChiM1 enzyme spray powder, Bacillus licheniformis powder, and Bacillus subtilis powder in a mass ratio of 5:3:2, the enzyme activity of the ChiM1 enzyme spray powder is greater than 2000U / g, the bacterial content of Bacillus licheniformis is greater than 10 billion cfu / g, and the bacterial content of Bacillus subtilis powder is greater than 10 billion cfu / g.

[0013] Further: The fermentation medium formula of the mutant ChiM1 recombinant bacteria fermentation is: 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%.

[0014] The present invention also provides the use of the shrimp shell biomass charcoal in removing drug residues in aquaculture water.

[0015] Furthermore, the drugs include florfenicol, enrofloxacin, doxycycline hydrochloride powder, and sulfadiazine.

[0016] Compared with the prior art, the advantages and technical effects of the present invention are as follows: the present invention mutates and improves chitinase derived from harzianase, and obtains a mutant ChiM1 with significantly improved enzyme activity by screening, which is twice the original gene, and its mutation site is K152E / E171A / I183V / N261D / L390Y. The ChiM1 recombinant bacteria is fermented to obtain a fermentation liquid, which is spray-dried to obtain ChiM1 enzyme spray-dried powder, and the ChiM1 enzyme spray-dried powder is compounded with Bacillus licheniformis powder and Bacillus subtilis powder in a mass ratio of 5:3:2 to obtain a composite preparation, which can effectively improve the degradation efficiency of shrimp shell powder and other wastes, and will have good application prospects in the fields of aquaculture, feed additives and biomass resource utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the amplification result of the chitinase gene; Figure 2 is the enzymatic activity of the chitinase mutant; Figure 3 The chitinase mutant is fermented in 30 L with Trichoderma reesei as a host; Figure 4 SDS-PAGE results of concentrated fermentation broth of ChiM1 chitinase; Figure 5 The enzyme spray-dried powder of the fermentation liquid of the chitinase mutant is compounded with Bacillus licheniformis powder and Bacillus subtilis powder to degrade shrimp shell powder; Figure 6is an electron microscope photograph of the shrimp shell biochar prepared by the present invention; Figure 7 The adsorption results of enrofloxacin and doxycycline hydrochloride by the shrimp shell biomass charcoal prepared in the present invention are as follows; Figure 8 The present invention compares the removal rates of commonly used antibiotics by the shrimp shell biomass charcoal prepared in the present invention. DETAILED DESCRIPTION

[0018] In order to facilitate the understanding of the present invention, the present invention will be described in more comprehensive and detailed manner with reference to the accompanying drawings and embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0019] The molecular biology experimental methods not specifically described in the following examples can be performed by referring to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd Edition) by J. Sambrook, or according to the kits and product instructions. The reagents and biological materials used in the specific examples can be obtained from commercial channels unless otherwise specified.

[0020] 1 Strains and vectors Trichoderma reesei QP4 (uracil-deficient), plasmid pCBH, plasmid pET-21a(+), Escherichia coli BL21 and the like were purchased from Invitrogen.

[0021] 2 Reagents and culture media Plasmid extraction kit, fragment purification and recovery kit, restriction endonuclease, etc. were purchased from Bao Biotechnology (Dalian) Co., Ltd.; ampicillin, kanamycin, uracil, etc. were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; protein marker: Blue Plus II Protein Marker (14-120 kDa) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0022] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl; Trichoderma reesei transformation medium (g / L): Transformation upper layer: Glucose 10g, MgSO4·7H2O 1g, KH2PO4 10g, (NH4)2SO4 6g, sodium citrate 3g, FeSO4·7H2O 0.005g, MnSO4·H2O 0.0016g, ZnSO4·7H2O 0.0014g, CoC12·2H2O 0.002g, sorbitol 182.18g, agarose 6g, sterilize at 115℃ for 30 min.

