MUTANT ENZYME, USE THEREOF AND PROCESS FOR PREPARING TRIPEPTIDE BY USING ENZYMATIC METHOD

Abstract

The present invention relates to the technical field of biochemistry. Disclosed are a mutant enzyme, the use thereof and a process for preparing a tripeptide by using an enzymatic method. The mutant enzyme comprises: glycine and L-histidine ligase GHS, and tripeptide ligase HKS; or a fusion enzyme of the two. Glycine and L-histidine ligase activity is achieved by means of modifying an Lal enzyme, so as to obtain the GHS enzyme; and the ability for synthesizing dipeptide glycine-L-histidine and L-lysine is achieved by means of using a gshB enzyme, so as to obtain the HKS enzyme. On this basis, the GHS enzyme is further fused with the HKS enzyme by means of using a polypeptide chain, and then a bifunctional enzyme GHKS that links glycine, L-histidine and L-lysine in one step can be constructed, so that a tripeptide is conveniently prepared with a high yield. With regard to the large amount of ATP required in an enzymatic reaction, polyphosphate kinase can be used for cyclic regeneration, such that the amount of ATP is greatly reduced.

Claims

1. A mutant enzyme, wherein it comprises glycine and L-histidine ligase GHS and tripeptide ligase HKS, or is a fusion enzyme of the two; wherein, the glycine and L-histidine ligase GHS is an enzyme with mutations on sites T244I, S290L, G292W, E84K, A158H, and G159D of a wild-type L-amino acid ligase Lal, and the tripeptide ligase HKS is an enzyme with mutations on sites V150F, S153E, E228I, N230H, D233T, R285V, D130Q, E146L, N148S, G387N, and I445D of a wild-type glutathione synthase gshB.

2-3. (canceled)

4. A method for producing prezatide by enzymatic catalysis method, comprising subjecting reaction raw materials of glycine, L-histidine, L-lysine and ATP or salt thereof to enzymatic catalysis reaction with the mutant enzyme according to claim 1 in a reaction medium within a pH range of the mutant enzyme according to claim 1 to produce prezatide.

5. The method according to claim 4, wherein the pH range of the mutant enzyme is 6.5-9.0.

6. The method according to claim 4, wherein the reaction medium is a buffer solution.

7. The method according to claim 4, further comprising adding reaction raw materials of PPK and ADK or a fusion protein of the two, polyphosphoric acid, magnesium chloride and potassium chloride.

8. The method according to claim 4, further comprising a purification step selected from the group consisting of removing protein impurities, removing residual reaction raw materials, desalting, removing phosphoric acid, crystallization and a combination thereof.

9. An enzyme preparation comprising the mutant enzyme according to claim 1, wherein the enzyme preparation is a host cell expressing the mutant enzyme, an enzyme solution of the mutant enzyme or an immobilized enzyme of the mutant enzyme.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0023] FIG. 1 shows the schematic diagram of the reaction principle of the present disclosure;

[0024] FIG. 2 shows the SDS-PAGE gel image of the purified enzyme; and

[0025] FIG. 3 shows the nuclear magnetic spectrum of the purified prezatide with 600 M Varian in D.sub.2O solution; where the upper panel shows .sup.1H-NMR, and the lower panel shows .sup.13C-NMR.

DETAILED DESCRIPTION

[0026] The present disclosure discloses a mutant enzyme and use thereof and a method for producing prezatide by enzymatic catalysis method. Those skilled in the art can learn from the content herein and appropriately improve the process parameters for realization. In particular, it should be noted that all similar replacements and modifications are apparent to those skilled in the art, which are all considered to be included in the present disclosure. The mutant enzyme and use thereof and the related method of the present disclosure have been described through preferred embodiments, and those skilled in the art can apparently make modifications or appropriate changes and combination to the mutant enzyme and use thereof and the related method herein without departing from the content, spirit and scope of the present disclosure to realize and apply the technology of the present disclosure.

[0027] The steps of the method described in the present disclosure are intended to clearly describe the core reaction route, and do not limit whether the whole reaction is carried out by one-step method or multi-step method.

[0028] In a specific embodiment of the present disclosure, all enzymes used can be artificially synthesized according to their sequences. The sequences of the enzymes mentioned in the present disclosure are summarized as shown in Table 1:

TABLE-US-00001 TABLE1 Sequence Abbreviation number Aminoacidsequenceofenzyme Lal SEQID MTQAKENILVVVDGYSSGSQLPTLMAESGWKCVHV No.1 SSSANPPEYYLRTYHKDEYIAHFEYQGDIQSLASAVE AWHPAAVLPGTESGVIVADLLAAALQLPGNDPSTSLA RRDKYTMHESLKAVGLRSMDHFLAVDRDALSAWAE RGSWPVVIKPQASAGTDSVTFCADQGELLESFDQLF GTVNQLGERNNAVLAQRLLVGPEYFINGVSGHGKHL ITEIWRADKLPAPDGGWIYDRAVLFDPTSPEMQEIVR YVHGVLDALGIRYGANHTELIVTADGPTLIECASRLS GGLHRPAANYAVGASQLDLVGKLVREGESAIDDILQT WQPHRYALWQVQFISNQEGVVARSSYDELLKTLKSN AWLQRAPKEGDTVVKTVDLFSSPGIVFMSHADGNVL HDDYRTVREWERTSRLFSVQ GHS SEQID MTQAKENILVVVDGYSSGSQLPTLMAESGWKCVHV No.2 SSSANPPEYYLRTYHKDEYIAHFEYQGDIQSLASAVE AWHPAAVLPGTKSGVIVADLLAAALQLPGNDPSTSLA RRDKYTMHESLKAVGLRSMDHFLAVDRDALSAWAE RGSWPVVIKPQASHDTDSVTFCADQGELLESFDQLF GTVNQLGERNNAVLAQRLLVGPEYFINGVSGHGKHL ITEIWRADKLPAPDGGWIYDRAVLFDPISPEMQEIVRY VHGVLDALGIRYGANHTELIVTADGPTLIECASRLLG WLHRPAANYAVGASQLDLVGKLVREGESAIDDILQT WQPHRYALWQVQFISNQEGVVARSSYDELLKTLKSN AWLQRAPKEGDTVVKTVDLFSSPGIVFMSHADGNVL HDDYRTVREWERTSRLFSVQ gshB SEQID MAHYPPSKDQLNELIQEVNQWAITNGLSMYPPKFEE No.3 NPSNASVSPVTIYPTPIPRKCFDEAVQIQPVFNELYARI TQDMAQPDSYLHKTTEALALSDSEFTGKLWSLYLAT LKSAQYKKQNFRLGIFRSDYLIDKKKGTEQIKQVEFN TVSVSFAGLSEKVDRLHSYLNRANKYDPKGPIYNDQ NMVISDSGYLLSKALAKAVESYKSQQSSSTTSDPIVAF IVQRNERNVFDQKVLELNLLEKFGTKSVRLTFDDVN DKLFIDDKTGKLFIRDTEQEIAVVYYRTGYTTTDYTSE KDWEARLFLEKSFAIKAPDLLTQLSGSKKIQQLLTDE GVLGKYISDAEKKSSLLKTFVKIYPLDDTKLGREGKR LALSEPSKYVLKPQREGGGNNVYKENIPNFLKGIEER HWDAYILMELIEPELNENNIILRDNKSYNEPIISELGIY GCVLFNDEQVLSNEFSGSLLRSKFNTSNEGGVAAGFG CLDSIILY HKS SEQID MAHYPPSKDQLNELIQEVNQWAITNGLSMYPPKFEE No.4 NPSNASVSPVTIYPTPIPRKCFDEAVQIQPVFNELYARI TQDMAQPDSYLHKTTEALALSDSEFTGKLWSLYLAT LKSAQYKKQNFRLGIFRSQYLIDKKKGTEQIKQVLFS TFSVEFAGLSEKVDRLHSYLNRANKYDPKGPIYNDQ NMVISDSGYLLSKALAKAVESYKSQQSSSTTSDPIVAF IVQRNIRHVFTQKVLELNLLEKFGTKSVRLTFDDVND KLFIDDKTGKLFIRDTEQEIAVVYYVTGYTTTDYTSE KDWEARLFLEKSFAIKAPDLLTQLSGSKKIQQLLTDE GVLGKYISDAEKKSSLLKTFVKIYPLDDTKLGREGKR LALSEPSKYVLKPQRENGGNNVYKENIPNFLKGIEER HWDAYILMELIEPELNENNIILRDNKSYNEPIISELGD YGCVLFNDEQVLSNEFSGSLLRSKFNTSNEGGVAAGF GCLDSIILY GHKS-1 SEQID MTQAKENILVVVDGYSSGSQLPTLMAESGWKCVHV No.5 SSSANPPEYYLRTYHKDEYIAHFEYQGDIQSLASAVE AWHPAAVLPGTKSGVIVADLLAAALQLPGNDPSTSLA RRDKYTMHESLKAVGLRSMDHFLAVDRDALSAWAE RGSWPVVIKPQASHDTDSVTFCADQGELLESFDQLF GTVNQLGERNNAVLAQRLLVGPEYFINGVSGHGKHL ITEIWRADKLPAPDGGWIYDRAVLFDPISPEMQEIVRY VHGVLDALGIRYGANHTELIVTADGPTLIECASRLLG WLHRPAANYAVGASQLDLVGKLVREGESAIDDILQT WQPHRYALWQVQFISNQEGVVARSSYDELLKTLKSN AWLQRAPKEGDTVVKTVDLFSSPGIVFMSHADGNVL HDDYRTVREWERTSRLFSVQGGGGSGGGGSGGGGSG GGGSMAHYPPSKDQLNELIQEVNQWAITNGLSMYPP KFEENPSNASVSPVTIYPTPIPRKCFDEAVQIQPVFNEL YARITQDMAQPDSYLHKTTEALALSDSEFTGKLWSLY LATLKSAQYKKQNFRLGIFRSQYLIDKKKGTEQIKQV LFSTFSVEFAGLSEKVDRLHSYLNRANKYDPKGPIYN DQNMVISDSGYLLSKALAKAVESYKSQQSSSTTSDPI VAFIVQRNIRHVFTQKVLELNLLEKFGTKSVRLTFDD VNDKLFIDDKTGKLFIRDTEQEIAVVYYVTGYTTTDY TSEKDWEARLFLEKSFAIKAPDLLTQLSGSKKIQQLLT DEGVLGKYISDAEKKSSLLKTFVKIYPLDDTKLGREG KRLALSEPSKYVLKPQRENGGNNVYKENIPNFLKGIE ERHWDAYILMELIEPELNENNIILRDNKSYNEPIISELG DYGCVLFNDEQVLSNEFSGSLLRSKFNTSNEGGVAA GFGCLDSIILY GHKS-2 SEQID MTQAKENILVVVDGYSSGSQLPTLMAESGWKCVHV No.6 SSSANPPEYYLRTYHKDEYIAHFEYQGDIQSLASAVE AWHPAAVLPGTKSGVIVADLLAAALQLPGNDPSTSLA RRDKYTMHESLKAVGLRSMDHFLAVDRDALSAWAE RGSWPVVIKPQASHDTDSVTFCADQGELLESFDQLF GTVNQLGERNNAVLAQRLLVGPEYFINGVSGHGKHL ITEIWRADKLPAPDGGWIYDRAVLFDPISPEMQEIVRY VHGVLDALGIRYGANHTELIVTADGPTLIECASRLLG WLHRPAANYAVGASQLDLVGKLVREGESAIDDILQT WQPHRYALWQVQFISNQEGVVARSSYDELLKTLKSN AWLQRAPKEGDTVVKTVDLFSSPGIVFMSHADGNVL HDDYRTVREWERTSRLFSVQGGGGSEAAAKEAAAKG GGGSMAHYPPSKDQLNELIQEVNQWAITNGLSMYPP KFEENPSNASVSPVTIYPTPIPRKCFDEAVQIQPVFNEL YARITQDMAQPDSYLHKTTEALALSDSEFTGKLWSLY LATLKSAQYKKQNFRLGIFRSQYLIDKKKGTEQIKQV LFSTFSVEFAGLSEKVDRLHSYLNRANKYDPKGPIYN DQNMVISDSGYLLSKALAKAVESYKSQQSSSTTSDPI VAFIVQRNIRHVFTQKVLELNLLEKFGTKSVRLTFDD VNDKLFIDDKTGKLFIRDTEQEIAVVYYVTGYTTTDY TSEKDWEARLFLEKSFAIKAPDLLTQLSGSKKIQQLLT DEGVLGKYISDAEKKSSLLKTFVKIYPLDDTKLGREG KRLALSEPSKYVLKPQRENGGNNVYKENIPNFLKGIE ERHWDAYILMELIEPELNENNIILRDNKSYNEPIISELG DYGCVLFNDEQVLSNEFSGSLLRSKFNTSNEGGVAA GFGCLDSIILY PPK SEQID MPMVAAVEFAKSPAEVLRVGSGFSLAGVDPESTPGYT No.7 GVKADGKALLAAQDARLAELQEKLFAEGKFGNPKR LLLILQAMDTAGKGGIVSHVVGAMDPQGVQLTAFKA PTDEEKSHDFLWRIEKQVPAAGMVGVFDRSQYEDVL IHRVHGWADAAELERRYAAINDFESRLTEQGTTIVKV MLNISKDEQKKRLIARLDDPSKHWKYSRGDLAERAY WDDYMDAYSVAFEKTSTEIAPWHVVPANKKWYARI AVQQLLLDALGGLQLDWPKADFDVAAERALVVES ADK SEQID MRIILLGAPGAGKGTQAQFIMEKYGIPQISTGDMLRA No.8 AVKSGSELGKQAKDIMDAGKLVTDELVIALVKERIAQ EDCRNGFLLDGFPRTIPQADAMKEAGINVDYVLEFD VPDELIVDRIVGRRVHAPSGRVYHVKFNPPKVEGKD DVTGEELTTRKDDQEETVRKRLVEYHQMTAPLIGYY SKEAEAGNTKYAKVDGTKPVAEVRADLEKILG

[0029] In Table 1, GHKS-1 and GHKS-2 are two fusion enzymes (GHS-HKSs with different linking peptides) provided by the present disclosure, bold and underlined amino acids indicate mutation sites and mutated amino acids, and italic and underlined sequences are linking peptide sequences.

