Application of expandase and its mutants in the production of G-7-ADCA

Abstract

The present invention provides an expandase and its mutants for use in producing G-7-ADCA. Its amino acid sequence is shown in one of SEQ ID No.5-13. The present invention generates a series of enzymes with expansion activity through NCBI sequence alignment, mining, and directed evolution techniques, and these enzymes can expand the substrate penicillin G into G-7-ADCA in one step; G-7-ADCA can then be converted into 7-ADCA by removing the side chain through the action of penicillin acylase. Therefore, these expandases have important application value in industry.

Description

Technical Field

[0001] The invention relates to application of expandase and mutants thereof in producing G-7-ADCA, belonging to the field of biotechnology. Background Art

[0002] Cephalosporin antibiotics are a class of anti-infective drugs widely used in clinical practice, with advantages such as low toxicity and broad spectrum. 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) is one of the important nuclei for the synthesis of cephalosporin antibiotics, which can be used to synthesize cephalexin, cephradine and cefadroxil, etc., and has a huge market demand.

[0003] At present, there are two main methods for industrial production of 7-ADCA: chemoenzymatic method and fermentation method. The process of preparing 7-ADCA by chemoenzymatic method uses penicillin G as raw material, and uses chemical methods to oxidize, expand and rearrange to generate phenylacetyl-7-aminodeacetoxycephalosporin acid (G-7-ADCA), and then removes the side chain under the action of penicillin acylase to generate 7-ADCA. This process is relatively complicated, costly, and produces a large amount of wastewater and pollution, which is very harmful to the environment (Wei CL et al,.Applied and Environmental Mirobiology, 2005). Under the pressure of national energy conservation, emission reduction and environmental protection, this process is gradually being eliminated.

[0004] Fermentation can also be used to produce 7-ADCA industrially. In 1998, Cho et al. found that expandase (DAOCS) has a certain activity on penicillin G, making it possible to produce G-7-ADCA from penicillin G (Cho et al., PNAS, 1998). Therefore, the commonly used method in some companies is to clone the expandase (DAOCS) gene into the yellow-producing Penicillium, add adipic acid during the fermentation process, directly ferment and produce G-7-ADCA, and then use penicillin acylase to remove the side chain to prepare 7-ADCA. However, the fermentation method has problems such as complex process and long fermentation cycle, and the core technology is mainly in the hands of foreign companies. At present, there is no industrial method for producing 7-ADCA by fermentation in China.

[0005] The whole enzyme method for preparing 7-ADCA has the advantages of being green, environmentally friendly and low cost. The whole enzyme method starts with the cheap potassium salt of penicillin G, generates G-7-ADCA through the catalytic ring expansion reaction of penicillin expandase, and then hydrolyzes the acyl side chain through the action of penicillin acylase to generate 7-ADCA (such as Figure 1As shown in Figure 1 ). The research on penicillin acylase is very mature and has been used in industry for many years. The most commonly used one is immobilized penicillin acylase; while the activity of expandase is relatively low and it is the rate-limiting step in the synthesis of 7-ADCA from penicillin G. Therefore, it is urgent to improve the activity of expandase.

[0006] Penicillin expandase (DAOCS), also known as deacetyloxycephalosporin C synthetase, is an Fe-dependent 2+ DAOCS is an enzyme that synthesizes 1,2-dihydro-1,4-dihydro-2-nitropropene and 1,4-dihydro-1,4-dihydro-2-nitropropene. It mainly catalyzes the conversion of the five-membered thiazole ring of penicillin to the six-membered thiazine ring (ring expansion), and is a key step in catalyzing the synthesis of cephalosporin antibiotics by Streptomyces clavuligerus and other prokaryotes. Therefore, DAOCS has important industrial application value.

[0007] However, in actual applications, it was found that the biological activity of DAOCS on penicillin G was low and could not meet the requirements of industrial applications, which became the rate-limiting step in the synthesis of 7-ADCA using the whole enzyme method. Therefore, it is urgent to modify the expandase in order to significantly improve its conversion efficiency to penicillin G and lay the foundation for industrial applications.

