A C family lactamase mutant and its immobilization method and application

Abstract

The present invention discloses a C-family beta-lactamase mutant, its immobilization method and application. The sequence of the C-family beta-lactamase mutant is as described in any one of SEQ ID NO: 1-5. The optimal immobilization conditions for this enzyme are: sodium alginate with a final concentration of 1.5%, CaCl2 with a concentration of 1.0%, and immobilization for 10 minutes. The C-family beta-lactamase mutant was immobilized under the optimal immobilization conditions and the enzymatic properties of the enzyme were studied. It was found that the optimal temperature of the immobilized enzyme is 50 °C and the optimal pH is 5.0. The removal rate of the immobilized C-family beta-lactamase for cefalexin at different concentrations can reach more than 95%. The present invention discovers that the C-family beta-lactamase mutant has a high rate of degrading cefalexin and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a C family lactamase mutant and an immobilization method and application thereof. Background Art

[0002] Lactam antibiotics, especially cephalexin, are widely used worldwide due to their broad antibacterial spectrum and effective therapeutic effects. These antibiotics are usually produced by microbial metabolism or artificially synthesized, and are mainly used to treat and prevent animal diseases and promote animal growth and development. However, the large-scale use of lactam antibiotics has brought serious environmental problems: untreated or poorly treated antibiotics are discharged into the environment through manure and sewage from livestock and poultry farming, resulting in the long-term accumulation of antibiotics in the environment, inducing the presence of a large number of drug-resistant genes and drug-resistant bacteria, which in turn has caused widespread environmental and public health concerns.

[0003] In particular, cephalexin, as a commonly used beta-lactam antibiotic, its continued presence in the environment not only poses a threat to water resource safety, but may also promote the emergence of drug-resistant bacteria. Therefore, the development of efficient cephalexin removal methods is of great significance to environmental protection. Currently, biodegradation technology, especially enzyme degradation technology, is regarded as an effective way to treat such pollutants due to its advantages of low cost, good effect, and no secondary pollution. However, there is currently a lack of enzymes with good stability, high catalytic activity and the ability to efficiently degrade cephalexin. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a C family lactamase mutant and an immobilization method and application thereof.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] In a first aspect, a C family lactamase mutant is provided, which is any of the following mutants:

[0007] Mutant 1, wherein, based on the amino acid sequence shown in SEQ ID NO: 1, Ser at position 71 is mutated to Arg, Asn at position 133 is mutated to Lys, Gln at position 144 is mutated to Lys, and Trp at position 265 is mutated to Cys; the amino acid sequence of mutant 1 is shown in SEQ ID NO: 2;

[0008] Mutant 2, wherein the mutant 2 is based on the amino acid sequence shown in SEQ ID NO: 1, with Asp at position 52 mutated to Asn, and Thr at position 151 mutated to Ser; the amino acid sequence of the mutant 2 is shown in SEQ ID NO: 3;

[0009] Mutant 3, wherein the mutant 3 is based on the amino acid sequence shown in SEQ ID NO: 1, with Gln at position 28 mutated to His, Met at position 33 mutated to Leu, Tyr at position 207 mutated to Ser, Ala at position 224 mutated to Gly, Met at position 270 mutated to Val, and Lys at position 295 mutated to Gln; the amino acid sequence of the mutant 3 is shown in SEQ ID NO: 4;

[0010] Mutant 4, wherein, based on the amino acid sequence shown in SEQ ID NO: 1, Gln at position 28 is mutated to His, Met at position 33 is mutated to Leu, Tyr at position 207 is mutated to Ser, Ala at position 224 is mutated to Gly, and Met at position 270 is mutated to Val; the amino acid sequence of mutant 4 is shown in SEQ ID NO: 5;

[0011] Mutant 5, wherein mutant 4 is based on the amino acid sequence shown in SEQ ID NO: 1, with Gln at position 28 mutated to His, Met at position 33 mutated to Leu, Thr at position 151 mutated to Ser, Tyr at position 207 mutated to Ser, Ala at position 224 mutated to Gly, and Met at position 270 mutated to Val; its amino acid sequence is shown in SEQ ID NO: 6.