[0030] The above enzymes can also be obtained by cell transformation with recombinant plasmids constructed with their coding genes respectively, for example:

[0031] The gene fragments of ADK, gshB and PPK were amplified with chromosomes of Escherichia coli K12, Saccharomyces cerevisiae (ATCC 204508) and Paenarthrobacter aurescens TC1 purchased from ATCC as templates by PCR using the primers in Table 2, subjected to enzyme digestion using the Nde I/Xho I purchased from NEB Company, and connected to a pET28a plasmid (purchased from Addgene) digested with the same enzyme. Then the plasmid was transformed into E. coli DH5a cells (purchased from Tsingke Biotechnology), and verified by colony PCR and gene sequencing. Lal gene fragment was synthesized by Anhui General Biology Co., Ltd., and subcloned into a pET28a plasmid. Then multi-site mutant enzyme genes GHS and HKS were constructed with Lal and gshB genes as templates using the mutation primers in Table 2 (by conventional PCR amplification). The above GHS, HKS, PPK and ADK plasmids constructed with the pET-28a vector were transferred into E. coli BL21 (DE3) (purchased from Anhui General Biology Co., Ltd.) strains, which were then cultured in a small-scale in 5 ml of LB culture medium containing 50 M Kanamycin at 37 C. When the cells grew to an OD value of 0.5-0.8, 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) was added to induce protein expression at 37 C. for 3 h. Finally the cells were collected, disrupted by freeze-thaw method, and centrifuged at high speed, and the collected supernatant was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the protein expression. The strains with correct protein expression were cultured step by step in a 5 liter fermenter to be induced for expression under the condition of 1.0 mM IPTG at 37 C. for 4 h, and wet cells were collected to be 35-55 g. Then after the cells and an appropriate amount of Tris.Math.HCl buffer solution (25 mM, pH=8.0) were mixed evenly, the cells were crushed with a high-pressure crusher at low temperature and centrifuged at high speed to remove the cell wall to obtain an enzyme solution, which was stored in a refrigerator at 4 C. for later use. The LB culture medium was composed of 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate and 5% glycerol.

TABLE-US-00002 TABLE2 Primername Primersequence InformationofprimersforconstructionofLalmutant:mutationatglycine bindingsite(theboldandunderlinedrepresentmutationsites) T244IForward CTGTTTGACCCGATCAGTCCCGAGATGCAG T244IReverse CTGCATCTCGGGACTGATCGGGTCAAACAG S290LForward GTGCCTCCCGTTTACTCGGGTGGCTGCATAGAC S290LReverse GTCTATGCAGCCACCCGAGTAAACGGGAGGCAC G292WForward GTGCCTCCCGTTTACTCGGGTGGCTGCATAGAC G292WReverse GTCTATGCAGCCACCCGAGTAAACGGGAGGCAC InformationofprimersforconstructionofLalmutant:mutationatL-histidine bindingsite E84KForward CTTCCTGGTACGAAAAGTGGAGTTATTGTC E84KReverse GACAATAACTCCACTTTTCGTACCAGGAAG A158HForward CAGGCTTCGCACGACACAGACAGTGTTACATTC A158HReverse GAATGTAACACTGTCTGTGTCGTGCGAAGCCTG G159DForward CAGGCTTCGCACGACACAGACAGTGTTACATTC G159DReverse GAATGTAACACTGTCTGTGTCGTGCGAAGCCTG InformationofprimersforconstructionofgshBmutant:mutationatglycine-L- histidinebindingsite V150FForward GATTAAGCAAGTCCTGTTTAGTACATTCTCAGTGGAATTTGCAGG V150FReverse CCTGCAAATTCCACTGAGAATGTACTAAACAGGACTTGCTTAATC S153EForward GATTAAGCAAGTCCTGTTTAGTACATTCTCAGTGGAATTTGCAGG S153EReverse CCTGCAAATTCCACTGAGAATGTACTAAACAGGACTTGCTTAATC E228IForward GCAAAGAAACATCAGACATGTGTTTACTCAAAAGGTC E228IReverse GACCTTTTGAGTAAACACATGTCTGATGTTTCTTTGC N230HForward GCAAAGAAACATCAGACATGTGTTTACTCAAAAGGTC N230HReverse GACCTTTTGAGTAAACACATGTCTGATGTTTCTTTGC D233TForward GCAAAGAAACATCAGACATGTGTTTACTCAAAAGGTC D233TReverse GACCTTTTGAGTAAACACATGTCTGATGTTTCTTTGC R285VForward GTGGTTTATTACGTAACGGGTTACACAACC R285VReverse GGTTGTGTAACCCGTTACGTAATAAACCAC InformationofprimersforconstructionofgshBmutant:mutationatL-lysine bindingsite D130QForward CTAGGTATATTTAGATCACAGTATTTGATTG D130QReverse CAATCAAATACTGTGATCTAAATATACCTAG E146LForward GATTAAGCAAGTCCTGTTTAGTACATTCTCAGTGGAATTTGCAGG E146LReverse CCTGCAAATTCCACTGAGAATGTACTAAACAGGACTTGCTTAATC N148SForward GATTAAGCAAGTCCTGTTTAGTACATTCTCAGTGGAATTTGCAGG N148SReverse CCTGCAAATTCCACTGAGAATGTACTAAACAGGACTTGCTTAATC G387NForward CCACAGCGGGAAAATGGCGGAAACAATGTTT G387NReverse AAACATTGTTTCCGCCATTTTCCCGCTGTGG I445DForward CAGTGAACTAGGAGATTATGGTTGCGTTC I445DReverse GAACGCAACCATAATCTCCTAGTTCACTG PrimersforcloningLal,gshB,PPKandADKgenes(theboldandunderlined representenzymedigestionsites) gshBForward GCACTACTCCTATAACATATGGCACACTATC gshBReverse GTTCTAGCATCATCTCTCGAGCATCTATGTG PPKForward GCAGAAACATATGCCAATGGTTGCTGCAG PPKReverse GATGCCAGCAGCGACCTCGAGACCCCAGC ADKForward GAGGCAATCGCCATATGGTGGTATCGTTTATC ADKReverse CTGCCCGAAAGCAGATCTGCTCGAGCTTG

[0032] Each enzyme can participate in catalysis reaction in the form of a crude enzyme solution containing the enzyme, a purified enzyme or an immobilized enzyme:

[0033] For example, the collected wet cells containing the enzyme were mixed in Tris-HCl buffer solution (25 mM, pH=8.0) (buffer solution A). After stirring evenly, the cells were crushed by high pressure and centrifuged at high speed to remove the cell wall. The collected supernatant was a crude enzyme solution, which was directly used for the subsequent catalysis reaction.

[0034] Alternatively, the above supernatant was gradually added with solid ammonium sulfate until the protein precipitated (35%-55%, w/v ammonium sulfate/buffer solution). The protein solid was then collected by high-speed centrifugation (10000 rpm, 12 min), slowly dissolved in Tris-HCl buffer solution (25 mM, pH=8.0), desalted by G25 desalting column (purchased from Sigma), separated and purified by DEAE Seplite FF (Xi'an Sunresin Co., Ltd.) anion exchange column to finally obtain a primarily purified enzyme. The SDS-PAGE gel image is shown in FIG. 2.