[0008] At present, DAOCS from C.acremonium, S.clavuligerus and N.lactamdurans have been expressed and purified, and the crystal structure of scDAOCS from S.clavuligerus has been obtained. This makes it possible to modify scDAOCS through protein engineering technology, improve its catalytic activity and broaden its substrate spectrum, and some progress has been made. Summary of the invention

[0009] The technical problem to be solved by the present invention is how to improve the ring-expansion catalytic activity of expandase on penicillin G.

[0010] In order to solve the above technical problems, the present invention first provides a protein.

[0011] The protein provided by the present invention is as follows (a) or (b) or (c) or (d):

[0012] (a) an amino acid sequence as shown in SEQ ID No. 4 (named H7), or a protein derived from (a) in which the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids and has expandase activity;

[0013] (b) an amino acid sequence as shown in SEQ ID No. 5 (named E727), or a protein derived from (b) having expandase activity, wherein the amino acid sequence in (b) is substituted, deleted or added with one or more amino acids;

[0014] (c) an amino acid sequence as shown in SEQ ID No. 6 (named E735), or a protein derived from (c) having expandase activity, wherein the amino acid sequence in (c) is substituted, deleted or added with one or more amino acids;

[0015] (d) An amino acid sequence as shown in SEQ ID No. 13 (named as scDAOCS-V303K), or a protein derived from (d) in which the amino acid sequence in (d) is substituted, deleted or added with one or more amino acids and has expandase activity.

[0016] Furthermore, the protein is any one of the following (1)-(6):

[0017] (1) a protein represented by the amino acid sequence of SEQ ID NO: 5, wherein position 155 is n1 and position 184 is n2;

[0018] (2) a protein represented by the amino acid sequence of SEQ ID NO:5, wherein the amino acid residue at position 275 is n3, the amino acid residue at position 281 is n4, and the amino acid residue at position 305 is n5;

[0019] (3) a protein represented by the amino acid sequence of SEQ ID NO: 5, wherein position 155 is n1, position 184 is n2, position 275 is n3, position 281 is n4, and position 305 is n5;

[0020] (4) a protein represented by the amino acid sequence of SEQ ID NO: 6, wherein position 155 is m1 and position 184 is m2;

[0021] (5) a protein represented by the amino acid sequence of SEQ ID NO: 6, wherein position 275 is m3, position 281 is m4, and position 305 is m5;

[0022] (6) a protein represented by the amino acid sequence of SEQ ID NO: 6, wherein position 155 is m1, position 184 is m2, position 275 is m3, position 281 is m4, and position 305 is m5;

[0023] Specifically, n1 at position 155 in the amino acid sequence shown in SEQ ID NO: 5 may be: cysteine, tyrosine, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, serine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, n2 at position 184 may be: tyrosine, histidine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine or arginine, and n3 at position 275 may be: valine, isoleucine, glycine, alanine, leucine, methionine, proline, tryptophan, serine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine or arginine. The n4 at position 281 may be: cysteine, tyrosine, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, serine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine; the n5 at position 305 may be: leucine, methionine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine;

[0024] In the amino acid sequence shown in SEQ ID NO: 6, m1 at position 155 may be phenylalanine, lysine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine or histidine, m2 at position 184 may be aspartic acid, glutamic acid, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine or histidine, and m3 at position 275 may be glutamic acid, histidine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, , proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, lysine or arginine, the m4 at position 281 can be: tyrosine, proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, serine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, and the m5 at position 305 can be: isoleucine, methionine, glycine, alanine, valine, leucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine.

[0025] Furthermore, the protein is any one of the following (1)-(6):

[0026] (1) A protein obtained by mutating the amino acid residue at position 155 of the amino acid sequence shown in SEQ ID NO:5 from cysteine ​​to tyrosine, and by mutating the amino acid residue at position 184 from tyrosine to histidine, named E727-M2 (C155Y / Y184H), and having an amino acid sequence as shown in SEQ ID NO:7;

[0027] (2) A protein obtained by mutating the amino acid residue at position 275 of the amino acid sequence shown in SEQ ID NO:5 from valine to isoleucine, mutating the amino acid residue at position 281 from cysteine ​​to tyrosine, and mutating the amino acid residue at position 305 from leucine to methionine was named E727-M3 (V275I / C281Y / L305M), and its amino acid sequence is shown in SEQ ID NO:8;

