A cephalosporinase mutant with improved enzyme activity and a method for constructing the same

Abstract

The present invention relates to a cephalosporinase mutant with improved enzyme activity and a construction method thereof, and belongs to the field of genetic engineering technology. The amino acid sequence of the cephalosporinase mutant with improved enzyme activity of the present invention is shown in SEQ ID NO: 1. The present invention adopts single-point mutation technology, based on a homology modeling method, optimizes the molecular structure of cephalosporinase by selecting specific amino acids, and improves enzyme activity, which is of great significance for meeting the industrial production demand of cephalosporinase and reducing production costs.

Description

Technical Field

[0001] The invention relates to a cephalosporinase mutant with improved enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering. Background Art

[0002] The enzyme system is mainly produced by chromosomes or plasmids of Gram-negative rods such as Enterobacter, and can hydrolyze and inactivate cephalosporin antibiotics. Most cephalosporinases (AMPC enzymes) can hydrolyze oxyimino β-lactam antibiotics and are not inhibited by clavulanic acid. Oxyimino β-lactam antibiotics include third-generation (such as ceftazidime, ceftriaxone), and monocyclic β-lactam antibiotics (such as aztreonam), all of which contain an oxyimino group in their molecular structures. Cefoxitin and ceftriaxone are extremely unstable to them, and bacteria that produce this enzyme can be highly resistant to them, but fourth-generation cephalosporins such as cefoperazone, cefpirome and carbapenem antibiotics such as imipenem and meropenem are stable to them.

[0003] The genes encoding enzymes can be divided into chromosome-mediated and plasmid-mediated according to the host-carrying mode. Chromosome-mediated enzymes can be divided into induced high-yield type, sustained high-yield type and sustained low-yield type according to their production mode. ① Induced high-yield type: Most Enterobacter cloacae have naturally existing genes on their chromosomes, but only produce a small amount of enzyme under normal conditions. When inducing β-lactam antibiotics such as penicillin and cephalosporin are present, the enzyme production is significantly doubled; ② Some enzyme-producing strains of the sustained high-yield type also have genes on their chromosomes. Through the mutation of the weak promoter of the regulatory gene and the attenuator of the hairpin structure or the mutation of the regulatory gene, they can produce high enzymes regardless of the presence of β-lactam antibiotics, which is also called derepressed high expression; ③ A very small number of enzyme-producing strains of the sustained low-yield type do not produce or produce defective proteins due to the lack of regulatory genes or gene mutations. They cannot act as activators in the presence of β-lactam antibiotics, nor can they act as inhibitors in the absence of β-lactam antibiotics, resulting in continuous low-level expression of the enzyme, which can be seen in Escherichia coli.

[0004] More than 60 plasmid-mediated AmpC enzymes have been discovered so far, which are divided into 6 different groups according to their sources, namely MOX, CIT, DHA, ACC, MIR (ACT-1), and FOX-type plasmid AmpC enzymes. Most plasmid-mediated AmpC enzymes are of continuous high-yield type, showing high-level continuous expression, and can be spread in the same or different species of bacteria through transformation, conjugation, etc., causing widespread spread of drug resistance and outbreaks of nosocomial infections. This is because the ampC gene in the plasmid lacks the regulatory gene ampR, or the ampR binding site is missing between ampR and ampC, and cannot produce transcriptional regulation and is continuously highly expressed. However, recent studies have shown that some plasmid-mediated ampC gene enzymes such as DHA-1, DHA-2, ACT-1, etc. may have regulatory sequences and regulatory mechanisms regulated by composite operons, and these plasmids can produce induced high-yield AmpC enzymes.

