Mutant, enzyme composition and application thereof in preparation of glutathione

Abstract

The invention relates to the field of bioengineering, in particular to a mutant, an enzyme composition and application of the enzyme composition in preparation of glutathione. The invention provides a mutant which comprises a mutant of glutathione reductase and / or a mutant of formate dehydrogenase. According to the method, a GSSG reduction system composed of glutathione reductase StGR-M4 and formate dehydrogenase LbFDH-M1 obtained through rational design transformation is introduced into a reaction system for synthesizing GSH with L-glutamic acid, L-cysteine hydrochloride monohydrate and glycine as substrates, the effects of reducing generation of impurities GSSG and improving the conversion rate of the product GSH are achieved, the material cost is low, and the product quality is high.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to mutants, enzyme compositions and their applications in the preparation of glutathione. Background Art

[0002] Glutathione is a tripeptide composed of glutamic acid, cysteine and glycine, in which the sulfhydryl group on cysteine is its active group (so it is often abbreviated as GSH), and it has the functions of antioxidant and detoxification. Since 1970, Kyowa Hakko in Japan has realized the industrial production of glutathione by extracting it from baker's yeast, making it widely used in health products, pharmaceuticals and skin care products. In 2009, Mitsubishi Kasei in Japan produced glutathione using Candida utilis and obtained FDA GRAS certification (GRN 293). In the same year, Shandong Jincheng Medicine obtained a patent license from Nippon Kayaku and produced glutathione by yeast fermentation, and its production capacity expanded from 30 tons / year to 500 tons / year in 2022. Domestic manufacturers also include Kaiping Qianniu (30 tons / year in 2020, immobilized enzyme method), Hunan Fulaige (10 tons / year in 2024, immobilized enzyme method), Anhui Gut (150 tons / year in 2025, crude liquid enzyme method), Jilin Bio (100 tons / year in 2024, crude liquid enzyme method), etc.

[0003] Compared with the fermentation method, the enzymatic synthesis of glutathione by γ-glutamylcysteine synthetase GshA and glutathione synthetase GshB or glutathione bifunctional synthetase GshF has the advantages of high product concentration, short production cycle and easy purification. At the same time, by coupling Saccharomyces cerevisiae, polyphosphate kinase to consume hexametaphosphate or acetate kinase to consume acetyl phosphate, the regeneration of relatively expensive adenosine triphosphate ATP can be achieved, reducing the material cost, and the application of polyphosphate kinase to consume hexametaphosphate is the most extensive.

[0004] However, the reducing substrates cysteine, the intermediate γ-glutamylcysteine and the product glutathione are easily oxidized into cystine, γ-glutamylcystine and oxidized glutathione GSSG respectively during the reaction process, which will cause waste of materials. Summary of the Invention

[0005] In view of this, the present invention provides mutants, enzyme compositions and their applications in the preparation of glutathione. The present invention introduces a GSSG reduction system composed of the rationally designed glutathione reductase StGR-M4 and formate dehydrogenase LbFDH-M1 into the reaction system for synthesizing GSH with L-glutamic acid, L-cysteine hydrochloride monohydrate and glycine as substrates, which plays a role in reducing the generation of impurity GSSG and increasing the conversion rate of product GSH, with low material cost and high product quality.

[0006] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The present invention provides mutants, including: mutants of glutathione reductase and / or mutants of formate dehydrogenase;

[0008] The mutation sites of the mutant of glutathione reductase include: one or more of the 197th, 198th, 199th, and 204th positions of the wild-type amino acid sequence of the glutathione reductase;

[0009] The mutation site of the mutant of formate dehydrogenase includes: the 222nd position of the wild-type amino acid sequence of the formate dehydrogenase.

[0010] In some embodiments of the present invention, in the above-mentioned mutants, the wild-type amino acid sequence of the glutathione reductase is as shown in SEQ ID NO:1.

[0011] In some embodiments of the present invention, in the above-mentioned mutants, the nucleotide sequence encoding the wild-type amino acid of the glutathione reductase is as shown in SEQ ID NO:2.

[0012] In some embodiments of the present invention, in the above-mentioned mutants, the wild-type amino acid sequence of the formate dehydrogenase is as shown in SEQ ID NO:5.

[0013] In some embodiments of the present invention, in the above-mentioned mutants, the nucleotide sequence encoding the wild-type amino acid of the formate dehydrogenase is as shown in SEQ ID NO:6.

[0014] In some embodiments of the present invention, in the above-mentioned mutants, the mutant of glutathione reductase includes: the 197th position is mutated from V to E, the 198th position is mutated from R to M, the 199th position is mutated from K to F, and the 204th position is mutated from R to P;

[0015] The mutant of formate dehydrogenase includes: the 222nd position is mutated from Q to D.

