WO2022095590A1 - 一种突变酶及其应用和酶催化法制备三胜肽的工艺 - Google Patents

一种突变酶及其应用和酶催化法制备三胜肽的工艺 Download PDF

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WO2022095590A1
WO2022095590A1 PCT/CN2021/117378 CN2021117378W WO2022095590A1 WO 2022095590 A1 WO2022095590 A1 WO 2022095590A1 CN 2021117378 W CN2021117378 W CN 2021117378W WO 2022095590 A1 WO2022095590 A1 WO 2022095590A1
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enzyme
tripeptide
histidine
glycine
ligase
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French (fr)
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于铁妹
潘俊锋
刘建
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深圳瑞德林生物技术有限公司
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Priority to US18/035,736 priority Critical patent/US20230407286A1/en
Priority to EP21888289.2A priority patent/EP4242300A1/en
Publication of WO2022095590A1 publication Critical patent/WO2022095590A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02003—Glutathione synthase (6.3.2.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the technical field of biochemistry, in particular to a mutant enzyme and its application and a process for preparing tripeptide by an enzyme-catalyzed method.
  • Tripeptide is a natural tripeptide compound (Gly-L-His-L-Lys) composed of glycyl, L-histidyl and L-lysine, and its molecular formula is C 14 H 24 N 6 O 4 , the molecular weight is 340, the tripeptide can be effectively complexed with equivalent copper to generate copper peptide. Copper is an important element in living organisms, and it participates in important physiological functions such as cellular respiration, anti-oxidation, detoxification, coagulation, melanin and connective tissue generation in living organisms; tripeptide can effectively complex and transport copper elements to play a corresponding role.
  • complexed copper peptides can effectively stimulate the biosynthesis of collagen in fibroblasts, thereby promoting rapid wound healing; copper peptides can also effectively prevent the nerve conduction of acetylcholine substances, thereby It has the functions of relaxing muscles and improving dynamic wrinkles. Tripeptides are now widely used in cosmetic additives.
  • tripeptide on the market are mainly separation method and chemical synthesis method. Since tripeptide exists in many animals, its initial discovery and preparation were extracted and separated from a large amount of animal viscera aqueous solution. The separation procedure of this method is cumbersome and the yield is extremely low, so its large-scale production cannot be realized. Chemical synthesis is a common method for the industrial production of tripeptides at this stage. Similar to the chemical synthesis of other peptides, tedious steps such as selective protection, condensation and deprotection of functional groups are inevitably required in the chemical preparation of tripeptides. It increases its production cost, and also leads to racemization of some chiral functional groups, thereby reducing product quality.
  • GHK glutathione
  • GHK tripeptide
  • L-amino acid ligase L-amino acid ligase
  • glycine and L-histidine ligase The activity of glycine and L-histidine ligase is very low (the maximum conversion rate is less than 1%), so it cannot be used for scale-up production; at the same time, glutathione synthase (gshB, EC 6.3.2.3) is also It has been widely reported to catalyze the attachment of specific dipeptides to amino acids to form tripeptide products, and the substrates are also diverse. However, through the practice of the present invention, the gshB enzyme has no ability to synthesize glycine-L-histidine and L-lysine. Therefore, how to obtain the corresponding amino acid ligase to produce GHK is the most critical issue.
  • the purpose of the present invention is to provide a kind of mutant enzyme, it can realize glycine, L-histidine ligase activity, and realize the synthesis ability of dipeptide glycine-L-histidine and L-lysine , so that the tripeptide can be produced efficiently by enzymatic method;
  • Another object of the present invention is to provide the application of the above mutant enzyme in the production of tripeptide
  • Another object of the present invention is to provide a method for producing tripeptide using the above mutant enzyme.
  • the present invention provides the following technical solutions:
  • a mutant enzyme characterized in that it comprises glycine and L-histidine ligase GHS and tripeptide ligase HKS; or a fusion enzyme of both;
  • the glycine and L-histidine ligase GHS is an enzyme with T244I, S290L, G292W, E84K, A158H, G159D site mutations on the basis of the wild-type L-amino acid ligase Lal;
  • the tripeptide ligase HKS is an enzyme with V150F, S153E, E228I, N230H, D233T, R285V, D130Q, E146L, N148S, G387, I445D site mutations based on the wild-type glutathione synthase gshB.
  • the present invention is aimed at the deficiency of the current lack of amino acid ligase to produce GHK, and carries out the position based on the existing L-amino acid ligase (Lal, EC 6.3.2.49) and glutathione synthase (gshB, EC 6.3.2.3).
  • Point mutation transformation can realize the linking activity of catalytic glycine and L-histidine and the ability to synthesize dipeptide glycine-L-histidine and L-lysine, and realize the purpose of generating tripeptide by enzymatic catalysis in one step.
  • the present invention also fuses two mutant enzymes, and the bifunctional enzyme GHKS that connects glycine, L-histidine and L-lysine at one time can be constructed by linking peptides to form a fusion enzyme, thereby realizing convenient and high yield.