[0005] GMP stipulates that the aseptic production process of sterile drugs (including biological products) must use environmental monitoring to evaluate whether the production environment control meets GMP requirements. Monitoring items include airborne particles, airborne planktonic bacteria, settling bacteria, microorganisms on the surface of facilities and equipment, and monitoring of the hygiene status of operators. The sterile filling room and functional room of cephalosporin antibiotics for injection are dust-producing functional rooms. Cephalosporin antibiotic dust falls into the monitoring culture medium plate, which will reduce the sensitivity of the culture medium in the plate, and thus affect the accuracy and reliability of the environmental monitoring data of the production process. The amount of cephalosporinase added to the monitoring culture medium plate is to ensure that the sensitivity of the monitoring plate culture medium for environmental monitoring is suitable for environmental monitoring of the production process. The development of highly active cephalosporinase has high commercial value.

[0006] The prominent problem of heterologous expression of cephalosporinase is low protein expression and low cephalosporinase activity. Therefore, site-directed mutagenesis of cephalosporinase to improve enzyme activity is of great significance for meeting the demand for industrial production of cephalosporinase and reducing production costs. The present invention adopts single-point mutation technology and optimizes the molecular structure of cephalosporinase and improves enzyme activity by selecting specific amino acids based on homology modeling. Summary of the invention

[0007] The object of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a cephalosporinase mutant with improved enzyme activity and a method for constructing the same. The cephalosporinase mutant of the present invention has higher enzyme activity, which is of great significance for meeting the demand for industrial production of cephalosporinase and reducing production costs.

[0008] To achieve the above object, the technical solution adopted by the present invention is: a cephalosporinase mutant with improved enzyme activity, the amino acid sequence of the mutant is shown in SEQ ID NO:1.

[0009] As a preferred embodiment of the cephalosporinase mutant of the present invention, the mutant has the proline at position 154 mutated to threonine based on the amino acid sequence shown in SEQ ID NO: 2.

[0010] As a preferred embodiment of the cephalosporinase mutant of the present invention, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 4. The engineered bacteria expressing the cephalosporinase shown in the nucleotide sequence of SEQ ID NO: 4 were obtained by the inventors in previous research.

[0011] In a second aspect, the present invention provides a cephalosporinase mutant gene with improved enzyme activity, wherein the gene encodes the cephalosporinase mutant with improved enzyme activity.

[0012] As a preferred embodiment of the cephalosporinase mutant gene of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0013] In a third aspect, the present invention provides an engineered bacterium, wherein the engineered bacterium expresses the cephalosporinase mutant with improved enzyme activity.

[0014] In a fourth aspect, the present invention provides a method for preparing the above-mentioned engineered bacteria, comprising the following steps: based on the nucleotide sequence shown in SEQ ID NO:4, mutating the proline at position 154 into threonine to obtain a mutant, connecting the recombinant gene to an expression vector to obtain a recombinant plasmid, and transforming the recombinant plasmid into a DH5ɑ host bacterium to obtain the engineered bacteria.

[0015] In a fifth aspect, the present invention provides use of the cephalosporinase mutant with improved enzyme activity in the preparation of cephalosporinase.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention adopts single-point mutation technology, based on homology modeling method, and optimizes the molecular structure of cephalosporinase by selecting specific amino acids and improving enzyme activity, which is of great significance for meeting the industrial production demand of cephalosporinase and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the fermentation curve of the cephalosporinase mutant engineered bacteria.

[0018] Figure 2 This is the enzyme SDS-PAGE detection diagram of the supernatant after the cephalosporinase mutant engineered bacteria were crushed and lysed. DETAILED DESCRIPTION

[0019] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0020] Example 1 Site-directed mutagenesis

[0021] Using the recombinant plasmid containing SEQ ID NO: 4 as the template plasmid, referring to the TaKaRa biological product and operation manual, using the TaKaRa MuTanBEST mutation kit, using the designed corresponding mutation primer pair, PCR amplified the site-directed mutation sequence of BmPGA, the specific operation steps are as follows:

[0022] (1) Mutation Primers

[0023] Forward primer BLAB-154-F is 5'-GACCCTGAACACCGCTATC RHT GGTGACAAACGTGACAC-3',

[0024] The reverse primer BLAB-154-R was 5′-GTGTCACGTTTGTCACCADYGATAGCGGTGTTCAGGGTC-3′.

