Preparation Method of Mutant Composition, Fusion Enzyme and Collagen Tripeptide

Abstract

The present invention relates to the field of bioengineering, and particularly to mutant compositions, fusion enzymes and methods for preparing collagen tripeptides. The present invention provides mutant compositions, including Mutant 1 and Mutant 2; Mutant 1 has an amino acid sequence as shown in SEQ ID NO: 1 and Mutant 2 has an amino acid sequence as shown in SEQ ID NO: 2. The preparation method provided by the present invention uses inexpensive amino acids as raw materials, and is highly efficiently converted into corresponding dipeptide or tripeptide products under the action of adenosine triphosphate (ATP) and the corresponding ligase. Introducing an ATP recycling system into the reaction system can greatly reduce its usage amount; at the same time, the amount of enzyme used in the actual production process is reduced by constructing fusion enzymes; the reaction and purification processes are further optimized by using immobilized enzyme catalysis, and the preparation scheme is very concise, with high yield, good product quality and easy to scale up.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to methods for preparing a mutant composition, a fusion enzyme and a collagen tripeptide. Background Art

[0002] Collagen is the main structural protein in the extracellular matrix found in various connective tissues of the human body. As the main component of connective tissue, it is the most abundant protein in mammals. Collagen is now widely used in cosmetic surgery, bone transplantation, tissue regeneration, etc. The share of collagen in the international beauty dietary supplement market has increased from 1% in 2014 to 9% in 2019.

[0003] The main active ingredient of collagen is collagen tripeptide (Gly-Pro-Hyp). Collagen tripeptide has a small molecular weight (the average molecular weight is about 280), so it is easily absorbed by the human body. At the same time, collagen tripeptide can also penetrate the stratum corneum and dermis very effectively, so it is widely used in skin moisturizing, anti-wrinkle, repair, breast augmentation and other materials.

[0004] The traditional preparation method of collagen tripeptide includes: (1) extraction of biological raw materials and degradation by corresponding hydrolase, which is a common method for collagen tripeptide sold on the market. Since animal skin (such as pig skin, fish skin, fish scales) contains a large amount of collagen, crude collagen can be obtained by processing and extracting these raw materials, and then it can be cut into a polypeptide mixture rich in collagen tripeptide using specific proteolytic enzymes. In addition, the source of collagen can also be obtained by fermentation of engineered bacteria, and then collagen tripeptide is prepared by extraction, purification and precise enzymatic hydrolysis of human-like collagen.

[0005] (2) Chemical synthesis: Similar to other peptide preparation methods, collagen tripeptide can also be prepared by chemical synthesis using amino acids as raw materials. In this preparation process, it is first necessary to protect the amino acid functional groups that are not desired to participate in the reaction with protective groups, then chemically couple the two molecules in sequence, and finally deprotect them. The chemical preparation reaction has many steps and the overall yield is low. At the same time, racemates will inevitably be produced during the chemical coupling process. Since the racemate has very similar properties to the product, it cannot be effectively separated, which greatly increases the purification cost.

[0006] At present, the main production process of collagen is to extract crude collagen from animal skins and then perform corresponding enzymatic cutting. Although this method is simple and cheap, it is inevitable that allergens will be mixed in the animal raw materials, which will affect its subsequent use; at the same time, this method obtains a mixture of various short peptides, and the content of the tripeptide Gly-Pro-Hyp with actual activity is too low. Therefore, in some special application scenarios (such as medical beauty) that require the addition of highly active tripeptides, the collagen tripeptide prepared by this method cannot meet the requirements. The chemical synthesis of pure collagen tripeptide is complex, environmentally polluting, and too costly. Summary of the invention

[0007] In view of this, the present invention provides a mutant composition, a fusion enzyme and a preparation method of collagen tripeptide. The preparation method provided by the present invention uses cheap amino acids as raw materials, and converts them into corresponding dipeptide or tripeptide products with high yield under the action of adenosine triphosphate (ATP) and corresponding ligase. In order to further save costs, the introduction of an ATP recycling regeneration system in the reaction system can greatly reduce its usage; at the same time, by constructing a fusion enzyme (GPHSynAB), the amount of enzyme used in the actual production process can be reduced; using immobilized enzyme catalysis can further optimize the reaction and purification process, so in general, the preparation scheme is very streamlined, high in yield, good in product quality, and easy to scale.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a mutant composition, including mutant 1 and mutant 2;