[0012] In a second aspect, a method for immobilizing a C family lactamase mutant is provided, wherein a sodium alginate carrier and a C family lactamase mutant solution are evenly mixed and then dropped onto a CaCl 2 The immobilization is carried out in the solution to obtain the immobilized C family lactamase mutant.

[0013] Furthermore, the immobilization conditions were: final concentration 1.5% sodium alginate, 1.0% CaCl 2， Fix for 10 minutes.

[0014] In a third aspect, a method for degrading cephalexin is provided.

[0015] The beneficial effects of the present invention are:

[0016] The present invention discloses a C family lactamase mutant and an immobilization method and application thereof. The C family lactamase mutant provided by the present invention is an enzyme with good stability, high catalytic activity and high efficiency in degrading cephalexin, thereby expanding the types of enzymes that can be used to hydrolyze C family lactam compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the relative activities of ACT-3 mutants immobilized with different concentrations of sodium alginate;

[0018] Figure 2 For different concentrations of CaCl 2 Schematic diagram of the relative activities of ACT-3 mutants under immobilization;

[0019] Figure 3 Schematic diagram of the relative activities of ACT-3 mutants at different immobilization times;

[0020] Figure 4 Schematic diagram of the temperature activity experimental results of immobilized ACT-3 mutants;

[0021] Figure 5 Schematic diagram of the pH activity experimental results of immobilized ACT-3 mutants;

[0022] Figure 6 Schematic diagram of the experimental results of degradation of 10 μg / mL cephalexin by ACT-3 mutant immobilized microspheres;

[0023] Figure 7 Schematic diagram of the experimental results of degradation of 30 μg / mL cephalexin by ACT-3 mutant immobilized microspheres;

[0024] Figure 8 Schematic diagram of the experimental results of degradation of 50 μg / mL cephalexin by ACT-3 mutant immobilized microspheres. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0026] Materials preparation:

[0027] (1) Deionized water

[0028] (2) Water bath

[0029] (3) 1.2mm bore needle and 1mL syringe

[0030] (4) Timer

[0031] (5) Funnel

[0032] (6) Beaker

[0033] (7) Graduated cylinder

[0034] (8) Snowflake Ice Machine

[0035] (9) Ice Box

[0036] (10) Pipette

[0037] (11) Gun head

[0038] (12) UV spectrophotometer

[0039] (13)Quartz cuvette

[0040] (14) 1mL EP tube

[0041] (15) 10mL centrifuge tube

[0042] (16) 50mL centrifuge tube

[0043] (17)EP pipe rack

[0044] (18) Centrifuge tube rack

[0045] (19) Long-handled spoon

[0046] (20) pH meter

[0047] (21) LB medium: Weigh 10.0 g of peptone, 5.0 g of yeast powder, and 10.0 g of sodium chloride, add 1 L of deionized water, mix thoroughly, and sterilize at 121 °C for 15 min.

[0048] (22) LB agar medium: Weigh 10.0 g of peptone, 5.0 g of yeast powder, 10.0 g of sodium chloride, and 15.0 g of agar powder, add 1 L of deionized water, mix thoroughly, and sterilize at 121 °C for 15 min.

[0049] (23) LB medium with kanamycin resistance: Weigh 10.0 g of peptone, 5.0 g of yeast powder, and 10.0 g of sodium chloride, add 1 L of deionized water, mix thoroughly, sterilize at 121 °C for 15 min, and then naturally cool to 50-60 °C before adding kanamycin to a final concentration of 100 μg / mL.

[0050] (24) 1x SDS-PAGE buffer: weigh 3.00 g of Tris, 14.40 g of glycine, and 1.00 g of SDS, and dissolve them in deionized water to make up to 1 L.

[0051] (25) 5x Loading Buffer: Component concentrations: 1.00 M Tris-HCl (pH 6.8), 10% SDS, 0.5% (W / V) BPB, 50% (V / V) glycerol, 5% (W / V) 2-ME. Take 1.25 mL 1 M Tris-HCl (pH 6.8), 5 mL SDS, 25 mg BPB, 2.5 mL glycerol, and 250 uL 2-ME and place them in a 15 mL plastic centrifuge tube. Dissolve them in deionized water, make up to 5 mL, divide into small portions (0.5 mL / portion), and store in a refrigerator at 4 °C for later use.

