A penicillin G acylase mutant with high hydrolytic activity

Abstract

This invention screened a penicillin G acylase mutant SEQ ID NO:3 through genetic engineering. Compared with the penicillin G acylase SEQ ID NO:1 derived from wild-type Escherichia coli, this mutant has significantly enhanced hydrolytic activity and can efficiently catalyze the hydrolysis of penicillin G potassium salt to generate 6-aminopenicillanic acid (6-APA).

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically, it relates to a method for obtaining penicillin G acylase mutants through random mutation and high-throughput screening, and their use in the production of 6-aminopenicillanic acid, a key parent nucleus of β-lactam antibiotics. Background Technology

[0002] Penicillin G acylase (EC3.5.1.11, abbreviated as PGA) is an important enzyme used in the antibiotic industry for the preparation of semi-synthetic β-lactam antibiotics. This enzyme is a reversible enzyme that participates in the hydrolysis and synthesis of β-lactam antibiotics. The hydrolytic function of this enzyme is mainly used industrially for the production of 6-aminopenicillinic acid (6-APA) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA), important parent nuclei of β-lactam antibiotics (Abian et al., Biotechnol Prog, 2003, 19(6), 1639-42, 2003); its synthetic function is mainly to catalyze the reaction of the parent nucleus 6-APA, 7-ADCA or other parent nuclei with various D-amino acid side chains to generate new semi-synthetic β-lactam antibiotics (semi-synthetic penicillins and cephalosporins) (Bruggink et al. 1998; Yang and Wei 2003; Youshko et al. 2004; Gabor et al. 2005). In addition, during the synthesis of chiral compounds, penicillin G acylase can protect hydroxyl and amino groups and resolve chiral compounds (Shewale et al., 1990).

[0003] For a long time, research on penicillin G acylase has largely focused on improving the enzyme activity of its synthesis reaction, with relatively little research on its hydrolytic function. Among these studies, there are relatively more reports on the application of PGA hydrolysis performance derived from Escherichia coli and Bacillus megaterium. With the increasing competition in the antibiotic raw material industry, improving enzyme performance, upgrading product preparation processes, and reducing production costs have become the most effective means of competition within the industry. Summary of the Invention

[0004] To obtain a penicillin G acylase with high hydrolytic activity, this invention utilizes genetic engineering technology to modify PGA (ecPGA) from *E. coli*, which is currently used in industrial applications, aiming to construct and screen mutant enzymes with further improved hydrolytic performance and apply them to the production of β-lactam antibiotics. After long-term experiments, using the wild-type penicillin G acylase with the amino acid sequence SEQ ID NO:1 (GenBank accession number X04114) as the modification target, a mutant with industrial application capability was finally screened. Specifically, this invention provides the following technical solution.

[0005] A penicillin G acylase, which is a mutant of wild-type penicillin G acylase SEQ ID NO:1, wherein F at position 227 is replaced with L, D at position 363 is replaced with N, D at position 582 is replaced with Y, and T at position 719 is replaced with P, and its amino acid sequence is SEQ ID NO:3:

[0006] MKNRNRMIVNCVTASLMYYWSLPALAEQSSSEIKIVRDEYGMPHIYANDTWHLFYGYGYVVAQDRLFQMEMARRSTQGTVAEVLGKDFVKFDKDIRRNYWPDAIRAQIAALSP EDMSILQGYADGMNAWIDKVNTNPETLLPKQFNTFGFTPKRWEPFDVAMIFVGTMANRFSDSTSEIDNLALLTALKDKYGVSQGMAVFNQLKWLVNPSAPTTIAVQESNYPLK L NQQNSQTAALLPRYDLPAPMLDRPAKGADGALLALTAGKNRETIVAQFAQGGANGLAGYPTTSNMWVIGKSKAQDAKAIMVNGPQFGWYAPAYTYGIGLHGAGYDVTGNTPFAYPGLVFGHNGVISWGSTAGFGD N VDIFAERLSAEKPGYYLHNGKWVKMLSREETITVKNGQAETFTVWRTVHGNILQTDQTTQTAYAKSRAWDGKEVASLLAWTHQMKAKNWQEWTQQAAKQALTINWYYAD VNGNIGYVHTGAYPDRQSGHDPRLPVPGTGKWDWKGLLPFEMNPKVYNPQSGYIANWNNSPQKDYPASDLFAFLWGGADRVTEIDRLLEQKPRLTADQAWDVIRQTSRQ YLNLRLFLPTLQAATSGLTQSDPRRQLVETLTRWDGINLLNDDGKTWQQPGSAILNVWLTSMLKRTVVAAVPMPFDKWYSASGYETTQDGPTGSLNISVGAKILYEAVQGDKSPIPQAVDLFAGKPQQEVVLAALED P WETLSKRYGNNVSNWKTPAMALTFRANNFFGVPQAAAEETRHQAEYQNRGTENDMIVFSPTTSDRPVLAWDVVAPGQSGFIAPDGTVDKHYEDQLKMYENFGRKSLWLTKQDVEAHKESQEVLHVQR (SEQ ID NO: 3).

[0007] A second aspect of the present invention provides a polynucleotide that is a gene encoding the above-mentioned penicillin G acylase SEQ ID NO:3.

[0008] Preferably, the nucleotide sequence of the above-mentioned polynucleotide can be SEQ ID NO:4 in the sequence listing. However, the polynucleotide of the present invention is not limited to this, and may also include other codon-optimized polynucleotides, preferably having a nucleotide sequence that has more than 80%, preferably more than 85%, preferably more than 90%, and preferably more than 95% homology with SEQ ID NO:4.

