CN115491361A - Application of expandase and mutant thereof in production of G-7-ADCA - Google Patents
Application of expandase and mutant thereof in production of G-7-ADCA Download PDFInfo
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- CN115491361A CN115491361A CN202110676893.1A CN202110676893A CN115491361A CN 115491361 A CN115491361 A CN 115491361A CN 202110676893 A CN202110676893 A CN 202110676893A CN 115491361 A CN115491361 A CN 115491361A
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Abstract
The invention provides an expandase and application of a mutant thereof in producing G-7-ADCA. The amino acid sequence is shown in one of SEQ ID No. 5-13. The invention generates a series of enzymes with the ring expansion activity by NCBI sequence comparison, excavation, directed evolution and other technologies, and the enzymes can expand the substrate penicillin G into G-7-ADCA in one step; G-7-ADCA can be converted into 7-ADCA by removing side chain under the action of penicillin acylase. Therefore, these expandases have important application values in industry.
Description
Technical Field
The invention relates to an application of expandase and a mutant thereof in producing G-7-ADCA, belonging to the technical field of biology.
Background
The cephalosporin antibiotics are anti-infective drugs widely applied clinically at present, and have the advantages of low toxicity, broad spectrum and the like. 7-amino-3-desacetoxycephalosporanic acid (7-ADCA) is one of the important mother nuclei for synthesis of cephalosporin antibiotics, can be used for synthesis of cefalexin, cefradine, cefadroxil and the like, and has huge market consumption.
Currently, there are two main methods for industrially producing 7-ADCA: chemoenzymatic and fermentation processes. The 7-ADCA preparing process with chemical enzyme process includes the steps of oxidizing penicillin G chemically to produce phenylacetyl-7-aminodesacetoxycephalosporanic acid (G-7-ADCA), and eliminating side chain under the action of penicillin amidase to produce 7-ADCA. The process is complex, costly, produces large amounts of waste water and pollution, and is environmentally damaging (Wei CL et al, applied and Environmental microbiology, 2005). Under the national pressure of energy conservation and emission reduction and environmental protection, the process is gradually eliminated.
The 7-ADCA can also be produced industrially by fermentation. In 1998, cho et al have found that expandase (DAOCS) has some activity on penicillin G, making it possible to produce G-7-ADCA from penicillin G (Cho et al, PNAS, 1998). Therefore, it is now common practice in some enterprises to clone the expandase (DAOCS) gene into Penicillium chrysogenum, produce G-7-ADCA by direct fermentation with the addition of adipic acid during fermentation, and then prepare 7-ADCA by side chain removal with penicillin acylase. However, the fermentation method has the problems of complex process, long fermentation period and the like, and the core technology is mainly mastered in foreign companies, so that no industrial method for producing 7-ADCA by adopting the fermentation method is seen in China at present.
The preparation of 7-ADCA by the holoenzyme method has the advantages of environmental protection, low cost and the like. The holoenzyme method starts from cheap penicillin G potassium salt, generates G-7-ADCA through the catalytic ring expansion reaction of penicillin expandase, and then hydrolyzes to remove an acyl side chain through the action of penicillin acylase to generate 7-ADCA (shown in figure 1). The study of penicillin acylases has been well established and has been in industrial use for many years. Most currently used are immobilized penicillin acylases; whereas the activity of expandase is lower and is the rate-limiting step in the pathway for the synthesis of 7-ADCA from penicillin G. Therefore, there is an urgent need to improve the activity of expandase.
Penicillin expandase (DAOCS), also known as deacetoxycephalosporin C synthetase, is a Fe-dependent enzyme 2+ 、O 2 And alpha-ketoglutaric acid, which mainly catalyzes the conversion (ring expansion) of five-membered thiazole ring to six-membered thiazide ring of penicillin, and is a key step for catalyzing streptomyces clavuligerus and other prokaryotes to synthesize cephalosporin antibiotics. Therefore, DAOCS has important industrial application value.