[0052] (26) 0.1% (w / v) Coomassie Brilliant Blue R-250: 0.25 L of methanol, 0.65 L of deionized water, 0.1 L of glacial acetic acid, and 1.0 g of Coomassie Brilliant Blue R250 were mixed and dissolved for later use.

[0053] (27) Coomassie staining decolorizing solution: Take 0.24 L of ethanol and 0.08 L of glacial acetic acid, add deionized water to make up to 1 L.

[0054] (28) 10% ammonium persulfate solution: Weigh 0.1 g of ammonium persulfate into a 2 mL centrifuge tube, add 1 mL of deionized water and pipette to dissolve, and store at 4 °C.

[0055] (29) Buffer A (20 mmol / L Tris, 500 mmol / L NaCl, pH 7.0): weigh 2.42 g Tris and 29.25 g NaCl into a 1 L reagent bottle, add 0.8 L deionized water to dissolve, adjust the pH to 7.0 with 30% HCl solution and a pH meter, make up to 1 L with deionized water, and filter with a 0.2 μm water filter.

[0056] (30) Buffer B (20 mmol / L Tris, 500 mmol / L NaCl, 500 mmol / imidazole, pH 7.0): weigh 2.42 g Tris, 29.25 g NaCl, and 34.04 g imidazole in a 1-L reagent bottle, add 0.8 L deionized water to dissolve, adjust the pH to 7.0 with 30% HCl solution and a pH meter, make up to 1 L with deionized water, and filter with a 0.2 μm water filter.

[0057] (31) 20 mmol / L tris-HCl: Weigh 2.42 g of Tris and dissolve it in 0.8 L of deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 1 L with deionized water. Filter with a 0.2 μm water filter.

[0058] (32) 20 mmol / L imidazole: Weigh 0.27 g imidazole, 0.48 g Tris, and 5.85 g NaCl and dissolve them in 0.15 L deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 0.2 L with deionized water. Filter with a 0.2 μm water filter.

[0059] (33) 50 mmol / L imidazole: Weigh 0.68 g imidazole, 0.48 g Tris, and 5.85 g NaCl and dissolve them in 0.15 L deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 0.2 L with deionized water. Filter with a 0.2 μm water filter.

[0060] (34) 200 mmol / L imidazole: Weigh 2.72 g imidazole, 0.48 g Tris, and 5.85 g NaCl and dissolve them in 0.15 L deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 0.2 L with deionized water. Filter with a 0.2 μm water filter.

[0061] (35)Plasmid extraction kit

[0062] (36) Preparation of pH buffer: Preparation of glycine-HCl buffer (pH 3.0, 4.0, 5.0): Take 2.5 mL of 0.2 mol / L glycine solution in a 50 mL centrifuge tube, add 0.2 mol / L hydrochloric acid solution to pH 3.0, and add pure water to make up to 10 mL. Prepare pH 4.0 and 5.0 buffers in the same way; Preparation of PBS buffer (pH 6.0): Take 1.23 mL of 0.2 mol / L disodium hydrogen phosphate and 8.77 mL of 0.2 mol / L sodium dihydrogen phosphate in a 50 mL centrifuge tube; Preparation of Tris-HCl buffer (pH 7.0, 8.0, 9.0): Weigh 2.42 g Dissolve Tris in 1000mL reagent bottle with 800mL pure water, adjust pH to 7.0 with HCl, and add pure water to 1000mL; weigh 0.48g Tris in 50mL reagent bottle, dissolve with 16mL pure water, adjust pH to 8.0 with HCl, and add pure water to 20mL; weigh 0.48g Tris in 50mL reagent bottle, dissolve with 16mL pure water, adjust pH to 9.0 with HCl, and add pure water to 20mL; prepare glycine-sodium hydroxide buffer (pH10.0, 11.0): take 2.5mL 0.2mol / L glycine solution in 50mL centrifuge tube, add 0.2mol / L sodium hydroxide solution to pH10.0. Prepare pH11.0 buffer in the same way.