  • the connecting peptide sequence is shown in SEQ ID No. 17 or 18;
  • L-amino acid ligase (Lal, EC 6.3.2.49) is derived from Pseudomonas syringae, and gshB enzyme (EC 6.3.2.3) is derived from Saccharomyces cerevisiae, which is a member of the PF02955&PF02951 enzyme family; PPK (EC 2.7.4.1) is derived from Paenarthrobacter aurescens, a member of the PF03976 enzyme family; ADK (EC 2.7.4.3) derived from Escherichia coli, a member of the PF05191 enzyme family.
  • the catalytic linking of glycine, L-histidine and L-lysine can be realized in one step to generate tripeptide, the yield reaches 62-91%, the purity is kept above 90%, and the product has less impurities , the reaction and purification process are simple and convenient.
  • the present invention provides the application of the mutant enzyme in catalyzing glycine, L-histidine, L-lysine to generate tripeptide or in the preparation of catalytic glycine, L-histidine, The application of L-lysine in the enzyme preparation for generating tripeptide.
  • 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.
  • the present invention provides a process for preparing tripeptide by enzymatic catalysis.
  • the reaction raw materials, glycine, L-histidine, L-lysine, ATP or its salt, are at the pH value of the mutant enzyme of the present invention.
  • the enzyme-catalyzed reaction is carried out with the mutant enzyme to generate the tripeptide.
  • the pH value of the system is maintained within the pH value range of the mutant enzyme; in a specific embodiment of the present invention, the pH value range of the mutant enzyme is selected from 6.5-9.0, but it is not excluded that others can exert the mutation pH range for enzyme function.
  • the ATP salt is a sodium ATP salt, such as adenosine triphosphate disodium salt, which can also provide ATP.
  • the reaction medium is a buffer; in a specific embodiment of the present invention, the buffer is Tris-HCl.
  • polyphosphate kinase (PPK, EC 2.7.4.1) can convert adenosine diphosphate ADP to adenosine triphosphate ATP using inexpensive polyphosphate as a raw material, while adenylate kinase (ADK, EC 2.7.4.3) can achieve The mutual conversion of the three adenosine phosphates (AMP, ADP, ATP), by using these two enzymes in combination or by expressing the two enzymes in fusion (PPK-ADK/ADK-PPK) can not only reduce the production cost of enzyme fermentation, but also Effectively speed up the regeneration of ATP.
  • the process of the present invention also includes the addition of reaction raw materials PPK and ADK or fusion proteins of both, polyphosphoric acid, magnesium chloride and potassium chloride (magnesium chloride and potassium chloride are used for ATP regeneration).
  • reaction raw materials PPK and ADK or fusion proteins of both, polyphosphoric acid, magnesium chloride and potassium chloride magnesium chloride and potassium chloride are used for ATP regeneration.
  • Magnnesium chloride and potassium chloride are used for ATP regeneration.
  • the mutant enzyme, PPK and ADK or both fusion enzymes (PPK-ADK/ADK-PPK) of the present invention can participate in the enzymatic catalysis reaction by using the host cell expressing each enzyme, the enzyme solution of each enzyme or the immobilized enzyme form of each enzyme .
  • the present invention also includes one or more purification steps selected from the group consisting of removing protein impurities, removing residual reaction raw materials, removing salt, removing phosphoric acid and crystallization. actual situation adjustment. Specifically, acidification treatment removes protein impurities, reverse osmosis removes salt, anion exchange resin removes phosphoric acid impurities, and crystallizes and purifies through ethanol aqueous solution, more preferably pure water: ethanol (1-3): 1v/v to crystallize and purify .
  • the present invention enables Lal enzyme to achieve glycine and L-histidine ligase activity, and gshB enzyme to achieve the synthesis ability of dipeptide glycine-L-histidine and L-lysine through modification.
  • the GHS enzyme and the HKS enzyme are further fused by the polypeptide chain, and the bifunctional enzyme GHKS can be constructed by linking glycine, L-histidine and L-lysine at one time, so as to achieve convenient and high yield.
  • the polyphosphate kinase PPK can be used for cyclic regeneration, thereby greatly reducing the amount of ATP.
  • Fig. 1 shows the reaction principle schematic diagram of the present invention
  • Figure 2 shows the SDS-PAGE gel image after enzyme purification
  • Figure 3 shows the nuclear magnetic spectrum of the purified tripeptide in 600M Varian solution in D 2 O; the upper picture is 1 H-NMR, and the lower picture is 13 C-NMR.
  • the invention discloses a mutant enzyme and its application and a process for preparing a tripeptide by an enzyme-catalyzed method.
  • Those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications will be apparent to those skilled in the art, and they are deemed to be included in the present invention.
