A B family lactamase mutant and its immobilization method and application

Abstract

The present invention discloses a B family lactamase mutant and an immobilization method and application thereof, wherein the sequence of the B family lactamase mutant is as described in any one of SEQ ID NOs: 1-5. The B family 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 1.5% sodium alginate, 1.0% CaCl2, and fixation for 10 minutes. The B family lactamase mutant is fixed under the optimal immobilization conditions and the enzymatic properties of the enzyme are studied. It is found that the optimum temperature of the immobilized enzyme is 30°C and the optimum pH is 7.0. The present invention finds that the removal rate of ampicillin degradation by the B family lactamase mutant can reach more than 95%, and has broad application prospects.

Description

Technical Field

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

[0002] Ampicillin, a type of lactam antibiotic, has been widely used around the world due to its wide antibacterial range and effective therapeutic properties. This antibiotic is mainly produced through microbial metabolism or artificial synthesis, and is mainly used for the treatment and prevention of animal diseases and to promote the growth and development of animals. However, the widespread use of ampicillin has had a significant impact on the environment: inadequately treated ampicillin is discharged into the environment through waste and sewage from the livestock and poultry industry, resulting in its long-term accumulation in the natural environment, contributing to the widespread distribution of drug-resistant genes and bacteria, which has aroused widespread concern about the environment and public health.

[0003] It is particularly noteworthy that, as a common beta-lactam antibiotic, the residue of ampicillin in the environment not only threatens the safety of water sources, but may also aggravate the development of drug-resistant bacteria. Therefore, the development of an efficient ampicillin removal method is crucial for environmental protection. At present, biodegradation, especially enzyme-catalyzed degradation technology, is considered to be a powerful means to deal with this type of pollution due to its advantages of low cost, high efficiency and no secondary pollution. However, the prior art lacks an enzyme with good stability, high catalytic activity and the ability to effectively degrade ampicillin. 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 B 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 family B lactamase mutant is provided, which is any of the following mutants:

[0007] Mutant 1, wherein the mutant 1 is based on the amino acid sequence shown in SEQ ID NO: 1, with Thr at position 107 mutated to Met, Thr at position 113 mutated to Ala, Phe at position 141 mutated to Tyr, and Val at position 160 mutated to Leu; the amino acid sequence of the 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 Leu at position 79 mutated to Val, Ala at position 84 mutated to Pro, and Tyr at position 148 mutated to His; the amino acid sequence of the mutant 2 is shown in SEQ ID NO: 3;

[0009] Mutant 3, wherein, based on the amino acid sequence shown in SEQ ID NO: 1, Leu at position 132 is mutated to Val, Ala at position 138 is mutated to Asp, Val at position 139 is mutated to Leu, His at position 153 is mutated to Gln, Asn at position 157 is mutated to Ser, and Cys at position 172 is mutated to Trp; the amino acid sequence of 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, lle at position 57 is mutated to Asn, Leu at position 79 is mutated to Pro, Thr at position 107 is mutated to Arg, Tyr at position 108 is mutated to Cys, Leu at position 129 is mutated to Val, Leu at position 158 is mutated to Val, Thr at position 180 is mutated to Ser, and Ala at position 207 is mutated to Gly; the amino acid sequence of mutant 4 is shown in SEQ ID NO: 5;

[0011] Mutant 5, based on the amino acid sequence shown in SEQ ID NO: 1, Ala at position 68 is mutated to Ser, Tyr at position 108 is mutated to Phe, Glu at position 192 is mutated to Gln, and Gly at position 213 is mutated to Glu; its amino acid sequence is shown in SEQ ID NO: 6.

[0012] In a second aspect, a method for immobilizing a family B lactamase mutant is provided, wherein a sodium alginate carrier and a family B lactamase mutant solution are evenly mixed and then dropped into a CaCl2 solution for fixation to obtain an immobilized family B 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 using an immobilized B family lactamase mutant in the degradation of ampicillin is provided.