[0035] For the immobilization, reference can be made to the conventional methods for producing immobilized enzyme in the art.

[0036] According to the reaction route of the method of the present disclosure, the amount of each reaction material used can be adjusted according to actual situation.

[0037] Although the coding gene sequences of the enzymes can be known according to their amino acid sequences provided by the present disclosure, the specific coding gene sequences are still provided in the present disclosure as shown in Table 3:

TABLE-US-00003 TABLE3 Sequence Abbreviation number Genesequenceofenzyme Lal SEQID ATGACGCAGGCCAAGGAAAACATCTTAGTT No.9 GTGGTCGATGGCTACTCCTCAGGCAGTCAG TTGCCTACCCTGATGGCGGAATCCGGCTGG AAGTGTGTTCATGTCTCGTCCTCGGCTAAC CCCCCGGAGTATTACCTGCGTACATACCAT AAGGATGAGTACATAGCTCACTTTGAGTAT CAAGGTGATATACAGTCACTTGCTAGTGCTGTT GAGGCGTGGCATCCCGCCGCAGTTCTTCCT GGTACGGAAAGTGGAGTTATTGTCGCTGAT TTACTGGCGGCTGCTTTGCAATTGCCAGGC AATGACCCGTCGACCTCCCTGGCGAGACGC GACAAATATACGATGCATGAGTCATTGAAA GCGGTGGGACTTCGGAGTATGGACCATTTC CTTGCCGTCGATCGTGATGCTTTATCAGCTTGG GCCGAGCGGGGATCTTGGCCAGTGGTAATT AAACCCCAGGCTTCGGCAGGCACAGACAGT GTTACATTCTGCGCCGATCAGGGAGAATTA TTAGAGTCGTTTGATCAATTGTTCGGCACTGTG AACCAATTGGGTGAACGCAATAATGCAGTG CTGGCTCAGCGTCTGTTGGTTGGTCCCGAG TACTTTATCAACGGAGTTTCTGGACATGGCAAA CACCTGATTACAGAGATTTGGCGTGCAGAC AAGCTGCCCGCACCGGACGGGGGTTGGATA TACGACCGGGCCGTGCTGTTTGACCCGACG AGTCCCGAGATGCAGGAGATTGTCCGCTAC GTGCATGGAGTATTAGATGCCCTTGGCATCCGC TATGGTGCGAACCATACAGAGCTGATCGTA ACGGCTGATGGCCCAACACTGATAGAATGT GCCTCCCGTTTATCCGGGGGACTGCATAGA CCTGCAGCGAACTATGCGGTTGGCGCATCA CAACTTGACTTGGTCGGTAAACTTGTACGG GAAGGGGAAAGCGCTATAGATGATATACTG CAAACTTGGCAACCTCACCGCTACGCATTG TGGCAAGTCCAATTCATATCGAATCAAGAG GGAGTAGTGGCTCGGAGTTCGTACGACGAA CTTCTTAAAACGTTGAAATCCAATGCCTGGTTG CAACGGGCTCCGAAGGAAGGCGATACCGTA GTCAAGACAGTCGACCTGTTCAGCTCGCCC GGAATAGTCTTTATGTCACACGCAGACGGTAAT GTTCTGCACGACGATTATCGGACGGTCCGG GAATGGGAGCGTACCTCGCGCCTGTTCTCG GTGCAGTAA GHS SEQID ATGACGCAGGCCAAGGAAAACATCTTAGTT No.10 GTGGTCGATGGCTACTCCTCAGGCAGTCAG TTGCCTACCCTGATGGCGGAATCCGGCTGG AAGTGTGTTCATGTCTCGTCCTCGGCTAAC CCCCCGGAGTATTACCTGCGTACATACCAT AAGGATGAGTACATAGCTCACTTTGAGTAT CAAGGTGATATACAGTCACTTGCTAGTGCTGTT GAGGCGTGGCATCCCGCCGCAGTTCTTCCT GGTACGAAAAGTGGAGTTATTGTCGCTGAT TTACTGGCGGCTGCTTTGCAATTGCCAGGC AATGACCCGTCGACCTCCCTGGCGAGACGC GACAAATATACGATGCATGAGTCATTGAAA GCGGTGGGACTTCGGAGTATGGACCATTTC CTTGCCGTCGATCGTGATGCTTTATCAGCTTGG GCCGAGCGGGGATCTTGGCCAGTGGTAATT AAACCCCAGGCTTCGCACGACACAGACAGT GTTACATTCTGCGCCGATCAGGGAGAATTA TTAGAGTCGTTTGATCAATTGTTCGGCACTGTG AACCAATTGGGTGAACGCAATAATGCAGTG CTGGCTCAGCGTCTGTTGGTTGGTCCCGAG TACTTTATCAACGGAGTTTCTGGACATGGCAAA CACCTGATTACAGAGATTTGGCGTGCAGAC AAGCTGCCCGCACCGGACGGGGGTTGGATA TACGACCGGGCCGTGCTGTTTGACCCGATC AGTCCCGAGATGCAGGAGATTGTCCGCTAC GTGCATGGAGTATTAGATGCCCTTGGCATCCGC TATGGTGCGAACCATACAGAGCTGATCGTA ACGGCTGATGGCCCAACACTGATAGAATGT GCCTCCCGTTTACTCGGGTGGCTGCATAGA CCTGCAGCGAACTATGCGGTTGGCGCATCA CAACTTGACTTGGTCGGTAAACTTGTACGG GAAGGGGAAAGCGCTATAGATGATATACTG CAAACTTGGCAACCTCACCGCTACGCATTG TGGCAAGTCCAATTCATATCGAATCAAGAG GGAGTAGTGGCTCGGAGTTCGTACGACGAA CTTCTTAAAACGTTGAAATCCAATGCCTGGTTG CAACGGGCTCCGAAGGAAGGCGATACCGTA GTCAAGACAGTCGACCTGTTCAGCTCGCCC GGAATAGTCTTTATGTCACACGCAGACGGTAAT GTTCTGCACGACGATTATCGGACGGTCCGG GAATGGGAGCGTACCTCGCGCCTGTTCTCG GTGCAGTAA gshB SEQID ATGGCACACTATCCACCTTCCAAGGATCAATTGAAT No.11 GAATTGATCCAGGAAGTTAACCAATGGGCTATCACT AATGGATTATCCATGTATCCTCCTAAATTCGAGGAG AACCCATCAAATGCATCGGTGTCACCAGTAACTATC