However, in practical application, it is found that DAOCS has low biological activity on penicillin G and cannot meet industrial application, which becomes the rate-limiting step for synthesizing 7-ADCA by using a holoenzyme method. Therefore, the modification of the expandase is urgently needed so as to greatly improve the conversion efficiency of the expandase on penicillin G and lay a foundation for industrial application.
Currently, DAOCS derived from cephalosporium acremonium (c.acremonium), streptomyces clavuligerus (s.clavuligerus) and nocardia (n.lactamdurans) have been expressed and purified, and scdaos derived from streptomyces clavuligerus has obtained a crystal structure, which makes it possible to modify scdaos by protein engineering techniques, improve its catalytic activity, broaden its substrate spectrum, and have made some progress.
Disclosure of Invention
The technical problem to be solved by the invention is how to improve the catalytic activity of the expandase on the expansion of penicillin G.
In order to solve the above technical problems, the present invention firstly provides a protein.
The protein provided by the invention is (a) or (b) or (c) or (d):
(a) The amino acid sequence of the protein is shown as SEQ ID No.4 (named as H7), or the protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a) and has the activity of expandase and is derived from (a);
(b) The amino acid sequence of the protein is shown as SEQ ID No.5 (named as E727), or the protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in the (b) and has the activity of expandase and is derived from the (b);
(c) A protein which has an amino acid sequence shown as SEQ ID No.6 (named as E735), or is derived from (c) by substituting, deleting or adding one or more amino acids in the amino acid sequence in (c) and has an expandase activity;
(d) The amino acid sequence of the protein is shown as SEQ ID No.13 (named as scDAOCS-V303K), or the protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in (d) and has the activity of expandase and is derived from (d).
Further, the protein is any one of the following (1) - (6):
(1) A protein represented by the amino acid sequence shown in SEQ ID NO.5 wherein n1 is at position 155 and n2 is at position 184;
(2) A protein represented by the amino acid sequence shown in SEQ ID NO.5 wherein n3 is at position 275, n4 is at amino acid residue 281, and n5 is at position 305;
(3) A protein represented by the amino acid sequence shown in 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 n 5;
(4) A protein represented by the amino acid sequence shown in SEQ ID NO.6 with m1 at position 155 and m2 at position 184;
(5) A protein represented by the amino acid sequence shown in SEQ ID NO.6, wherein m3 is at position 275, m4 is at position 281, and m5 is at position 305;
(6) A protein represented by the amino acid sequence shown in SEQ ID NO.6, wherein m1 is at position 155, m2 is at position 184, m3 is at position 275, m4 is at position 281, and m5 is at position 305;
specifically, SEQ ID NO: 5n 1 at position 155 may be: cysteine, tyrosine, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, serine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine, and n2 at position 184 can 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, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, and 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, n5 at position 305 can be: leucine, methionine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine;
SEQ ID NO:6, m1 at position 155 can 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 can be aspartic acid, glutamic acid, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine, or histidine, m3 at position 275 can be glutamic acid, histidine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, lysine, or arginine, 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 m5 at position 305 may be isoleucine, methionine, glycine, alanine, valine, leucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine.