[0063] (37) Preparation of sodium alginate solution: To prepare 3.0% sodium alginate solution, weigh 0.60 g of sodium alginate into a 50 mL centrifuge tube, add 20 mL of 20 mM Tris-HCl (pH 7.0), and stir until completely dissolved. To prepare 4.2% sodium alginate solution, weigh 0.84 g of sodium alginate into a 50 mL centrifuge tube, add 20 mL of 20 mM Tris-HCl (pH 7.0), and stir until completely dissolved.

[0064] (38)CaCl 2 Solution preparation: Prepare 0.5% CaCl 2 Solution, weigh 0.10g CaCl 2 In a 50 mL centrifuge tube, dissolve with 20 mL of pure water; prepare 1.0% CaCl 2 Solution, weigh 0.20g CaCl 2 In a 50 mL centrifuge tube, dissolve with 20 mL of pure water; prepare 1.5% CaCl 2 Solution, weigh 0.30g CaCl 2 In a 50 mL centrifuge tube, dissolve with 20 mL of pure water; prepare 2.0% CaCl 2 Solution, weigh 0.40g CaCl 2 In a 50 mL centrifuge tube, dissolve with 20 mL of pure water; prepare 2.5% CaCl 2 Solution, weigh 0.50gCaCl 2 Dissolve in 20 mL of pure water in a 50 mL centrifuge tube.

[0065] (39) 1 mg / mL cephalexin: Weigh 0.0200 g of cephalexin into a 50 mL centrifuge tube, dissolve in 20 mL of 20 mM Tris-HCl (pH 7.0), and place on ice until ready for use.

[0066] Example 1

[0067] Plasmid extraction: Inoculate the bacteria containing the plasmid into LB agar medium with kanamycin resistance, culture in a 37°C constant temperature incubator for 8-12 hours, pick a single colony and inoculate it into 5 mL LB liquid culture medium, culture in a 37°C shaker for 8-12 hours, collect the bacteria, and extract the plasmid according to the steps in the instructions of the plasmid extraction kit.

[0068] Conversion:

[0069] (1) Make ice: Take out a tube (100uL) of competent bacteria from a -70℃ ultra-low temperature freezer and place it on ice to wait for the competent cells to thaw.

[0070] (2) Add 5 μL of the ligated plasmid to 100 μL of competent medium, shake gently, and place on ice for 30 min.

[0071] (3) After gently shaking, place the tube in a 42°C water bath for 90 seconds, then quickly place it back on ice and let it stand for 10 minutes.

[0072] (4) Add 800uL of LB medium to each tube in a clean bench, mix gently, and then place on a 37°C shaker for 1 hour.

[0073] (5) After centrifugation, take 50uL of the above transformation mixture in an ultra-clean workbench and drop it onto solid LB medium containing kanamycin, and spread it evenly with a coating rod.

[0074] (6) Mark the coated culture dish and place it in a 37°C constant temperature incubator for 30-60 min until the liquid on the surface has penetrated into the culture medium. Then turn it upside down and place it in a 37°C constant temperature incubator overnight.

[0075] Induced expression: Pick a positive monoclone and culture it in 5mL LB medium at 37℃ overnight as seed solution. Take 200uL seed solution and inoculate it in 200mL medium (500mL conical flask), culture it in a shaking incubator at 37℃ until OD600=0.6-0.8, add IPTG (0.5mM) for induction, the induction temperature is 25℃, and the induction time is 6h. Collect the bacteria by centrifugation at 6500rpm for 5min to obtain C family lactamase ACT-3 and mutants.