  • the mutant enzyme of the present invention and its application and related processes have been described through preferred embodiments, and relevant persons can obviously make changes or appropriate changes to the mutant enzyme, its application and related processes herein without departing from the content, spirit and scope of the present invention and combinations to implement and apply the technology of the present invention.
  • the steps of the process of the present invention are intended to clearly describe the core reaction route, and do not limit whether the entire reaction is carried out by a one-step method or a multi-step method.
  • each enzyme used can be artificially synthesized according to the sequence, and the sequence of each enzyme mentioned in the present invention is summarized in Table 1 below:
  • GHKS-1 and GHKS-2 are two fusion enzymes (GHS-HKS, different connecting peptides) provided by the present invention, the bold and underlined amino acid represents the mutation site and the amino acid after mutation, and the italic and underlined sequence is the connection peptide sequence.
  • the above-mentioned enzymes can also construct recombinant plasmids through their respective coding genes for cell transformation, for example:
  • Escherichia coli Escherichia coli K12
  • Saccharomyces cerevisiae ATCC 204508 and Paenarthrobacter aurescens TC1 chromosomes purchased by ATCC as templates
  • the ADK, gshB and PPK genes were amplified by PCR with the primers in Table 2.
  • Fragment then use Nde I/Xho I purchased by NEB Company to carry out corresponding enzyme digestion, and connect to the pET28a plasmid (purchased from Addgene) of the same cut point, and finally carry out plasmid transformation (transfer into Ecoli Dh5a cells, purchased from Qing Branch Biotechnology) and colony PCR and gene sequencing verification.
  • the La1 gene fragment was synthesized by the company (Anhui General Bio) and subcloned into the pET28a plasmid. Finally, using the La1 and gshB genes as templates, the mutagenic primers in Table 2 (implemented by conventional PCR amplification) were used to construct the multi-site mutant enzyme genes GHS and HKS.
  • the above-mentioned GHS, HKS, PPK and ADK plasmids constructed on the pET-28a vector were transferred into E.coli BL21 (DE3) (purchased from Anhui General Bio) strain, and 5ml contained 50uM kanamycin (Kanamycin) at 37°C.
  • the LB medium was composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.
  • Each enzyme can catalyze the reaction in the form of crude enzyme solution containing enzyme, purified enzyme or immobilized enzyme:
  • the above supernatant was gradually added with solid ammonium sulfate until the precipitated protein was precipitated (35%-55%, w/v ammonium sulfate/buffer).
  • the amount of each reaction substance can be adjusted according to the actual situation.
  • Example 1 Catalytic preparation of tripeptide (GHS, HKS, PPK, ADK combination)
  • the La1 gene fragment was synthesized by the company (Anhui Universal Bio) and subcloned into the pET28a plasmid. Finally, using the La1 and gshB genes as templates, the mutagenic primers in Table 2 (implemented by conventional PCR amplification) were used to construct the multi-site mutant enzyme genes GHS and HKS.
  • the above-mentioned GHS, HKS, PPK and ADK plasmids constructed on the pET-28a vector were transferred into E.coli BL21 (DE3) (purchased from Anhui General Bio) strain, and 5ml contained 50uM kanamycin (Kanamycin) at 37°C.
  • the LB medium was composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.
  • the reaction system was slowly stirred at room temperature for 6 hours (the pH value of the system was maintained at 6.5-9.0 by adding HCl or NaOH aqueous solution during the reaction process), and most of the raw materials were consumed (using the ProFoldin company L-histidine detection kit to detect in the reaction solution residual histidine starting material).
  • reaction was terminated by adjusting the pH of the reaction solution and the protein in the reaction solution was sedimented (add acid to adjust the pH value of the solution to 1.5 and stir quickly), filter to remove the protein precipitate, and then adjust the solution to pH 7.0; use reverse osmosis to remove salt , D201 anion exchange resin removes impurities containing phosphoric acid (use deionized water as eluent, tripeptide GHK directly flows out due to its weak binding ability to resin), glycine-L-histidine-L-lysine after lyophilization
  • the crude product was crystallized from pure water and ethanol 1:(1-3) v/v to give 59 g of an off-white solid (87% yield, 96.0% purity).
  • the nuclear magnetic spectrum of the purified tripeptide in 600M Varian solution in D 2 O is shown in Figure 3 , the upper picture is 1 H-NMR, and the lower picture is 13 C-NMR.
  • Example 2 Catalytic preparation of tripeptide (GHKS-1, PPK, ADK combination)
  • the tripeptide GHK synthase gene fragment GHKS-1 was synthesized by Gene Company (Anhui General Bio) and subcloned into the pET28a plasmid; the same as in Example 1, the plasmid was transferred into the Ecoli BL21 (DE3) strain for a small amount of After protein expression, it was amplified into a 5L fermenter for fermentation production, and the collected wet cells were about 40 grams; while the polyphosphate kinase PPK and adenylate kinase ADK enzyme solutions prepared in Example 1 were directly used for subsequent preparation reactions.