[0009] A third aspect of the invention provides a plasmid on which one of the aforementioned polynucleotides is cloned for expressing penicillin G acylase SEQ ID NO:3.

[0010] As the backbone of the aforementioned plasmid, the plasmid vector can be from the PET series, such as pET22b, pET24a, pET28a, etc., but is not limited to this. A more preferred vector is pET24a, so as to express penicillin G acylase SEQ ID NO:3 in bacteria such as Escherichia coli.

[0011] A third aspect of the present invention provides a microorganism that is a transformant transformed with the above-described plasmid.

[0012] For example, the microorganisms mentioned above can be Escherichia coli, with Escherichia coli BL21(DE3) being the preferred host.

[0013] It should be understood that the microorganism used to express the above-mentioned penicillin G acylase SEQ ID NO:3 is not limited to bacteria, but can also be other fungi with rapid proliferation rates and easily controllable fermentation processes, such as yeast.

[0014] A third aspect of the invention provides the use of the above-described penicillin G acylase SEQ ID NO:3 or its expressing microorganism in the hydrolysis of potassium penicillin G to produce 6-aminopenicillanic acid (6-APA).

[0015] The penicillin G acylase SEQ ID NO:3 of the present invention can not only be used in the form of an enzyme to catalyze the hydrolysis of penicillin G potassium salt or penicillin G solution, but can also be used directly in the form of its expressed microbial cells to catalyze the hydrolysis reaction.

[0016] The penicillin G acylase SEQ ID NO:3 of this invention exhibits a significantly higher specific activity for hydrolyzing penicillin G potassium salt than the wild-type enzyme, reaching 2.1 times that of the wild-type enzyme. Therefore, compared to existing technologies, it can catalyze the generation of 6-aminopenicillanic acid (6-APA), an important parent nucleus of β-lactamase antibiotics, with higher catalytic efficiency, demonstrating great application potential. Attached Figure Description

[0017] Figure 1 Map of pET24a-ecPGA plasmid constructed in this invention for expressing wild-type enzyme (ecPGA).

[0018] Figure 2 These are SDS-PAGE electrophoresis images of the wild-type enzyme ecPGA and the mutant enzyme ecPGA-M2139. Lane 1: ecPGA-M2139 affinity chromatography elution; Lanes 2-3: ecPGA-M2139 affinity chromatography elution; Lane 4: ecPGA-M2139 affinity chromatography sample flow-through; Lane 5: ecPGA affinity chromatography sample elution; M: protein marker (molecular weights from top to bottom: 116 kDa, 66.2 kDa, 45 kDa, 35 kDa, 25 kDa). Detailed Implementation

[0019] The purpose of this invention is to modify the penicillin G acylase used in the industrial production of 6-APA from hydrolyzed penicillin G. By comparing wild-type enzymes from different microbial sources, *E. coli*-derived PGA (ecPGA) was selected as the primary wild-type enzyme (or initiating enzyme, original enzyme) for mutation and screening. This resulted in a mutant with further enhanced hydrolytic activity, thereby reducing the production cost of 6-APA.

[0020] The wild-type penicillin G acylase with the amino acid sequence SEQ ID NO:1 (GenBank accession number X04114) was used as the target for modification in order to obtain penicillin G acylase with high hydrolytic activity.

[0021] In this article, the terms "wild-type", "wild-type enzyme" and "wild-type enzyme" have the same meaning, all referring to wild-type penicillin G acylase ecPGA.

[0022] Similarly, the terms “penicillin G acylase mutant,” “mutant penicillin G acylase,” “mutant penicillin G acylase,” and “mutant enzyme” have the same meaning, all referring to the mutant penicillin G acylase SEQ ID NO:3.

[0023] For the sake of convenience, the wild-type enzyme SEQ ID NO:1 and its mutant SEQ ID NO:3 may be referred to as “penicillin G acylase” in this article.

[0024] To obtain a penicillin G acylase mutant with high hydrolytic activity, enzyme engineering was performed on wild-type penicillin G acylase. In this invention, error-prone PCR was performed on the coding gene sequence SEQ ID NO:2 of SEQ ID NO:1, and through high-throughput screening, the mutant amino acid sequence SEQ ID NO:3, with mutations at four sites relative to SEQ ID NO:1 (phenylalanine at position 227, aspartic acid at position 363, aspartic acid at position 582, and threonine at position 719), was finally obtained.

[0025] The penicillin G acylase mutant of the present invention, SEQ ID NO:3, contains 846 amino acids, of which positions 1-26 are signal peptides, positions 27-235 are α subunits, positions 236-289 are spacer peptides, and positions 290-846 are β subunits. The penicillin G acylase expressed by microorganisms is a complex composed of α and β subunits, with a well-defined protein sequence structure. Therefore, those skilled in the art can easily obtain its encoding gene, expression cassettes and plasmids containing these genes, and transformants containing the plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0026] The host for the aforementioned transformant can be any microorganism suitable for expressing penicillin G acylase, including bacteria and fungi. Preferred microorganisms are Escherichia coli and yeast, with Escherichia coli or Pichia pastoris being particularly preferred.

[0027] When used as a biocatalyst in production, the penicillin G acylate of the present invention can be in the form of an enzyme or in the form of microbial fermentation cells. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, and enzymes immobilized on a carrier; the cell form includes live cells and dead cells.

[0028] For ease of operation, control, and post-processing, it is preferable to use an enzyme as a catalyst to catalyze the hydrolysis of penicillin G to produce 6-APA.

[0029] The enzyme separation and purification techniques of the present invention, including the immobilized enzyme preparation techniques, are well known to those skilled in the art.