Further, the protein is any one of the following (1) - (6):
(1) The 155 th amino acid residue of the amino acid sequence shown in SEQ ID NO.5 is mutated from cysteine to tyrosine, and the 184 th amino acid residue is mutated from tyrosine to histidine to obtain a protein which is named as E727-M2 (C155Y/Y184H), and the amino acid sequence of the protein is shown in SEQ ID NO. 7;
(2) The protein obtained by mutating the 275 th amino acid residue of the amino acid sequence shown in SEQ ID NO.5 from valine to isoleucine, the 281 th amino acid residue from cysteine to tyrosine and the 305 th amino acid residue from leucine to methionine is named as E727-M3 (V275I/C281Y/L305M), and the amino acid sequence of the protein is shown in SEQ ID NO. 8;
(3) The protein obtained by mutating the 155 th amino acid residue of the amino acid sequence shown in SEQ ID NO.5 from cysteine to tyrosine, mutating the 184 th amino acid residue from tyrosine to histidine, mutating the 275 th amino acid residue from valine to isoleucine, mutating the 281 th amino acid residue from cysteine to tyrosine, and mutating the 305 th amino acid residue from leucine to methionine is named as E727-M5 (C155Y/Y184H/V275I/C281Y/L305M), and the amino acid sequence thereof is shown in SEQ ID NO. 9;
(4) The protein obtained by mutating the 155 th amino acid residue of the amino acid sequence shown in SEQ ID NO.6 from cysteine to tyrosine and mutating the 184 th amino acid residue from tyrosine to histidine is named as E735-M2 (C155Y/Y184H), and the amino acid sequence thereof is shown in SEQ ID NO. 10;
(5) The protein obtained by mutating the 275 th amino acid residue of the amino acid sequence shown in SEQ ID NO.6 from valine to isoleucine, the 281 th amino acid residue from cysteine to tyrosine and the 305 th amino acid residue from isoleucine to methionine is named as E735-M3 (V275I/C281Y/I305M), and the amino acid sequence of the protein is shown in SEQ ID NO. 11;
(6) The protein obtained by mutating the 155 th amino acid residue of the amino acid sequence shown in SEQ ID NO.6 from cysteine to tyrosine, mutating the 184 th amino acid residue from tyrosine to histidine, mutating the 275 th amino acid residue from valine to isoleucine, mutating the 281 th amino acid residue from cysteine to tyrosine, and mutating the 305 th amino acid residue from isoleucine to methionine is named as E735-M5 (C155Y/Y184H/V275I/C281Y/I305M), and the amino acid sequence thereof is shown in SEQ ID No. 12;
nucleic acid molecules which code for the above proteins also belong to the scope of protection of the present invention.
The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be an RNA, such as an mRNA, hnRNA, or tRNA, and the like.
The nucleic acid molecule is specifically a gene encoding the protein.
The recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the nucleic acid molecule also belongs to the protection scope of the invention.
The recombinant vector can be a recombinant expression vector and can also be a recombinant cloning vector.
The recombinant expression vector can be constructed using an existing expression vector. The expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., contain the polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.
The expression cassette consists of a promoter capable of driving expression of the gene, and a transcription termination sequence.
The application of the protein as penicillin expandase also belongs to the protection scope of the invention.
The application of the protein in catalyzing penicillin G to generate G-7-ADCA by expanding penicillin G as penicillin expandase to improve the conversion rate of penicillin G also belongs to the protection scope of the invention.
The application of the protein or the nucleic acid molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium in any one of the following is also within the protection scope of the invention:
(b1) Preparing a product having penicillin expandase activity;
(b2) Preparation of G-7-ADCA.
The invention also provides a method for preparing G-7-ADCA.
The method for preparing G-7-ADCA provided by the invention specifically comprises the following steps: preparing the protein; the protein is used as penicillin expandase to catalyze penicillin G to expand ring to generate G-7-ADCA.
The invention also provides a method for improving the conversion rate of penicillin G in the reaction of penicillin expandase enzyme catalyzing penicillin G to expand ring to generate G-7-ADCA.
The method for improving the conversion rate of penicillin G in the reaction of catalyzing penicillin G to expand ring to generate G-7-ADCA by using the penicillin expandase, which is provided by the invention, is characterized in that the protein is used as the penicillin expandase to catalyze penicillin G to expand ring to generate G-7-ADCA.
The invention generates a series of enzymes with ring expanding activity by NCBI sequence comparison, excavation, directed evolution and other technologies, and the enzymes can expand a substrate penicillin G into G-7-ADCA in one step; G-7-ADCA can be converted into 7-ADCA by removing side chain under the action of penicillin acylase. Therefore, these expandases have important application values in industry.
Drawings
FIG. 1 is a scheme showing the reaction scheme of the 7-ADCA catalyzed by the expandase enzyme.
FIG. 2 shows the sequence alignment information used in SCHEMA technology.