  • the reaction conditions were similar to those in Example 1. 11.2 g of glycine (150 mM), 23.2 g of L-histidine (150 mM) and 21.9 g of L-lysine were added to 1 L of 100 mM pH 8.0 tris hydrochloric acid solution, respectively. (150mM), 5.6 g of adenosine triphosphate disodium salt ATP (10 mM), 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 monophosphate); pH was adjusted to 8.0 and added with 2000 U of GHKS-1 enzyme solution, 1500U PPK and 1000U ADK enzyme solution.
  • Example 3 Catalytic preparation of tripeptide (GHKS-1, PPK, ADK combination)
  • the tripeptide synthase gene fragment GHKS-2 was synthesized by Gene Company (Anhui General Bio), and subcloned into the pET28a plasmid; the protein was directly amplified and prepared after a small amount of expression was verified, and the overexpressed cell was stored in the disrupted solution. Freeze at 4° C. for later use; the polyphosphate kinase PPK and adenylate kinase ADK enzyme solutions prepared in Example 1 can be directly used in this enzyme reaction.
  • the reaction was similar to that in Example 2, and 15 g of glycine (200 mM), 31 g of L-histidine (200 mM), 29.2 g of L-lysine ( 200mM), 5.6g adenosine triphosphate disodium salt ATP (10mM), 1.8g magnesium chloride (20mM), 3.75g potassium chloride (20mM) and 51.6g polyphosphoric acid (500mM monophosphate); add 2000U after the pH is adjusted to 8.0 GHKS-2 enzyme solution, 2000U PPK and 1500U ADK enzyme solution, the reaction system was stirred at room temperature for 7 hours (maintaining the pH value of the reaction system at 6.5-9.0), and it was found that most of the histidine raw materials were completely converted after monitoring.

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Abstract

涉及生化技术领域，公开了一种突变酶及其应用和酶催化法制备三胜肽的工艺。包括甘氨酸和L-组氨酸连接酶GHS以及三肽连接酶HKS；或为两者的融合酶。通过改造让Lal酶实现甘氨酸、L-组氨酸连接酶活力从而获得GHS酶，以及让gshB酶实现二肽甘氨酸-L-组氨酸与L-赖氨酸的合成能力从而获得HKS酶。在此基础上，进一步利用多肽链将GHS酶与HKS酶进行融合，则可以构造一次性连接甘氨酸、L-组氨酸以及L-赖氨酸的双功能酶GHKS，从而实现方便的、高收率的制备三胜肽；而对于该酶催化反应中所需要的大量ATP，可以采用多聚磷酸激酶进行循环再生，进而大大降低ATP的用量。

Description

一种突变酶及其应用和酶催化法制备三胜肽的工艺
本申请要求于2020年11月06日提交中国专利局、申请号为202011231194.8、发明名称为“一种突变酶及其应用和酶催化法制备三胜肽的工艺”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生化技术领域，具体涉及一种突变酶及其应用和酶催化法制备三胜肽的工艺。