[0023] Transformation lower layer: Glucose 10g, MgSO4·7H2O 1g, KH2PO4 10g, (NH4)2SO4 6g, sodium citrate 3g, FeSO4·7H2O 0.005g, MnSO4·H2O 0.0016g, ZnSO4·7H2O 0.0014g, CoC12·2H2O0.002g, agar powder 12g, sterilize at 115℃ for 30 min.

[0024] The above culture media need to be supplemented with 10 mM uracil when culturing uracil-deficient cells.

[0025] PDA medium: 20% potato extract, 2% glucose.

[0026] 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.

[0027] Transformation-related reagents: Solution 1 (200 mL): 43.72 g sorbitol, 2.72 g potassium dihydrogen phosphate, adjusted to pH 5.5 with sodium hydroxide solution.

[0028] Solution 2 (100 mL): sorbitol 18.22 g, calcium chloride dihydrate 0.735 g, 10 mM Tris-Hcl, adjust pH to 7.48.

[0029] Solution 3 (100 mL): PEG6000 25 g, calcium chloride dihydrate 0.735 g, 10 mM Tris-Hcl, pH adjusted to 7.5.

[0030] 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%.

[0031] 3 Chitinase activity was detected by spectrophotometry, referring to GB / T34799-2017 Chitinase activity detection method.

[0032] 4. Transformation method of Trichoderma reesei (1) Experimental preparation: PDA plate, sterile saline (0.9% sodium chloride, 0.05% Tween-20), funnel with four layers of lens paper or filter paper (sterilized), and cellophane. Cut the cellophane into the size of a culture dish, place a piece of filter paper in the middle of each layer, place in the culture dish, and sterilize.

[0033] (2) Bacterial culture: Take QP4 spores on a PDA (containing uracil) plate and culture at 30°C until spores form; take fresh spores, add 5 ml of sterile saline, wash the spores to obtain a spore suspension; add a layer of cellophane on the PDA (containing uracil) plate and spread it flat with a spreading stick, add 100 ul of spore suspension on the cellophane, spread it evenly, and culture at 30°C until more hyphae grow.

[0034] (3) Preparation and transformation of protoplasts: Take a clean plate, add 0.1g lytic enzyme, then add 20mL solution 1, and mix well; wash the cultured mycelium into the plate containing lytic enzyme, and lyse at 30℃ for 90 minutes; after lysis, filter the mycelium with a funnel containing filter paper (lens paper), and collect the protoplasts by centrifugation; resuspend with 4mL solution 2, centrifuge at 2500rpm and 4℃ for 10min, and discard the supernatant; resuspend with solution 2 and divide into 200ul per tube, add 15ul transformation plasmid respectively, and place on ice for 5 minutes; then add 2mL solution 3 and 4mL solution 2 respectively, add 30mL transformation upper layer, and transfer to the plate; culture at 30℃ until transformants grow.

[0035] Example 1: Construction of chitinase mutant genes and expression verification in Trichoderma reesei After screening and verification in the early stage, a chitinase was obtained, which has high chitinase activity. The sequence was translated into the corresponding nucleotide sequence, and after gene sequence optimization, it was 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.

[0036] Primers were designed as follows: an Nco I restriction enzyme site was designed at the 5' end and a Sac I restriction enzyme site was designed at the 3' end.

[0037] GeneMorph II random mutation PCR kit was used to perform random mutation using the artificially synthesized gene as a template. The primer sequences used are as follows. The PCR amplification results are as follows: Figure 1As shown, and cloned into the Nco I and Sac I sites of plasmid pCBH, according to the genetic transformation method of Trichoderma reesei, the recombinant plasmid was transformed into Trichoderma reesei QP4 to obtain recombinant bacteria. According to the 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%. Under 30℃, shake flask fermentation was carried out for 4-5 days, and the culture solution was centrifuged to obtain the supernatant for use.

[0038] Chi-F1: CATGCCATGGATGCTCAGCTTCCTCGGCAA (SEQ ID NO: 5) Chi-R1: CGAGCTCCTAGTTGAGGCCGTTCTTAATG (SEQ ID NO: 6).