[0076] Protein purification

[0077] (1) Column Packing

[0078] (2) Buffer A balance

[0079] (3) Sample loading

[0080] (4) Imidazole elution and collection of protein samples

[0081] (5) Buffer B rinse

[0082] Table ACT-3 and its relative enzyme activities of different mutants

[0083] Protein name Relative enzyme activity ACT-3 100％ ACT-3(S71R-N133K-Q144K-W265C) 103％ ACT-3(D52N-T151S) 88％ ACT-3(Q28H-M33L-Y207S-A224G-M270V-K295Q) 98％ ACT-3(Q28H-M33L-Y207S-A224G-M270V) 109％ ACT-3(Q28H-M33L-T151S-Y207S-A224G-M270V) 110％

[0084] The following examples were tested using mutants with higher enzyme activity.

[0085] Example 2 Immobilization Optimization

[0086] 1.1 Optimization of sodium alginate concentration:

[0087] (1) Prepare a mixture of sodium alginate (final concentration 0.5%, 1.0%, 1.5%, 2.0%, 2.5%) and mutants according to the following table, taking 1 mL as an example:

[0088]

[0089] (2) Take 0.75 mL of 1 mg / mL cephalexin solution in a 50 mL centrifuge tube and add 24.25 mL of 20 mM Tris-HCl (pH 7.0) to dilute the cephalexin to 30 μg / mL.

[0090] (3) Take two small beakers and mark them as blank and experimental groups. Add 5 mL of 2.0% CaCl 2 Solution, slowly drip the above 0.5% mixed solution into the beaker with a syringe 1mm away from the liquid surface, let it stand for 10min, rinse it with 20mMTris-HCl (pH7.0) 3 times, then store the microspheres in EP tubes, mark them, and put them on ice for later use. Take 4 1mL EP tubes, add 2 blank microspheres and 1mL30μg / mL cephalexin to the first tube, and add 2 enzyme-containing microspheres and 1mL30μg / mL cephalexin to the other three tubes. Quickly put the four EP tubes into a 40℃ water bath to react for 3min, quickly measure the absorbance value in a UV spectrophotometer, and record the data. The 1.0%, 1.5%, 2.0%, and 2.5% sodium alginate experimental groups were carried out in the same way.

[0091] The experimental results are as follows Figure 1 As shown, in 2% CaCl 2 Under the condition of fixation for 10 minutes, the activity of the enzyme showed an upward trend when it was fixed with sodium alginate at a final concentration of 0.5%-1.5%, the enzyme activity was the best when it was fixed with sodium alginate at a final concentration of 1.5%, and the activity gradually decreased when it was fixed with sodium alginate at a final concentration of 1.5%-2.5%.

[0092] 1.2CaCl 2 Concentration optimization:

[0093] (1) Prepare 5 mL of a mixture of sodium alginate (final concentration 1.5%) and mutant enzyme solution: 2.5 mL of 3.0% sodium alginate, 2 mL of mutant enzyme solution, and 0.5 mL of 20 mM Tris-HCl (pH 7.0); prepare 1 mL of a blank group mixture: 0.5 mL of 3.0% sodium alginate and 0.5 mL of 20 mM Tris-HCl (pH 7.0).

[0094] (2) Take 0.75 mL of 1 mg / mL cephalexin solution in a 50 mL centrifuge tube and add 24.25 mL of 20 mM Tris-HCl (pH 7.0) to dilute the cephalexin to 30 μg / mL.

[0095] (3) Take two small beakers and mark them as blank and experimental groups. Add 5 mL of 0.5% CaCl2 Solution, slowly drip the above 1.5% mixed solution into the beaker with a syringe 1mm away from the liquid surface, let it stand for 10min, rinse it with 20mMTris-HCl (pH7.0) 3 times, store the microspheres in EP tubes, mark them, and put them on ice for later use. Take 4 1mL EP tubes, add 2 blank microspheres and 1mL30μg / mL cephalexin to the first tube, and add 2 enzyme-containing microspheres and 1mL30μg / mL cephalexin to the other three tubes. Quickly put the four EP tubes into a 40℃ water bath to react for 3min, quickly measure the absorbance value in a UV spectrophotometer, and record the data.

[0096] The experimental results are as follows Figure 2 As shown, when fixed in 1.5% sodium alginate for 10 min, the enzyme 2 The enzyme activity is better when fixed under the conditions.