背景技术
三胜肽(GHK)是由甘氨酰、L-组氨酰与L-赖氨酸连接而成的一种天然三肽化合物(Gly-L-His-L-Lys)，其分子式为C 14H 24N 6O 4、分子量为340，三胜肽能与等当量铜有效络合而生成铜胜肽。铜是生物体内重要元素，它参与细胞呼吸作用、生物体内抗氧化、解毒、凝血、黑色素以及结缔组织的生成等等重要的生理功能；三胜肽能通过有效络合、转运铜元素而发挥相应的作用，譬如络合的铜胜肽能有效刺激成纤维细胞(fibroblasts)内胶原蛋白的生物合成，从而促进伤口的快速愈合；铜胜肽还能有效阻止乙醯胆碱物质的神经传导，从而具有放松肌肉，改善动态性皱纹等功效。三胜肽现被广泛应用于化妆品添加剂。
市面上三胜肽制备方法主要是分离法与化学合成法。由于三胜肽存在于不少动物体内，其最初的发现及制备则是从大量动物内脏水溶液中提取、分离，该方法分离程序繁琐、产率极低，因而无法实现其规模化生产。化学合成法是现阶段三胜肽工业生产的常见方法，与其它多肽化学合成类似，三胜肽化学制备过程中不可避免需要采用繁琐的官能团选择性保护、缩合以及脱保护等步骤，这不仅大大的增加了其生产成本，同时也会导致部分手性官能团消旋，从而降低产品品质。
与谷胱甘肽(GSH)不同，三胜肽(GHK)虽然也存在于不少动物体内，但是至今没有发现对应的氨基酸连接酶来生产GHK，其来源可能是通过 体内多肽或蛋白分级降解而成。但是自然界存在大量的短肽合成酶，如L-氨基酸连接酶(Lal，EC 6.3.2.49)，它们被发现能直接连接或者改造后连接多种多样的氨基酸，但经过本发明HPLC检测Lal仅具有很低的甘氨酸及L-组氨酸连接酶活力(最高转化率低于1％)，所以不能用来放大生产；与此同时，谷胱甘肽合成酶(gshB，EC 6.3.2.3)也被广泛报道能催化特定二肽与氨基酸的连接从而形成三肽产物，其底物也同样具有多样性。然而经过本发明的实践验证，gshB酶没有甘氨酸-L-组氨酸与L-赖氨酸的合成能力。因此，如何获得对应的氨基酸连接酶来生产GHK是其中最为关键的问题。
发明内容
有鉴于此，本发明的目的在于提供一种突变酶，使其能够实现甘氨酸、L-组氨酸连接酶活力，以及实现二肽甘氨酸-L-组氨酸与L-赖氨酸的合成能力，从而可以利用酶法高效率生产三胜肽；
本发明的另外一个目的在于提供上述突变酶在生产三胜肽中的应用；
本发明的另外一个目的在于提供利用上述突变酶生产三胜肽的方法。
为实现上述目的，本发明提供如下技术方案：
一种突变酶，其特征在于，包括甘氨酸和L-组氨酸连接酶GHS以及三肽连接酶HKS；或为两者的融合酶；
其中，所述甘氨酸和L-组氨酸连接酶GHS为在野生型L-氨基酸连接酶Lal基础上具有T244I、S290L、G292W、E84K、A158H、G159D位点突变的酶；所述三肽连接酶HKS为在野生型谷胱甘肽合成酶gshB基础上具有V150F、S153E、E228I、N230H、D233T、R285V、D130Q、E146L、N148S、G387、I445D位点突变的酶。
本发明针对目前缺少氨基酸连接酶来生产GHK的缺陷，在现有L-氨基酸连接酶(Lal，EC 6.3.2.49)以及谷胱甘肽合成酶(gshB，EC 6.3.2.3)的基础上进行位点突变改造，可实现催化甘氨酸与L-组氨酸连接活力和对二肽甘氨酸-L-组氨酸与L-赖氨酸的合成能力，一步实现酶法催化生成三胜肽的目的。
同时，本发明还将两种突变酶融合，通过连接肽组成融合酶则可以构 造一次性连接甘氨酸、L-组氨酸以及L-赖氨酸的双功能酶GHKS，从而实现方便的、高收率的制备三胜肽。在本发明具体实施方式中，所述连接肽序列如SEQ ID No.17或18所示；
本发明中，L-氨基酸连接酶(Lal，EC 6.3.2.49)来源于Pseudomonas syringae、gshB酶(EC 6.3.2.3)来源于Saccharomyces cerevisiae，属于PF02955&PF02951酶家族成员；PPK(EC 2.7.4.1)来源于Paenarthrobacter aurescens，属于PF03976酶家族成员；ADK(EC 2.7.4.3)来源于Escherichia coli，属于PF05191酶家族成员。
通过利用本发明提供的突变酶可以一步实现甘氨酸、L-组氨酸与L-赖氨酸的催化连接生成三胜肽，收率达到62-91％，纯度保持在90％以上，产物杂质少，反应、纯化工艺简便。基于这种优异的技术效果，本发明提供了所述突变酶在催化甘氨酸、L-组氨酸、L-赖氨酸生成三胜肽中的应用或在制备催化甘氨酸、L-组氨酸、L-赖氨酸生成三胜肽的酶制剂中的应用。作为优选，所述酶制剂为表达突变酶的宿主细胞、突变酶的酶液或突变酶的固定化酶。
依据应用，本发明提供了一种酶催化法制备三胜肽的工艺，反应原料甘氨酸、L-组氨酸、L-赖氨酸、ATP或其盐，在本发明所述突变酶的pH值范围内的反应介质中，与所述突变酶进行酶催化反应生成三胜肽。