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

[0016] The present invention discloses a B family lactamase mutant and an immobilization method and application thereof. The B family lactamase mutant provided by the present invention is an enzyme with good stability and high catalytic activity and can effectively degrade ampicillin, thereby expanding the types of enzymes that can be used for metal B-lactam compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the relative activity of the immobilized enzyme VIM-1 mutant under the condition of changing the sodium alginate concentration while keeping other conditions unchanged;

[0018] Figure 2 It is a schematic diagram of the relative activity of the immobilized enzyme VIM-1 mutant under the condition of changing the CaCl2 concentration while keeping other conditions unchanged;

[0019] Figure 3 It is a schematic diagram of the relative activity of the enzyme VIM-1 mutants when other conditions remain unchanged and the immobilization time is changed;

[0020] Figure 4 It is a schematic diagram of the optimal action temperature of the immobilized enzyme VIM-1 mutant;

[0021] Figure 5 This is a schematic diagram of the optimal action pH of the immobilized enzyme VIM-1 mutant;

[0022] Figure 6 This is a diagram showing the relationship between the relative residual rate of ampicillin and time, showing the degradation experiment of 50 μg / mL, 100 μg / mL, and 150 μg / mL ampicillin by VIM-1 mutant-immobilized microspheres. DETAILED DESCRIPTION

[0023] 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.

[0024] (1) Deionized water

[0025] (2) Water bath

[0026] (3) 1.2mm bore needle and 1mL syringe

[0027] (4) Timer

[0028] (5) Funnel

[0029] (6) Beaker

[0030] (7) Graduated cylinder

[0031] (8) Snowflake Ice Machine

[0032] (9) Ice Box

[0033] (10) Pipette

[0034] (11) Gun head

[0035] (12) UV spectrophotometer

[0036] (13)Quartz cuvette

[0037] (14) 1mL EP tube

[0038] (15) 10mL centrifuge tube

[0039] (16) 50mL centrifuge tube

[0040] (17)EP pipe rack

[0041] (18) Centrifuge tube rack

[0042] (19) Long-handled spoon

[0043] (20) pH meter

[0044] (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.

[0045] (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.

[0046] (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.

[0047] (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.

[0048] (25) 5x Loading Buffer: Component concentrations: 1.00M Tris-HCl (pH 6.8), 10% SDS, 0.5% (W / V) BPB, 50% (V / V) glycerol, 5% (W / V) 2-ME. Measure 1.25mL 1M Tris-HCl (pH 6.8), 5mL SDS, 25mg BPB, 2.5mL glycerol, and 250uL 2-ME into a 15mL plastic centrifuge tube, add deionized water to dissolve, dilute to 5mL, divide into small portions (0.5mL / portion), and store in a refrigerator at 4℃ for later use.

[0049] (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.

[0050] (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.

[0051] (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.

[0052] (29) Buffer A (20mmol / L Tris, 500mmol / L NaCl, pH 7.0): Weigh 2.42g Tris and 29.25g NaCl into a 1L reagent bottle, add 0.8L deionized water to dissolve, adjust the pH to 7.0 with 30% HCl solution and a pH meter, make up to 1L with deionized water, and filter with a 0.2μm water filter.

[0053] (30) Buffer B (20mmol / L Tris, 500mmol / L NaCl, 500mmol / L imidazole, pH=7.0): weigh 2.42g Tris, 29.25g NaCl, and 34.04g imidazole in a 1L reagent bottle, add 0.8L deionized water to dissolve, adjust the pH to 7.0 with 30% HCl solution and a pH meter, make up to 1L with deionized water, and filter with a 0.2μm water filter.

[0054] (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.

[0055] (32) 20 mmol / L imidazole: Weigh 0.27 g imidazole, 0.48 g Tris and 5.85 g NaCl, dissolve 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.

[0056] (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.

[0057] (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.