[0030] Example

[0031] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0033] Materials and methods

[0034] In the embodiments, the whole gene synthesis, primer synthesis and sequencing were all completed by Suzhou Genewiz Biotechnology Co., Ltd.

[0035] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions can be determined through simple experiments if necessary.

[0036] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0037] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium with an additional 20 g / L agar powder.)

[0038] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K₂HPO₄·3H₂O, 2.31 g / L KH₂PO₄, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium with an additional 20 g / L agar powder.)

[0039] The potassium penicillin G salt was donated by Sinopharm Weichida Pharmaceutical Co., Ltd.

[0040] NIPAB (6-nitro-3-aminobenzoic acid) was purchased from Weiben Technology & Trade Co., Ltd.

[0041] Example 1: Construction of recombinant Escherichia coli with initial penicillin G acylase gene

[0042] The amino acid sequence of the penicillin G acylase ecPGA from *Escherichia coli* is SEQ ID NO:1. Based on the gene sequence X04114 published in Genebank, its coding gene sequence SEQ ID NO:2 was synthesized. Restriction endonuclease sites Nde I and Xho I were designed at both ends of the gene, and subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain the recombinant plasmid pET24a-ecPGA, the structural map of which is shown below. Figure 1 As shown.

[0043] The recombinant plasmid pET24a-ecPGA was transformed into the expression host Escherichia coli BL21(DE3) (Invitrogen) by electroporation to obtain recombinant Escherichia coli pET24a-ecPGA / BL21(DE3) expressing wild-type penicillin G acylase SEQ ID NO:1.

[0044] Example 2: Establishment and Screening of Error-Prone Mutation Library

[0045] 2.1 Establishment of a library of error-prone mutations

[0046] Using plasmids extracted from pET24a-ecPGA / BL21(DE3) strain as templates, a library of error-prone mutations of penicillin G acylase was established.

[0047] The forward primer ecPGA-F is 5'-GCCGCATATTTATGCCAATGATACATG-3'.

[0048] The reverse primer ecPGA-R is 5'-ACTTCCTGCGACTCCTTATGCGCCTC-3'.

[0049] Error-prone PCR reaction system: 100 ng plasmid template, 20 μM primer pair ecPGA-F and ecPGA-R, 1×Taq buffer, 0.2 mM dGTP, 1 mM dATP, 1 mM dCTP, 0.2 mM dTTP, 7 mM MgCl2, and 5 units of Taq enzyme (Thermo).

[0050] The PCR reaction conditions were: 95℃ for 5 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 2 min / kbp; 25-30 cycles; 72℃ for 10 min.

[0051] A 2.5 kbp random mutant fragment was extracted using an Axygen DNA Gel Extraction Kit (AP-GX-50) as a large primer. MegaPrimer PCR was performed using KOD-plus DNA polymerase: 94℃ for 5 min; 98℃ for 20 s, 60℃ for 40 s, 68℃ for 2 min / kbp, 30 cycles; 68℃ for 10 min. The plasmid template was digested with Dpn I restriction endonuclease (Thermo Fisher Scientific) and electroporated into *E. coli* BL21(DE3) (Invitrogen), yielding more than 10... 4 A random mutation library of clones.

[0052] 2.2 High-throughput screening of mutant libraries

[0053] Transformants from the mutant library were inoculated into 500 μL of LB liquid medium containing 50 μg / mL kanamycin in a 96-well deep-well plate and cultured overnight. Then, 80 μL of the overnight culture was transferred to 800 μL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 3 h. After that, 0.5 mM IPTG was added to a final concentration, the temperature was lowered to 25°C, and the culture was incubated overnight. 600 μL of the culture was centrifuged at 4500 rpm for 15 min, the supernatant was discarded, and the cells were resuspended in 100 μL of 50 mM potassium phosphate buffer (pH 7.5) for viability assay.

[0054] 2.3 High-throughput enzyme activity assay

[0055] NIPAB stock solution: Dissolve 90 mg NIPAB in 100 ml of 50 mM potassium phosphate buffer (pH 7.5) and heat to boiling.

[0056] Add 20 μL of bacterial suspension to 200 μL of NIPAB stock solution, react at 37℃ for 60 min, add 0.2 ml of anhydrous ethanol to terminate the reaction, centrifuge at 5000 rpm for 20 min, and take 200 μL to measure the change in absorbance at 405 nm.

[0057] Screening of approximately 6,000 mutant clones revealed that one clone strain exhibited a significantly enhanced ability to hydrolyze the substrate NIPAB, approximately 3.9 times that of the wild-type enzyme strain pET24a-ecPGA / BL21(DE3). This clone was designated pET24a-ecPGA-M2139 / BL21(DE3).

[0058] Plasmids were extracted from the screened mutant strain pET24a-ecPGA-M2139 / BL21(DE3) and sequenced by Suzhou Genewiz Biotechnology Co., Ltd. Through genome sequencing comparison, it was determined that the penicillin G acylase encoding gene was mutated to SEQ ID NO:4, which has led to changes in the amino acid sequence, with four amino acid mutations: F227L, D363N, D582Y, and T719P.

[0059] Example 3: Shake-flask fermentation culture of strain

[0060] Single colonies were picked from LB agar plates of strain pET24a-ecPGA / BL21(DE3) and mutant strain pET24a-ecPGA-M2139 / BL21(DE3) and inoculated into 5 mL of LB liquid medium containing 50 mg / L kanamycin sulfate. The cultures were incubated overnight at 37°C and 250 rpm. Then, the overnight cultures were inoculated into 1 L of TB medium at a 0.5 v / v% inoculation rate and incubated at 37°C and 250 rpm for 2–3 h. When the OD600 reached 0.8–1.2, 0.2 mM IPTG was added, and the cultures were incubated overnight at 28°C and 200 rpm. Finally, the cells were collected by centrifugation at 10,000 rpm for 10 min at 4°C and frozen for later use.