[0025] (2) Reaction system (10ul)

[0026] 10ul of 10xPfu polymerase buffer, 2ul of 10mmol / L dNTP solution, 25pmoles of forward primer, 25pmoles of reverse primer, 5U of Pfu DNA polymerase, 100-200pg of plasmid DNA template, add water to 100ul.

[0027] (3) PCR conditions: 94°C for 3 min, 94°C for 30 s, 55°C for 30 s, 72°C for 300 s, 30 cycles, 72°C for 10 min.

[0028] Refer to the TAKARA operation manual, phosphorylate the 5' end of the DNA fragment obtained by PCR, then cyclize the phosphorylated mutant primer, transform DH5α competent cells, and spread on the LB selective plate with kanasulfomycin antibiotics, and invert and culture at 37℃ for about 20h. The transformants that can grow on the LB plate with kanasulfomycin antibiotics are the transformants transformed with the recombinant plasmid. Finally, the transformants are further cultured to amplify the recombinant plasmid, and the amplified recombinant plasmid is extracted.

[0029] The screened mutant plasmids were sequenced to determine that the obtained plasmids had mutated at the target mutation site but not at the non-target mutation site.

[0030] Example 2 Obtaining Engineering Bacteria

[0031] Referring to the method in the Molecular Cloning Experiment Guide, the mutated plasmid was transformed into Escherichia coli competent cells or yeast competent cells to obtain a genetically engineered strain expressing the mutant enzyme. Finally, the engineered strain was spread on an LB selective plate with kanamycin sulfate antibiotics, and inverted and cultured at 37°C for about 12-18 hours. The transformants that can grow on the LB plate with kanamycin sulfate antibiotics are the transformants that have been transformed with the recombinant plasmid.

[0032] Example 3 Fermentation culture of engineering bacteria

[0033] Pick a single colony from the LB selective culture plate, inoculate it into 3mL of LB liquid medium, add kanamycin to a final concentration of 100μg / mL, and culture it at 37℃ and 250r / min overnight; take 2mL of the culture solution cultured overnight, inoculate it into 200mL of LB liquid medium, culture it at 37℃ and 250r / min for 4-6h, and obtain the seed bacterial solution when the OD600 reaches between 1.0-1.6. Inoculate it into a 5L fermenter containing 3L of TB medium at a ratio of 1:15, and ferment it at 37℃, 400rpm, and 1.3vvm until the OD600 reaches 25, then adjust the temperature to 30℃, induce it with IPTG at a final concentration of 1%, continue to culture for 2 hours, then add IPTG at a final concentration of 0.5% to induce it, and continue to culture for about 14-16 hours, and the fermentation ends.

[0034] The fermentation curve of the engineered bacteria is shown in Figure 1 As shown, the horizontal axis is time, and the vertical axis is OD600 value. The higher the value, the higher the bacterial concentration. When the bacteria are collected, OD600 reaches 85, which meets the requirements of industrial production. The fermentation broth is centrifuged at 8000rpm for 20min, and the bacteria are collected and stored at -20℃.

[0035] Take 1 ml of the bacterial solution that has been fermented for 14-16 hours, centrifuge at 12000rpm for 1min, discard the supernatant, and then resuspend the bacteria with 1 ml of 20mM potassium phosphate (PH7.0), then ultrasonically disrupt the cells and perform SDS-PAGE detection. SDS-PAGE uses the Laemmli electrophoresis method, with a concentration of 5% for the concentrated gel and 8% for the separation gel. After the reduced sample is mixed with the loading buffer, it is boiled at 100℃ for 5 minutes for loading electrophoresis.

[0036] The results are as follows Figure 2 As shown, the molecular weight of cephalosporinase is about 25KD. 3-B is the sample taken before induction, and 3-A is the sample taken after induction. The results show that a positive band appeared at 25KD after induction, indicating that cephalosporinase was successfully expressed.