[0058] (35)Plasmid extraction kit

[0059] (36) Preparation of pH buffer: Prepare glycine-hydrochloric acid 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 = 2.9-3.1, and add purified water to make up to 10 mL. Prepare pH=4.0 and 5.0 buffers in the same way; prepare PBS buffer (pH=6.0): take 1.23mL 0.2mol / L disodium hydrogen phosphate and 8.77mL 0.2mol / L sodium dihydrogen phosphate in a 50mL centrifuge tube; prepare Tris-HCl buffer (pH=7.0, 8.0, 9.0): weigh 2.42g Tris in a 1000mL reagent bottle, dissolve it in 800mL pure water, adjust the pH to 7.0 with HCl, and add pure water to 1000mL; weigh 0.48g Tris in a 50mL reagent bottle, dissolve it in 16mL pure water, adjust the pH to 8.0 with HCl, and add pure water to 20mL; weigh 0.48g Dissolve Tris in a 50mL reagent bottle with 16mL pure water, adjust the pH to 9.0 with HCl, and add pure water to 20mL; prepare glycine-sodium hydroxide buffer (pH = 10.0, 11.0): take 2.5mL 0.2mol / L glycine solution in a 50mL centrifuge tube, add 0.2mol / L sodium hydroxide solution to pH = 10.0. Prepare pH = 11.0 buffer in the same way.

[0060] (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 pure water, 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 pure water, and stir until completely dissolved.

[0061] (38) Preparation of CaCl2 solution:

[0062] To prepare 0.5% CaCl2 solution, weigh 0.10 g of CaCl2 into a 50 mL centrifuge tube and dissolve it in 20 mL of pure water;

[0063] To prepare 1.0% CaCl2 solution, weigh 0.20 g of CaCl2 into a 50 mL centrifuge tube and dissolve it in 20 mL of pure water;

[0064] To prepare 1.5% CaCl2 solution, weigh 0.30 g of CaCl2 into a 50 mL centrifuge tube and dissolve it in 20 mL of pure water;

[0065] Prepare 2.0% CaCl2 solution by weighing 0.40 g of CaCl2 into a 50 mL centrifuge tube and dissolving it with 20 mL of pure water;

[0066] Prepare 2.5% CaCl2 solution by weighing 0.50 g CaCl2 into a 50 mL centrifuge tube and dissolving it with 20 mL pure water.

[0067] (39) 500 ug / mL, 5 mg / mL ampicillin: Weigh 0.0100 g and 1.00 g of ampicillin respectively into a 50 mL centrifuge tube, dissolve in 20 mL of 20 mM Tris-HCl (pH = 7.0), and place on ice until used.

[0068] Example 1

[0069] 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, place it in a 37°C shaker for 8-12 hours, collect the bacteria by centrifugation, and extract the plasmid according to the steps in the instructions of the plasmid extraction kit.

[0070] Conversion:

[0071] (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.

[0072] (2) Add 5 μL of the ligated plasmid to 100 μL of competent medium, shake gently, and place on ice for 30 min.

[0073] (4) 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.

[0074] (5) In a clean bench, add 800uL of LB medium to each tube and mix gently. Then fix it on a 37°C shaker and shake for 1 hour.

[0075] (6) 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.

[0076] (7) 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.

[0077] Induced expression: Pick a positive monoclone and culture it in 5 mL LB medium at 37°C overnight as seed solution. Take 100uL of seed solution and inoculate it into 100 mL medium (250 mL conical flask), culture it in a shaking incubator at 37°C until the OD value is 0.6-0.8, then add IPTG (0.5mM) for induction, the induction temperature is 25°C, the induction time is 6h, and B-lactamase VIM-1 and mutants are obtained.

[0078] Protein purification

[0079] (1) After induction, the bacterial liquid was centrifuged to collect the bacterial cells. The centrifugation conditions were: 6500 rpm, 5 min.

[0080] (2) Resuspending and breaking the bacterial cells: Resuspend the centrifuged bacterial liquid in Buffer A and place it in a cell-breaking machine to break the bacterial cells until the resuspended liquid is translucent.