[0061] Example 4: Extraction of pure enzyme solution

[0062] Two types of bacterial cells collected from shake-flask fermentation in Example 3 were resuspended in 100mM potassium phosphate buffer (pH 8.0) until the bacterial concentration reached 20% (w / v). The cells were then disrupted using an ultrasonic cell disruptor (300W energy, 2 seconds on, 5 seconds off, for 20-40 min). The cell disruption solution was centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was collected. The supernatant was added at a rate of 1 ml / min to an affinity chromatography column containing 10 ml of Ni-NAT matrix. The column was then washed with equilibration buffer containing 10 mM imidazole to remove impurities. Finally, the column was washed with equilibration buffer containing 500 mM imidazole to remove the target protein, and the peak value was collected.

[0063] The eluent was desalted using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to obtain two pure enzyme proteins, ecPGA and ecPGA-M2139. Figure 2 Their SDS-PAGE electrophoresis images are shown.

[0064] Example 5: Specific activity determination of two purified enzymes

[0065] Pierce using Thermo TMThe protein quantification kit was used to determine the protein concentration of the two purified enzyme proteins obtained in Example 4. Then, 1 mL of the purified enzyme solution was accurately measured and added to 40 mL of 5% penicillin G potassium salt solution (5 g of penicillin G potassium salt dissolved in 100 mL of 50 mM potassium phosphate buffer (pH 7.8)) preheated to 28 °C. The temperature was maintained at 28 °C, and the mixture was stirred rapidly. The pH was adjusted to 8.0 with 0.1 M NaOH titrant. The reaction time was recorded, and the pH was kept constant for 3-5 min. The amount of NaOH added and the reaction time (in minutes) were recorded.

[0066] The enzyme hydrolysis activity unit (U) is defined as: the amount of penicillin G acylase required to hydrolyze 1 μmol of penicillin G potassium salt per minute under the conditions of 28℃ and pH 8.0 is 1 unit.

[0067] Activity assays of the two purified enzymes showed that the specific activity of the mutant protein purified enzyme ecPGA-M2139 was significantly higher than that of the wild-type enzyme ecPGA, by approximately 2.1 times. Specific data are shown in the table below.

[0068]