[0016] In some embodiments of the present invention, in the above-mentioned mutants, the mutant of glutathione reductase has:

[0017] (1), the amino acid sequence as shown in SEQ ID NO:13; or

[0018] (2), the amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence as shown in (1), and the amino acid sequence having the same or similar function as the amino acid sequence as shown in (1); or

[0019] (3) An amino acid sequence having at least 80% identity with the amino acid sequence shown in (1) or (2); and / or

[0020] The mutant of the formate dehydrogenase has:

[0021] (4) The amino acid sequence shown in SEQ ID NO:15; or

[0022] (5) An amino acid sequence obtained by substituting, deleting or adding one or more amino groups to the amino acid sequence shown in (4), and having the same or similar function as the amino acid sequence shown in (4); or

[0023] (6) An amino acid sequence having at least 80% identity with the amino acid sequence shown in (4) or (5).

[0024] The present invention also provides an enzyme composition, comprising: the above mutant, glutathione bifunctional synthetase and polyphosphate kinase.

[0025] In some embodiments of the present invention, in the above enzyme composition, the amino acid sequence of the glutathione bifunctional synthetase is as shown in SEQ ID NO:17.

[0026] In some embodiments of the present invention, in the above enzyme composition, the amino acid sequence of the polyphosphate kinase is as shown in SEQ ID NO:19.

[0027] The present invention also provides a nucleic acid molecule encoding the above mutant or the above enzyme composition, characterized in that the nucleic acid molecule encoding the mutant of glutathione reductase has:

[0028] (7) The nucleotide sequence shown in SEQ ID NO:14; or

[0029] (8) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (7), and having the same or similar function as the nucleotide sequence shown in (7); or

[0030] (9) A nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (7) or (8); and / or

[0031] The nucleic acid molecule encoding the mutant of the formate dehydrogenase has:

[0032] (10) The nucleotide sequence shown in SEQ ID NO:16; or

[0033] (11) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (10), and a nucleotide sequence having the same or similar function as the nucleotide sequence as shown in (10); or

[0034] (12) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (10) or (11); and / or

[0035] The nucleic acid molecule encoding the glutathione bifunctional synthetase has:

[0036] (13) A nucleotide sequence as shown in SEQ ID NO:18; or

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

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

[0039] The nucleic acid molecule encoding the polyphosphate kinase has:

[0040] (16) A nucleotide sequence as shown in SEQ ID NO:20; or

[0041] (17) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (13), and a nucleotide sequence having the same or similar function as the nucleotide sequence as shown in (13); or

[0042] (18) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (13) or (14).

[0043] The present invention also provides primers for amplifying the above nucleic acid molecule, including: primer set 1 and / or primer set 2;

[0044] The primer set 1 is used for amplifying the nucleic acid molecule encoding the mutant of glutathione reductase, and has: sequences as shown in SEQ ID NO:7 to SEQ ID NO:10;

[0045] The primer set 2 is used for amplifying the nucleic acid molecule encoding the mutant of formate dehydrogenase, and has: sequences as shown in SEQ ID NO:11 and SEQ ID NO:12.

[0046] The present invention also provides a recombinant vector, including: the above nucleic acid molecule.

[0047] The present invention also provides a host for transforming and / or transfecting the above recombinant vector.

[0048] The present invention also provides the use of the above mutant, the above enzyme composition, the above nucleic acid molecule, the above recombinant vector and / or the above host in the preparation of glutathione.

[0049] The present invention also provides a method for preparing glutathione, wherein the raw material is converted into glutathione by any of the following;

[0050] (I), the above mutant; or

[0051] (II), the above enzyme composition; or

[0052] (III), the above nucleic acid molecule; or

[0053] (IV), the above recombinant vector; or

[0054] (V), the above host;

[0055] The raw material includes: glutamic acid, glycine, cysteine, sodium hexametaphosphate and AMP.

[0056] In some embodiments of the present invention, in the above preparation method, the raw material further includes: NAD, magnesium chloride hexahydrate and sodium formate.

[0057] In some embodiments of the present invention, in the above preparation method, the cysteine is L-cysteine hydrochloride monohydrate.

[0058] In some embodiments of the present invention, in the above preparation method, the raw material includes: L-glutamic acid, glycine, sodium hexametaphosphate, magnesium chloride hexahydrate, sodium formate, NAD, adenosine monophosphate and L-cysteine hydrochloride monohydrate.