[0081] (3) After the cell wall of the resuspension is broken, the aliquots are centrifuged and the supernatant is collected.

[0082] (4) Column Packing

[0083] (5) Buffer A balance

[0084] (6) Sample loading

[0085] (7) Buffer A rinse

[0086] (8) 50mM imidazole to wash out impurities

[0087] (9) Elute the target protein with 200 mM imidazole and collect the target protein eluate.

[0088] (10) Buffer B rinse

[0089] (11) Table Relative enzyme activities of Vim-1 and its different mutants

[0090] Protein name Relative enzyme activity VIM-1 100％ VIM-1(T107M-T113A-F141Y-V160L) 21％ VIM-1(L79V-A84P-Y148H) 105％ VIM-1(L132V-A138D-V139L-H153Q-N157S-C172W) 113％ VIM-1(I57N-L79P-T107R-Y108C-L129V-L158V-T180S-A207G) 108％ VIM-1(A68S-Y108F-E192Q-G213E) 121％

[0091] The following examples were tested using mutants with higher enzyme activity.

[0092] Example 2 Immobilization Optimization

[0093] 1.1 Optimization of sodium alginate concentration:

[0094] (1) Preparation of blank group and experimental group microspheres

[0095] The final concentrations of sodium alginate were 2.5%, 2%, 1.5%, 1%, and 0.5%, respectively. The following are the different proportions added to 1 mL of sodium alginate-enzyme mixture of different concentrations:

[0096] 2.5% SA: 600μL 4.17% sodium alginate solution + 400μL mutant enzyme solution

[0097] 2% SA: 479.6 μL 4.17% sodium alginate solution + 120.4 μL Tris-HCl (pH=7.0) buffer + 400 μL mutant enzyme solution

[0098] 1.5% SA: 500 μL 3% sodium alginate solution + 100 μL Tris-HCl (pH=7.0) buffer + 400 μL mutant enzyme solution

[0099] 1% SA: 333 μL 3% sodium alginate solution + 267 μL Tris-HCl (pH=7.0) buffer + 400 μL mutant enzyme solution

[0100] 0.5% SA: 167 μL 3% sodium alginate solution + 433 μL Tris-HCl (pH=7.0) buffer + 400 μL mutant enzyme solution

[0101] For the blank group, the mutant enzyme solution was replaced with Tris-HCl (pH = 7.0) buffer.

[0102] Preparation of microspheres: prepare 5 culture dishes, add 5 mL of 2.0% CaCl2 solution into the dishes, and slowly drip the above sodium alginate-enzyme mixture into the culture dishes containing CaCl2 solution 1 mm from the liquid surface with a syringe. After standing for 10 minutes to immobilize, rinse with 20 mM Tris-HCl (pH = 7.0) three times and store the microspheres in EP tubes, mark them, and put them on ice for later use. The blank group is operated in the same way as above.

[0103] (2) Detection

[0104] The final concentration of the substrate (ampicillin) is fixed at 150 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group of experiments, for a total of four groups, i.e., 4 EP tubes as one group of experiments. Number the EP tubes and mark them. Use a 1 mL pipette to draw 700 μL Tris-HCl (pH = 7.0) buffer into the EP tube, then draw 300 μL 500 μg / mL ampicillin into the EP tube, preheat it in a 40°C water bath for two minutes, take out the EP tube after preheating, take out the prepared blank group and experimental group microspheres, quickly add 3 microspheres, and immediately put it back into the 40°C water bath to react for 3 minutes after adding the microspheres. Use a spectrophotometer with a wavelength of 235 nm to detect the corresponding OD value of each group and record it. Repeat the above operation for each group of experiments. After obtaining the data, perform calculations.