[0097] Example 3 Experiments with different immobilization times

[0098] (1) Prepare a mixture of 2 mL of sodium alginate (final concentration 1.5%) and mutant enzyme solution: 1.0 mL of 3.0% sodium alginate, 0.8 mL of mutant enzyme solution, and 0.2 mL of 20 mM Tris-HCl (pH 7.0).

[0099] (2) Prepare 1 mL of blank mixed solution: 0.5 mL of 3.0% sodium alginate and 0.5 mL of 20 mM Tris-HCl (pH 7.0).

[0100] (3) Take 0.75 mL of 1 mg / mL cephalexin solution in a 50 mL centrifuge tube and add 24.25 mL of 20 mM Tris-HCl (pH 7.0) to dilute the cephalexin to 30 μg / mL.

[0101] (4) Take two small beakers and mark them as blank and experimental groups. Add 5 mL of 1.0% CaCl 2 Solution, slowly drip the above 1.5% mixed solution into the beaker with a syringe at a distance of 1mm from the liquid surface. After standing for 5min, rinse with 20mM Tris-HCl (pH7.0) 3 times and store the microspheres in EP tubes, mark them, and put them on ice for later use. Take 4 1mL EP tubes, add 2 blank microspheres and 1mL30μg / mL cephalexin to the first tube, and add 2 enzyme-containing microspheres and 1mL30μg / mL cephalexin to the other three tubes. Quickly put the four EP tubes into a 40℃ water bath to react for 3 minutes, quickly measure the absorbance value in a UV spectrophotometer, and record the data. The 10min, 30min, 60min, and 90min experimental groups were carried out in the same way.

[0102] The experimental results are as follows Figure 3 As shown, in 1.5% sodium alginate, 1% CaCl 2 Under the given conditions, the enzyme activity was the best when the fixation time was 10 minutes. As the fixation time increased, the enzyme activity gradually decreased. When the fixation time was 90 minutes, the relative enzyme activity was less than 30%.

[0103] Example 4 Immobilized Enzyme Properties

[0104] 1.1 Optimum temperature

[0105] (1) Prepare a mixture of 3 mL of sodium alginate (final concentration 1.5%) and mutant enzyme solution: 1.5 mL of 3.0% sodium alginate, 1.2 mL of mutant enzyme solution, and 0.3 mL of 20 mM Tris-HCl (Ph7.0).

[0106] (2) Prepare 1 mL of blank mixed solution: 0.5 mL of 3.0% sodium alginate and 0.5 mL of 20 mM Tris-HCl (pH 7.0).

[0107] (3) Take 1.5 mL of 1 mg / mL cephalexin solution in a 50 mL centrifuge tube and add 48.5 mL of 20 mM Tris-HCl (pH 7.0) to dilute the cephalexin to 30 μg / mL.

[0108] (4) Take two small beakers and mark them as blank and experimental groups. Add 5 mL of 1.0% CaCl 2 Solution, slowly drip the above 1.5% mixed solution into the beaker with a syringe at a distance of 1mm from the liquid surface. After standing for 10 minutes, rinse with 20mMTris-HCl (pH7.0) three times and store the microspheres in EP tubes, mark them, and put them on ice for later use. Take 4 1mL EP tubes, add 1mL 30μg / mL cephalexin to them, preheat at 10℃ for 5 minutes, then quickly add 2 blank microspheres to the first tube, add 2 enzyme-containing microspheres to the other three tubes, and then quickly put the four EP tubes into a 10℃ water bath to react for 15 minutes, quickly measure the absorbance value in the UV spectrophotometer, and record the data. The same method was used for the experimental groups at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃. The experimental results are as follows Figure 4 As shown, the activity of the enzyme gradually increased at 10-50°C, reached the highest activity at 50°C, gradually decreased at 50-80°C, and could not be detected at 80-100°C.

[0109] 1.2 Optimum pH experiment

[0110] (1) Prepare a mixture of 3 mL of sodium alginate (final concentration 1.5%) and mutant enzyme solution: 1.5 mL of 3.0% sodium alginate, 1.2 mL of mutant enzyme solution, and 0.3 mL of 20 mM Tris-HCl (Ph7.0).