[0059] In some embodiments of the present invention, in the above preparation method, the enzyme activity of the mutant of glutathione reductase is: 468 U / mL; the enzyme activity of the mutant of formate dehydrogenase is: 695 U / mL; the enzyme activity of glutathione bifunctional synthetase is: 600 U / mL; the enzyme activity of polyphosphate kinase is: 360 U / mL.

[0060] The present invention also provides a product, including: the above mutant, the above enzyme composition, the above nucleic acid molecule, the above recombinant vector and / or the above host.

[0061] The present invention introduces glutathione reductase GR and formate dehydrogenase FDH into the reaction system to further avoid the oxidation of glutathione, and changes the coenzyme selectivity of GR by rational design and modification, replacing expensive nicotinamide adenine dinucleotide phosphate NADP with relatively inexpensive nicotinamide adenine dinucleotide NAD, thereby reducing the material cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] 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 the description of the embodiments or the prior art.

[0063] Figure 1 Showing the electrophoresis results of StGR and BcGDH proteins expressed in shake flasks;

[0064] Figure 2 Showing the reaction formulas of the enzymes StGR and BcGDH reducing GSSG;

[0065] Figure 3 Showing the electrophoresis results of LbFDH protein expressed in shake flasks;

[0066] Figure 4 Showing the reaction formulas of the enzymes StGR and LbFDH reducing GSSG;

[0067] Figure 5 Showing the reaction of reducing GSSG to generate GSH with NADP as a coenzyme;

[0068] Figure 6 Showing the reaction of reducing GSSG to generate GSH with NAD as a coenzyme;

[0069] Figure 7 Showing the electrophoresis results of StGshF and SlPPK proteins expressed in shake flasks;

[0070] Figure 8 Showing the enzyme reaction of synthesizing GSH with L-glutamic acid, L-cysteine hydrochloride monohydrate, and glycine as substrates;

[0071] Figure 9 Showing the liquid phase chromatogram at 4 h of the two groups of enzyme reactions for synthesizing GSH. The blue is the StGshF_SlPPK group, and the black is the StGshF_SlPPK_StGR-M4_LbFDH-M1 group;

[0072] Figure 10 Showing the mass spectrum of glutathione, the ion peak 308.000 is [M+H] + , the ion peak 306.000 is [M-H] - , the ion peak 613.000 is [2M-H] - . DETAILED DESCRIPTION OF THE EMBODIMENTS

[0073] The present invention discloses mutants, enzyme compositions and their applications in the preparation of glutathione.

[0074] It should be understood that the expression "one or more of..." individually includes each of the recited objects following 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.

[0075] The terms "comprising", "having", or "containing", including the use of their grammatical synonyms, should generally be understood to be open and non-limiting, e.g., not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0076] It should be understood that the order of steps or the order of performing certain actions is not important as long as the present invention remains operable. In addition, two or more steps or actions can be carried out simultaneously.

[0077] 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 present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0078] 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 numerical 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, numerical 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.

[0079] For the first time, the present invention attempted to use glutathione reductase StGR and glucose dehydrogenase BcGDH to reduce oxidized glutathione GSSG with glucose Glucose to generate glutathione GSH and by-product glucono-1,5-lactone, but it was abandoned due to unsatisfactory conversion rate.

[0080] Then, an attempt was made to use StGR and formate dehydrogenase LbFDH to reduce GSSG with sodium formate Sodium formate to generate GSH and by-product carbon dioxide CO2, and the generated CO2 would be released into the air without residue. Further, the coenzyme selectivity of StGR was changed through rational design and modification, and relatively inexpensive nicotinamide adenine dinucleotide NAD was used to replace expensive nicotinamide adenine dinucleotide phosphate NADP.

[0081] Finally, the engineered glutathione reductase StGR-M4 and formate dehydrogenase LbFDH-M1 were introduced into the reaction system for synthesizing GSH using L-glutamic acid, L-cysteine hydrochloride monohydrate, and glycine as substrates, which played a role in reducing the production of GSSG and increasing the conversion rate of GSH.

[0082] Amino acid sequence of glutathione reductase StGR from Streptococcus thermophilus: MVKEYDYIVIGGGSGGIASANRAAMHGAKVILFEGKEVGGTCVNVGCVPKKVMWYGAQVAETLHRYAGEYGFDVTINNFDFATLKANRQAYIDRIHGSFERGFDSNGVERVYEYARFVDPHTVEVAGELYTAPHILIATGGHPLYPNIPGSEYGITSDGFFELDEVPKRTAVIGAGYIAVEVAGVLNALGSDTHLFVRKDRPLRTFDKDIIDVLVDEMAKSGPTLHMHANATEVVKNADDSLTISFDNEETITVDCLIWAVGRAANTSGFGLEKTGVELTERGNIYSDEFENTSVPGIYALGDVTGKLDLTPVAVKAGRQLSERLFNNKVDAKLDYTDVATVVFSHPAIGAIGLTEEKAIAKYGAENIKVYKSSFTPMYTALGDNRQLSTMKLVTLGEDEKIIGLHGIGYGVDEMIQGFSVAIKMGATKADFDNTVAIHPTGSEEFVTMR (SEQ ID NO:1);