[0069] Experimental results show that the specific activity of the penicillin G acylase mutant ecPGA-M2139 of this invention in hydrolyzing penicillin G potassium salt is significantly higher than that of the wild-type enzyme ecPGA, reaching 2.1 times that of the wild-type enzyme. Therefore, compared with the prior art, it can catalyze the generation of 6-aminopenicillanic acid (6-APA), an important parent nucleus of β-lactamase antibiotics, with higher catalytic efficiency, showing great potential for industrial application. sequence list <110> Shanghai Banglin Biotechnology Co., Ltd. <120> A penicillin G acylase mutant with high hydrolytic activity <130> SHPI2210074 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 846 <212> PRT <213> Escherichia coli <400> 1 Met Lys Asn Arg Asn Arg Met Ile Val Asn Cys Val Thr Ala Ser Leu 1 5 10 15 Met Tyr Tyr Trp Ser Leu Pro Ala Leu Ala Glu Gln Ser Ser Ser Glu 20 25 30 Ile Lys Ile Val Arg Asp Glu Tyr Gly Met Pro His Ile Tyr Ala Asn 35 40 45 Asp Thr Trp His Leu Phe Tyr Gly Tyr Gly Tyr Val Val Ala Gln Asp 50 55 60 Arg Leu Phe Gln Met Glu Met Ala Arg Arg Ser Thr Gln Gly Thr Val 65 70 75 80 Ala Glu Val Leu Gly Lys Asp Phe Val Lys Phe Asp Lys Asp Ile Arg 85 90 95 Arg Asn Tyr Trp Pro Asp Ala Ile Arg Ala Gln Ile Ala Ala Leu Ser 100 105 110 Pro Glu Asp Met Ser Ile Leu Gln Gly Tyr Ala Asp Gly Met Asn Ala 115 120 125 Trp Ile Asp Lys Val Asn Thr Asn Pro Glu Thr Leu Leu Pro Lys Gln 130 135 140 Phe Asn Thr Phe Gly Phe Thr Pro Lys Arg Trp Glu Pro Phe Asp Val 145 150 155 160 Ala Met Ile Phe Val Gly Thr Met Ala Asn Arg Phe Ser Asp Ser Thr 165 170 175 Ser Glu Ile Asp Asn Leu Ala Leu Leu Thr Ala Leu Lys Asp Lys Tyr 180 185 190 Gly Val Ser Gln Gly Met Ala Val Phe Asn Gln Leu Lys Trp Leu Val 195 200 205 Asn Pro Ser Ala Pro Thr Thr Ile Ala Val Gln Glu Ser Asn Tyr Pro 210 215 220 Leu Lys Phe Asn Gln Gln Asn Ser Gln Thr Ala Ala Leu Leu Pro Arg 225 230 235 240 Tyr Asp Leu Pro Ala Pro Met Leu Asp Arg Pro Ala Lys Gly Ala Asp 245 250 255 Gly Ala Leu Leu Ala Leu Thr Ala Gly Lys Asn Arg Glu Thr Ile Val 260 265 270 Ala Gln Phe Ala Gln Gly Gly Ala Asn Gly Leu Ala Gly Tyr Pro Thr 275 280 285 Thr Ser Asn Met Trp Val Ile Gly Lys Ser Lys Ala Gln Asp Ala Lys 290 295 300 Ala Ile Met Val Asn Gly Pro Gln Phe Gly Trp Tyr Ala Pro Ala Tyr 305 310 315 320 Thr Tyr Gly Ile Gly Leu His Gly Ala Gly Tyr Asp Val Thr Gly Asn 325 330 335 Thr Pro Phe Ala Tyr Pro Gly Leu Val Phe Gly His Asn Gly Val Ile 340 345 350 Ser Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala 355 360 365 Glu Arg Leu Ser Ala Glu Lys Pro Gly Tyr Tyr Leu His Asn Gly Lys 370 375 380 Trp Val Lys Met Leu Ser Arg Glu Glu Thr Ile Thr Val Lys Asn Gly 385 390 395 400 Gln Ala Glu Thr Phe Thr Val Trp Arg Thr Val His Gly Asn Ile Leu 405 410 415 Gln Thr Asp Gln Thr Thr Gln Thr Ala Tyr Ala Lys Ser Arg Ala Trp 420 425 430 Asp Gly Lys Glu Val Ala Ser Leu Leu Ala Trp Thr His Gln Met Lys 435 440 445 Ala Lys Asn Trp Gln Glu Trp Thr Gln Gln Ala Ala Lys Gln Ala Leu 450 455 460 Thr Ile Asn Trp Tyr Tyr Ala Asp Val Asn Gly Asn Ile Gly Tyr Val 465 470 475 480 His Thr Gly Ala Tyr Pro Asp Arg Gln Ser Gly His Asp Pro Arg Leu 485 490 495 Pro Val Pro Gly Thr Gly Lys Trp Asp Trp Lys Gly Leu Leu Pro Phe 500 505 510 Glu Met Asn Pro Lys Val Tyr Asn Pro Gln Ser Gly Tyr Ile Ala Asn 515 520 525 Trp Asn Asn Ser Pro Gln Lys Asp Tyr Pro Ala Ser Asp Leu Phe Ala 530 535 540 Phe Leu Trp Gly Gly Ala Asp Arg Val Thr Glu Ile Asp Arg Leu Leu 545 550 555 560 Glu Gln Lys Pro Arg Leu Thr Ala Asp Gln Ala Trp Asp Val Ile Arg 565 570 575 Gln Thr Ser Arg Gln Asp Leu Asn Leu Arg Leu Phe Leu Pro Thr Leu 580 585 590 Gln Ala Ala Thr Ser Gly Leu Thr Gln Ser Asp Pro Arg Arg Gln Leu 595 600 605 Val Glu Thr Leu Thr Arg Trp Asp Gly Ile Asn Leu Leu Asn Asp Asp 610 615 620 Gly Lys Thr Trp Gln Gln Pro Gly Ser Ala Ile Leu Asn Val Trp Leu 625 630 635 640 Thr Ser Met Leu Lys Arg Thr Val Val Ala Ala Val Pro Met Pro Phe 645 650 655 Asp Lys Trp Tyr Ser Ala Ser Gly Tyr Glu Thr Thr Gln Asp Gly Pro 660 665 670 Thr Gly Ser Leu Asn Ile Ser Val Gly Ala Lys Ile Leu Tyr Glu Ala 675 680 685 