FIG. 3 is a schematic diagram of the position of the Loop-associated mutation site obtained according to the Rossetta Design method.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: expandase mutant generated based on SCHEMA technology
1. Gene acquisition and vector construction
The mimic DNA shuffling was analyzed for recombination using SCHEMA technology. According to the result of computational simulation, 20 sequences were selected. After codon optimization, a full-length gene is obtained by using a full-gene synthesis mode and is constructed on a pET-24a expression vector. The three expandase sequences (E632, SEQ ID No.1,8MUT SEQ ID No.2, and E633 SEQ ID No. 3) used in SCHEMA technology and the alignment are shown in FIG. 2.
Comparing the sequences by SCHEMA technology to obtain the sequences shown in the table 1, carrying out gene synthesis according to the obtained sequences, and carrying out screening according to the catalytic efficiency after expression.
2. Protein expression and Activity detection
Transforming the expression vector into a host bacterium BL21 (DE 3), setting a negative control group (empty vector for transforming pET-24 a) and a positive control group (pET-24 a-scDAOCS) at the same time, and culturing at 37 ℃ overnight; selecting a single colony, transferring the single colony into an LB culture medium containing 100 mu g/mL kanamycin, and culturing for 8-10h at 37 ℃ and 220 rpm; then, the cells were inoculated into 50mL of LTB medium containing 100. Mu.g/mL in an amount of 1/100, and cultured at 37 ℃ until OD600 reached about 0.6, and then 0.1mM IPTG was added to induce protein expression for 16 hours, thereby obtaining recombinant cells expressing expandase.
3. Reaction system
And collecting the thallus. Pouring the thalli into a 50mL centrifuge tube, and centrifuging at 8000rpm for 10min to obtain thalli; the mycelia were washed once with 50mM phosphate buffer pH 7.4; centrifuging at 8000rpm for 10min to collect thallus; and (5) weighing.
The formula of the reaction solution is as follows: 50mM phosphate buffer pH7.4, 50. Mu.g/mL FeSO 4 0.4mM ascorbic acid, 5mM or 10mM alpha-ketoglutaric acid, 5mM or 10mM penicillin G.
And (4) reacting. The reaction solution was added to the cells at a ratio of 10mL to 1g of wet cells, and then resuspended. The mixture was placed on a shaker at 25 ℃ and 220rpm and reacted for 2 hours.
Detecting the amount of G-7-ADCA produced. After reacting for 2h, 12000rpm, centrifuging for 1min, and taking the supernatant for HPLC detection.
The detection conditions of the liquid chromatogram are as follows: a chromatographic column: agilent ZORBAX SB-C18 Stable bond analytical4.6 x 250mm; mobile phase: 20mM pH 3.0 sodium phosphate buffer: methanol = 55. Flow rate: 1mL/min, and the detection wavelength is 215nm.
4. Conversion data results for catalytic penicillin G conversion
TABLE 1 conversion of expandase mutants produced using SCHEMA technology
The reaction conditions are as follows:
50mM phosphate buffer pH7.4, 50. Mu.g/mL FeSO4,0.4mM ascorbic acid, 10mM alpha-ketoglutaric acid, 10mM penicillin G, 200. Mu.L crude enzyme solution. The mixture was placed on a shaker at 25 ℃ and 220rpm for 30min.
5. Modification of enzymes
E727 and E735 with higher activity are subjected to double mutation, triple mutation and five-mutant transformation, mutation sites in a high-activity mutant H7 are selected and transplanted into E727 and E735, and 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/Y H/V275I/C281Y/I305M) mutants are obtained. The activity is as follows:
TABLE 2 conversion of expandase mutants generated using SCHEMA program
As can be seen from Table 2, the conversion of penicillin G catalyzed by E727-M2 was the highest and better than that of H7.
6. Pure enzyme reaction and HPLC detection thereof
The pure enzyme activity was verified in 1mL system. PBS buffer pH7.4, 50. Mu.g/mL FeSO4,0.4mM ascorbic acid, 5mM alpha-ketoglutaric acid, 5mM penicillin G, 100. Mu.L enzyme solution. The mixture was placed on a shaker at 25 ℃ and 220rpm for 30min. The HPLC detection results are shown in Table 3.