在反应过程中维持体系pH值在所述突变酶pH值范围内；在本发明具体实施方式中，所述突变酶pH值范围选自为6.5-9.0，但不排除其他的能够发挥所述突变酶功能的pH值范围。作为优选，所述ATP盐为ATP钠盐，如三磷酸腺苷二钠盐，其同样可以提供ATP。
作为优选，所述反应介质为缓冲液；在本发明具体实施方式中，所述缓冲液为为Tris-HCl。
同时，多聚磷酸激酶(PPK，EC 2.7.4.1)能利用廉价的多聚磷酸作为原料将二磷酸腺苷ADP转化成三磷酸腺苷ATP，而腺苷酸激酶(ADK，EC 2.7.4.3)则能实现三种磷酸腺苷(AMP、ADP、ATP)的相互转化，通过联合使用这两个酶或将两个酶融合表达生成(PPK-ADK/ADK-PPK)不仅可以降低酶发酵生产成本，还能有效加快ATP再生速度。因此，本 发明工艺还包括添加反应原料PPK和ADK或两者融合蛋白、多聚磷酸、氯化镁和氯化钾(氯化镁和氯化钾用于ATP再生)。具体的反应原理示意图见图1。
本发明所述突变酶、PPK和ADK或两者融合酶(PPK-ADK/ADK-PPK)可以利用表达各酶的宿主细胞、各酶的酶液或各酶的固定化酶形式参与酶催化反应。
与大多数的反应一样，本发明还包括选自除蛋白杂质、除残留反应原料、除盐、除磷酸和结晶中的一种或两种以上操作的纯化步骤，具体选择何种纯化步骤，根据实际情况调整。具体地，酸化处理除去蛋白杂质，反渗透除盐，阴离子交换树酯除含磷酸杂质，结晶通过乙醇水溶液来结晶纯化，更优选为纯水:乙醇(1-3):1v/v来结晶纯化。
由以上技术方案可知，本发明通过改造让Lal酶实现甘氨酸、L-组氨酸连接酶活力，以及让gshB酶实现二肽甘氨酸-L-组氨酸与L-赖氨酸的合成能力。在此基础上，进一步利用多肽链将GHS酶与HKS酶进行融合，则可以构造一次性连接甘氨酸、L-组氨酸以及L-赖氨酸的双功能酶GHKS，从而实现方便的、高收率的制备三胜肽；而对于该酶催化反应中所需要的大量三磷酸腺苷，可以采用多聚磷酸激酶PPK进行循环再生，进而大大降低ATP的用量。
附图说明
图1所示为本发明反应原理示意图；
图2所示为酶纯化后的SDS-PAGE凝胶图；
图3所示为纯化后三胜肽在600M Varian在D 2O溶液核磁谱图；上图为 1H-NMR，下图为 13C-NMR。
具体实施方式
本发明公开了一种突变酶及其应用和酶催化法制备三胜肽的工艺，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们 都被视为包括在本发明。本发明突变酶及其应用和相关工艺已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文突变酶及其应用和相关工艺进行改动或适当变更与组合，来实现和应用本发明技术。
本发明所述工艺的步骤旨在清楚的描述核心的反应路线，并不限制整个反应采用一步法还是多步法进行。
在本发明具体实施方式中，所采用的各酶可以根据序列进行人工合成，本发明中所提及的各酶的序列汇总如下表1：
表1
Figure PCTCN2021117378-appb-000001
Figure PCTCN2021117378-appb-000002
Figure PCTCN2021117378-appb-000003
表1中，GHKS-1和GHKS-2为本发明提供的两种融合酶(GHS-HKS，连接肽不同)，加粗加下划线氨基酸表示突变位点以及突变后氨基酸，斜体加下划线序列为连接肽序列。
上述各酶也可以通过各自的编码基因构建重组质粒进行细胞转化，例如：
以ATCC购买的大肠杆菌(Escherichia coli K12)，酿酒酵母(Saccharomyces cerevisiae ATCC 204508)以及保氏杆菌(Paenarthrobacter aurescens TC1)染色体为模板，利用表2引物PCR扩增出扩增出ADK，gshB以及PPK基因片段，然后利用NEB公司购买的Nde I/Xho I进行相应的酶切，并连接到相同切点的pET28a质粒上(购于Addgene)，最后进行质粒转化(转入E coli Dh5a细胞，购于擎科生物)并菌落PCR及基因测序验证。La1基因片段是通过公司合成(安徽通用生物)并亚克隆到pET28a质粒上。最后以La1与gshB基因为模板，利用表2突变引物(常规的PCR扩增实现)构建多位点突变酶基因GHS与HKS。将上述构建到pET-28a载体上的GHS，HKS，PPK以及ADK质粒转入E.coli BL21(DE3)(购于安徽通用生物)菌株里，在37℃、5ml含50uM卡那霉素(Kanamycin)的LB培养液中进行小量培养，当细胞生长至OD 0.5-0.8加入0.5mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，37℃诱导蛋白表达3小时，最后收集细胞、冻融法进行细胞破碎、高速离心，收集到的上清液再利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确认蛋白表达。蛋白表达正确的菌株逐级培养至5升发酵罐，在1.0mM IPTG条件下37℃诱导表达4小时，收集湿细胞近35-55克；最后将细胞与适量的Tris-HCl缓冲液(25mM，pH＝8.0)混合均匀后用高压破碎仪低温破碎，高速离心除去细胞壁后酶液保存在4℃冰箱备用。LB培养基构成为：1％胰蛋白胨、0.5％酵母粉，1％NaCl，1％磷酸氢二钾、1％磷酸氢二钾以及5％的甘油。