[0028] (3) A protein obtained by mutating the amino acid residue at position 155 of the amino acid sequence shown in SEQ ID NO:5 from cysteine ​​to tyrosine, mutating the amino acid residue at position 184 from tyrosine to histidine, mutating the amino acid residue at position 275 from valine to isoleucine, mutating the amino acid residue at position 281 from cysteine ​​to tyrosine, and mutating the amino acid residue at position 305 from leucine to methionine was named E727-M5 (C155Y / Y184H / V275I / C281Y / L305M), and the amino acid sequence thereof is shown in SEQ ID NO:9;

[0029] (4) A protein obtained by mutating the amino acid residue at position 155 of the amino acid sequence shown in SEQ ID NO:6 from cysteine ​​to tyrosine, and by mutating the amino acid residue at position 184 from tyrosine to histidine, named E735-M2 (C155Y / Y184H), and having an amino acid sequence shown in SEQ ID NO:10;

[0030] (5) A protein obtained by mutating the amino acid residue at position 275 of the amino acid sequence shown in SEQ ID NO:6 from valine to isoleucine, mutating the amino acid residue at position 281 from cysteine ​​to tyrosine, and mutating the amino acid residue at position 305 from isoleucine to methionine was named E735-M3 (V275I / C281Y / I305M), and its amino acid sequence is shown in SEQ ID NO:11;

[0031] (6) A protein obtained by mutating the amino acid residue at position 155 of the amino acid sequence shown in SEQ ID NO:6 from cysteine ​​to tyrosine, mutating the amino acid residue at position 184 from tyrosine to histidine, mutating the amino acid residue at position 275 from valine to isoleucine, mutating the amino acid residue at position 281 from cysteine ​​to tyrosine, and mutating the amino acid residue at position 305 from isoleucine to methionine, and named E735-M5 (C155Y / Y184H / V275I / C281Y / I305M), and its amino acid sequence is shown in SEQ ID No. 12;

[0032] Nucleic acid molecules encoding the above proteins also fall within the protection scope of the present invention.

[0033] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA, hnRNA or tRNA.

[0034] The nucleic acid molecule is specifically a gene encoding the above protein.

[0035] The recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the above nucleic acid molecule also fall within the protection scope of the present invention.

[0036] The recombinant vector may be a recombinant expression vector or a recombinant cloning vector.

[0037] The recombinant expression vector can be constructed using an existing expression vector. The expression vector may also contain the 3' non-translated region of the exogenous gene, i.e., a polyadenylic acid signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor. When the gene is used to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, which can be used alone or in combination with other promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0038] The expression cassette consists of a promoter capable of initiating the expression of the gene, the gene, and a transcription termination sequence.

[0039] The use of the protein as penicillin expandase also falls within the protection scope of the present invention.

[0040] The use of the protein as penicillin expandase to catalyze the ring expansion of penicillin G to generate G-7-ADCA and improve the conversion rate of penicillin G also falls within the protection scope of the present invention.

[0041] The use of the protein or the nucleic acid molecule or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in any of the following also falls within the protection scope of the present invention:

[0042] (b1) preparing a product having penicillin expandase activity;

[0043] (b2) Preparation of G-7-ADCA.

[0044] The present invention also protects a method for preparing G-7-ADCA.

[0045] The method for preparing G-7-ADCA provided by the present invention may specifically include the steps of: preparing the protein; using the protein as penicillin expandase to catalyze the ring expansion of penicillin G to generate G-7-ADCA.

[0046] The present invention also protects a method for improving the conversion rate of penicillin G in the reaction of penicillin expandase catalyzing the ring expansion of penicillin G to generate G-7-ADCA.

[0047] The invention provides a method for improving the conversion rate of penicillin G in the reaction of penicillin expandase catalyzing the ring expansion of penicillin G to generate G-7-ADCA, wherein the protein is used as penicillin expandase to catalyze the ring expansion of penicillin G to generate G-7-ADCA.

[0048] The present invention generates a series of enzymes with ring expansion activity by using NCBI sequence alignment, mining, directed evolution and other technologies, and these enzymes can expand the substrate penicillin G into G-7-ADCA in one step; G-7-ADCA can then be converted into 7-ADCA by removing the side chain through the action of penicillin acylase. Therefore, these expandases have important application value in industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Figure 2 is a flow chart of the 7-ADCA reaction catalyzed by expandase.