Val Gln Gly Asp Lys Ser Pro Ile Pro Gln Ala Val Asp Leu Phe Ala 690 695 700 Gly Lys Pro Gln Gln Glu Val Val Leu Ala Ala Leu Glu Asp Thr Trp 705 710 715 720 Glu Thr Leu Ser Lys Arg Tyr Gly Asn Asn Val Ser Asn Trp Lys Thr 725 730 735 Pro Ala Met Ala Leu Thr Phe Arg Ala Asn Asn Phe Phe Gly Val Pro 740 745 750 Gln Ala Ala Ala Glu Glu Thr Arg His Gln Ala Glu Tyr Gln Asn Arg 755 760 765 Gly Thr Glu Asn Asp Met Ile Val Phe Ser Pro Thr Thr Ser Asp Arg 770 775 780 Pro Val Leu Ala Trp Asp Val Val Ala Pro Gly Gln Ser Gly Phe Ile 785 790 795 800 Ala Pro Asp Gly Thr Val Asp Lys His Tyr Glu Asp Gln Leu Lys Met 805 810 815 Tyr Glu Asn Phe Gly Arg Lys Ser Leu Trp Leu Thr Lys Gln Asp Val 820 825 830 Glu Ala His Lys Glu Ser Gln Glu Val Leu His Val Gln Arg 835 840 845 <210> 2 <211> 2538 <212> DNA <213> Escherichia coli <400> 2 atgaaaaata gaaatcgtat gatcgtgaac tgtgttactg cttccctgat gtattattgg 60 agcttacctg cactggctga gcagtcgtca agtgagataa agattgttcg cgatgaatac 120 ggcatgccgc atatttatgc caatgataca tggcacctat tttatggcta tggctatgta 180 gtagcacaag atcgcctttt tcagatggaa atggcacgtc gcagtactca agggactgtc 240 gcggaagtgc ttggcaaaga ttttgtgaaa tttgataaag ataccgtcg taactactgg 300 ccggatgcta tccgggcgca aattgctgcc ctttccccag aggatatgtc cattctgcaa 360 ggctacgctg atggaatgaa tgcctggatt gataaggtaa ataccaatcc agagacgctc 420 ttaccaaaac agtttaatac atttggcttt actcctaagc gctgggaacc gtttgatgtc 480 gcgatgatat ttgtgggcac catggcaaac cgcttctctg atagcactag cgaaattgat 540 aatctggcac tgctaacggc tttaaaagat aaatatggtg tatcacaagg catggcggta 600 tttaatcagt tgaaatggct ggtaaaccca tcagcgccaa ccactattgc cgtacaagag 660 agtaactacc cacttaaatt taatcagcaa aactcgcaaa cagcagctct gttgccacgc 720 tacgatttac ctgcaccaat gcttgaccga ccagcaaaag gggcggatgg cgcactgctg 780 gcgttaacag cagggaagaa ccgggaaact attgttgcac aatttgcaca gggtggtgcc 840 aatggtctgg cggggtatcc aacgaccagc aatatgtggg ttatcggcaa aagcaaagcc 900 caggatgcga aagcaatcat ggtaaatggt ccgcagtttg gctggtatgc gcctgcgtat 960 acttatggta ttggtctgca cggtgctggt tatgatgtca ctggcaatac accatttgcc 1020 tatcctgggc tggtttttgg tcataatggt gtgatttcct ggggatcaac ggcaggtttc 1080 ggcgatgatg tcgatatttt tgctgaacgg ctgtcggcag agaaaccagg ctactacttg 1140 cataatggta agtgggtgaa aatgttaagc cgtgaggaaa ccattacggt gaaaaatggt 1200 caggcagaga cctttactgt ctggcgtacg gtgcatggca acattctcca aactgaccag 1260 acgacacaaa cggcttacgc taaatcccgc gcatgggatg gtaaagaggt ggcgtctttg 1320 ctggcctgga ctcatcagat gaaggccaaa aattggcagg agtggacaca gcaggcagcg 1380 aaacaagcac tgaccatcaa ctggtactat gctgatgtaa acggcaatat tggttatgtt 1440 catactggtg cttatccaga tcgtcaatca ggccatgatc cgcgattacc cgttcctggt 1500 acgggaaaat gggactggaa agggctattg ccttttgaaa tgaaccctaa ggtgtataac 1560 ccccagtcgg gatatattgc taactggaac aattctcccc aaaaagatta tcccgcttca 1620 gatctgtttg cctttttgtg gggtggtgca gatcgcgtta cggagatcga ccgactgctt 1680 gagcaaaagc cacgcttaac tgctgatcag gcatgggatg ttattcgcca aaccagtcgt 1740 caggatctta acctgaggct ttttttacct actctgcaag cagcgacatc tggtttgaca 1800 cagagcgatc cgcgtcgtca gttggtagaa acattaacac gttgggatgg catcaatttg 1860 cttaatgatg atggtaaaac ctggcagcag ccaggctctg ccatcctgaa cgtttggctg 1920 accagtatgt tgaagcgtac cgtagtggct gccgtaccta tgccatttga taagtggtac 1980 agcgccagtg gctacgaaac aacccaggac ggcccaactg gttcgctgaa tataagtgtt 2040 ggagcaaaaa ttttgtatga ggcggtgcag ggagacaaat caccaatccc acaggcggtt 2100 gatctgtttg ctgggaaacc acagcaggag gttgtgttgg ctgcgctgga agatacctgg 2160 gagactcttt ccaaacgcta tggcaataat gtgagtaact ggaaaacacc tgcaatggcc 2220 ttaacgttcc gggcaaataa tttctttggt gtaccgcagg ccgcagcgga agaaacgcgt 2280 catcaggcgg agtatcaaaa ccgtggaaca gaaaacgata tgattgtttt ctcaccaacg 2340 acaagcgatc gtcctgtgct tgcctgggat gtggtcgcac ccggtcagag tgggtttatt 2400 gctcccgatg gaacagttga taagcactat gaagatcagc tgaaaatgta cgaaaatttt 2460 ggccgtaagt cgctctggtt aacgaagcag gatgtggagg cgcataagga gtcgcaggaa 2520 gtgttgcacg ttcagaga 2538 <210> 3 <211> 846 <212> PRT <213> Artificial Sequence <400> 3 Met Lys Asn Arg Asn Arg Met Ile Val Asn Cys Val Thr Ala Ser Leu 1 5 10 15 Met Tyr Tyr Trp Ser Leu Pro Ala Leu Ala Glu Gln Ser Ser Ser Glu [[ID=,11]]20 25 30 Ile Lys Ile Val Arg Asp Glu