TATCCAACCCCAATTCCTAGGAAATGTTTTGATGAG GCCGTTCAAATACAACCGGTATTCAATGAATTATAC GCCCGTATTACCCAAGATATGGCCCAACCTGATTCT TATTTACATAAAACAACTGAAGCGTTAGCTCTATCA GATTCCGAGTTTACTGGAAAACTGTGGTCTCTATAC CTTGCTACCTTAAAATCTGCACAGTACAAAAAGCA GAATTTTAGGCTAGGTATATTTAGATCAGATTATTTG ATTGATAAGAAAAAGGGTACTGAACAGATTAAGCA AGTCGAGTTTAATACAGTGTCAGTGTCATTTGCAGG CCTTAGCGAGAAAGTTGATAGATTGCACTCTTATTT AAATAGGGCAAACAAGTACGATCCTAAAGGACCAA TTTATAATGATCAAAATATGGTCATTTCTGATTCAGG ATACCTTTTGTCTAAGGCATTGGCCAAAGCTGTGGA ATCGTATAAGTCACAACAAAGTTCTTCTACAACTAG TGATCCTATTGTCGCATTCATTGTGCAAAGAAACGA GAGAAATGTGTTTGATCAAAAGGTCTTGGAATTGA ATCTGTTGGAAAAATTCGGTACCAAATCTGTTAGGT TGACGTTTGATGATGTTAACGATAAATTGTTCATTGA TGATAAAACGGGAAAGCTTTTCATTAGGGACACAG AGCAGGAAATAGCGGTGGTTTATTACAGAACGGGT TACACAACCACTGATTACACGTCCGAAAAGGACTG GGAGGCAAGACTATTCCTCGAAAAAAGTTTCGCAA TAAAGGCCCCAGATTTACTCACTCAATTATCTGGCT CCAAGAAAATTCAGCAATTGTTGACAGATGAGGGC GTATTAGGTAAATACATCTCCGATGCTGAGAAAAAG AGTAGTTTGTTAAAAACTTTTGTCAAAATATATCCCT TGGATGATACGAAGCTTGGCAGGGAAGGCAAGAG GCTGGCATTAAGTGAGCCCTCTAAATACGTGTTAAA ACCACAGCGGGAAGGTGGCGGAAACAATGTTTATA AAGAAAATATTCCTAATTTTTTGAAAGGTATCGAAG AACGTCACTGGGATGCATATATTCTCATGGAGTTGA TTGAACCAGAGTTGAATGAAAATAATATTATATTACG TGATAACAAATCTTACAACGAACCAATCATCAGTGA ACTAGGAATTTATGGTTGCGTTCTATTTAACGACGA GCAAGTTTTATCGAACGAATTTAGTGGCTCATTACT AAGATCCAAATTTAATACTTCAAATGAAGGTGGAGT GGCGGCAGGATTCGGATGTTTGGACAGTATTATTCT TTACTAG HKS SEQID ATGGCACACTATCCACCTTCCAAGGATCAATTGAAT No.12 GAATTGATCCAGGAAGTTAACCAATGGGCTATCACT AATGGATTATCCATGTATCCTCCTAAATTCGAGGAG AACCCATCAAATGCATCGGTGTCACCAGTAACTATC TATCCAACCCCAATTCCTAGGAAATGTTTTGATGAG GCCGTTCAAATACAACCGGTATTCAATGAATTATAC GCCCGTATTACCCAAGATATGGCCCAACCTGATTCT TATTTACATAAAACAACTGAAGCGTTAGCTCTATCA GATTCCGAGTTTACTGGAAAACTGTGGTCTCTATAC CTTGCTACCTTAAAATCTGCACAGTACAAAAAGCA GAATTTTAGGCTAGGTATATTTAGATCACAGTATTTG ATTGATAAGAAAAAGGGTACTGAACAGATTAAGCA AGTCCTGTTTAGTACATTCTCAGTGGAATTTGCAG GCCTTAGCGAGAAAGTTGATAGATTGCACTCTTATT TAAATAGGGCAAACAAGTACGATCCTAAAGGACCA ATTTATAATGATCAAAATATGGTCATTTCTGATTCAG GATACCTTTTGTCTAAGGCATTGGCCAAAGCTGTGG AATCGTATAAGTCACAACAAAGTTCTTCTACAACTA GTGATCCTATTGTCGCATTCATTGTGCAAAGAAACA TCAGACATGTGTTTACTCAAAAGGTCTTGGAATTG AATCTGTTGGAAAAATTCGGTACCAAATCTGTTAGG TTGACGTTTGATGATGTTAACGATAAATTGTTCATTG ATGATAAAACGGGAAAGCTTTTCATTAGGGACACA GAGCAGGAAATAGCGGTGGTTTATTACGTAACGGG TTACACAACCACTGATTACACGTCCGAAAAGGACT GGGAGGCAAGACTATTCCTCGAAAAAAGTTTCGCA ATAAAGGCCCCAGATTTACTCACTCAATTATCTGGC TCCAAGAAAATTCAGCAATTGTTGACAGATGAGGG CGTATTAGGTAAATACATCTCCGATGCTGAGAAAAA GAGTAGTTTGTTAAAAACTTTTGTCAAAATATATCC CTTGGATGATACGAAGCTTGGCAGGGAAGGCAAGA GGCTGGCATTAAGTGAGCCCTCTAAATACGTGTTAA AACCACAGCGGGAAAATGGCGGAAACAATGTTTAT AAAGAAAATATTCCTAATTTTTTGAAAGGTATCGAA GAACGTCACTGGGATGCATATATTCTCATGGAGTTG ATTGAACCAGAGTTGAATGAAAATAATATTATATTAC GTGATAACAAATCTTACAACGAACCAATCATCAGTG AACTAGGAGATTATGGTTGCGTTCTATTTAACGACG AGCAAGTTTTATCGAACGAATTTAGTGGCTCATTAC TAAGATCCAAATTTAATACTTCAAATGAAGGTGGAG TGGCGGCAGGATTCGGATGTTTGGACAGTATTATTC TTTACTAG GHKS-1 SEQID ATGACGCAGGCCAAGGAAAACATCTTAGTT No.13 GTGGTCGATGGCTACTCCTCAGGCAGTCAG TTGCCTACCCTGATGGCGGAATCCGGCTGG AAGTGTGTTCATGTCTCGTCCTCGGCTAAC CCCCCGGAGTATTACCTGCGTACATACCAT AAGGATGAGTACATAGCTCACTTTGAGTAT CAAGGTGATATACAGTCACTTGCTAGTGCTGTT GAGGCGTGGCATCCCGCCGCAGTTCTTCCT GGTACGAAAAGTGGAGTTATTGTCGCTGAT TTACTGGCGGCTGCTTTGCAATTGCCAGGC AATGACCCGTCGACCTCCCTGGCGAGACGC GACAAATATACGATGCATGAGTCATTGAAA GCGGTGGGACTTCGGAGTATGGACCATTTC CTTGCCGTCGATCGTGATGCTTTATCAGCTTGG GCCGAGCGGGGATCTTGGCCAGTGGTAATT AAACCCCAGGCTTCGCACGACACAGACAGT