[0050] Figure 2 It is the sequence alignment information used by SCHEMA technology.

[0051] Figure 3 Schematic diagram of the location of loop-related mutation sites obtained based on the Rosseta Design method. DETAILED DESCRIPTION

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0053] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0054] Example 1: Expandase mutants generated based on SCHEMA technology

[0055] 1. Gene acquisition and vector construction

[0056] The SCHEMA technique was used to analyze and simulate DNA shuffling for recombination. According to the results of computational simulation, 20 sequences were screened. After codon optimization, the full-length gene was obtained by whole gene synthesis and constructed into the pET-24a expression vector. The three expandase sequences used in the SCHEMA technique (E632, SEQ ID No.1, 8MUT SEQ ID No.2, and E633 SEQ ID No.3) and the alignment are shown in Figure 2. Figure 2 shown.

[0057] After SCHEMA technology alignment, the sequences shown in Table 1 were obtained, and gene synthesis was performed according to the obtained sequences. After expression, screening was performed according to the catalytic efficiency.

[0058] 2. Protein expression and activity detection

[0059] The above expression vector was transformed into the host bacteria BL21 (DE3), and a negative control group (empty vector of pET-24a) and a positive control group (pET-24a-scDAOCS) were set up at the same time, and cultured overnight at 37°C; a single colony was picked and transferred to LB medium containing 100 μg / mL kanamycin, and cultured at 37°C and 220rpm for 8-10h; then, it was transferred to 50mL TB medium containing 100 μg / mL according to an inoculum size of 1 / 100, and cultured at 37°C until OD600 reached about 0.6, and 0.1mM IPTG was added to induce protein expression, and the induction time was 16h to obtain recombinant bacteria expressing expandase.

[0060] 3. Reaction system

[0061] Collect the cells. Pour the cells into a 50 mL centrifuge tube and centrifuge at 8000 rpm for 10 min to obtain the cells; resuspend and wash the cells once with 50 mM pH 7.4 phosphate buffer; centrifuge at 8000 rpm for 10 min to collect the cells; and weigh them.

[0062] Reaction solution formula: 50mM pH 7.4 phosphate buffer, 50μg / mL FeSO4, 0.4mM ascorbic acid, 5mM or 10mM α-ketoglutaric acid, 5mM or 10mM penicillin G.

[0063] Reaction. Add 10 mL of liquid to 1 g of wet bacteria, add the reaction solution to the bacteria, resuspend. Place in a shaker at 25°C, 220 rpm, and react for 2 hours.

[0064] The amount of G-7-ADCA produced was detected. After 2 hours of reaction, the mixture was centrifuged at 12,000 rpm for 1 minute, and the supernatant was taken for HPLC detection.

[0065] The liquid chromatography detection conditions are as follows: chromatographic column: Agilent ZORBAX SB-C18 Stable Bond Analytical 4.6×250 mm; mobile phase: 20 mM pH 3.0 sodium phosphate buffer: methanol = 55:45. Flow rate: 1 mL / min, detection wavelength 215 nm.

[0066] 4. Results of conversion rate of catalytic penicillin G conversion

[0067] Table 1 Conversion rates of expandase mutants generated using SCHEMA technology

[0068]

[0069] Reaction conditions:

[0070] 50mM pH 7.4 phosphate buffer, 50μg / mL FeSO4, 0.4mM ascorbic acid, 10mM α-ketoglutaric acid, 10mM penicillin G, 200μL crude enzyme solution. Place in a 25℃ shaker, 220rpm, and react for 30min.

[0071] 5. Enzyme modification

[0072] The more active E727 and E735 were transformed into double, triple and five mutants, and the mutation sites in the highly active mutant H7 were selected and transplanted into E727 and E735 to obtain E727-M2 (C155Y / Y184H), E727-M3 (V275I / C281Y / L305M), E727-M5 (C155Y / Y184H / V275I / C281Y / L305M), E735-M2 (C155Y / Y184H), E735-M3 (V275I / C281Y / I305M), and E727-M5 (C155Y / Y184H / V275I / C281Y / I305M) mutants. Their activities are as follows:

[0073] Table 2 Conversion rates of expandase mutants generated using the SCHEMA program

[0074]

[0075] As shown in Table 2, the conversion rate of penicillin G catalyzed by E727-M2 was the highest and better than that of H7.