[0039] According to the above determination method, the supernatant after centrifugation of the fermentation broth was taken to determine the enzyme activity of the supernatant of each mutant fermentation broth. After repeated verification, 5 mutants with higher enzyme activity than the control group were screened out, such as Figure 2 As shown, the recombinant bacteria with the highest enzyme activity were selected for gene amplification and sequencing analysis, and the chitinase mutant ChiM1 was obtained after sequencing, 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 about twice that of the original gene.

[0040] SEQ ID NO: 3 MLSFLGKSVALLAALQATLSSPKPGHRRASVEKRANGYANSVYFTNWGIYDRNFQPADLV ASDVTHVIYSFMNLQADGTVISGDTYADYEKHYADDSWNDVGTNAYGCVKQLFKVKKANR GLKVLLSIGGWTWSTNFPSAASTDANRKNFAETAITFMKDWGFDGIDIDWAYPADATQAS NMVLLLKEVRSQLDAYAAQYAPGYHFLLTIAAPAGKDNYSKLRLADLGQVLDYINLMAYD YAGSFSPLTGHDANLFNNPSDPNATPFNTDSAVKDYINGGVPANKIVLGMPIYGRSFQNT AGIGQTYNGVGSGSWEAGIWDYKALPKAGATVQYDSVAKGYYSYNSATKELISFDTPDMI NTKVAYLKSLGLGGSMFWEASADKKGADSYIGTSHRALGGLDTTQNLLSYPNSKYDNIKNGLN SEQ ID NO:4 ATGCTCAGCTTCCTCGGCAAGAGCGTCGCCCTCCTCGCCGCCCTCCAGGCCACCCTCAGC AGCCCGAAGCCGGGCCACCGCCGCGCCAGCGTCGAGAAGCGCGCCAACGGCTACGCCAAC AGCGTCTACTTCACCAACTGGGGCATTTACGACCGCAACTTCCAGCCGGCCGACCTCGTC GCCAGCGACGTCACCCACGTCATTTACAGCTTCATGAACCTCCAGGCCGACGGCACCGTC ATTAGCGGCGACACCTACGCCGACTACGAGAAGCACTACGCCGACGACAGCTGGAACGAC GTCGGCACCAACGCCTACGGCACTGTCAAGCAGCTCTTCAAGGTCAAGAAGGCCAACCGC GGCCTCAAGGTCCTCCTCAGCATTGGCGGCTGGACCTGGAGCACCAACTTCCCGAGCGCC GCCAGCACCGACGCCAACCGCAAGAACTTCGCCGAGACCGCCATTACCTTCATGAAGGAC TGGGGCTTCGACGGCATTGACATTGACTGGGCCTACCCGGCCGACGCCACCCAGGCCAGC AACATGGTCCTCCTCCTCAAGGAGGTCCGCAGCCAGCTCGACGCCTACGCCGCCCAGTAC GCCCCGGGCTACCACTTCCTCCTCACCATTGCCGCCCCGGCCGGCAAGGACAACTACAGC AAGCTCCGCCTCGCCGACCTCGGCCAGGTCCTCGACTACATTAACCTCATGGCCTACGAC TACGCCGGCAGCTTCAGCCCGCTCACCGGCCACGACGCCAACCTCTTCAACAACCCGAGC GACCCGAACGCCACCCCGTTCAACACCGACAGCGCCGTCAAGGACTACATTAACGGCGGC GTCCCGGCCAACAAGATTGTCCTCGGCATGCCGATTTACGGCCGCAGCTTCCAGAACACC GCCGGCATTGGCCAGACCTACAACGGCGTCGGCAGCGGCAGCTGGGAGGCCGGCATTTGG GACTACAAGGCCCTCCCGAAGGCCGGCGCCACCGTCCAGTACGACAGCGTCGCCAAGGGC TACTACAGCTACAACAGCGCCACCAAGGAGCTCATTAGCTTCGACACCCCGGACATGATT AACACCAAGGTCGCCTACCTCAAGAGCCTCGGCCTCGGCGGCAGCATGTTCTGGGAGGCC AGCGCCGACAAGAAGGGCGCCGACAGCTACATTGGCACCAGCCACCGCGCCCTCGGCGGC CTCGACACCACCCAGAACCTCCTCAGCTACCCGAACAGCAAGTACGACAACATTAAGAAC GGCCTCAACTAG Example 2: Fermentation and preparation of ChiM1 recombinant bacteria in a 30L fermenter The genetically engineered bacteria expressing the ChiM1 recombinant bacteria in the above example were streaked on a PDA plate and cultured at 30°C until spores were formed. After about 7 days of culture, the spores were washed off with sterile water and single spores were isolated. The spores were diluted and applied to MM (containing Triton-X100) medium until the colonies grew out, and then cultured to form spores. Pure strains were isolated in three generations. The pure spores were washed off with 100 mL of sterile water and used as seed liquid to inoculate the fermenter.