GTTACATTCTGCGCCGATCAGGGAGAATTA TTAGAGTCGTTTGATCAATTGTTCGGCACTGTG AACCAATTGGGTGAACGCAATAATGCAGTG CTGGCTCAGCGTCTGTTGGTTGGTCCCGAG TACTTTATCAACGGAGTTTCTGGACATGGCAAA CACCTGATTACAGAGATTTGGCGTGCAGAC AAGCTGCCCGCACCGGACGGGGGTTGGATA TACGACCGGGCCGTGCTGTTTGACCCGATC AGTCCCGAGATGCAGGAGATTGTCCGCTAC GTGCATGGAGTATTAGATGCCCTTGGCATCCGC TATGGTGCGAACCATACAGAGCTGATCGTA ACGGCTGATGGCCCAACACTGATAGAATGT GCCTCCCGTTTACTCGGGTGGCTGCATAGA CCTGCAGCGAACTATGCGGTTGGCGCATCA CAACTTGACTTGGTCGGTAAACTTGTACGG GAAGGGGAAAGCGCTATAGATGATATACTG CAAACTTGGCAACCTCACCGCTACGCATTG TGGCAAGTCCAATTCATATCGAATCAAGAG GGAGTAGTGGCTCGGAGTTCGTACGACGAA CTTCTTAAAACGTTGAAATCCAATGCCTGGTTG CAACGGGCTCCGAAGGAAGGCGATACCGTA GTCAAGACAGTCGACCTGTTCAGCTCGCCC GGAATAGTCTTTATGTCACACGCAGACGGTAAT GTTCTGCACGACGATTATCGGACGGTCCGG GAATGGGAGCGTACCTCGCGCCTGTTCTCG GTGCAGGGTGGTGGTGGATCAGGGGGT GGGGGTTCAGGCGGTGGCGGATCCGGC GGGGGTGGTTCCATGGCACACTATCCACCT TCCAAGGATCAA TTGAATGAATTGATCCAGGAAGTTAACCAATGGGCT ATCACTAATGGATTATCCATGTATCCTCCTAAATTCG AGGAGAACCCATCAAATGCATCGGTGTCACCAGTA ACTATCTATCCAACCCCAATTCCTAGGAAATGTTTTG ATGAGGCCGTTCAAATACAACCGGTATTCAATGAAT TATACGCCCGTATTACCCAAGATATGGCCCAACCTG ATTCTTATTTACATAAAACAACTGAAGCGTTAGCTCT ATCAGATTCCGAGTTTACTGGAAAACTGTGGTCTCT ATACCTTGCTACCTTAAAATCTGCACAGTACAAAAA GCAGAATTTTAGGCTAGGTATATTTAGATCACAGTAT TTGATTGATAAGAAAAAGGGTACTGAACAGATTAA GCAAGTCCTGTTTAGTACATTCTCAGTGGAATTTG CAGGCCTTAGCGAGAAAGTTGATAGATTGCACTCTT ATTTAAATAGGGCAAACAAGTACGATCCTAAAGGA CCAATTTATAATGATCAAAATATGGTCATTTCTGATT CAGGATACCTTTTGTCTAAGGCATTGGCCAAAGCTG TGGAATCGTATAAGTCACAACAAAGTTCTTCTACAA CTAGTGATCCTATTGTCGCATTCATTGTGCAAAGAA ACATCAGACATGTGTTTACTCAAAAGGTCTTGGAA TTGAATCTGTTGGAAAAATTCGGTACCAAATCTGTT AGGTTGACGTTTGATGATGTTAACGATAAATTGTTC ATTGATGATAAAACGGGAAAGCTTTTCATTAGGGAC ACAGAGCAGGAAATAGCGGTGGTTTATTACGTAAC GGGTTACACAACCACTGATTACACGTCCGAAAAGG ACTGGGAGGCAAGACTATTCCTCGAAAAAAGTTTC GCAATAAAGGCCCCAGATTTACTCACTCAATTATCT GGCTCCAAGAAAATTCAGCAATTGTTGACAGATGA GGGCGTATTAGGTAAATACATCTCCGATGCTGAGAA AAAGAGTAGTTTGTTAAAAACTTTTGTCAAAATATA TCCCTTGGATGATACGAAGCTTGGCAGGGAAGGCA AGAGGCTGGCATTAAGTGAGCCCTCTAAATACGTGT TAAAACCACAGCGGGAAAATGGCGGAAACAATGT TTATAAAGAAAATATTCCTAATTTTTTGAAAGGTATC GAAGAACGTCACTGGGATGCATATATTCTCATGGAG TTGATTGAACCAGAGTTGAATGAAAATAATATTATAT TACGTGATAACAAATCTTACAACGAACCAATCATCA GTGAACTAGGAGATTATGGTTGCGTTCTATTTAACG ACGAGCAAGTTTTATCGAACGAATTTAGTGGCTCAT TACTAAGATCCAAATTTAATACTTCAAATGAAGGTG GAGTGGCGGCAGGATTCGGATGTTTGGACAGTATTA TTCTTTACTAG GHKS-2 SEQID ATGACGCAGGCCAAGGAAAACATCTTAGTT No.14 GTGGTCGATGGCTACTCCTCAGGCAGTCAG TTGCCTACCCTGATGGCGGAATCCGGCTGG AAGTGTGTTCATGTCTCGTCCTCGGCTAAC CCCCCGGAGTATTACCTGCGTACATACCAT AAGGATGAGTACATAGCTCACTTTGAGTAT CAAGGTGATATACAGTCACTTGCTAGTGCTGTT GAGGCGTGGCATCCCGCCGCAGTTCTTCCT GGTACGAAAAGTGGAGTTATTGTCGCTGAT TTACTGGCGGCTGCTTTGCAATTGCCAGGC AATGACCCGTCGACCTCCCTGGCGAGACGC GACAAATATACGATGCATGAGTCATTGAAA GCGGTGGGACTTCGGAGTATGGACCATTTC CTTGCCGTCGATCGTGATGCTTTATCAGCTTGG GCCGAGCGGGGATCTTGGCCAGTGGTAATT AAACCCCAGGCTTCGCACGACACAGACAGT GTTACATTCTGCGCCGATCAGGGAGAATTA TTAGAGTCGTTTGATCAATTGTTCGGCACTGTG AACCAATTGGGTGAACGCAATAATGCAGTG CTGGCTCAGCGTCTGTTGGTTGGTCCCGAG TACTTTATCAACGGAGTTTCTGGACATGGCAAA CACCTGATTACAGAGATTTGGCGTGCAGAC AAGCTGCCCGCACCGGACGGGGGTTGGATA TACGACCGGGCCGTGCTGTTTGACCCGATC AGTCCCGAGATGCAGGAGATTGTCCGCTAC GTGCATGGAGTATTAGATGCCCTTGGCATCCGC TATGGTGCGAACCATACAGAGCTGATCGTA ACGGCTGATGGCCCAACACTGATAGAATGT GCCTCCCGTTTACTCGGGTGGCTGCATAGA CCTGCAGCGAACTATGCGGTTGGCGCATCA CAACTTGACTTGGTCGGTAAACTTGTACGG GAAGGGGAAAGCGCTATAGATGATATACTG CAAACTTGGCAACCTCACCGCTACGCATTG