[0076] 6. Pure enzyme reaction and HPLC detection

[0077] 1mL system was used to verify the pure enzyme activity. PBS buffer solution of pH 7.4, 50μg / mL FeSO4, 0.4mM ascorbic acid, 5mM α-ketoglutaric acid, 5mM penicillin G, 100μL enzyme solution. Place in a 25℃ shaker, 220rpm, and react for 30min. The HPLC test results are shown in Table 3.

[0078] Table 3 Enzyme activity catalyzed by expandase mutants

[0079]

[0080]

[0081] Enzyme activity (U) is defined as the amount of enzyme that can convert 1 micromole of substrate in 1 minute at 25°C; specific enzyme activity (U / mg) is the enzyme activity per mg of protein.

[0082] Experimental verification showed that the enzyme activity of mutant E727-M2 was 25% higher than that of scH7, and it has good application prospects.

[0083] Example 2: Mutants obtained by modifying expandase H7 based on the Rosetta Design method

[0084] 1. Acquisition of genes.

[0085] All computational designs were completed using Rosetta design software developed by David Baker's team at the University of Washington. The top 20 conformations that met all the necessary conditions were selected for analysis and experimental verification. Using the expandase 7 mutant scDAOCS H7 as a template, four combinatorial mutation libraries were constructed after dynamic simulation of loop-related mutation sites. The mutation libraries are shown in Table 4. The positions of the mutation sites on the expandase are shown in Table 4. Figure 3 shown.

[0086] Table 4 Mutant library established according to RosettaDesign method

[0087]

[0088] 2. Establishment of mutant library.

[0089] The corresponding primers of the selected residues were used for site-by-site mutagenesis. The corresponding primers are shown in Table 5.

[0090] Table 5 Primer sequence list

[0091]

[0092]

[0093] Using plasmid scDAOCS H7 as template, primers T72-F1, T72-F2, T72-F3, T72-F4, T73-F1, T73-F2, T73-F3, and T73-F4 in Table 5 were mixed in a ratio of 3:1:1:1:3:1:1:1, 2 μL of the above mixture was taken as the upstream primer, and 2 μL of T72-T73-R74-R in Table 5 was taken as the downstream primer to amplify gene fragment L1. Using plasmid scDAOCS H7 as a template, Y94-D95-Y97-F1, Y94-D95-Y97-F2, and Y94-D95-Y97-F3 in Table 5 were mixed at a ratio of 2:1:1, 2 μL was taken as the upstream primer, and Y94-D95-Y97-R2 μL in Table 4 was taken as the downstream primer to amplify gene fragment L2. Using plasmid scDAOCS H7 as a template, L179-F1, L179-F2, and L179-F3 in Table 5 were mixed at a ratio of 2:1:1, 2 μL was taken as the upstream primer, R242-H244-R1 and R242-H244-R2 in Table 5 were mixed at a ratio of 2:1, and 2 μL of the mixture was taken as the downstream primer to amplify gene fragment L3. Using plasmid scDAOCS H7 as template, N301-F1, N301-F2, Y302-F1, Y302-F2, Y302-F3, V303-F1, V303-F2 in Table 4 were mixed in a ratio of 2:2:6:6:3:6:2, 2 μL was taken as the upstream primer, and 2 μL of N301-Y302-V303-R in Table 5 was taken as the downstream primer to amplify gene fragment L4. The first round of PCR program: 94°C for 2 min, (98°C for 15 s, 55°C for 30 s, 72°C for 30 s) × 28 cycles, 72°C for 5 min.

[0094] Take 2 μL of the first round PCR products L1, L2, L3, L4 as primers, and use plasmid scDAOCS H7 as template to perform the second PCR to construct ML1, ML2, ML3, ML4 mutant libraries. The second round PCR program: 94℃ for 2 min, (98℃ for 15 s, 60℃ for 30 s, 72℃ for 5 min) × 28 cycles, 72℃ for 10 min.