[0010] The mutant 1 has:

[0011] (1), the amino acid sequence shown in SEQ ID NO: 1; or

[0012] (2) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or

[0013] (3) a sequence having a homology of more than 90% with the amino acid sequence shown in (1); and

[0014] The mutant 2 has:

[0015] (4) the amino acid sequence shown in SEQ ID NO: 2; or

[0016] (5) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (4); or

[0017] (6) A sequence having a homology of more than 90% with the amino acid sequence shown in (4).

[0018] In some embodiments of the present invention, the mutant 1 in the above-mentioned mutant composition is a ligase (GPSynA): the mother plate is derived from Salmonella enterica (Uniprot ID: A0A756LAZ2), and is mutated at the following sites: S11L, L81M, S83A, L85F, D238T, M241I, Q242E, G287Y, T289A, I291Q, L337W.

[0019] In some embodiments of the present invention, the mutant 2 in the above mutant composition is a ligase (GPHSynB): the mother plate is derived from Torulaspora delbrueckii (Uniprot ID: G9A034), and is mutated at the following sites: D126Q, E140V, N142E, I144L, S147N, E218Y, N220E, D223S, R275Q, G377I, I435L.

[0020] In some embodiments of the present invention, the sequence of SEQ ID NO: 1 in the mutant composition is: MKKYIVVVEALFLGVEYVAKAIRKIGYEPVFLTNYRSQEGDALVQLATERAIFCETTETKDIISVIDLLGRSNIVGVTTFMDARFSIIASVNKLLGLPGVSESLLKLKDKSYVNGIIPEFVPKSLAVEWGKTPKDKIEDFLLNSDANKYIAKPSFTAGAIGTFVFKKYEELSNGIDSSVDKIPKHLEPNKYVVQEFFDGNLV SIEGYTYKDRIEFIGATLRYKFDNTEVKHKFPYQDTMGIENYEKCKSIICKLINRSNYEYGFFHVEFMVSDNEVRLIDANMGRPYGANQMELISFSYEIDPVLMFEHAISIAVFQKPTIDSQFFIDTPCVEMTGVWYGQKETARLLRFESPTLKSTHHLAVNMGTTVPKLGESNWSVIGTLTGRPNDVFTDLKNIKLITDKGVCSAVISE.

[0021] In some embodiments of the present invention, the sequence of SEQ ID NO: 2 of the mutant composition is: MVSAYPQLSERARDAILPEVHQWALTNGLIMYPRNFTSEQATIAPTTLYPTLLPRSSVESVVSLQKAYNELYARIIRGENDDWLAKETVKLASYDTEFTGKLWSLYLKTKELGTTQNLRLGIFRSQYLIDKKNSQAKQVVFETLSVNFGGSSTKTGELHNYLNNSGKYCPDSGMPFYQTQIPVSKSSSLLAKGIAEAVNYYQGLNDERIVAFIVQENYREAFSQRIIEYALLQNHG IKSVRVTLGDVPHLTVVEGKSKRLFYKKTGQEIAVVYYQAGYSPTEYKDEKDWDSRLLLETSYAIKAPDLLTQLSGTKKIQQLLTNENILTRFVPDSDTRSKLISTFVEIYPLDDSPLGQEGK KLAFESPSKFVLKPQREIGGNNIYKENIPSFLKEIDEKDWSAYILMELIQPFETTENVVIRGNESFNEPITSELGLFGCILFDDSKIHFNEYSGWLLRSKFSRSDEGGVAAGFGCVDSFVLY.

[0022] The present invention also provides a fusion enzyme, comprising the mutant composition and a linker.

[0023] In some embodiments of the present invention, the fusion enzyme is a ligase (GPHSynAB): the GPSynA enzyme and GPHSynB are fused and expressed at the gene level, and the amino acid peptide chain connecting the two enzymes is: GGGGSLVPRGSGGGGS.