[0041] Fermentation production 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%. The pH is natural, the temperature is 30℃, the stirring rate is 600rpm, the ventilation volume is 1.5 (v / v), and the dissolved oxygen is controlled at more than 20%. The pH of the fermentation process is natural. The enzyme activity is measured after 24 hours of fermentation. After the fermentation is completed (generally 48 hours), the fermentation liquid is processed by a plate and frame filter to obtain a crude enzyme liquid, which is spray-dried into a powder preparation through a spray tower for application testing.

[0042] The fermentation process curve is as follows Figure 3 As shown: samples were taken every 8 hours to measure the enzyme production level. The enzyme activity level reached the highest point after 60 hours of fermentation.

[0043] Example 3: SDS-PAGE detection of ChiM1 chitinase expression After the fermentation broth in Example 2 was centrifuged and filtered, the supernatant was collected, and then the supernatant was passed through a protein purification column to filter out molecular components such as proteins below 10KD, and the fermentation broth was further concentrated and stored at -20 degrees.

[0044] Electrophoresis: 100 μL of the concentrated fermentation enzyme solution was added to 100 μL Loading Buffer, boiled for 10 min, and 20 μL was taken for loading.

[0045] Electrophoresis conditions: voltage 80-90mv, current 30mA for stacking gel; voltage 110-120mv, current 40mA for separation gel. Stop electrophoresis when the sample is 1cm away from the edge of the gel. Strip the gel, stain for 2h, decolorize for 24h, and change the decolorizing solution during this period.

[0046] The test results are as follows Figure 4 As shown, it is basically consistent with the theoretical molecular weight of 46KD, and there is a relatively dense protease band, indicating that the expression level of ChiM1 chitinase is high, which is convenient for subsequent testing and application.

[0047] Example 4: Application experiment of ChiM1 recombinant bacterial fermentation broth and Bacillus licheniformis and Bacillus subtilis composite preparation 1. Composite preparation: After centrifuging the ChiM1 recombinant bacterial fermentation liquid, obtain the supernatant and spray dry it to obtain ChiM1 enzyme spray powder (enzyme activity greater than 2000U / g), add 50% ChiM1 enzyme spray 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 mass ratio, and add it in an amount of 0.1-2% of the mass ratio of shrimp shell powder when using.

[0048] 2. Compound preparations to degrade shrimp shell powder Experimental group: 200g of shrimp shell powder (passed through 80 mesh sieve) was placed in a sealed bag, and the compound preparation was added to the shrimp shell powder according to the addition amount of 1%, that is, 2g of the compound preparation was weighed, dissolved in 40mL of deionized water, and then mixed into the shrimp shell powder and mixed thoroughly. Placed in a 37℃ incubator for anaerobic fermentation for 3 days, after the fermentation was completed, dissolved and filtered, and the remaining solids were dried and weighed.

[0049] Control group: Take 200g of shrimp shell powder (passed through 80 mesh sieve) in a sealed bag, add 40mL of deionized water directly, mix into the shrimp shell powder, and mix thoroughly. Place in a 37℃ incubator for anaerobic fermentation for 3 days. After the fermentation is completed, dissolve and filter, dry the remaining solids, and weigh them.