[0037] Example 4 Purification of mutant enzyme

[0038] Measure 5mL of pretreated affinity carrier FP-IDA-Ni2+ and load it into the purification column. The sample passes through the column at a rate of 1.0BV / h. Use 3-5BV of Washing buffer at a flow rate of 1.0BV / h to remove impurities, and use a protein nucleic acid detector to detect the protein content of the effluent at 280nm until the Washing buffer is clarified and the value of the protein nucleic acid detector no longer changes. Finally, under the monitoring of the protein nucleic acid detector at 280nm, use about 1-2BV of Elution buffer at a rate of 1-2BV / h to elute the target enzyme, and store the enzyme solution at 4℃.

[0039] Example 5 Determination of enzyme hydrolysis activity

[0040] 1. Determination method

[0041] Preparation of penicillin solution: Weigh an appropriate amount of sodium (potassium) penicillin and dissolve it in phosphate buffer (pH 7.0) to make a solution containing 10,000 units of penicillin per 1 ml.

[0042] Preparation of cephalosporinase dilution: Take cephalosporinase fermentation broth / purified enzyme, dilute 1000-5000 times, and preheat at 37°C.

[0043] Determination method: Accurately measure 50 ml of penicillin solution, place it in a 100 ml volumetric flask, preheat it to 37°C, then accurately add 25 ml of preheated cephalosporinase diluent, mix quickly, and place it at 37°C for 1 hour. Accurately measure 3 ml and immediately add it to 25 ml of accurately measured iodine titrant (0.005 mol / L) [Accurately measure 10 ml of iodine titrant (0.05 mol / L), place it in a 100 ml volumetric flask, and dilute it to the scale with sodium acetate buffer (pH 4.5)], place it in a dark place at room temperature for 15 minutes, and titrate with sodium thiosulfate titrant (0.01 mol / L). When it is close to the end point, add starch indicator solution and continue titrating until the blue color disappears.

[0044] Blank test: Take 2 ml of preheated penicillin solution, place it at 37°C for 1 hour, accurately add 25 ml of the above iodine titration solution (0.005 mol / L), then accurately add 1 ml of cephalosporinase diluent, place it in the dark at room temperature for 15 minutes, and titrate with sodium thiosulfate titration solution (0.01 mol / L). Calculate according to the following formula:

[0045] E=(BA)×M×F×D×100

[0046] Where, E is the cephalosporinase activity, unit / (ml·hour);

[0047] B is the volume of the sodium thiosulfate titration solution consumed in the blank titration, ml;

[0048] A is the volume of the sodium thiosulfate titration solution consumed by the test sample titration, ml;

[0049] M is the concentration of sodium thiosulfate titrant, mol / L;

[0050] F is the potency of penicillin per 1 ml of the above iodine titration solution (0.005 mol / L) under the same conditions, in units;

[0051] D is the dilution factor of cephalosporinase solution.

[0052] Phosphate buffer (pH 7.0): Take 7.36 g of dipotassium hydrogen phosphate and 3.14 g of potassium dihydrogen phosphate, add water to make 1000 ml.

[0053] Sodium acetate buffer (pH 4.5): Take 13.86 ml of glacial acetic acid and add water to make 250 ml; take 27.30 g of crystalline sodium acetate and add water to make 200 ml, and mix the two liquids evenly.

[0054] 2. Test results

[0055] The test results of the enzyme hydrolysis activity are shown in Table 1. As shown in Table 1, the enzyme activity of the mutant enzyme in hydrolyzing the substrate is 9.52×10 11 U / Lh, relative to the original strain (1.77×10 11 U / Lh) increased by 5.36 times.