[0111] (2) Prepare 1 mL of blank mixed solution: 0.5 mL of 3.0% sodium alginate and 0.5 mL of 20 mM Tris-HCl (pH 7.0).

[0112] (3) Take nine 10 mL centrifuge tubes, add 0.15 mL of 1 mg / mL cephalexin solution to each tube, and then add 9.85 mL of nine pH buffers to dilute the cephalexin to 30 μg / mL.

[0113] (4) Take two small beakers and mark them as blank and experimental groups. Add 5 mL of 1.0% CaCl 2 Solution, slowly drip the above 1.5% mixed solution into the beaker with a syringe at a distance of 1mm from the liquid surface. After standing for 10 minutes, rinse with 20mMTris-HCl (pH7.0) 3 times and store the microspheres in EP tubes, mark them, and put them on ice for later use. Take 4 1mL EP tubes, add 2 blank microspheres and 1mL of the above pH3.0 cephalexin solution to the first tube, and add 2 enzyme-containing microspheres and 1mL of the above pH3.0 cephalexin solution to the other three tubes. Quickly place the four EP tubes in a 40℃ water bath to react for 15 minutes, measure the absorbance in a UV spectrophotometer, and record the data. The same method was used for the pH4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 experimental groups. The experimental results are as follows Figure 5 As shown, the enzyme activity was undetectable at pH 3.0, the enzyme activity showed an increasing trend at pH 4.0-5.0, and gradually decreased at pH 5.0-11.0, but the relative activity was above 60%.

[0114] Example 5 Removal Experiment

[0115] (1) Prepare 10 mL of a mixture of sodium alginate (final concentration 1.5%) and mutant enzyme solution: 5.0 mL of 3.0% sodium alginate, 4.0 mL of mutant enzyme solution, and 1.0 mL of 20 mM Tris-HCl (pH 7.0).

[0116] (2) Prepare 2 mL of blank mixed solution: 1.0 mL of 3.0% sodium alginate and 1.0 mL of 20 mM Tris-HCl (pH 7.0).

[0117] (3) Take two small beakers and mark them as blank and experimental groups. Add 5 mL of 1.0% CaCl 2Solution, slowly drip the above 1.5% mixed solution into the beaker with a syringe at a distance of 1mm from the liquid surface. After standing for 10 minutes, rinse with 20mM Tris-HCl (pH7.0) for 3 times and store the microspheres in EP tubes, mark them and keep them on ice for later use.

[0118] 1.1 10 μg / mL Cephalexin Removal Experiment

[0119] (1) Take a 50 mL centrifuge tube and add 0.5 mL of 1 mg / mL cephalexin solution to the tube. Then add 49.5 mL of 20 mM Tris-HCl (pH 7.0) to dilute the cephalexin to 10 μg / mL.

[0120] (2) Take 4 1mL EP tubes, add 1mL 30μg / mL cephalexin into each tube, quickly measure the absorbance in a UV spectrophotometer, and record the value at 0min. Take 4 1mL EP tubes, add 2 blank microspheres and 1mL 10μg / mL cephalexin to the first tube, and add 2 enzyme-containing microspheres and 1mL 10μg / mL cephalexin to the other three tubes. Quickly place the 4 EP tubes in a 40℃ water bath to react for 1min, then quickly measure the absorbance in a UV spectrophotometer and record the data. Use the same method to measure the absorbance after 3, 5, 7, 9, 11, 13, 15, and 30min of reaction, and record the data. The experimental results are as follows: Figure 6 As shown, the enzyme rapidly degraded 10 μg / mL of cephalexin within 0-5 min, causing the antibiotic residue rate to drop rapidly to about 10%. At 11 min, the degradation rate of 10 μg / mL of cephalexin reached more than 99%.

[0121] 1.2 30 μg / mL Cephalexin Removal Experiment

[0122] (1) Take a 50 mL centrifuge tube and add 1.5 mL of 1 mg / mL cephalexin solution to the tube. Then add 48.5 mL of 20 mM Tris-HCl (pH 7.0) to dilute the cephalexin to 30 μg / mL.