Tyr Gly Met Pro His Ile Tyr Ala Asn 35 40 45 Asp Thr Trp His Leu Phe Tyr Gly Tyr Gly Tyr Val Val Ala Gln Asp 50 55 60 Arg Leu Phe Gln Met Glu Met Ala Arg Arg Ser Thr Gln Gly Thr Val 65 70 75 80 Ala Glu Val Leu Gly Lys Asp Phe Val Lys Phe Asp Lys Asp Ile Arg 85 90 95 Arg Asn Tyr Trp Pro Asp Ala Ile Arg Ala Gln Ile Ala Ala Leu Ser 100 105 110 Pro Glu Asp Met Ser Ile Leu Gln Gly Tyr Ala Asp Gly Met Asn Ala 115 120 125 Trp Ile Asp Lys Val Asn Thr Asn Pro Glu Thr Leu Leu Pro Lys Gln 130 135 140 Phe Asn Thr Phe Gly Phe Thr Pro Lys Arg Trp Glu Pro Phe Asp Val 145 150 155 160 Ala Met Ile Phe Val Gly Thr Met Ala Asn Arg Phe Ser Asp Ser Thr 165 170 175 Ser Glu Ile Asp Asn Leu Ala Leu Leu Thr Ala Leu Lys Asp Lys Tyr 180 185 190 Gly Val Ser Gln Gly Met Ala Val Phe Asn Gln Leu Lys Trp Leu Val 195 200 205 Asn Pro Ser Ala Pro Thr Thr Ile Ala Val Gln Glu Ser Asn Tyr Pro 210 215 220 Leu Lys Leu Asn Gln Gln Asn Ser Gln Thr Ala Ala Leu Leu Pro Arg 225 230 235 240 Tyr Asp Leu Pro Ala Pro Met Leu Asp Arg Pro Ala Lys Gly Ala Asp 245 250 255 Gly Ala Leu Leu Ala Leu Thr Ala Gly Lys Asn Arg Glu Thr Ile Val 260 265 270 Ala Gln Phe Ala Gln Gly Gly Ala Asn Gly Leu Ala Gly Tyr Pro Thr 275 280 285 Thr Ser Asn Met Trp Val Ile Gly Lys Ser Lys Ala Gln Asp Ala Lys 290 295 300 Ala Ile Met Val Asn Gly Pro Gln Phe Gly Trp Tyr Ala Pro Ala Tyr 305 310 315 320 Thr Tyr Gly Ile Gly Leu His Gly Ala Gly Tyr Asp Val Thr Gly Asn 325 330 335 Thr Pro Phe Ala Tyr Pro Gly Leu Val Phe Gly His Asn Gly Val Ile 340 345 350 Ser Trp Gly Ser Thr Ala Gly Phe Gly Asp Asn Val Asp Ile Phe Ala 355 360 365 Glu Arg Leu Ser Ala Glu Lys Pro Gly Tyr Tyr Leu His Asn Gly Lys 370 375 380 Trp Val Lys Met Leu Ser Arg Glu Glu Thr Ile Thr Val Lys Asn Gly 385 390 395 400 Gln Ala Glu Thr Phe Thr Val Trp Arg Thr Val His Gly Asn Ile Leu 405 410 415 Gln Thr Asp Gln Thr Thr Gln Thr Ala Tyr Ala Lys Ser Arg Ala Trp 420 425 430 Asp Gly Lys Glu Val Ala Ser Leu Leu Ala Trp Thr His Gln Met Lys 435 440 445 Ala Lys Asn Trp Gln Glu Trp Thr Gln Gln Ala Ala Lys Gln Ala Leu 450 455 460 Thr Ile Asn Trp Tyr Tyr Ala Asp Val Asn Gly Asn Ile Gly Tyr Val 465 470 475 480 His Thr Gly Ala Tyr Pro Asp Arg Gln Ser Gly His Asp Pro Arg Leu 485 490 495 Pro Val Pro Gly Thr Gly Lys Trp Asp Trp Lys Gly Leu Leu Pro Phe 500 505 510 Glu Met Asn Pro Lys Val Tyr Asn Pro Gln Ser Gly Tyr Ile Ala Asn 515 520 525 Trp Asn Asn Ser Pro Gln Lys Asp Tyr Pro Ala Ser Asp Leu Phe Ala 530 535 540 Phe Leu Trp Gly Gly Ala Asp Arg Val Thr Glu Ile Asp Arg Leu Leu 545 550 555 560 Glu Gln Lys Pro Arg Leu Thr Ala Asp Gln Ala Trp Asp Val Ile Arg 565 570 575 Gln Thr Ser Arg Gln Tyr Leu Asn Leu Arg Leu Phe Leu Pro Thr Leu 580 585 590 Gln Ala Ala Thr Ser Gly Leu Thr Gln Ser Asp Pro Arg Arg Gln Leu 595 600 605 Val Glu Thr Leu Thr Arg Trp Asp Gly Ile Asn Leu Leu Asn Asp Asp 610 615 620 Gly Lys Thr Trp Gln Gln Pro Gly Ser Ala Ile Leu Asn Val Trp Leu 625 630 635 640 Thr Ser Met Leu Lys Arg Thr Val Val Ala Ala Val Pro Met Pro Phe 645 650 655 Asp Lys Trp Tyr Ser Ala Ser Gly Tyr Glu Thr Thr Gln Asp Gly Pro 660 665 670 Thr Gly Ser Leu Asn Ile Ser Val Gly Ala Lys Ile Leu Tyr Glu Ala 675 680 685 Val Gln Gly Asp Lys Ser Pro Ile Pro Gln Ala Val Asp Leu Phe Ala 690 695 700 Gly Lys Pro Gln Gln Glu Val Val Leu Ala Ala Leu Glu Asp Pro Trp 705 710 715 720 Glu Thr Leu Ser Lys Arg Tyr Gly Asn Asn Val Ser Asn Trp Lys Thr 725 730 735 Pro Ala Met Ala Leu Thr Phe Arg Ala Asn Asn Phe Phe Gly Val Pro 740 745 750 Gln Ala Ala Ala Glu Glu Thr Arg His Gln Ala Glu Tyr Gln Asn Arg 755 760 765 Gly Thr Glu Asn Asp Met Ile Val Phe Ser Pro Thr Thr Ser Asp Arg 770 775 780 Pro Val Leu Ala Trp Asp Val Val Ala Pro Gly Gln Ser Gly Phe Ile 785 790 795 800 Ala Pro Asp Gly Thr Val Asp Lys His Tyr Glu Asp Gln Leu Lys Met 805 810 815 Tyr Glu Asn Phe Gly Arg Lys Ser Leu Trp Leu Thr Lys Gln Asp Val 820 825 830 Glu Ala His Lys Glu Ser Gln Glu Val Leu His Val Gln Arg 835 840 845 <210> 4 <211> 2538 <212> DNA <213> Artificial Sequence <400> 4 atgaaaaata gaaatcgtat gatcgtgaac tgtgttactg cttccctgat gtattattgg 60 agcttacctg cactggctga gcagtcgtca agtgagataa agattgttcg