TABLE 3 enzymatic Activity catalyzed by expandase mutants
Enzyme activity (U) is defined as the amount of enzyme that can convert 1 micromole of substrate in 1 minute at 25 ℃; the specific enzyme activity (U/mg) is the enzyme activity per mg of protein.
Experiments prove that the enzyme activity of the mutant E727-M2 is improved by 25 percent compared with the enzyme activity of scH7, and the mutant has better application prospect.
Example 2 mutant obtained by modification of expandase H7 based on Rosetta Design method
1. And (5) obtaining the gene.
All computational designs were performed using Rosetta design software developed by David Baker group, washington university, usa. The first 20 conformations that meet all the necessary conditions were selected for analysis and experimental validation. 4 combined mutation libraries are constructed by using the 7-mutant scDAOCS H7 of the expandase as a template and simulating loop-related mutation sites through dynamics, wherein the mutation libraries are shown in the following table 4. The position of the mutation site on the expandase is shown in FIG. 3.
TABLE 4 mutant library based on RosettaDesign method
2. And (4) establishing a mutant library.
Site combinatorial mutagenesis was performed with the corresponding primers to the selected residues as in table 5.
TABLE 5 primer sequence Listing
Taking the plasmid scDAOCS H7 as a template, the primers T72-F1, T72-F2, T72-F3, T72-F4, T73-F1, T73-F2, T73-F3 and T73-F4 in the table 5 are as follows: 1:1:1:3:1:1:1, and 2. Mu.L of the mixture was used as an upstream primer and 2. Mu.L of T72-T73-R74-R2 in Table 5 was used as a downstream primer to amplify the gene fragment L1. Taking the plasmid scDAOCS H7 as a template, mixing Y94-D95-Y97-F1, Y94-D95-Y97-F2 and Y94-D95-Y97-F3 in the table 5 according to a ratio of 2. The plasmid scDAOCS H7 is used as a template, L179-F1, L179-F2 and L179-F3 in the table 5 are mixed according to a ratio of 2. The gene fragment L4 was amplified by mixing the following 2 parts of N301-F1, N301-F2, Y302-F1, Y302-F2, Y302-F3, V303-F1, V303-F2 in Table 4 with the plasmid scDAOCS H7 as a template, and taking 2. Mu.L of N301-Y302-V303-R2. Mu.L in Table 5 as a downstream primer, in a ratio of 2. First round PCR procedure: heat preservation at 94 ℃ for 2min, (heat preservation at 98 ℃ for 15s, heat preservation at 55 ℃ for 30s, heat preservation at 72 ℃ for 30 s) x 28 cycles, and heat preservation at 72 ℃ for 5min.
And (3) respectively taking 2 mu L of first round PCR products L1, L2, L3 and L4 as primers, taking the plasmid scDAOCS H7 as a template, and performing second PCR to construct ML1, ML2, ML3 and ML4 mutant libraries. Second round PCR program: heat preservation at 94 ℃ for 2min, (heat preservation at 98 ℃ for 15s, heat preservation at 60 ℃ for 30s, heat preservation at 72 ℃ for 5 min) x 28 cycles, and heat preservation at 72 ℃ for 10min.
The second round of PCR products obtained were subjected to the following operations:
to the product of the second round of PCR, 1. Mu.L of Dpn I enzyme was added for digesting the plasmid template, and the mixture was treated at 37 ℃ for 3 hours. And (3) performing electric transformation on 2 mu L of the second round PCR product after enzyme digestion to Escherichia coli BL21 (DE 3), uniformly coating the bacterial liquid of the electric transformed Escherichia coli BL21 (DE 3) on an LB (Lankanamycine) -resistant (with the concentration of 50 mu g/mL) plate, and culturing at the constant temperature of 37 ℃ for 14h to grow a single colony, namely a mutant library. All single colonies on one plate were scraped off for sequencing to verify the diversity of the mutant pool.