表2
Figure PCTCN2021117378-appb-000004
Figure PCTCN2021117378-appb-000005
各酶可以以含酶的粗酶液、纯化后的酶或固定化酶的方式进行催化反应：
例如：收集的含酶的湿细胞混合在Tris-HCl缓冲液(25mM，pH＝8.0)(缓冲液A)，搅拌均匀后通过高压破碎细胞，高速离心除去细胞壁，收集到的清液为粗酶液，直接进行后续催化反应；
或者，上述上清液逐渐加入硫酸铵固体直至析出蛋白析出(35％-55％，w/v硫酸铵/缓冲液)。该蛋白固体随后通过高速离心收集(10000rpm，12min)，并缓慢溶入Tris-HCl缓冲液(25mM，pH＝8.0)，经G25脱盐柱(购于Sigma)除盐后经DEAE Seplite FF(西安蓝晓公司)阴离子交换柱分离纯化，最终得到初纯化酶，SDS-PAGE凝胶图见图2。
在进行固定化时，可参照本领域常规的固定化酶制备方式。
依照本发明工艺的反应路线，各反应物质的用量可以根据实际情况调整。
虽然根据本发明提供的酶的序列可以知晓其编码序列，但在本发明中还是提供了如下表3的具体的编码序列：
表3
Figure PCTCN2021117378-appb-000006
Figure PCTCN2021117378-appb-000007
Figure PCTCN2021117378-appb-000008
Figure PCTCN2021117378-appb-000009
Figure PCTCN2021117378-appb-000010
Figure PCTCN2021117378-appb-000011
Figure PCTCN2021117378-appb-000012
Figure PCTCN2021117378-appb-000013
Figure PCTCN2021117378-appb-000014
下面结合实施例，进一步阐述本发明。
实施例1：催化制备三胜肽(GHS，HKS，PPK，ADK组合)
以ATCC购买的大肠杆菌(Escherichia coli K12)，酿酒酵母(Saccharomyces cerevisiae ATCC 204508)以及保氏杆菌(Paenarthrobacter aurescens TC1)染色体为模板用上述对应引物PCR扩增出ADK，gshB以及PPK基因片段，然后利用NEB公司购买的Nde I/Xho I进行相应的酶切，并连接到相同切点的pET28a质粒上(购于Addgene)，最后进行质粒转化(转入E coli Dh5a细胞，购于擎科生物)并菌落PCR及基因测序验证。La1基因片 段是通过公司合成(安徽通用生物)并亚克隆到pET28a质粒上。最后以La1与gshB基因为模板，利用表2突变引物(常规的PCR扩增实现)构建多位点突变酶基因GHS与HKS。将上述构建到pET-28a载体上的GHS，HKS，PPK以及ADK质粒转入E.coli BL21(DE3)(购于安徽通用生物)菌株里，在37℃、5ml含50uM卡那霉素(Kanamycin)的LB培养液中进行小量培养，当细胞生长至OD 0.5-0.8加入0.5mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，37℃诱导蛋白表达3小时，最后收集细胞、冻融法进行细胞破碎、高速离心，收集到的上清液再利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确认蛋白表达。蛋白表达正确的菌株逐级培养至5升发酵罐，在1.0mM IPTG条件下37℃诱导表达4小时，收集湿细胞近35-55克；最后将细胞与适量的Tris.HCl缓冲液(25mM，pH＝8.0)混合均匀后用高压破碎仪低温破碎，高速离心除去细胞壁后酶液保存在4℃冰箱备用。LB培养基构成为：1％胰蛋白胨、0.5％酵母粉，1％NaCl，1％磷酸氢二钾、1％磷酸氢二钾以及5％的甘油。
在1L 100mM pH 8.0的三羟甲基氨基甲烷盐酸(Tris.HCl)溶液中分别加入15克甘氨酸(200mM)，31克L-组氨酸(200mM)，29.2克L-赖氨酸(200mM)，5.6克三磷酸腺苷二钠盐ATP(10mM)，1.8克氯化镁(20mM)，3.75克氯化钾(20mM)以及51.6克多聚磷酸(Sigma，25聚，500mM单磷酸)，待pH值调节到8.0后；加入1000U的GHS酶液，1500U的HKS酶液，2000U的PPK以及1200U的ADK酶液。该反应体系在室温缓慢搅拌6小时(反应过程中通过添加HCl或NaOH水溶液维持体系pH值在6.5-9.0)后检测原料大部分消耗(利用ProFoldin公司L-组氨酸检测试剂盒检测反应液中残留的组氨酸原料)。最后通过调节反应液pH终止反应并沉底反应液中的蛋白(加酸调节溶液pH值到1.5后并快速搅拌)，过滤除去蛋白沉淀，随后将溶液调回pH值7.0；利用反渗透除盐，D201阴离子交换树酯除含磷酸杂质(用去离子水做洗脱液，三胜肽GHK由于与树脂结合能力弱直接流出)，冻干后甘氨酸-L-组氨酸-L-赖氨酸粗品通过纯水和乙醇1:(1-3)v/v结晶得到59克灰白色固体(收率87％，纯度96.0％)。纯化后三 胜肽在600M Varian在D 2O溶液核磁谱图见图3，上图为 1H-NMR，下图为 13C-NMR。