cgatgaatac 120 ggcatgccgc atatttatgc caatgataca tggcacctat tttatggcta tggctatgta 180 gtagcacaag atcgcctttt tcagatggaa atggcacgtc gcagtactca agggactgtc 240 gcggaagtgc ttggcaaaga ttttgtgaaa tttgataaag ataccgtcg taactactgg 300 ccggatgcta tccgggcgca aattgctgcc ctttccccag aggatatgtc cattctgcaa 360 ggctacgctg atggaatgaa tgcctggatt gataaggtaa ataccaatcc agagacgctc 420 ttaccaaaac agtttaatac atttggcttt actcctaagc gctgggaacc gtttgatgtc 480 gcgatgatat ttgtgggcac catggcaaac cgcttctctg atagcactag cgaaattgat 540 aatctggcac tgctaacggc tttaaaagat aatatggtg tatcacaagg catggcggta 600 tttaatcagt tgaaatggct ggtaaaccca tcagcgccaa ccactattgc cgtacaagag 660 agtaactacc cactaatt aaatcagcaa aactcgcaaa cagcagctct gttgccacgc 720 tacgatttac ctgcaccaat gcttgaccga ccagcaaaag gggcggatgg cgcactgctg 780 gcgttaacag cagggaagaa ccgggaaact attgttgcac aatttgcaca gggtggtgcc 840 aatggtctgg cggggtatcc aacgaccagc aatatgtggg ttatcggcaa aagcaaagcc 900 caggatgcga aagcaatcat ggtaaatggt ccgcagtttg gctggtatgc gcctgcgtat 960 actatggta ttggtctgca cggtgctggt tatgatgtca ctggcaatac accatttgcc 1020 tatcctgggc tggtttttgg tcataatggt gtgatttcct ggggatcaac ggcaggtttc 1080 ggcgataatg tcgatatttt tgctgaacgg ctgtcggcag agaaaccagg ctactacttg 1140 cataatggta agtgggtgaa aatgttaagc cgtgaggaaa cattacggt gaaaaatggt 1200 caggcagaga cctttactgt ctggcgtacg gtgcatggca acattctcca aactgaccag 1260 acgacacaaa cggcttacgc taaatcccgc gcatgggatg gtaaagaggt ggcgtctttg 1320 ctggcctgga ctcatcagat gaaggccaaa aattggcagg agtggacaca gcaggcagcg 1380 aaacaagcac tgaccatcaa ctggtactat gctgatgtaa acggcaatat tggttatgtt 1440 catactggtg cttatccaga tcgtcaatca ggccatgatc cgcgattacc cgttcctggt 1500 acgggaaaat gggactggaa agggctattg ccttttgaaa tgaaccctaa ggtgtataac 1560 ccccagtcgg gatatattgc taactggaac aattctcccc aaaaagatta tcccgcttca 1620 gatctgtttg cctttttgtg gggtggtgca gatcgcgtta cggagatcga ccgactgctt 1680 gagcaaaagc cacgcttaac tgctgatcag gcatgggatg ttattcgcca aaccagtcgt 1740 cagtatctta acctgaggct ttttttacct actctgcaag cagcgacatc tggtttgaca 1800 cagagcgatc cgcgtcgtca gttggtagaa acattaacac gttgggatgg catcaatttg 1860 cttaatgatg atggtaaaac ctggcagcag ccaggctctg ccatcctgaa cgtttggctg 1920 accagtatgt tgaagcgtac cgtagtggct gccgtaccta tgccatttga taagtggtac 1980 agcgccagtg gctacgaaac aacccaggac ggcccaactg gttcgctgaa tataagtgtt 2040 ggagcaaaaa ttttgtatga ggcggtgcag ggagacaaat caccaatccc acaggcggtt 2100 gatctgtttg ctgggaaacc acagcaggag gttgtgttgg ctgcgctgga agatccctgg 2160 gagactcttt ccaaacgcta tggcaataat gtgagtaact ggaaaacacc tgcaatggcc 2220 ttaacgttcc gggcaaataa tttctttggt gtaccgcagg ccgcagcgga agaaacgcgt 2280 catcaggcgg agtatcaaaa ccgtggaaca gaaaacgata tgattgtttt ctcaccaacg 2340 acaagcgatc gtcctgtgct tgcctgggat gtggtcgcac ccggtcagag tgggtttatt 2400 gctcccgatg gaacagttga taagcactat gaagatcagc tgaaaatgta cgaaaatttt 2460 ggccgtaagt cgctctggtt aacgaagcag gatgtggagg cgcataagga gtcgcaggaa 2520 gtgttgcacg ttcagaga 2538

Claims

1. A penicillin G acylase, characterized in that, The amino acid sequence is SEQ ID NO:

3.

2. A polynucleotide, characterized in that, The enzyme is coded as described in claim 1, which is a penicillin G acylase.

3. The polynucleotide as described in claim 2, characterized in that, The nucleotide sequence is SEQ ID NO:

4.

4. A plasmid, characterized in that, The polynucleotide described in claim 2 or 3 is cloned thereon.

5. The plasmid as described in claim 4, characterized in that, The carrier is PET series.

6. The plasmid as described in claim 4, characterized in that, The carrier is pET24a.

7. A microorganism, characterized in that, It is a transformant that has been transformed with the plasmid as described in claim 5.

8. The microorganism as described in claim 7, characterized in that, It's E. coli.

9. The microorganism as described in claim 7, characterized in that, The host is Escherichia coli BL21(DE3).

10. Use of the penicillin G acylase as described in claim 1 or the microorganism as described in claim 7 in the hydrolysis of potassium penicillin G to produce 6-aminopenicillanic acid (6-APA).

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