[0024] In some embodiments of the present invention, the Linker of the above fusion enzyme has:

[0025] (7), the amino acid sequence shown in SEQ ID NO: 3; or

[0026] (8) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (7); or

[0027] (9) A sequence having a homology of more than 90% with the amino acid sequence shown in (7).

[0028] In some embodiments of the present invention, the sequence of SEQ ID NO: 3 in the fusion enzyme is: GGGGSLVPRGSGGGGS.

[0029] In some embodiments of the present invention, the above fusion enzyme has:

[0030] (10), the amino acid sequence shown in SEQ ID NO: 4; or

[0031] (11) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (10); or

[0032] (12) A sequence having a homology of more than 90% with the amino acid sequence shown in (10).

[0033] In some embodiments of the present invention, the sequence of the above SEQ ID NO:4 in the above fusion enzyme is:.

[0034] The present invention also provides a nucleic acid molecule encoding the mutant composition.

[0035] The nucleic acid molecule encoding the mutant 1 has:

[0036] (13), the nucleotide sequence shown in SEQ ID NO: 5; or

[0037] (14) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (13), and having the same or similar function as the nucleotide sequence shown in (13); or

[0038] (15) a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (13) or (14); and

[0039] The nucleic acid molecule encoding the mutant 2 has:

[0040] (16), the nucleotide sequence shown in SEQ ID NO: 6; or

[0041] (17) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (16), and having the same or similar function as the nucleotide sequence shown in (16); or

[0042] (18) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (16) or (17).

[0043]

[0044]

[0045] The present invention also provides a nucleic acid molecule encoding the above fusion enzyme having:

[0046] (19), the nucleotide sequence shown in SEQ ID NO:7; or

[0047] (20) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (19), and having the same or similar function as the nucleotide sequence shown in (19); or

[0048] (21) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (19) or (20).

[0049]

[0050] The present invention also provides an expression vector, comprising the above nucleic acid molecule.

[0051] The present invention also provides a host for transforming and / or transfecting the above expression vector.

[0052] The present invention also provides a method for preparing collagen tripeptide, which uses proline, glycine and hydroxyproline as raw materials and is catalyzed by the mutant composition and / or the fusion protein to prepare the collagen tripeptide.

[0053] In some embodiments of the present invention, in the above preparation method, the raw materials also include sodium hexaphosphate and polyphosphate kinase.

[0054] In some embodiments of the present invention, the polyphosphate kinase PPK in the above preparation method is derived from Deinococcus radiodurans, Uniprot ID: Q9RY20.

[0055] In some embodiments of the present invention, the polyphosphate kinase in the above preparation method has an amino acid sequence as shown in SEQ ID NO: 8; the sequence of SEQ ID NO: 8 is MDIDNYRVKPGKRVKLSDWATNDDAGL SKEEGQAQTAKLAGELAEWQERLYAEGKQSLLLILQARDAAGKDGAVKKVIGAFNPAGVQITSFKQPSAEELSHDFLWRIHQKAPAKGYVGVFNRSQYEDVLVTRVYDMIDDKTAKRRLEHIRHFEELLTDNATRIVKVYLHISPEEQKERLQARLDNPGKHWKFNPGDLKDRSNWDKFNDVYEDALTTSTDDAPWYVVPADRKWYRDLVLSHILLGALKDMNPQFPAIDYDPSKVVIH.

[0056] In some embodiments of the present invention, the nucleic acid molecule encoding the polyphosphate kinase in the above preparation method has a nucleotide sequence as shown in SEQ ID NO: 9; the sequence of SEQ ID NO: 9 is.

[0057] In some embodiments of the present invention, in the above preparation method, the catalysis time is 2 hours or 4 hours.

[0058] In some embodiments of the present invention, in the above preparation method, after catalysis, the steps of denaturation and centrifugation are further included.

[0059] The present invention also provides the use of the above mutant composition, the above fusion protein, the above expression vector, and the above host in directly or indirectly preparing collagen tripeptide and / or products containing collagen tripeptide.

[0060] The present invention provides a mutant composition, including mutant 1 and mutant 2;

[0061] The mutant 1 has:

[0062] (1) The amino acid sequence shown in SEQ ID NO: 1; or

[0063] (2) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in (1); or

[0064] (3) A sequence having a homology of more than 90% with the amino acid sequence shown in (1); and

[0065] The mutant 2 has:

[0066] (4) The amino acid sequence shown in SEQ ID NO: 2; or

[0067] (5) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in (4); or

[0068] (6) A sequence having a homology of more than 90% with the amino acid sequence shown in (4).