实施例2：催化制备三胜肽(GHKS-1，PPK，ADK组合)
三胜肽GHK合成酶基因片段GHKS-1是通过基因公司合成(安徽通用生物)，并亚克隆到pET28a质粒上；与实施例1相同，该质粒转入E coli BL21(DE3)菌株进行小量蛋白表达后放大到5L发酵罐内发酵生产，收集到的湿细胞约为40克；而实施例1制备的多聚磷酸激酶PPK与腺苷酸激酶ADK酶液则直接用于后续制备反应。
反应条件与实施例1类似，在1L 100mM pH 8.0的三羟甲基氨基甲烷盐酸溶液中分别加入11.2克甘氨酸(150mM)，23.2克L-组氨酸(150mM)，21.9克L-赖氨酸(150mM)，5.6克三磷酸腺苷二钠盐ATP(10mM)，1.8克氯化镁(20mM)，3.75克氯化钾(20mM)以及20.6克多聚磷酸(200mM单磷酸)；pH调节到8.0后加入2000U的GHKS-1酶液，1500U的PPK以及1000U的ADK酶液。室温反应10个小时后监测到反应液中L-组氨酸消耗完全，最后通过在反应液中加入HCl溶液至pH 1.5终止反应并沉淀蛋白，过滤除去蛋白沉淀，清液pH值调回7.0后反渗透法除盐，最后用阴离子交换柱除溶液中含磷酸杂质，流出液浓缩后用乙醇与水结晶纯化得31.6克三胜肽纯品(收率62％，纯度91.2％)。纯化后三胜肽在600M Varian在D 2O溶液核磁谱图同实施例1。
实施例3：催化制备三胜肽(GHKS-1，PPK，ADK组合)
与实施例2类似，三胜肽合成酶基因片段GHKS-2通过基因公司合成(安徽通用生物)，并亚克隆到pET28a质粒上；蛋白小量表达验证后直接放大制备，过表达细胞破碎液保存在4℃冻存备用；实施例1制备的多聚磷酸激酶PPK与腺苷酸激酶ADK酶液可直接用于此次酶反应。
反应与实施例2类似，往1L 100mM pH 8.0的三羟甲基氨基甲烷盐酸溶液中分别加入15克甘氨酸(200mM)，31克L-组氨酸(200mM)，29.2克L-赖氨酸(200mM)，5.6克三磷酸腺苷二钠盐ATP(10mM)，1.8克氯化 镁(20mM)，3.75克氯化钾(20mM)以及51.6克多聚磷酸(500mM单磷酸)；待pH值调节到8.0后加入2000U的GHKS-2酶液，2000U的PPK以及1500U的ADK酶液,反应体系室温搅拌7小时(维持反应体系pH值在6.5-9.0)后监测发现大部分组氨酸原料转化完全。加HCl水溶液终止反应并变性沉淀蛋白，与上相同，最后除盐并用阴离子交换柱除去反应中的含磷酸杂质，三胜肽粗液浓缩后结晶，最终得到61.8克灰白色固体(收率91％，纯度94.5％)。纯化后三胜肽在600M Varian在D 2O溶液核磁谱图同实施例1。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 一种突变酶，其特征在于，包括甘氨酸和L-组氨酸连接酶GHS以及三肽连接酶HKS；或为两者的融合酶；
    其中，所述甘氨酸和L-组氨酸连接酶GHS为在野生型L-氨基酸连接酶Lal基础上具有T244I、S290L、G292W、E84K、A158H、G159D位点突变的酶；所述三肽连接酶HKS为在野生型谷胱甘肽合成酶gshB基础上具有V150F、S153E、E228I、N230H、D233T、R285V、D130Q、E146L、N148S、G387、I445D位点突变的酶。
  2. 权利要求1所述突变酶在催化甘氨酸、L-组氨酸、L-赖氨酸生成三胜肽中的应用或在制备催化甘氨酸、L-组氨酸、L-赖氨酸生成三胜肽的酶制剂中的应用。
  3. 根据权利要求2所述应用，其特征在于，所述酶制剂为表达突变酶的宿主细胞、突变酶的酶液或突变酶的固定化酶。
  4. 一种酶催化法制备三胜肽的工艺，其特征在于，反应原料甘氨酸、L-组氨酸、L-赖氨酸、ATP或其盐，在权利要求1所述突变酶的pH值范围内的反应介质中，与权利要求1所述突变酶进行酶催化反应生成三胜肽。
  5. 根据权利要求4所述工艺，其特征在于，所述突变酶的pH值范围为6.5-9.0。
  6. 根据权利要求4所述工艺，其特征在于，所述反应介质为缓冲液。
  7. 根据权利要求4所述工艺，其特征在于，还包括添加反应原料PPK和ADK或两者融合蛋白、多聚磷酸、氯化镁和氯化钾。
  8. 根据权利要求4-7任意一项所述工艺，其特征在于，还包括选自除蛋白杂质、除残留反应原料、除盐、除磷酸和结晶中的一种或两种以上操作的纯化步骤。
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