3. Mutant screening and reaction system
Single colonies from the mutant pool were inoculated into 300. Mu.L LB medium containing 50. Mu.g/mL kanamycin at the final concentration in 96 deep-well plates, respectively, cultured at 37 ℃ for 8-12 hours while setting a negative control group (empty vector of pET-24 a) and a positive control group (pET-24 a-scDAOCSH 7), and then 200. Mu.L of seed solution was inoculated into 800. Mu.L LB medium containing IPTG at the final concentration of 0.1-0.2mM and 50. Mu.g/mL kanamycin. After 16 hours at 25 ℃, the cells were collected by centrifugation, and 400. Mu.L of 50mM phosphate buffer pH7.4 containing 50. Mu.g/mL FeSO4,0.4mM ascorbic acid, 5mM or 10mM alpha-ketoglutaric acid, 5mM or 10mM penicillin G was added thereto and reacted at 25 ℃ for 2 hours. After reacting for 2h, 12000rpm, centrifuging for 1min, and taking the supernatant for HPLC detection. The detection conditions of the liquid chromatography are shown in the first embodiment. The following enzyme-catalyzed reaction conversion data were obtained:
TABLE 6 mutant conversion
And taking pET24a as a negative control and scDAOCS-H7 as a positive control, carrying out shake flask rescreening on the scDAOCS-V303K mutant, and sequencing the mutant.
The results are shown in Table 7, where the mutant scdaos-H7-V303K has improved activity in whole cells and in crude enzyme compared to scdaos-H7.
TABLE 7 conversion rates of rescreened pools of high activity mutants
In conclusion, compared with the scDAOCS-H7 expandase, the expandase mutant is constructed, so that the enzyme activity is greatly improved, the conversion rate is improved by 25%, and the method has a wide application prospect.
Sequence listing
Wild type WT-scDAOCS
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTMRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA
SEQ IDNo.1(E632)
MDTTVPTFSLDELQEGLHQDEFRRCLTEKGVFYLTDSGLSEADHKSAKDVAVDFFEHGTEEEKRAVTSPIPTIRRGFSGLESESTAQITNTGTYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVAAPGRDQRAGSSRTSSVFFLRPKSDFSFSVPLARECGFDVSLDGETATFGDWIGGNYVNIRRTSKA
SEQ IDNo.2(8MUT)
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLLMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYVNMRRTSKA
SEQ IDNo.3(E633)
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVVDFFEHGTEEEKRAVTSPVPTIRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.4(ScDAOCSH7)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYVNMRRTSKA
SEQ ID No.5(E727)：
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.6(E735)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.7(E727-M2)：
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.8(E727-M3)：
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA
SEQ ID No.9(E727-M5)：
MDTTVPTFHLAELQEGLHQDEFRSCLMEKGVFYLTGSSLSEADQKSAKDVVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEH
RSAEEEPLRMAPHHDLSIVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPRHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA
SEQ ID No.10(E735-M2)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSVPLAKRCGFDIGLDGDTAAFQDWIAGNYVNLRTKTKA
SEQ ID No.11(E735-M3)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA
SEQ ID No.12(E735-M5)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYTDYSMCYSMGTSDNLFPTADFERVWTHYFDRMYDASREVARQVLKATGTEPDGGVDAFLDYEPLLRFRYFPEVPEHRSAEEEPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVDGTFVDLPARPDAVLVFCGAVATLVTGGKVKAPKHHVVAPARDRIAGSSRTSSVFFLRPNADFTFSIPLAKRYGFDIGLDGDTAAFQDWIAGNYVNMRTKTKA
SEQ ID No.13(scDAOCS-V303K)：
MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYKNMRRTSKA。
SEQUENCE LISTING
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> expandase and application of mutant thereof in 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
Met Arg Arg Thr Ser Lys Ala
305 310
Claims (10)
1. A protein which is (b), (c) or (d) below:
(b) The amino acid sequence of the protein is shown as SEQ ID No.5, or the protein which is derived from the protein (b) and has the activity of expandase, wherein the amino acid sequence in the protein (b) is substituted, deleted or added with one or more amino acids;
(c) The amino acid sequence of the protein is shown as SEQ ID No.6, or the protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in (c) and has the activity of expandase and is derived from (c);
(d) The amino acid sequence of the protein is shown as SEQ ID No.13, or the protein which is derived from (d) and has the activity of expandase, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence in (d).