[0105] according to Figure 1As shown, the relative activity of the enzyme after immobilization showed a normal distribution when the sodium alginate concentration changed in the range of 0.5%-2.5%. At 1.5%, the relative activity of the enzyme was the highest. Under the conditions of other sodium alginate concentrations, the relative enzyme activity of the immobilized VIM-1 mutant enzyme solution was above 70%. That is, the optimal concentration of sodium alginate for the immobilization of the VIM-1 mutant enzyme solution was 1.5%.

[0106] 1.2 Optimization of CaCl2 concentration

[0107] (1) Preparation of blank group and experimental group microspheres

[0108] The final concentration of sodium alginate is 1.5%, and the immobilization time is fixed at 10 min. Because the CaCl2 concentration is different during immobilization, the prepared CaCl2 concentration is 3%. Different concentrations of CaCl2 need to be diluted with purified water. The following gives the dilution ratios of 10 mL of different concentrations of CaCl2:

[0109] 2.5%CaCl2: 10mL 2.5%CaCl2

[0110] 2% CaCl2: 8 mL 2.5% CaCl2 + 2 mL purified water

[0111] 1.5% CaCl2: 6mL 2.5% CaCl2 + 4mL purified water

[0112] 1% CaCl2: 4mL 2.5% CaCl2 + 6mL purified water

[0113] 0.5% CaCl2: 2 mL 2.5% CaCl2 + 8 mL purified water

[0114] For example, 1 mL of sodium alginate-enzyme mixture was prepared: 500 μL of 3% sodium alginate solution + 400 μL of mutant enzyme solution + 100 μL of 20 mM Tris-HCl (pH = 7.0) buffer; for the blank group, the mutant enzyme solution was replaced with 20 mM Tris-HCl (pH = 7.0) buffer.

[0115] Preparation of microspheres: Prepare 5 culture dishes, pour different concentrations of CaCl2 solution into the culture dishes respectively, and slowly drip the above sodium alginate-enzyme mixture into the culture dishes containing different concentrations of CaCl2 with a syringe 1mm away from the liquid surface. After standing for 10 minutes, rinse with 20mM Tris-HCl (pH=7.0) for 3 times, store the microspheres in EP tubes, mark them, and put them on ice for standby. The blank group is operated in the same way as above. Repeat the above operation for different concentrations of CaCl2 experimental groups and blank groups, and the preparation is completed. The microspheres are placed on ice for standby.

[0116] (2) Detection: The final concentration of the substrate (ampicillin) is fixed at 150 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group of experiments, for a total of four groups, i.e., 4 EP tubes as one group of experiments. Number the EP tubes and mark them. Use a 1 mL pipette to draw 700 μL Tris-HCl (pH = 7.0) buffer into the EP tube, then draw 300 μL 500 μg / mL ampicillin into the EP tube, preheat it in a 40°C water bath for two minutes, take out the EP tube after preheating, take out the prepared blank group and experimental group microspheres, quickly add 3 microspheres, and immediately put it back into the 40°C water bath for reaction for 3 minutes. Use a spectrophotometer with a wavelength of 235 nm to detect the corresponding OD value of each group and record it. Repeat the above operation for each group of experiments. After obtaining the data, perform calculations.

[0117] according to Figure 2 As shown, when the CaCl2 concentration is 1.0%, the relative activity of the enzyme is the highest; at 1.5% and 2.0%, the relative enzyme activities of the two groups of immobilized VIM-1 are close to each other, about 80%, and at 0.5% and 2.5%, the relative enzyme activities of the immobilized VIM-1 are close to each other, about 60%. That is, the optimal CaCl2 concentration for immobilizing the VIM-1 mutant enzyme is 1.0%.

[0118] 1.3 Optimization of the optimal fixed time

[0119] (1) Preparation of blank group and experimental group microspheres

[0120] The final concentration of sodium alginate was 1.5%, and the concentration of CaCl2 was 1%. Because the immobilization time was different, a timer was prepared to collect the microspheres at 5min, 10min, 30min, 60min, and 90min. The proportion of the sodium alginate-enzyme mixture remained unchanged, such as preparing 1mL of sodium alginate-enzyme mixture, 500μL 3% sodium alginate solution + 400μL enzyme + 100μL 20mM Tris-HCl (pH=7.0) buffer; for the blank group, the enzyme was replaced with 20mM Tris-HCl (pH=7.0) buffer.