[0069] The beneficial effects of the present invention include:

[0070] (1) The preparation method provided by the present invention uses inexpensive amino acids as raw materials and is highly efficiently converted into corresponding dipeptide or tripeptide products under the action of adenosine triphosphate (ATP) and the corresponding ligase. To further save costs, an ATP recycling system is introduced into the reaction system, which can greatly reduce its usage amount; at the same time, by constructing a fusion enzyme (GPHSynAB), the amount of enzyme used in the actual production process can be reduced; using immobilized enzyme catalysis can further optimize the reaction and purification processes. Therefore, generally speaking, this preparation scheme is very concise, has a high yield, good product quality, and is easy to scale up.

[0071] (2) The present invention uses two amino acid ligases to sequentially link L-glycine, L-proline, and hydroxyproline to generate collagen tripeptide with a high conversion rate. This process uses bulk and inexpensive amino acids as the initial raw materials and can rapidly produce the product through mild and simple enzymatic reactions. Compared with the common processes of purifying and extracting from animal skins and enzymatic cleavage on the market, the large-scale production process of this patent is stable, the product quality is also stable, with high purity and few by-products; at the same time, this method is also significantly superior to chemical synthesis methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art.

[0073] Figure 1 Showing the preparation route of the amino acid ligase method of the present invention;

[0074] Figure 2 Showing the preparation route of the dipeptide glycine-proline;

[0075] Figure 3 Showing the route of the GPHSynB enzyme to prepare collagen tripeptide;

[0076] Figure 4 Showing the preparation route of the dipeptide glycine-proline;

[0077] Figure 5 Showing the liquid phase chromatogram of Example 1; wherein: the upper figure shows the reaction of the GPSynA crude enzyme solution to prepare Gly-Pro for 0 h; the lower figure shows the reaction of the GPSynA crude enzyme solution to prepare Gly-Pro for 3 h;

[0078] Figure 6 Showing the route of the GPHSynB enzyme to prepare collagen tripeptide;

[0079] Figure 7 Showing the liquid phase chromatogram of Example 2; wherein: the upper figure shows the reaction of the GPHSynB crude enzyme solution to prepare Gly-Pro-Hyp for 0 h; the lower figure shows the reaction of the GPHSynB crude enzyme solution to prepare Gly-Pro-Hyp for 2 h;

[0080] Figure 8 Showing the NMR spectrum of H-Gly-Pro-Hyp-OH;

[0081] Fig. 9 Showing the control, the standard NMR spectrum (citing Patent CN 114901670 A);

[0082] Fig.10 Showing the route of the fusion enzyme solution to prepare tripeptide;

[0083] Fig.11Liquid chromatogram of Example 3; wherein: the upper figure shows the reaction of preparing Gly-Pro-Hyp from the crude enzyme solution of GPHSynAB for 0 h; the lower figure shows the reaction of preparing Gly-Pro-Hyp from the crude enzyme solution of GPHSynAB for 4 h;

[0084] Fig.12 Shows the route for preparing tripeptide by adding an ATP regeneration system to the fusion enzyme solution

[0085] Fig.13 Liquid chromatogram of Example 4; wherein: the upper figure shows the reaction of preparing Gly-Pro-Hyp from the crude enzyme solution of GPHSynAB - ATP regeneration for 0 h; the lower figure shows the reaction of preparing Gly-Pro-Hyp from the crude enzyme solution of GPHSynAB - ATP regeneration for 4 h;

[0086] Fig.14 Shows the route for preparing tripeptide by adding an ATP regeneration system to the immobilized fusion enzyme

[0087] Fig.15 Liquid chromatogram of Example 5; wherein: the upper figure shows the reaction of preparing Gly-Pro-Hyp from the immobilized enzyme for 0 h; the lower figure shows the reaction of preparing Gly-Pro-Hyp from the immobilized enzyme for 2 h;

[0088] Fig.16 Gel electrophoresis pattern of the protein in the present invention; wherein: from left to right are: PPK, GPSynA, GPHSynB, GPHSynAB. Detailed implementation mode

[0089] The present invention discloses a mutant composition, a fusion enzyme and a preparation method of collagen tripeptide.