2. The protein of claim 1, wherein: the protein is any one of the following (1) to (6):
(1) A protein represented by the amino acid sequence shown in SEQ ID NO.5 wherein n1 is at position 155 and n2 is at position 184;
(2) A protein represented by the amino acid sequence shown by SEQ ID NO.5 wherein the 275 th position is n3, the 281 th amino acid residue is n4, and the 305 th position is n 5;
(3) A protein represented by the amino acid sequence shown in SEQ ID NO.5 wherein the amino acid sequence at position 155 is n1, the amino acid sequence at position 184 is n2, the amino acid sequence at position 275 is n3, the amino acid sequence at position 281 is n4, and the amino acid sequence at position 305 is n 5;
(4) A protein represented by the amino acid sequence shown in SEQ ID NO.6 with m1 at position 155 and m2 at position 184;
(5) A protein represented by the amino acid sequence shown in SEQ ID NO.6, wherein m3 is at position 275, m4 is at position 281, and m5 is at position 305;
(6) M1 at position 155, m2 at position 184, m3 at position 275, m4 at position 281, and m5 at position 305, and a protein represented by the amino acid sequence of SEQ ID NO. 6.
3. The protein of claim 2, wherein: SEQ ID NO:5 wherein n1 at position 155 is: 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 is: tyrosine, histidine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine or arginine, n3 at position 275 is: valine, isoleucine, glycine, alanine, leucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, n4 at position 281 is: cysteine, tyrosine, glycine, alanine, valine, leucine, isoleucine, proline, tryptophan, serine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine, n5 at position 305 is: leucine, methionine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine;
SEQ ID NO:6, wherein m1 at position 155 in the amino acid sequence is 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 is aspartic acid, glutamic acid, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine or histidine, m3 at position 275 is glutamic acid, histidine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, lysine or arginine, m4 at position 281 is: tyrosine, proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, serine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, and m5 at position 305 is isoleucine, methionine, glycine, alanine, valine, leucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine.
4. The protein of any one of claims 1-3, wherein: the protein is any one of the following (1) to (6):
(1) The amino acid sequence of the protein is shown in SEQ ID NO. 7, wherein the 155 th amino acid residue of the amino acid sequence shown in SEQ ID NO.5 is mutated from cysteine to tyrosine, and the 184 th amino acid residue is mutated from tyrosine to histidine;
(2) The amino acid sequence of the protein is shown in SEQ ID NO. 8, wherein the 275 th amino acid residue of the amino acid sequence shown in SEQ ID NO.5 is mutated from valine to isoleucine, the 281 th amino acid residue is mutated from cysteine to tyrosine, and the 305 th amino acid residue is mutated from leucine to methionine;
(3) The amino acid sequence of the protein is shown in SEQ ID NO.5, wherein the 155 th amino acid residue of the amino acid sequence is mutated from cysteine to tyrosine, the 184 th amino acid residue is mutated from tyrosine to histidine, the 275 th amino acid residue is mutated from valine to isoleucine, the 281 th amino acid residue is mutated from cysteine to tyrosine, and the 305 th amino acid residue is mutated from leucine to methionine, and the amino acid sequence is shown in SEQ ID NO. 9;
(4) The amino acid sequence of the protein is shown in SEQ ID NO. 10, wherein the 155 th amino acid residue of the amino acid sequence shown in SEQ ID NO.6 is mutated from cysteine to tyrosine, and the 184 th amino acid residue is mutated from tyrosine to histidine;
(5) The protein is obtained by mutating the 275 th amino acid residue of the amino acid sequence shown in SEQ ID NO.6 from valine to isoleucine, the 281 th amino acid residue from cysteine to tyrosine, and the 305 th amino acid residue from isoleucine to methionine, and the amino acid sequence of the protein is shown in SEQ ID NO. 11;
(6) The protein is obtained by mutating the 155 th amino acid residue of the amino acid sequence shown in SEQ ID NO.6 from cysteine to tyrosine, mutating the 184 th amino acid residue from tyrosine to histidine, mutating the 275 th amino acid residue from valine to isoleucine, mutating the 281 th amino acid residue from cysteine to tyrosine, and mutating the 305 th amino acid residue from isoleucine to methionine, and the amino acid sequence of the protein is shown in SEQ ID No. 12.