TGGCAAGTCCAATTCATATCGAATCAAGAG GGAGTAGTGGCTCGGAGTTCGTACGACGAA CTTCTTAAAACGTTGAAATCCAATGCCTGGTTG CAACGGGCTCCGAAGGAAGGCGATACCGTA GTCAAGACAGTCGACCTGTTCAGCTCGCCC GGAATAGTCTTTATGTCACACGCAGACGGTAAT GTTCTGCACGACGATTATCGGACGGTCCGG GAATGGGAGCGTACCTCGCGCCTGTTCTCG GTGCAGGGTGGTGGGGGATCTGAGGCTGCG GCTAAAGAGGCGGCAGCAAAAGGAGGAGGC GGAAGCATGGCACACTATCCACCTTCCAAG GATCAA TTGAATGAATTGATCCAGGAAGTTAACCAATGGGCT ATCACTAATGGATTATCCATGTATCCTCCTAAATTCG AGGAGAACCCATCAAATGCATCGGTGTCACCAGTA ACTATCTATCCAACCCCAATTCCTAGGAAATGTTTTG ATGAGGCCGTTCAAATACAACCGGTATTCAATGAAT TATACGCCCGTATTACCCAAGATATGGCCCAACCTG ATTCTTATTTACATAAAACAACTGAAGCGTTAGCTCT ATCAGATTCCGAGTTTACTGGAAAACTGTGGTCTCT ATACCTTGCTACCTTAAAATCTGCACAGTACAAAAA GCAGAATTTTAGGCTAGGTATATTTAGATCACAGTAT TTGATTGATAAGAAAAAGGGTACTGAACAGATTAA GCAAGTCCTGTTTAGTACATTCTCAGTGGAATTTG CAGGCCTTAGCGAGAAAGTTGATAGATTGCACTCTT ATTTAAATAGGGCAAACAAGTACGATCCTAAAGGA CCAATTTATAATGATCAAAATATGGTCATTTCTGATT CAGGATACCTTTTGTCTAAGGCATTGGCCAAAGCTG TGGAATCGTATAAGTCACAACAAAGTTCTTCTACAA CTAGTGATCCTATTGTCGCATTCATTGTGCAAAGAA ACATCAGACATGTGTTTACTCAAAAGGTCTTGGAA TTGAATCTGTTGGAAAAATTCGGTACCAAATCTGTT AGGTTGACGTTTGATGATGTTAACGATAAATTGTTC ATTGATGATAAAACGGGAAAGCTTTTCATTAGGGAC ACAGAGCAGGAAATAGCGGTGGTTTATTACGTAAC GGGTTACACAACCACTGATTACACGTCCGAAAAGG ACTGGGAGGCAAGACTATTCCTCGAAAAAAGTTTC GCAATAAAGGCCCCAGATTTACTCACTCAATTATCT GGCTCCAAGAAAATTCAGCAATTGTTGACAGATGA GGGCGTATTAGGTAAATACATCTCCGATGCTGAGAA AAAGAGTAGTTTGTTAAAAACTTTTGTCAAAATATA TCCCTTGGATGATACGAAGCTTGGCAGGGAAGGCA AGAGGCTGGCATTAAGTGAGCCCTCTAAATACGTGT TAAAACCACAGCGGGAAAATGGCGGAAACAATGT TTATAAAGAAAATATTCCTAATTTTTTGAAAGGTATC GAAGAACGTCACTGGGATGCATATATTCTCATGGAG TTGATTGAACCAGAGTTGAATGAAAATAATATTATAT TACGTGATAACAAATCTTACAACGAACCAATCATCA GTGAACTAGGAGATTATGGTTGCGTTCTATTTAACG ACGAGCAAGTTTTATCGAACGAATTTAGTGGCTCAT TACTAAGATCCAAATTTAATACTTCAAATGAAGGTG GAGTGGCGGCAGGATTCGGATGTTTGGACAGTATTA TTCTTTACTAG PPK SEQID ATGCCAATGGTTGCTGCAGTTGAGTTCGCCAAAAG No.15 TCCGGCCGAAGTACTGAGGGTTGGATCGGGGTTTT CGCTGGCAGGCGTGGATCCCGAATCCACACCCGGC TACACCGGTGTGAAAGCTGATGGCAAGGCGTTGCT TGCCGCGCAGGACGCGCGGCTGGCGGAACTGCAG GAAAAGCTCTTCGCCGAAGGAAAGTTCGGCAACC CCAAACGGCTCCTGCTCATCCTTCAGGCCATGGATA CTGCGGGCAAGGGCGGCATTGTCAGCCACGTTGTT GGCGCCATGGACCCGCAAGGCGTACAACTGACCGC CTTCAAAGCGCCTACGGACGAGGAAAAGTCGCAC GACTTCCTCTGGAGAATCGAAAAACAAGTCCCTGC CGCCGGAATGGTGGGGGTGTTCGACCGCTCGCAGT ACGAAGACGTGCTGATCCACCGTGTCCATGGCTGG GCGGACGCTGCCGAACTGGAGCGCCGGTACGCCGC GATCAACGACTTTGAGTCACGCCTGACCGAGCAGG GAACCACCATCGTCAAGGTCATGCTCAATATCAGCA AGGACGAACAGAAAAAGCGTTTGATCGCCCGGTTG GACGATCCCAGCAAGCACTGGAAATACAGTCGCGG CGACCTCGCGGAACGTGCGTACTGGGATGACTACA TGGACGCCTACAGCGTTGCCTTCGAGAAGACCTCC ACAGAGATTGCTCCTTGGCACGTGGTGCCGGCAAA CAAGAAGTGGTACGCACGCATCGCAGTCCAGCAAC TGCTGCTGGATGCCCTCGGCGGTTTGCAGCTGGAC TGGCCCAAAGCTGACTTCGATGTCGCCGCTGAGCG CGCCCTCGTGGTGGAATCCTAG ADK SEQID ATGCGTATCATTCTGCTTGGCGCTCCGGGCGCGGGG No.16 AAAGGGACTCAGGCTCAGTTCATCATGGAGAAATA TGGTATTCCGCAAATCTCCACTGGCGATATGCTGCG TGCTGCGGTCAAATCTGGCTCCGAGCTGGGTAAAC AAGCAAAAGACATTATGGATGCTGGCAAACTGGTC ACCGACGAACTGGTGATCGCGCTGGTTAAAGAGCG CATTGCTCAGGAAGACTGCCGTAATGGTTTCCTGTT GGACGGCTTCCCGCGTACCATTCCGCAGGCAGACG CGATGAAAGAAGCGGGCATCAATGTTGATTACGTTC TGGAATTCGACGTACCGGACGAACTGATCGTTGAC CGTATCGTCGGTCGCCGCGTTCATGCGCCGTCTGGT CGTGTTTATCACGTTAAATTCAATCCGCCGAAAGTA GAAGGCAAAGACGACGTTACCGGTGAAGAACTGA CTACCCGTAAAGATGATCAGGAAGAGACCGTACGT AAACGTCTGGTTGAATACCATCAGATGACAGCACC GCTGATCGGCTACTACTCCAAAGAAGCAGAAGCGG GTAATACCAAATACGCGAAAGTTGACGGCACCAAG CCGGTTGCTGAAGTTCGCGCTGATCTGGAAAAAAT CCTCGGCTAA