[0090] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0091] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open and non-restrictive, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0092] It should be understood that as long as the present invention is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0093] The use of any and all examples or exemplary language such as "for example" or "including" in this document is merely intended to better illustrate the invention and does not limit the scope of the invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0094] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0095] The preparation route of the amino acid ligase method of the present invention is as Figure 1 shown.

[0096] In the present invention, the GPSynA enzyme can first link glycine and proline in an aqueous solution to generate the Gly-Pro dipeptide, and then the GPHSynB enzyme can be used to link the Gly-Pro dipeptide and hydroxyproline to obtain the GPH tripeptide; alternatively, the fusion enzyme GPHSynAB can be used to link the three amino acid raw materials into a collagen tripeptide at one time. The ATP in the reaction process can be equimolar, or the catalytic amount can be used in the way of ATP regeneration. The reaction can use the crude enzyme solution obtained by cell disruption or immobilized enzyme.

[0097] In Examples 1 to 5 and the effect examples of the present invention, the required enzymes were all prepared by synthesizing the corresponding genes by the company, constructing them on a specific expression plasmid, and then fermenting and producing them by Escherichia coli.

[0098] Regarding the fermentation production of enzymes in Examples 1 to 5 and the effect examples of the present invention:

[0099] Specifically, it includes the following steps: After optimizing the sequences of the genes corresponding to the enzymes in Examples 1 to 5, place an order for synthesis with General Biosystems (Chuzhou, Anhui). Then introduce NdeI / XhoI restriction enzyme sites and subclone them onto the pET 28a expression vector. Transfer the plasmid with correct sequence into E. coli (BL21) competent cells for plate culture (GenScript) and monoclonal small-scale liquid culture. The bacteria with correct protein expression are finally subjected to stepwise scaled-up liquid culture. Specifically, transfer a single colony into 5 mL of LB culture medium containing 50 μM kanamycin (at 37 °C) for culture. When the cells grow to the logarithmic phase, inoculate them into 250 mL of LB culture medium containing the same antibiotic. When they grow to the logarithmic phase again, transfer them into a 5 L culture fermenter for culture and final protein expression. During the 5 L fermenter culture, when the cell OD reaches ~20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25 °C to induce protein expression for 6 hours. Finally, collect the cells by high-speed centrifugation (4000 rpm, 20 min) to obtain 25 - 50 g of wet cells with overexpressed enzyme. Take a small amount of cells and first mix them evenly with Tris-HCl buffer (50 mM, pH 8.0) on an ice bath. Then use the freeze-thaw method to break the cells, remove the cell walls by high-speed centrifugation, and run SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on the supernatant to determine protein expression. The bacterial cells with correct protein expression are used for the next catalytic experiment. Specifically, mix the remaining cells evenly with Tris-HCl buffer (50 mM, pH 8.0) at low temperature (mixing ~10 g of wet cells: 200 mL of buffer), then break the cell walls by low-temperature high-pressure, and remove the cell walls by high-speed centrifugation (16,000 rpm, 45 min) to obtain the enzyme-containing supernatant for standby (the obtained enzyme activity is 250 - 500 U / mL, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature). The composition of the LB medium is: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 5% glycerol.

[0100] In Examples 1 to 5 and the effect examples of the present invention regarding the immobilization of the enzyme:

[0101] The specific steps are as follows: Take the crude enzyme supernatant collected in the fermentation production of the enzyme, slowly add solid ammonium sulfate until protein solids precipitate (20 - 50% w / v ammonium sulfate: buffer), collect the protein solids by high-speed centrifugation (10000 rpm, 10 min), and then slowly dissolve them in 25 mM Tris buffer at pH 8.0 (buffer A). Then dialyze in 50 volumes of buffer A (twice, with a 4-hour interval each time) to remove ammonium sulfate from the enzyme solution. Finally, load the dialysate onto a DEAE Seplite FF (Xi'an LX Biotechnology Co., Ltd.) anion exchange column (NaCl gradient elution in buffer: 0 - 1 N NaCl) to obtain the preliminarily purified GPHSynAB and PPK enzyme solutions; then, GPHSynAB and PPK enzymes are immobilized in one step using LX-1000EP epoxy resin (Xi'an LX Biotechnology Co., Ltd.) at an activity unit ratio of 1:(2 - 3) as follows: Dissolve 1500 U of the purified mixed enzymes in 1 L of 50 mM potassium phosphate buffer at pH 8.0 (buffer B), then add 30 - 50 mM phenoxyacetic acid and 500 g of LX-1000EP epoxy resin. Stir at room temperature for 10 hours, then filter out the immobilized enzyme. Finally, wash it twice with water and buffer B each, and store it at low temperature for later use. The immobilized enzyme has 70 - 92% of the initial activity.

[0102] In Examples 1 - 5 and the Effect Example of the present invention, the raw materials and reagents used can be purchased from the market.

[0103] The present invention will be further described below in conjunction with the examples:

[0104] Example 1 Preparation of dipeptide glycine-proline Gly-Pro and collagen tripeptide Gly-Pro-Hyp using crude enzyme solutions of wild ligase (GPSynA0) and wild ligase (GPHSynB0)

[0105] (1) The preparation route is as Figure 2As shown, 12.4 g of L-glycine (165 mM), 17.3 g of L-proline (150 mM), and 83.0 g of adenosine 5'-triphosphate monosodium salt (ATP, 157 mM) were added to 1 L of 100 mM Tris-HCl solution at pH 8.0. Then, after adjusting the pH of the reaction system to 8.0 with an aqueous NaOH solution, 3000 U of ligase GPSynA was added to initiate the reaction. The reaction was gently stirred at 30 °C while maintaining the pH of the reaction system between 7.0 and 9.0. After 3 hours, the target product was detected by HPLC, but the conversion rate of the raw materials was relatively low. Then, the pH was adjusted to 1.0 with an aqueous HCl solution to denature and precipitate the enzyme in the reaction system, which was removed by centrifugation. After adjusting the pH of the reaction solution to 7.0, it was directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: water, 2:1, v:v) to obtain approximately 11.7 - 13.9 g of L-glycine-L-proline (Gly-Pro) dipeptide (yield 45% - 54%). Due to the small proportion of the product, crystallization and purification were very difficult.

[0106] (2) Similar to the preparation method of Gly-Pro in step (1) above, the preparation route is as Figure 3 As shown, 17.2 g of L-glycine-L-proline (100 mM), 14.4 g of trans-4-OH-L-proline (110 mM), and 55.5 g of adenosine 5'-triphosphate monosodium salt (ATP, 105 mM) were added to 1 L of 100 mM Tris-HCl solution at pH 7.5. Then, after adjusting the pH of the reaction system to 7.5 with an aqueous NaOH solution, 3000 U of ligase GPHSynB was added to initiate the reaction. The reaction was gently stirred at 30 °C while maintaining the pH of the reaction system between 7.0 and 9.0. After 2 hours, the target product was detected by HPLC. Then, the pH was adjusted to 1.0 with an aqueous HCl solution to denature and precipitate the enzyme in the reaction system, which was removed by centrifugation. After adjusting the pH of the reaction solution to 7.0, it was directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: water, 3:1, v:v) to obtain only 8.6 - 9.9 g of collagen tripeptide (yield only 30% - 35%). As described in the previous step, crystallization and purification in this step were even more difficult.

[0107] Example 2 Preparation of collagen tripeptide Gly-Pro-Hyp using crude enzyme solutions of mutant ligases (GPSynA) and mutant ligases (GPHSynB)