[0095] The obtained second round PCR products were subjected to the following operations:

[0096] Add 1 μL of Dpn I enzyme to the product of the second round of PCR to digest the plasmid template and treat it at 37°C for 3 hours. Take 2 μL of the second round of PCR product after enzyme digestion and electrotransform it into E. coli BL21 (DE3). Evenly spread the electrotransformed E. coli BL21 (DE3) on a kanamycin-resistant LB plate (concentration of 50 μg / mL). After culturing at 37°C for 14 hours, a single colony will grow, which is the mutant library. Scrape all the single colonies on a plate and send them for sequencing to verify the diversity of the mutant library.

[0097] 3. Mutant screening and reaction system

[0098] The single colonies in the mutant library were inoculated into 300 μL LB medium containing a final concentration of 50 μg / mL kanamycin in 96 deep-well plates and cultured at 37°C for 8-12 hours. A negative control group (empty vector of pET-24a) and a positive control group (pET-24a-scDAOCSH7) were set up at the same time. Then 200 μL of seed solution was inoculated into 800 μL TB medium containing a final concentration of 0.1-0.2 mM IPTG and 50 μg / mL kanamycin. After expressing at 25°C for 16 hours, the cells were collected by centrifugation, 400 μL of 50 mM, pH 7.4 phosphate buffer containing 50 μg / mL FeSO4, 0.4 mM ascorbic acid, 5 mM or 10 mM α-ketoglutaric acid, and 5 mM or 10 mM penicillin G were added, and the reaction was carried out at 25°C for 2 hours. After reacting for 2 hours, centrifuge at 12000 rpm for 1 minute, and the supernatant was taken for HPLC detection. The liquid chromatography detection conditions are shown in Example 1. The enzyme catalytic reaction conversion rate data obtained are as follows:

[0099] Table 6. Mutant transformation rate

[0100]

[0101] Using pET24a as a negative control and scDAOCS-H7 as a positive control, the scDAOCS-V303K mutant was screened in shake flasks and sequenced.

[0102] The results are shown in Table 7. The whole cells and crude enzyme of the mutant scDAOCS-H7-V303K had improved activity compared with scDAOCS-H7.

[0103] Table 7. Transformation rate of the multiple screening high activity mutant library

[0104]

[0105] In summary, the present invention constructs an expandase mutant, and compared with the scDAOCS-H7 expandase, the enzyme activity is greatly improved, the conversion rate is increased by 25%, and it has broad application prospects.

[0106] Sequence Listing

[0107] Wild-type WT-scDAOCS

[0108] MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTMRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDC EPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA

[0109] SEQ ID No.1(E632)

[0110] MDTTVPTFSLDELQEGLHQDEFRRCLTEKGVFYLTDSGLSEADHKSAKDVAVDFFEHGTEEEKRAVTSPIPTIRRGFSGLESESTAQITNTGTYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDC EPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVAAPGRDQRAGSSRTSSVFFLRPKSDFSFSVPLARECGFDVSLDGETATFGDWIGGNYVNIRRTSKA

[0111] SEQ ID No.2(8MUT)

[0112] MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDY EPLLRFRYFPQVPEHRSAEEQPLLMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYVNMRRTSKA

[0113] SEQ ID No. 3(E633)

[0114] MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVVDFFEHGTEEEKRAVTSPVPTIRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDC EPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA

[0115] SEQ ID No.4(ScDAOCSH7):

[0116] MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYVNMRRTSKA

[0117] SEQ ID No.5(E727):

[0118] MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA

[0119] SEQ ID No.6(E735):

[0120] MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA

[0121] SEQ ID No.7(E727-M2):

[0122] MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA

[0123] SEQ ID No.8(E727-M3):

[0124] MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA

[0125] SEQ ID No.9(E727-M5):

[0126] MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEH

[0127] RSAEEEPLRMAPHHDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA

[0128] SEQ ID No.10(E735-M2):

[0129] MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA

[0130] SEQ ID No.11(E735-M3):

[0131] MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA

[0132] SEQ ID No.12(E735-M5):

[0133] MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA

[0134] SEQ ID No.13 (scDAOCS-V303K):