[0121] (2) Detection: The final concentration of the substrate (ampicillin) is fixed at 150 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group of experiments, for a total of four groups, i.e., 4 EP tubes as one group of experiments. Number the EP tubes and mark them. Use a 1 mL pipette to draw 700 μL Tris-HCl (pH = 7.0) buffer into the EP tube, then draw 300 μL 500 μg / mL ampicillin into the EP tube, preheat it in a 40°C water bath for two minutes, take out the EP tube after preheating, take out the prepared blank group and experimental group microspheres, quickly add 3 microspheres, and immediately put it back into the 40°C water bath for reaction for 3 minutes. Use a spectrophotometer with a wavelength of 235 nm to detect the corresponding OD value of each group and record it. Repeat the above operation for each group of experiments. After obtaining the data, perform calculations.

[0122] according to Figure 3 As shown, when the immobilization time is 10 minutes, the relative activity of the enzyme is the highest; when the immobilization time is 5 minutes and 10 minutes, the activity expressed by the immobilized VIM-1 mutant enzyme is relatively high. When the immobilization time exceeds 30 minutes, the relative activity expressed by the immobilized VIM-1 mutant enzyme is significantly reduced, less than 20%. That is, the optimal immobilization time for the immobilization of the VIM-1 mutant enzyme is 10 minutes.

[0123] Example 3 Immobilized Enzyme Properties

[0124] 1.1 Optimum temperature

[0125] (1) Preparation of blank group and experimental group microspheres

[0126] From the first three experiments, the optimal conditions for VIM-1 immobilization are: 1.5% sodium alginate, 1% CaCl2, and a fixation time of 10 min; if 1 mL of sodium alginate-enzyme mixture is prepared, 500 μL of 3% sodium alginate solution + 400 μL of enzyme + 100 μL of 20 mM Tris-HCl (pH = 7.0) buffer; for the blank group, the enzyme is replaced with 20 mM Tris-HCl (pH = 7.0) buffer.

[0127] Preparation of microspheres: prepare 5 culture dishes, pour 1% CaCl2 solution into the culture dishes, slowly drip the above 1.5% sodium alginate-enzyme mixture into the culture dishes containing 1% CaCl2 solution 1mm away from the liquid surface with a syringe, let it stand for 10 minutes to fix, then rinse it with 20mM Tris-HCl (pH=7.0) for 3 times and store the microspheres in EP tubes, mark them, and put them on ice for later use. Perform the same operation for the blank group.

[0128] (2) Detection:

[0129] The final concentration of the substrate (ampicillin) was fixed at 150 μg / mL. Prepare an EP tube rack and 1.5 mL EP tubes. Perform three parallel experiments and one blank experiment for each group of experiments, for a total of four groups, i.e., 4 EP tubes per group of experiments. Number the EP tubes and mark them well.

[0130] It is necessary to detect the activity of the enzyme at different temperatures after immobilization. It is necessary to conduct the experiment at a temperature gradient of 10°C to 100°C. Use a 1mL pipette to draw 700μL 20mM Tris-HCl (pH=7.0) buffer into the EP tube, and then draw 300μL 500μg / mL ampicillin into the EP tube. Put it in the experimental temperature environment for preheating for two minutes. After preheating, take out the prepared blank group and experimental group microspheres, quickly add 3 microspheres, and immediately put it back into the experimental temperature environment after adding the microspheres for reaction timing for 10 minutes. Use a spectrophotometer with a wavelength of 235nm to detect the corresponding OD value of each group and record it. Repeat the above operation for each group of experiments. After obtaining the data, perform calculation processing.