[0050] After repeated verification, the results are as follows Figure 5 As shown in the data, the degradation rate of shrimp shell powder in the experimental group was 45.5%, while that in the control group was only 15%. This indicates that after adding a compound preparation containing ChiM1 chitinase, Bacillus licheniformis and Bacillus subtilis to the shrimp shell powder, the degradation rate can be increased by more than 2 times, significantly improving the decomposition of shrimp shells and other materials containing a large amount of chitin, and promoting the recyclable utilization of biomass resources.

[0051] Example 5: Preparation of biochar from Procambarus clarkii shells 1. The method for preparing shrimp shell biomass charcoal by degrading Procambarus clarkii shrimp shells using the composite preparation comprises the following steps: (1) Washing and drying shrimp shells: The shrimp shells of Procambarus clarkii are washed with clean water, detergent and deionized water for multiple times to remove impurities (residual grease, remaining meat), and the washed shrimp shells are dried in an oven at 105° C. for about 3 hours and cooled to room temperature; (2) Crushing: The dried shrimp shells are crushed with a crusher and then passed through an 80-mesh sieve; (3) Take 200g of shrimp shell powder (passed through an 80-mesh sieve) in 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 and mix it thoroughly. Place it in a 37℃ incubator for anaerobic fermentation for 3 days. After the fermentation is completed, dissolve and filter it; (4) The solid obtained in step (3) was placed in a quartz boat, and under nitrogen protection, a tube furnace was used at a temperature of 700° C. for 2 hours. After cooling to room temperature, the solid was passed through an 80-mesh sieve to obtain shrimp shell biochar.

[0052] 2. Characterization of shrimp shell biochar After the shrimp shell biochar production was completed, the elemental composition of the shrimp shell biochar was measured by X-ray photoelectron spectroscopy (XPS). The specific surface area of ​​the adsorbent was determined by Brunauer-Emmett-Teller (BET) analysis. The surface morphology of the sample was scanned by scanning electron microscopy (SEM). The electron microscope photo of the shrimp shell biochar is shown in Figure 2. Figure 6 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.

[0053] Table 1 Elemental composition and specific surface area of ​​crayfish shell biochar

[0054] Example 6: Application of Procambarus clarkii shell biochar in removing residual drugs from aquaculture water In aquaculture, some antibiotics are often mixed in feed and fed, but excessive drugs will cause serious water pollution. The shrimp shell biomass charcoal prepared in Example 5 is used to improve the adsorption efficiency of residual drugs.

[0055] 1. Adsorption experiments of enrofloxacin and doxycycline hydrochloride by shrimp shell biochar: Experimental group: 1L of aquaculture water (pH 5.0-8.0) containing 0.5‰ antibiotics (enrofloxacin or doxycycline hydrochloride) was added with 0.2% of the shrimp shell biochar treated with the composite preparation, and the adsorption efficiency was measured at constant temperature of 15 to 35 degrees for 24 hours; Control group: Take 1L of aquaculture water (pH 5.0-8.0), containing 0.5‰ antibiotics (enrofloxacin or doxycycline hydrochloride), add shrimp shell biochar that has not been treated with composite preparations at an addition rate of 0.2%, and measure the adsorption efficiency after 24 hours at constant temperature and stirring at 15 to 35 degrees; After adsorption, drug residues were detected by high performance liquid chromatography (HPLC) and the removal rate of antibiotics was calculated.

[0056] Experimental results: Figure 7 As shown, the shrimp shell biochar treated with the composite 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%.

[0057] 2. Comparison of removal rates of commonly used antibiotics by shrimp shell biochar 1L of aquaculture water (pH 5.0-8.0) was taken, containing 0.5‰ of antibiotics (florfenicol powder, enrofloxacin powder, doxycycline hydrochloride powder, sulfadiazine), and stirred at a constant temperature of 35 degrees. After 24 hours, the removal rate of the four antibiotics by the shrimp shell biochar treated with the composite preparation was determined.