[0108] (1) The preparation route is as Figure 4As shown, 12.4 g of L-glycine (165 mM), 17.3 g of L-proline (150 mM), and 83.0 g of adenosine 5'-triphosphate monosodium salt (ATP, 157 mM) were added to 1 L of 100 mM Tris.HCl solution at pH 8.0. Then, after adjusting the pH of the reaction system to 8.0 with an aqueous NaOH solution, 2000 U of ligase GPSynA was added to initiate the reaction. The reaction was gently stirred at 30 °C while maintaining the pH of the reaction system between 7.0 and 9.0. After 3 hours, HPLC detection showed that the raw materials had basically reacted completely (as Figure 5 shown). Then, the pH was adjusted to 1.0 with an aqueous HCl solution to denature and precipitate the enzyme in the reaction system, which was removed by centrifugation. After adjusting the pH of the reaction solution to 7.0, it was directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:water, 2:1, v:v) to obtain 21.9 - 22.4 g of L-glycine-L-proline (Gly-Pro) dipeptide (yield 85% - 87%). The crystallized and purified product Gly-Pro was submitted for NMR inspection during the process, and the correct structure was confirmed: 1H NMR (400 MHz, D2O): δ = 4.54–4.62 (m, 1H), 3.50 (s, 2H), 3.40–3.70 (m, 2H), 1.87–2.35 (m, 4H).

[0109] (2) The preparation route is as Figure 6 shown, similar to the preparation method of Gly-Pro in step (1). 17.2 g of L-glycine-L-proline (100 mM), 14.4 g of trans-4-OH-L-proline (110 mM), and 55.5 g of adenosine 5'-triphosphate monosodium salt (ATP, 105 mM) were added to 1 L of 100 mM Tris.HCl solution at pH 7.5. Then, after adjusting the pH of the reaction system to 7.5 with an aqueous NaOH solution, 2000 U of ligase GPHSynB was added to initiate the reaction. The reaction was gently stirred at 30 °C while maintaining the pH of the reaction system between 7.0 and 9.0. After 2 hours, HPLC detection showed that the raw materials had basically reacted completely (as Figure 7 shown). Then, the pH was adjusted to 1.0 with an aqueous HCl solution to denature and precipitate the enzyme in the reaction system, which was removed by centrifugation. After adjusting the pH of the reaction solution to 7.0, it was directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol:water, 3:1, v:v) to obtain 23.1 - 24.2 g of collagen tripeptide (yield 81% - 85%). The crystallized and purified product Gly-Pro-Hyp was submitted for NMR inspection, and the NMR spectrum is as Figure 8 shown, and the chromatogram of the standard product is as Fig. 9 The correct structure was confirmed by comparison (as shown). 1H NMR (400 MHz, D2O) δ = 4.79 (m, 1H), 4.62 (m, 1H), 4.53 (t, J = 8.4 Hz, 1H), 3.99 (s, 2H), 3.92–3.69 (m, 2H), 3.57 (m, 2H), 2.36 (m, 2H), 2.15 (m, 1H), 1.98 (m, 3H).

[0110] Example 3: One-time preparation of collagen tripeptide Gly-Pro-Hyp using the crude enzyme solution of the fusion enzyme (GPHSynAB)

[0111] The preparation route is as Fig.10 shown. Similarly, 8.3 g of L-glycine (110 mM), 12.1 g of L-proline (105 mM), 13.1 g of trans-4-OH-L-proline (100 mM), and 111 g of adenosine monophosphate sodium salt (ATP, 210 mM) were added to 1 L of 100 mM Tris-HCl solution with pH 7.5. Then, after adjusting the pH of the reaction system to 7.5 with an aqueous NaOH solution, 2000 U of the ligase GPHSynAB was added to initiate the reaction. The reaction was gently stirred at 30 °C and the pH of the reaction system was maintained between 6.5 and 9.0. After 4 hours, it was detected by HPLC that the raw materials had basically reacted completely (as Fig.11 shown). Then, the pH was adjusted to 1.0 with an aqueous HCl solution to denature and precipitate the enzyme in the reaction system, which was removed by centrifugation. After adjusting the pH of the reaction solution to 7.0, it was directly loaded onto a D201 anion exchange resin to remove impurities such as adenosine diphosphate and free phosphoric acid. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: water, 3:1, v:v) to obtain 22–23 g of collagen tripeptide (yield 77%–80%).