[0131] according to Figure 4 As shown, the relative activity of VIM-1 after immobilization at 10℃-30℃ increased linearly and dramatically. At 30℃, the relative activity of the enzyme was the highest. At 40℃ to 60℃, the relative activity of VIM-1 after immobilization tended to be consistent, with a slight downward trend, but the relative activity was >80%. When the temperature was >60℃, the activity of the enzyme decreased significantly. That is, the optimal reaction temperature for the immobilized VIM-1 mutant enzyme was 30℃.

[0132] 1.2 Optimum pH

[0133] (1) Preparation of blank group and experimental group microspheres

[0134] From the first three experiments, the optimal conditions for VIM-1 immobilization are: 1.5% sodium alginate, 1% CaCl2, and a fixation time of 10 min. If 1 mL of sodium alginate-enzyme mixture is prepared, 500 μL of 3% sodium alginate solution + 400 μL of enzyme + 100 μL of 20 mM Tris-HCl (pH = 7.0) buffer; for the blank group, the enzyme is replaced with 20 mM Tris-HCl (pH = 7.0) buffer.

[0135] Preparation of microspheres: prepare 5 culture dishes, pour 1% CaCl2 solution into the culture dishes, slowly drip the above 1.5% sodium alginate-enzyme mixture into the culture dishes containing 1% CaCl2 solution 1mm away from the liquid surface with a syringe, let it stand for 10 minutes, rinse it with 20mM Tris-HCl (pH=7.0) for 3 times, then store the microspheres in EP tubes, mark them, and put them on ice for later use. Perform the same operation for the blank group.

[0136] (2) Detection:

[0137] The final concentration of the substrate (ampicillin) is 150 μg / mL. Since it is necessary to detect the activity of the immobilized enzyme under different pH environments, high-concentration ampicillin (5 mg / mL ampicillin) is diluted with each buffer to make the final concentration of the substrate 150 μg / mL, and the dilution ratio is 1 mL: 30 μL ampicillin + 970 μL buffer. Three parallel experiments and one blank experiment are conducted for each group of experiments, a total of four groups. To ensure that factors other than pH have an impact, the substrate and buffer conditions need to be the same for the same group of experiments. Prepare 15 mL centrifuge tubes, dilute according to the ratio, and make good marks after dilution and store them on ice for later use (to prevent antibiotic degradation).

[0138] After preparing different pH environments and EP tubes, use a 1 mL pipette to aspirate 1 mL of the diluted ampicillin-buffer into the EP tube. Take out the prepared blank group and experimental group microspheres, quickly add 3 microspheres with a small long spoon, and immediately put them back into a 30 °C water bath for reaction timing for 10 min. Use a spectrophotometer with a wavelength of 235 nm to detect the corresponding OD value of each group and record it. Repeat the above operations for each group of experiments. After obtaining the data, perform calculation and processing.

[0139] According to Figure 5 As shown, when 2.0 < pH < 12.0, the relative activity of immobilized VIM-1 generally shows a normal distribution. When pH = 7.0, the relative activity of the immobilized VIM-1 mutant enzyme is the highest; when pH ≤ 4.0 and pH ≥ 10.0, the activity of the immobilized VIM-1 mutant enzyme is relatively low; when 5.0 ≤ pH ≤ 9.0, the immobilized VIM-1 mutant enzyme shows relatively high relative activity, and the relative activity is all higher than 50%. That is, the optimal reaction pH for the immobilized VIM-1 mutant enzyme is 7.0.

[0140] Example 4 Removal experiment after immobilization of enzyme VIM-1

[0141] (1) Preparation of experimental group microspheres

[0142] From the above experiments, the optimal conditions for VIM-1 immobilization are as follows: 1.5% sodium alginate, 1% CaCl2, the immobilization time is 10 min, the optimal temperature is 30 °C, and the optimal pH is 7.0. The enzyme content in the prepared sodium alginate-enzyme mixture is fixed at 40%. If preparing 1 mL of sodium alginate-enzyme mixture, 500 μL of 3% sodium alginate solution + 400 μL of enzyme + 100 μL of 20 mM Tris-HCl (pH = 7.0) buffer.