[0135] MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYKNMRRTSKA SEQUENCE LISTING <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> Application of Ring-Expansion Enzyme and Its Mutants in the Production of G-7-ADCA <130> GNCAQ211831 <160> 1 <170> PatentIn version 3.5 <210> WT-scDAOCS <211> 311 <212> PRT <213> C. acremonium <400> WT-scDAOCS Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Met Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr 115 120 125 Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln 165 170 175 Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala 225 230 235 240 Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp 275 280 285 Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn 290 295 300 Ile Arg Arg Thr Ser Lys Ala 305 310 <210> 1 <211> 311 <212> PRT <213> Artificial sequence <400> 1 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Asp Glu Leu Gln Glu Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Thr Glu Lys Gly Val Phe 20 25 30 Tyr Leu Thr Asp Ser Gly Leu Ser Glu Ala Asp His Lys Ser Ala Lys 35 40 45 Asp Val Ala Val Asp Phe Phe Glu His Gly Thr Glu Glu Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Ile Pro Thr Ile Arg Arg Gly Phe Ser Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Thr Tyr Thr Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr 100 105 110 Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Ile Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Arg His His Val Ala Ala Pro Gly Arg Asp Gln Arg Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Lys Ser Asp Phe Ser 260 265 270 Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp 275 280 285 Gly Glu Thr Ala Thr Phe Gly Asp Trp Ile Gly Gly Asn Tyr Val Asn 290 295 300 Ile Arg Arg Thr Ser Lys Ala 305 310 <210> 2 <211> 311 <212> PRT <213> Artificial sequence <400> 2 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr 115 120 125 Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln 165 170 175 Pro Leu Leu Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Gly Gly Ala Phe Val Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala 225 230 235 240 Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Ile Pro Leu Ala Arg Glu Tyr Gly Phe Asp Val Ser Leu Asp 275 280 285 Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn 290 295 300 Met Arg Arg Thr Ser Lys Ala 305 310 <210> 3 <211> 311 <212> PRT <213> Artificial sequence <400> 3 Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe 20 25 30 Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys 35 40 45 Asp Val Val Val Asp Phe Phe Glu His Gly Thr Glu Glu Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Ile Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr 115 120 125 Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala 225 230 235 240 Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Leu Arg Thr Lys Thr Lys Ala 305 310 <210> 4 <211> 311 <212> PRT <213> Artificial sequence <400> 4 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr 115 120 125 Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln 165 170 175 Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Gly Gly Ala Phe Val Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala 225 230 235 240 Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Ile Pro Leu Ala Arg Glu Tyr Gly Phe Asp Val Ser Leu Asp 275 280 285 Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn 290 295 300 Met Arg Arg Thr Ser Lys Ala 305 310 <210> 5 <211> 311 <212> PRT <213> Artificial sequence <400> 5 Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe 20 25 30 Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys 35 40 45 Asp Val Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Ile Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Arg His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Leu Arg Thr Lys Thr Lys Ala 305 310 <210> 6 <211> 311 <212> PRT <213> Artificial sequence <400> 6 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Thr Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr 100 105 110 Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Leu Arg Thr Lys Thr Lys Ala 305 310 <210> 7 <211> 311 <212> PRT <213> Artificial sequence <400> 7 Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe 20 25 30 Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys 35 40 45 Asp Val Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His His Asp Leu Ser Ile Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Arg His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Leu Arg Thr Lys Thr Lys Ala 305 310 <210> 8 <211> 311 <212> PRT <213> Artificial sequence <400> 8 Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe 20 25 30 Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys 35 40 45 Asp Val Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Ile Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Arg His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Ile Pro Leu Ala Lys Arg Tyr Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Met Arg Thr Lys Thr Lys Ala 305 310 <210> 9 <211> 311 <212> PRT <213> Artificial sequence <400> 9 Met Asp Thr Thr Val Pro Thr Phe His Leu Ala Glu Leu Gln Glu Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Ser Cys Leu Met Glu Lys Gly Val Phe 20 25 30 Tyr Leu Thr Gly Ser Ser Leu Ser Glu Ala Asp Gln Lys Ser Ala Lys 35 40 45 Asp Val Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His His Asp Leu Ser Ile Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Arg His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Ile Pro Leu Ala Lys Arg Tyr Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Met Arg Thr Lys Thr Lys Ala 305 310 <210> 10 <211> 311 <212> PRT <213> Artificial sequence <400> 10 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Thr Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr 100 105 110 Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Val Pro Leu Ala Lys Arg Cys Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Leu Arg Thr Lys Thr Lys Ala 305 310 <210> 11 <211> 311 <212> PRT <213> Artificial sequence <400> 11 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Thr Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr 100 105 110 Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Ile Pro Leu Ala Lys Arg Tyr Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Met Arg Thr Lys Thr Lys Ala 305 310 <210> 12 <211> 311 <212> PRT <213> Artificial sequence <400> 12 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Thr Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ser Asp Asn Leu Phe Pro Thr 100 105 110 Ala Asp Phe Glu Arg Val Trp Thr His Tyr Phe Asp Arg Met Tyr Asp 115 120 125 Ala Ser Arg Glu Val Ala Arg Gln Val Leu Lys Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Asp Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ser Ala Glu Glu Glu 165 170 175 Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Asp Gly Thr Phe Val Asp Leu Pro Ala Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Val Ala Thr Leu Val Thr Gly Gly Lys Val Lys Ala 225 230 235 240 Pro Lys His His Val Val Ala Pro Ala Arg Asp Arg Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Ile Pro Leu Ala Lys Arg Tyr Gly Phe Asp Ile Gly Leu Asp 275 280 285 Gly Asp Thr Ala Ala Phe Gln Asp Trp Ile Ala Gly Asn Tyr Val Asn 290 295 300 Met Arg Thr Lys Thr Lys Ala 305 310 <210> 13 <211> 311 <212> PRT <213> Artificial sequence <400> 13 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Thr Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr 115 120 125 Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Tyr Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln 165 170 175 Pro Leu Arg Met Ala Pro His His Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Gly Gly Ala Phe Val Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala 225 230 235 240 Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Ile Pro Leu Ala Arg Glu Tyr Gly Phe Asp Val Ser Leu Asp 275 280 285 Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Lys Asn 290 295 300 MetArgArgThrSerLysAla 305 310