[0038] The present disclosure is further illustrated below in conjunction with examples.

Example 1: Preparation of Prezatide by Catalysis (Combination of GHS, HKS, PPK and ADK)

[0039] The gene fragments of ADK, gshB and PPK were amplified with chromosomes of Escherichia coli K12, Saccharomyces cerevisiae (ATCC 204508) and Paenarthrobacter aurescens TC1 purchased from ATCC as templates by PCR using the above corresponding primers, subjected to enzyme digestion using the Nde I/Xho I purchased from NEB Company, and connected to a pET28a plasmid (purchased from Addgene) digested with the same enzyme. Then the plasmid was transformed into E. coli DH5a cells (purchased from Tsingke Biotechnology), and verified by colony PCR and gene sequencing. Lal gene fragment was synthesized by Anhui General Biology Co., Ltd., and subcloned into a pET28a plasmid. Then multi-site mutant enzyme genes GHS and HKS were constructed with Lal and gshB genes as templates using the mutation primers in Table 2 (by conventional PCR amplification). The above GHS, HKS, PPK and ADK plasmids constructed with the pET-28a vector were transferred into E. coli BL21 (DE3) (purchased from Anhui General Biology Co., Ltd.) strains, which were then cultured in a small-scale in 5 ml of LB culture medium containing 50 M Kanamycin at 37 C. When the cells grew to an OD value of 0.5-0.8, 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) was added to induce protein expression at 37 C. for 3 h. Finally the cells were collected, disrupted by freeze-thaw method, and centrifuged at high speed, and the collected supernatant was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the protein expression. The strains with correct protein expression were cultured step by step in a 5 liter fermenter to be induced for expression under the condition of 1.0 mM IPTG at 37 C. for 4 h, and wet cells were collected to be 35-55 g. Then after the cells and an appropriate amount of Tris.Math.HCl buffer solution (25 mM, pH=8.0) were mixed evenly, the cells were crushed with a high-pressure crusher at low temperature and centrifuged at high speed to remove the cell wall to obtain an enzyme solution, which was stored in a refrigerator at 4 C. for later use. The LB culture medium was composed of 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate and 5% glycerol.

[0040] 1 L of 100 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris.Math.HCl) solution at pH 8.0 was added with 15 g of glycine (200 mM), 31 g of L-histidine (200 mM), 29.2 g of L-lysine (200 mM), 5.6 g of adenosine disodium triphosphate (10 mM) providing ATP, 1.8 g of magnesium chloride (20 mM), 3.75 g of potassium chloride (20 mM) and 51.6 g of polyphosphoric acid (Sigma, 25 poly, 500 mM monophosphoric acid). After adjusting pH to 8.0, 1000 U of GHS enzyme solution, 1500 U of HKS enzyme solution, 2000 U of PPK and 1200 U of ADK enzyme solution were added. The reaction system was stirred slowly at room temperature for 6 h. During the reaction, the system was maintained at pH of 6.5-9.0 by adding aqueous solutions of HCl and NaOH. Then it was detected that most of the raw materials were consumed by detecting the residual raw material histidine in the reaction solution using L-histidine detection kit of ProFoldin. Finally, the pH of the reaction solution was adjusted to terminate the reaction and precipitate the protein in the reaction solution by adding acid to adjust the solution to pH of 1.5 and stirring rapidly. The protein precipitate was removed by filtration, and then the solution was adjusted back to pH 7.0. The salt was removed by reverse osmosis. The impurities containing phosphoric acid were removed by D201 anion exchange resin, where the deionized water was used as eluent, prezatide GHK was directly eluted out due to its weak binding ability to resin. After lyophilization, the crude product of glycine-L-histidine-L-lysine was crystallized with pure water and ethanol of 1:(1-3) v/v to obtain 59 g of grey-white solid with a yield of 87% and a purity of 96.0%. The nuclear magnetic spectrum of the purified prezatide with 600 M Varian in D.sub.2O solution is shown in FIG. 3, where the upper panel shows .sup.1H-NMR, and the lower panel shows .sup.13C-NMR.

Example 2: Preparation of Prezatide by Catalysis (Combination of GHKS-1, PPK and ADK)

[0041] The gene fragment GHKS-1 of prezatide GHK synthetase was synthesized by Anhui General Biology Co., Ltd., and subcloned into a pET28a plasmid. Similar to Example 1, the plasmid was transformed into E. coli BL21 (DE3) strain for protein expression in a small amount, and then amplified in a 5 L fermenter for fermentation, and wet cells were collected to be about 40 g. The enzyme solutions of polyphosphate kinase PPK and adenylate kinase ADK prepared in Example 1 were directly used for the subsequent preparation reactions.

[0042] Under the similar reaction conditions to those in Example 1, 1 L of 100 mM tris(hydroxymethyl)aminomethane hydrochloride solution at pH 8.0 was added with 11.2 g of glycine (150 mM), 23.2 g of L-histidine (150 mM), 21.9 g of L-Lysine (150 mM), 5.6 g of adenosine disodium triphosphate (10 mM) providing ATP, 1.8 g of magnesium chloride (20 mM), 3.75 g of potassium chloride (20 mM) and 20.6 g of polyphosphoric acid (200 mM monophosphoric acid). After adjusting pH to 8.0, 2000 U of GHKS-1 enzyme solution, 1500 U of PPK and 1000 U of ADK enzyme solution were added. The reaction was performed for 10 h at room temperature, and it was detected that L-histidine in the reaction solution was completely consumed. Finally, HCl solution was added to the reaction to adjust pH to 1.5 to terminate the reaction and precipitate the protein. The protein precipitate was removed by filtration. Then the supernatant was adjusted back to pH 7.0, and the salt was removed by reverse osmosis. Finally the impurities containing phosphoric acid in the solution were removed by an anion exchange column. The eluent was concentrated and purified by crystallization with ethanol and water to obtain 31.6 g of pure prezatide with a yield of 62% and a purity of 91.2%. The nuclear magnetic spectrum of the purified prezatide with 600 M Varian in D.sub.2O solution is the same as in Example 1.

Example 3: Preparation of Prezatide by Catalysis (Combination of GHKS-1, PPK and ADK)

[0043] Similar to Example 2, the gene fragment GHKS-2 of prezatide GHK synthetase was synthesized by Anhui General Biology Co., Ltd., and subcloned into a pET28a plasmid. After the protein was verified through expression in a small amount, the preparation was directly amplified, and the overexpressed cell lysate was stored at 4 C. for later use. The enzyme solutions of polyphosphate kinase PPK and adenylate kinase ADK prepared in Example 1 can be directly used in this enzymatic reaction.

[0044] Similar to Example 2, 1 L of 100 mM tris(hydroxymethyl)aminomethane hydrochloride solution at pH 8.0 was added with 15 g of glycine (200 mM), 31 g of L-histidine (200 mM), 29.2 g of L-Lysine (200 mM), 5.6 g of adenosine disodium triphosphate ATP (10 mM), 1.8 g of magnesium chloride (20 mM), 3.75 g of potassium chloride (20 mM) and 51.6 g of polyphosphoric acid (500 mM monophosphoric acid). After adjusting pH to 8.0, 2000 U of GHKS-2 enzyme solution, 2000 U of PPK and 1500 U of ADK enzyme solution were added. The reaction system was stirred at room temperature for 7 h while maintaining the pH value of the reaction system at 6.5-9.0. Then it was detected that most of the raw material histidine was converted completely. HCl aqueous solution was added to terminate the reaction and precipitate the protein by denaturation. Similar to the above, the salt was finally removed, and the impurities containing phosphoric acid in the reaction was removed with an anion exchange column. The crude solution of prezatide was concentrated and crystallized to finally obtain 61.8 g of grey-white solid with a yield of 91% and a purity of 94.5%. The nuclear magnetic spectrum of the purified prezatide with 600 M Varian in D.sub.2O solution is the same as in Example 1.

[0045] The above are only preferred embodiments of the present disclosure, and it should be noted that for those of ordinary skill in the art, several improvements and modifications can also be made without departing from the principle of the present disclosure, and these improvements and modifications should also be considered as the protection scope of the present disclosure.