[0056] Table 1

[0057] Bacteria No. Amino acid mutation site Relative activity (%) Original strain —— 100％ Cephalosporinase P154T P154T 536％

[0058] Example 6 Preparation of TSA solid culture medium with enzyme and sensitivity test

[0059] Prepare TSA solid culture medium, including 15.0g / L tryptone, 5.0g / L soy peptone, 5.0g / L sodium chloride, 14g / L agar, and pH 7.5. Autoclave the prepared culture medium at 121°C for 25 minutes, wait until it cools to 50°C, add cephalosporinase, and mix well to make the concentration of cephalosporinase dissolved in TSA culture medium 300,000 units / mL. On the clean bench, spread 20ml of TSA enzyme culture medium evenly on the bottom plate of the plate, cool and solidify, and cover the plate with the top cover.

[0060] Add 10 mg of cephalosporin antibiotics to the dish, and perform the test according to the coating method in the 1105 plate method of the 2015 edition of the Chinese Pharmacopoeia. The recovery rates of Micrococcus luteus (G+ cocci), Ralstonia pekinensis (G- bacilli) and molds were 100%, 100%, and 100%, respectively. The results showed that they all effectively neutralized 10 mg of cephalosporin antibiotic dust, and the sensitivity met the suitability test of the culture medium, which is suitable for the daily environmental sedimentation bacteria monitoring of cephalosporin antibiotics.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention. SEQUENCE LISTING <110> Guangzhou Baiyunshan Baidi Biopharmaceutical Co., Ltd. <120> A cephalosporinase mutant with improved enzyme activity and a method for constructing the same <130> 20181221 <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 269 <212> PRT <213> Artificial sequence <400> 1 Met Gln Pro Ala Asn Ala Lys Ala Asn Ile Gln Gln Gln Leu Ser Glu 1 5 10 15 Leu Glu Lys Asn Ser Gly Gly Arg Leu Gly Val Ala Leu Ile Asp Thr 20 25 30 Ala Asp Asn Ser Gln Ile Leu Tyr Arg Gly Asp Glu Arg Phe Pro Met 35 40 45 Cys Ser Thr Ser Lys Val Met Ala Val Ser Ala Leu Leu Lys Gln Ser 50 55 60 Glu Thr Asp Lys Asn Leu Leu Ala Lys Arg Met Glu Ile Lys Gln Ser 65 70 75 80 Asp Leu Val Asn Tyr Asn Pro Ile Ala Glu Lys His Leu Asp Thr Gly 85 90 95 Met Thr Leu Ala Glu Phe Ser Ala Ala Thr Ile Gln Tyr Ser Asp Asn 100 105 110 Thr Ala Met Asn Lys Ile Leu Glu His Leu Gly Gly Pro Ala Lys Val 115 120 125 Thr Glu Phe Ala Arg Thr Ile Gly Asp Lys Thr Phe Arg Leu Asp Arg 130 135 140 Thr Glu Pro Thr Leu Asn Thr Ala Ile Thr Gly Asp Lys Arg Asp Thr 145 150 155 160 Thr Ser Pro Gln Ala Met Ala Ile Ser Leu Gln Asn Leu Thr Leu Gly 165 170 175 Lys Ala Leu Ala Glu Pro Gln Arg Ala Gln Leu Val Glu Trp Met Lys 180 185 190 Gly Asn Thr Thr Gly Gly Ala Ser Ile Arg Ala Gly Leu Pro Thr Thr 195 200 205 Trp Val Val Gly Asp Lys Thr Gly Ser Gly Asp Tyr Gly Thr Thr Asn 210 215 220 Asp Ile Ala Val Ile Trp Pro Ala Asn His Ala Pro Leu Val Leu Val 225 230 235 240 Thr Tyr Phe Thr Gln Pro Gln Gln Asn Ala Glu Ala Arg Lys Asp Val 245 250 255 Leu Ala Ala Ala Ala Lys Ile Val Thr Ala Gly Leu Leu 260 265 <210> 2 <211> 269 <212> PRT <213> artificial sequence <400> 2 Met Gln Pro Ala Asn Ala Lys Ala Asn Ile Gln Gln Gln Leu Ser Glu 1 5 10 15 Leu Glu Lys Asn Ser Gly Gly Arg Leu Gly Val Ala Leu Ile Asp Thr 20 25 30 Ala Asp Asn Ser Gln Ile Leu Tyr Arg Gly Asp Glu Arg Phe Pro Met 35 40 45 Cys Ser Thr Ser Lys Val Met Ala Val Ser Ala Leu Leu Lys Gln Ser 50 55 60 Glu Thr Asp Lys Asn Leu Leu Ala Lys Arg Met Glu Ile Lys Gln Ser 65 70 75 80 Asp Leu Val Asn Tyr Asn Pro Ile Ala Glu Lys His Leu Asp Thr Gly 85 90 95 Met Thr Leu Ala Glu Phe Ser Ala Ala Thr Ile Gln Tyr Ser