[0123] (2) Take 4 1mL EP tubes, add 1mL 30μg / mL cephalexin into each tube, quickly measure the absorbance in a UV spectrophotometer, and record the value at 0min. Take 4 1mL EP tubes, add 2 blank microspheres and 1mL 30μg / mL cephalexin to the first tube, and add 2 enzyme-containing microspheres and 1mL 30μg / mL cephalexin to the other three tubes. Quickly place the four EP tubes in a 40℃ water bath to react for 1min, then quickly measure the absorbance in a UV spectrophotometer and record the data. Use the same method to measure the absorbance after 3, 5, 7, 9, 11, 13, 15, and 30min of reaction, and record the data. The experimental results are as follows. Figure 7 As shown, the enzyme degraded 30 μg / mL of cephalexin at a relatively uniform rate within 0-15 min. At 30 min, the degradation rate of 30 μg / mL of cephalexin reached more than 99%.

[0124] 1.3 50 μg / mL Cephalexin Removal Experiment

[0125] (1) Take a 50 mL centrifuge tube and add 2.5 mL of 1 mg / mL cephalexin solution to the tube. Then add 47.5 mL of 20 mM Tris-HCl (pH 7.0) to dilute the cephalexin to 50 μg / mL.

[0126] (2) Take 4 1mL EP tubes, add 1mL 30μg / mL cephalexin into each tube, quickly measure the absorbance in a UV spectrophotometer, and record the value at 0min. Take 4 1mL EP tubes, add 2 blank microspheres and 1mL 50μg / mL cephalexin to the first tube, and add 2 enzyme-containing microspheres and 1mL 50μg / mL cephalexin to the other three tubes. Quickly place the four EP tubes in a 40℃ water bath to react for 1min, then quickly measure the absorbance in a UV spectrophotometer and record the data. Use the same method to measure the absorbance after 3, 5, 7, 9, 11, 13, 15, and 30min of reaction, and record the data. The experimental results are as follows. Figure 8 As shown, the enzyme degraded 50 μg / mL of cephalexin at a relatively uniform rate within 0-15 min, reducing its residual rate to about 30%, and reduced the residual rate of 50 μg / mL of cephalexin to below 10% in 30 min.

[0127] The present invention discloses a C family lactamase mutant capable of degrading cephalexin, and uses the immobilized enzyme to remove cephalexin from chicken farm wastewater. The lactamase mutant is immobilized with sodium alginate, and the immobilization conditions are optimized. It is found that the optimal immobilization conditions of the enzyme are: final concentration of 1.5% sodium alginate, 1.0% CaCl 2， Fix for 10 minutes.

[0128] The C family lactamase mutant was immobilized under the optimal immobilization conditions and the enzymatic properties of the enzyme were studied. It was found that the optimal temperature of the immobilized enzyme was 50°C and the optimal pH was 5.0. The immobilized C family lactamase mutant was used to perform a removal experiment on cephalexin at different concentrations. The present invention utilizes the C family lactamase mutant to degrade cephalexin.

[0129] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0130] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A family C lactamase mutant, characterized in that: It is mutant 1, and the mutant 1 is based on the amino acid sequence shown in SEQ ID NO: 1, with Ser at position 71 mutated to Arg, Asn at position 133 mutated to Lys, Gln at position 144 mutated to Lys, and Trp at position 265 mutated to Cys; its amino acid sequence is shown in SEQ ID NO:

2.

2. A method for immobilizing a family C lactamase mutant according to claim 1, characterized in that: The sodium alginate carrier and the C family lactamase mutant solution were evenly mixed and then dropped into a CaCl2 solution for fixation to obtain an immobilized C family lactamase mutant.

3. The method for immobilizing a family C lactamase mutant according to claim 2, characterized in that: The immobilization conditions were: final concentration 1.5% sodium alginate, 1.0% CaCl 2， Fix for 10 minutes.

4. Use of the immobilized family C lactamase mutant according to any one of claims 2-3 in the degradation of cephalexin.

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