[0058] The results are as follows Figure 8 As shown in the figure, the removal rates of florfenicol powder, enrofloxacin powder, doxycycline hydrochloride powder and sulfadiazine by shrimp shell biochar treated with the composite preparation were 72%, 81%, 84% and 70%, respectively, among which the removal effect of doxycycline hydrochloride powder was the best.

[0059] 3. Application of the shrimp shell biochar to remove drug residues in water under different conditions (1) Add enrofloxacin to pond water at a final concentration of 1 mg / L; (2) Treating enrofloxacin in water with the shrimp shell biochar, the experimental temperature is 25°C, 1h, and the addition amount is 0.05g / 50ml; (3) At pH 7, the average removal rates of enrofloxacin by shrimp shell biochar at the addition amounts of 0.05 g / 50 ml, 0.1 g / 50 ml and 0.15 g / 50 ml were 85.36%, 92.57% and 84.26%, respectively, and the addition amount of 0.1 g / 50 ml had the best effect. (4) The average removal rates at pH values ​​of 5, 7, and 9 were 65.25%, 77.62%, and 59.65%, respectively. The best adsorption effect was obtained under neutral conditions (pH=7).

[0060] 4. The application steps of the present invention for removing drug residues in water using the shrimp shell biochar are as follows: adding the shrimp shell biochar to the water at an addition amount of 2 g / 1L, and performing adsorption treatment at pH=7.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A composite preparation for preparing shrimp shell biochar, characterized in that: The composite preparation comprises chitinase, Bacillus licheniformis and Bacillus subtilis. The chitinase is 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 152nd amino acid of the chitinase with the amino acid sequence of SEQ ID NO: 1 from lysine to glutamic acid, the 171st amino acid from glutamic acid to alanine, the 183rd amino acid from isoleucine to valine, the 261st amino acid from asparagine to aspartic acid, and the 390th amino acid from leucine to tyrosine.

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

4.

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

5. A genetically engineered bacterium comprising the recombinant expression vector according to 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 preparation according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Washing and drying shrimp shells: The shrimp shells are washed with clean water, detergent and deionized water for multiple times to remove impurities. The washed shrimp shells are dried and cooled to room temperature. (2) Crushing: The dried shrimp shells are crushed with a crusher and then passed through a 40-80 mesh sieve; (3) adding the composite preparation to shrimp shell powder at a mass ratio of 0.1% to 2%, adding water to mix thoroughly, and fermenting. After the fermentation is completed, dissolving and filtering; (4) The solid obtained in step (3) is placed in a quartz boat, and heated in a tube furnace at 700° C. for 1.5-3 hours under nitrogen protection. After cooling to room temperature, the solid is sieved to obtain shrimp shell biochar.

7. The preparation method according to claim 6, characterized in that: The composite preparation contains ChiM1 enzyme spray powder, Bacillus licheniformis powder and Bacillus subtilis powder in a mass ratio of 5:3:

2. The enzyme activity of the ChiM1 enzyme spray powder is greater than 2000U / g, the bacterial content of Bacillus licheniformis is greater than 10 billion cfu / g, and the bacterial content of Bacillus subtilis powder is greater than 10 billion cfu / g.

8. The preparation method according to claim 7, characterized in that: The ChiM1 enzyme spray-dried powder is obtained by spray-drying the fermentation liquid of the ChiM1 recombinant bacteria. The formula of the fermentation medium of the ChiM1 recombinant bacteria is: 1-3% soybean meal, 1000U / g neutral protease, 1-3% glucose, 0.1-0.5% ammonium sulfate, 0.01-0.1% magnesium sulfate, 0.01-0.1% calcium chloride, 0.2-0.8% potassium dihydrogen phosphate, 0.02-0.05% Tween 80, and 3-10% corn steep liquor.

9. Use of the shrimp shell biochar prepared according to claim 6 in removing drug residues in aquaculture water.

10. The use according to claim 9, characterized in that: The drugs include florfenicol, enrofloxacin, doxycycline hydrochloride, and sulfadiazine.

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