Asp Asn 100 105 110 Thr Ala Met Asn Lys Ile Leu Glu His Leu Gly Gly Pro Ala Lys Val 115 120 125 Thr Glu Phe Ala Arg Thr Ile Gly Asp Lys Thr Phe Arg Leu Asp Arg 130 135 140 Thr Glu Pro Thr Leu Asn Thr Ala Ile Pro Gly Asp Lys Arg Asp Thr 145 150 155 160 Thr Ser Pro Gln Ala Met Ala Ile Ser Leu Gln Asn Leu Thr Leu Gly 165 170 175 Lys Ala Leu Ala Glu Pro Gln Arg Ala Gln Leu Val Glu Trp Met Lys 180 185 190 Gly Asn Thr Thr Gly Gly Ala Ser Ile Arg Ala Gly Leu Pro Thr Thr 195 200 205 Trp Val Val Gly Asp Lys Thr Gly Ser Gly Asp Tyr Gly Thr Thr Asn 210 215 220 Asp Ile Ala Val Ile Trp Pro Ala Asn His Ala Pro Leu Val Leu Val 225 230 235 240 Thr Tyr Phe Thr Gln Pro Gln Gln Asn Ala Glu Ala Arg Lys Asp Val 245 250 255 Leu Ala Ala Ala Ala Lys Ile Val Thr Ala Gly Leu Leu 260 265 <210> 3 <211> 804 <212> DNA <213> Artificial Sequence <400> 3 atgcagccgg ctaacgctaa agctaacatc cagcagcagc tgtctgaact ggaaaaaaac 60 tctggtggtc gtctgggtgt tgctctgatc gacaccgctg acaactctca gatcctgtac 120 cgtggtgacg aacgtttccc gatgtgctct acctctaaag ttatggctgt ttctgctctg 180 ctgaaacagt ctgaaaccga caaaaacctg ctggctaaac gtatggaaat caaacagtct 240 gacctggtta actacaaccc gatcgctgaa aaacacctgg acaccggtat gaccctggct 300 gaattctctg ctgctaccat ccagtactct gacaacaccg ctatgaacaa aatcctggaa 360 cacctgggtg gtccggctaa agttaccgaa ttcgctcgta ccatcggtga caaaaccttc 420 cgtctggacc gtaccgaacc gaccctgaac accgctatca ctggtgacaa acgtgacacc 480 acctctccgc aggctatggc tatctctctg cagaacctga ccctgggtaa agctctggct 540 gaaccgcagc gtgctcagct ggttgaatgg atgaaaggta acaccaccgg tggtgcttct 600 gaaccgcagc gtgctcagct ggttgaatgg atgaaaggta acaccaccgg tggtgcttct 600 atccgtgctg gtctgccgac cacctgggtt gttggtgaca aaaccggttc tggtgactac 660 atccgtgctg gtctgccgac cacctgggtt gttggtgaca aaaccggttc tggtgactac 660 ggtaccacca acgacatcgc tgttatctgg ccggctaacc acgctccgct ggttctggtt 720 ggtaccacca acgacatcgc tgttatctgg ccggctaacc acgctccgct ggttctggtt 720 acctacttca cccagccgca gcagaacgct gaagctcgta aagacgttct ggctgctgct 780 acctacttca cccagccgca gcagaacgct gaagctcgta aagacgttct ggctgctgct 780 gctaaaatcg ttaccgctgg tctg 804 gctaaaatcg ttaccgctgg tctg 804 <210> 4<210> 4 <211> 804<211> 804 <212> DNA<212> DNA <213> 人工序列<213> Artificial sequence <400> 4 <400> 4 atgcagccgg ctaacgctaa agctaacatc cagcagcagc tgtctgaact ggaaaaaaac 60 atgcagccgg ctaacgctaa agctaacatc cagcagcagc tgtctgaact ggaaaaaaac 60 tctggtggtc gtctgggtgt tgctctgatc gacaccgctg acaactctca gatcctgtac 120 tctggtggtc gtctgggtgt tgctctgatc gacaccgctg acaactctca gatcctgtac 120 cgtggtgacg aacgtttccc gatgtgctct acctctaaag ttatggctgt ttctgctctg 180 cgtggtgacg aacgtttccc gatgtgctct acctctaaag ttatggctgt ttctgctctg 180 ctgaaacagt ctgaaaccga caaaaacctg ctggctaaac gtatggaaat caaacagtct 240 ctgaaacagt ctgaaaccga caaaaacctg ctggctaaac gtatggaaat caaacagtct 240 gacctggtta actacaaccc gatcgctgaa aaacacctgg acaccggtat gaccctggct 300 gacctggtta actacaaccc gatcgctgaa aaacacctgg acaccggtat gaccctggct 300 gaattctctg ctgctaccat ccagtactct gacaacaccg ctatgaacaa aatcctggaa 360 gaattctctg ctgctaccat ccagtactct gacaacaccg ctatgaacaa aatcctggaa 360 cacctgggtg gtccggctaa agttaccgaa ttcgctcgta ccatcggtga caaaaccttc 420 cgtctggacc gtaccgaacc gaccctgaac accgctatcc cgggtgacaa acgtgacacc 480 acctctccgc aggctatggc tatctctctg cagaacctga ccctgggtaa agctctggct 540 gaaccgcagc gtgctcagct ggttgaatgg atgaaaggta acaccaccgg tggtgcttct 600 atccgtgctg gtctgccgac cacctgggtt gttggtgaca aaaccggttc tggtgactac 660 ggtaccacca acgacatcgc tgttatctgg ccggctaacc acgctccgct ggttctggtt 720 acctacttca cccagccgca gcagaacgct gaagctcgta aagacgttct ggctgctgct 780 gctaaaatcg ttaccgctgg tctg 804