Claims

1. Protein, which is (b) or (c) or (d) or (e) as follows: (b) a protein having an amino acid sequence as shown in SEQ ID No. 5; (c) a protein obtained by mutating the amino acid residue at position 155 of the amino acid sequence shown in SEQ ID NO:5 from cysteine ​​to tyrosine, and by mutating the amino acid residue at position 184 from tyrosine to histidine, the amino acid sequence of which is shown in SEQ ID NO:7; (d) a protein obtained by mutating the amino acid residue at position 275 of the amino acid sequence shown in SEQ ID NO:5 from valine to isoleucine, mutating the amino acid residue at position 281 from cysteine ​​to tyrosine, and mutating the amino acid residue at position 305 from leucine to methionine, the amino acid sequence of which is shown in SEQ ID NO:8; (e) A protein obtained by mutating the amino acid residue at position 155 of the amino acid sequence shown in SEQ ID NO:5 from cysteine ​​to tyrosine, mutating the amino acid residue at position 184 from tyrosine to histidine, mutating the amino acid residue at position 275 from valine to isoleucine, mutating the amino acid residue at position 281 from cysteine ​​to tyrosine, and mutating the amino acid residue at position 305 from leucine to methionine, the amino acid sequence of which is shown in SEQ ID NO:

9.

2. A nucleic acid molecule encoding the protein of claim 1, wherein the nucleic acid molecule is a gene encoding the protein of claim 1.

3. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the nucleic acid molecule of claim 2.

4. Use of the protein according to claim 1 as penicillin expandase to catalyze the ring expansion of penicillin G to produce G-7-ADCA.

5. Use of the protein according to claim 1 as penicillin expandase to catalyze the ring expansion of penicillin G to generate G-7-ADCA and improve the conversion rate of penicillin G.

6. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2, or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria according to claim 3 in any of the following: (b1) preparing a product having penicillin expandase activity; (b2) Preparation of G-7-ADCA.

7. A method for preparing G-7-ADCA or a method for improving the conversion rate of penicillin G in the reaction of penicillin expandase catalyzing the ring expansion of penicillin G to generate G-7-ADCA, which is one of the following methods: 1) preparing the protein of claim 1; using the protein as penicillin expandase to catalyze the ring expansion of penicillin G to generate G-7-ADCA; 2) fermenting Escherichia coli containing a gene encoding the protein of claim 1, so that the obtained fermentation product catalyzes the ring expansion of penicillin G to produce G-7-ADCA; 3) The gene encoding the protein of claim 1 is cloned into penicillin G-producing Escherichia coli to obtain a recombinant bacterium, and the recombinant bacterium is fermented to catalyze the ring expansion of penicillin G to obtain G-7-ADCA.

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