5. A nucleic acid molecule encoding the protein according to any one of claims 1 to 4, which is a gene encoding the protein according to any one of claims 1 to 4.
6. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the nucleic acid molecule of claim 5.
7. Use of a protein according to any one of claims 1 to 4 as penicillin expandase.
8. Use of a protein as defined in any one of claims 1-4 as penicillin expandase for catalyzing the expansion of penicillin G to G-7-ADCA for increasing the conversion of penicillin G.
9. Use of the protein of any one of claims 1 to 4 or the nucleic acid molecule of claim 5 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 6 in any one of:
(b1) Preparing a product having penicillin expandase activity;
(b2) Preparation of G-7-ADCA.
10. A process for the preparation of G-7-ADCA or a process for increasing the conversion of penicillin G in a penicillin expandase catalyzed penicillin G expansion to G-7-ADCA, wherein the penicillin G conversion is one of the following:
1) Preparing a protein according to any one of claims 1 to 4; catalyzing penicillin G to expand ring by using the protein as penicillin expandase to generate G-7-ADCA;
2) Fermenting a microorganism containing a gene encoding a protein according to any of claims 1-4, so that the fermentation product obtained catalyzes the expansion of penicillin G to G-7-ADCA;
3) Cloning the gene encoding the protein of any one of claims 1-4 into a strain producing penicillin G to obtain a recombinant strain, and fermenting the recombinant strain to catalyze the ring expansion of penicillin G to obtain G-7-ADCA.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IN188444B (en) * | 1996-03-29 | 2002-09-28 | Werner Grabher | |
| CN1448506A (en) * | 2002-04-01 | 2003-10-15 | 骏翰生化股份有限公司 | Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same |
| US20090087893A1 (en) * | 2004-04-22 | 2009-04-02 | Orchid Chemicals & Pharmaceutical Ltd. | Modified Expandase Enzyme and its use |
| CN107304418A (en) * | 2016-04-18 | 2017-10-31 | 百瑞全球有限公司 | Penicillin ring enlargement enzyme mutant, the DNA for encoding the mutant, the kit containing the mutant and its application |
| CN112852913A (en) * | 2020-04-17 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Deacetoxycephalosporin C synthetase mutant and application thereof in synthesis of beta-lactam antibiotic parent nucleus |
-
2021
- 2021-06-18 CN CN202110676893.1A patent/CN115491361A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IN188444B (en) * | 1996-03-29 | 2002-09-28 | Werner Grabher | |
| CN1448506A (en) * | 2002-04-01 | 2003-10-15 | 骏翰生化股份有限公司 | Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same |
| US20090087893A1 (en) * | 2004-04-22 | 2009-04-02 | Orchid Chemicals & Pharmaceutical Ltd. | Modified Expandase Enzyme and its use |
| CN107304418A (en) * | 2016-04-18 | 2017-10-31 | 百瑞全球有限公司 | Penicillin ring enlargement enzyme mutant, the DNA for encoding the mutant, the kit containing the mutant and its application |
| CN112852913A (en) * | 2020-04-17 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Deacetoxycephalosporin C synthetase mutant and application thereof in synthesis of beta-lactam antibiotic parent nucleus |
Non-Patent Citations (2)
| Title |
|---|
| LIN等: "Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G", PNAS, vol. 112, no. 32, 11 August 2015 (2015-08-11), pages 9855 * |
| 季俊杰;张红梅;: "定点突变改造棒状链霉菌青霉素扩环酶", 江苏农业科学, no. 08, 25 August 2013 (2013-08-25), pages 53 - 54 * |
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