[0143] (2) Preparation of microspheres: prepare a culture dish, pour 1% CaCl2 solution into the culture dish, and slowly drip the above 1.5% sodium alginate-enzyme mixture into the culture dish containing 1% CaCl2 solution 1 mm from the liquid surface with a syringe. After standing for 10 minutes to immobilize, rinse with 20mM Tris-HCl (pH=7.0) three times and store the microspheres in an EP tube, mark it, and put it on ice for later use. The blank group is operated in the same way as above.

[0144] Detection:

[0145] The final concentration of the substrate (ampicillin) is divided into three gradients: 50μg / mL, 100μg / mL, and 150μg / mL. Taking 50μg / mL ampicillin as an example, a total of 10 time gradients were designed: 0min, 1min, 3min, 5min, 7min, 9min, 11min, 13min, 15min, and 30min.

[0146] Take 50μg / mL as an example, dilute ampicillin to 50μg / mL. Take 4 1mL EP tubes, add 1mL 50μg / mL ampicillin to the first tube, quickly measure the absorbance value in the UV spectrophotometer, and record the data as the value of 0min. Take 4 1mL EP tubes, add 3 blank microspheres and 1mL 50μg / mL ampicillin to the first tube, add 3 enzyme-containing microspheres and 1mL 50μg / mL ampicillin to the other three tubes, quickly put the four EP tubes into a 40℃ water bath to react for 1min, and quickly measure the absorbance value in the UV spectrophotometer and record the data. Use the same method to measure the absorbance values ​​after 3min, 5min, 7min, 9min, 11min, 13min, 15min, and 30min of reaction, and record the data. Similarly, test the removal results of 100μg / mL and 150μg / mL in turn.

[0147] In order to detect the removal of different concentrations of ampicillin substrate after the optimal immobilization conditions and optimal reaction conditions of VIM-1 mutant enzyme were obtained, the residual rate of ampicillin was used as the dependent variable and time as the independent variable. Figure 6 From left to right, 50ug / ml, 100ug / ml, and 150ug / ml ampicillin were used as substrates respectively.

[0148] With 50ug / ml and 100ug / ml ampicillin as substrate, the immobilized VIM-1 mainly completed the reaction in the first 5 minutes, and the relative residual rate of ampicillin showed a significant downward trend. The relative residual rate of ampicillin measured after 5 minutes was close to 0. With 150ug / ml ampicillin as substrate, the immobilized VIM-1 mainly completed the reaction in the first 7 minutes, and the relative residual rate of ampicillin showed a significant downward trend. The relative residual rate of ampicillin measured after 7 minutes was close to 0.

[0149] The present invention discloses a B family lactamase mutant, and uses the immobilized enzyme to remove ampicillin from pig farm wastewater. The B family lactamase mutant is immobilized with sodium alginate, and the immobilization conditions are optimized. It is found that the optimal immobilization conditions for the enzyme are: final concentration 1.5% sodium alginate, 1.0% CaCl 2， Fix for 10 minutes.

[0150] The B 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 30°C and the optimal pH was 7.0. The B family lactamase mutant was immobilized under the optimal immobilization conditions and the removal experiment of different concentrations of ampicillin was carried out under the optimal conditions. The present invention utilizes the B family lactamase mutant to degrade ampicillin.

[0151] 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.

[0152] 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 B lactamase mutant, characterized in that: It is mutant 2, and the mutant 2 is based on the nucleotide sequence shown in SEQ ID NO: 1, with Leu at position 79 mutated to Val, Ala at position 84 mutated to Pro, and Tyr at position 148 mutated to His; its nucleotide sequence is shown in SEQ ID NO:

3.

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

3. The method for immobilizing a family B 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 min.

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

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