Claims

1. A cephalosporinase mutant with improved enzyme activity, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO:

1. Based on the amino acid sequence shown in SEQ ID NO: 2, the proline at position 154 of the mutant is mutated into threonine. The enzyme activity of the mutant in hydrolyzing penicillin substrate is 9.52×10 11 U / Lh.

2. The cephalosporinase mutant according to claim 1, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:

4.

3. A cephalosporinase mutant gene with improved enzyme activity, characterized in that: The gene encodes the cephalosporinase mutant with improved enzyme activity according to any one of claims 1 to 2.

4. The cephalosporinase mutant gene according to claim 3, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

3.

5. An engineered bacterium, characterized in that: The engineered bacteria expresses the cephalosporinase mutant with improved enzyme activity as claimed in any one of claims 1 to 2.

6. The method for preparing the engineered bacteria according to claim 5, characterized in that: The following steps are involved: Based on the nucleotide sequence shown in SEQ ID NO: 4, the proline codon at position 154 of its coding sequence is mutated into a threonine codon to obtain a recombinant gene, the recombinant gene is connected to an expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a DH5ɑ host bacterium to obtain an engineered bacterium.

7. Use of the cephalosporinase mutant with improved enzyme activity according to any one of claims 1 to 2 in the preparation of cephalosporinase.

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