[0112] Example 4: One-time preparation of collagen tripeptide Gly-Pro-Hyp using the crude enzyme solution of the fusion enzyme (GPHSynAB) and an ATP regeneration system

[0113] The preparation route is as Fig.12As shown, similar to Example 3, 8.3 g of L-glycine (110 mM), 12.1 g of L-proline (105 mM), 13.1 g of trans-4-OH-L-proline (100 mM), 5.4 g of adenosine monophosphate monosodium salt (ATP, 10 mM), and 32.2 g of sodium hexametaphosphate (52.6 mM) were added to 1 L of 100 mM pH 7.5 tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) solution. After adjusting the pH of the solution to 7.5, 3000 U of polyphosphate kinase PPK enzyme and 2000 U of ligase GPHSynAB were added. The reaction system was gently stirred at room temperature (25 °C) and the pH of the reaction system was maintained between 7.0 and 9.0. After 4 hours, it was detected by HPLC that the raw materials had basically reacted completely (as Fig.13 shown). Then, the pH was adjusted to 1.0 with an aqueous HCl solution to denature and precipitate the enzymes in the reaction system, which were removed by centrifugation. After adjusting the pH of the reaction solution to 7.0, it was directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated, and crystallized using a reverse osmosis membrane (ethanol: water, 3:1, v:v) to obtain 23.6 - 24.2 g of collagen tripeptide (yield 83% - 85%).

[0114] Example 5 Preparation of collagen tripeptide Gly-Pro-Hyp using immobilized enzymes in one step

[0115] The preparation route is as Fig.14 shown. The reaction is similar to Example 4. 4.2 g of L-glycine (55 mM), 5.7 g of L-proline (50 mM), 6.5 g of trans-4-OH-L-proline (50 mM), 5.4 g of adenosine monophosphate monosodium salt (ATP, 10 mM), and 16.1 g of sodium hexametaphosphate (26.3 mM) were added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) solution. After adjusting the pH of the solution to 8.0, 3000 U of the immobilized GPHSynAB / PPK mixture was added. The reaction system was gently stirred at 30 °C and the pH of the reaction system was maintained between 7.0 and 9.0. After 2 hours, it was detected by HPLC that the raw materials had basically reacted completely (as Fig.15Then directly filter and recover the immobilized GPHSynAB / PPK enzyme. The solid is rinsed three times with 25 mM Tris pH 8.0 buffer for standby. The reaction solution is adjusted to pH 1.0 with HCl aqueous solution to precipitate proteins and removed by centrifugation. Then the pH of the reaction solution is adjusted to 7.0 and directly loaded onto D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product is desalted, concentrated and crystallized (ethanol: water, 3:1, v:v) using a reverse osmosis membrane to obtain 12.8 - 13.5 g of collagen tripeptide (yield 89.6% - 94.5%). The recovered immobilized GPHSynAB / PPK enzyme has 88% of its initial activity.

[0116] Effect Example

[0117] Table 1 Some statistical data of preliminary experiments and examples

[0118]

[0119]

[0120] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Mutant composition, characterized in that, it is composed of mutant 1 and mutant 2; the amino acid sequence of the mutant 1 is shown as SEQ ID NO:1; the amino acid sequence of the mutant 2 is shown as SEQ ID NO:

2.

2. Fusion enzyme, characterized in that, its amino acid sequence is shown as SEQ ID NO:

4.

3. Nucleic acid molecule encoding the mutant composition according to claim 1, characterized in that, the nucleotide sequence of the nucleic acid molecule encoding the mutant 1 is shown as SEQ ID NO:5; and the nucleotide sequence of the nucleic acid molecule encoding the mutant 2 is shown as SEQ ID NO:

6.

4. Nucleic acid molecule encoding the fusion enzyme according to claim 2, characterized in that, its nucleotide sequence is shown as SEQ ID NO:

7.

5. Expression vector, characterized in that, it comprises the nucleic acid molecule according to claim 3 or 4.

6. Host, characterized in that, it is transformed and / or transfected with the expression vector according to claim 5.

7. Preparation method of collagen tripeptide, characterized in that, using proline, glycine and hydroxyproline as raw materials, and catalyzed by the mutant composition according to claim 1 and / or the fusion enzyme according to claim 2, the collagen tripeptide is prepared.

8. Use of the mutant composition according to claim 1, the fusion enzyme according to claim 2, the expression vector according to claim 5, and the host according to claim 6 in directly or indirectly preparing collagen tripeptide and / or products containing collagen tripeptide.

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