[0083]

[0084] Amino acid sequence of glucose dehydrogenase BcGDH derived from Burkholderia cenocepacia: MSKLAGKVAIVTGASKGIGAAIAKALADEGAAVVVNYASSKAGADAVVSAITEAGGRAVAVGGDVSKAADAQRIVDTAIETYGRLDVLVNNSGVYEFAPIEAITEEHYRRQFDTNVFGVLLTTQAAVKHLGEGASIINISSVVTSITPPASAVYSGTKGAVDAITGVLALELGPRKIRVNAINPGMIVTEGTHSAGIIGSDLEAQVLGQTPLGRLGEPNDIASVAVFLASDDARWMTGEHLVVSGGLN (SEQ ID NO:3);

[0085] Nucleotide sequence (codon-optimized) of glucose dehydrogenase BcGDH derived from Burkholderia cenocepacia: ATGAGCAAACTGGCAGGTAAAGTTGCAATTGTTACCGGTGCAAGCAAAGGTATTGGTGCAGCAATTGCAAAAGCACTGGCAGATGAAGGTGCAGCAGTTGTTGTTAATTATGCAAGCAGCAAAGCCGGTGCAGATGCAGTTGTTAGCGCAATTACCGAAGCCGGTGGTCGTGCAGTTGCCGTTGGTGGTGATGTTAGCAAAGCAGCAGATGCACAGCGTATTGTTGATACCGCAATTGAAACCTATGGTCGTCTGGATGTTCTGGTGAATAATAGCGGTGTTTATGAATTTGCACCGATTGAAGCCATTACCGAGGAACATTATCGTCGTCAGTTTGATACCAATGTTTTTGGTGTTCTGCTGACCACACAGGCAGCAGTTAAACATTTAGGTGAAGGTGCCTCCATTATTAACATTAGCAGCGTTGTTACCAGCATTACCCCTCCGGCAAGCGCAGTTTATAGCGGCACCAAAGGTGCAGTTGATGCGATTACCGGTGTTCTGGCACTGGAACTGGGTCCGCGTAAAATTCGTGTTAATGCAATTAATCCGGGTATGATTGTGACCGAAGGCACCCATAGCGCAGGTATTATTGGTAGCGATCTGGAAGCACAGGTTCTGGGTCAGACACCGCTGGGTCGCCTGGGTGAACCGAATGATATTGCAAGCGTTGCAGTTTTTCTGGCAAGTGATGATGCACGTTGGATGACCGGTGAACATCTGGTTGTGAGCGGTGGTCTGAATTAA (SEQ ID NO:4);

[0086] Amino acid sequence of formate dehydrogenase LbFDH derived from Lentilactobacillus buchneri: MTKVLAVLYPDPVDGFPPKYVRDDIPKITHYPDGSTVPTPEGIDFKPGELLGSVSGGLGLKKYLESKGVEFVVTSDKEGPDSVFEKELPTADVVISQPFWPAYLTADLIDKAKKLKLAITAGIGSDHVDLNAANEHNITVAEVTYSNSVSVAEAEVMQLLALVRNFIPAHDIVKAGGWNIADAVSRAYDLEGMTVGVIGAGRIGRAVLERLKPFGVKLVYNQRHQLPDEVENELGLTYFPDVHEMVKVVDAVVLAAPLHAQTYHLFNDEVLATMKRGAYIVNNSRGEEVDRDAIVRALNSGQIGGYSGDVWYPQPAPKDHPWRTMPNEAMTPHMSGTTLSAQARYAAGAREILEDFLEDKPIRPEYLIAQGGSLAGTGAKSYTVKKGEETPGSGEAEK (SEQ ID NO:5);

[0087]

[0088] The nucleotide sequence of the upstream primer for constructing the StGR-V197E_R198M_K199F triple mutant: GCGATACCCACCTGTTTGAAATGTTTGATCGTCCGCTGCGTACC (SEQ ID NO:7);

[0089] The nucleotide sequence of the downstream primer for constructing the StGR-V197E_R198M_K199F triple mutant: GGTACGCAGCGGACGATCAAACATTTCAAACAGGTGGGTATCGC (SEQ ID NO:8);

[0090] The nucleotide sequence of the upstream primer for constructing the StGR-R204P mutant: GATCGTCCGCTGCCTACCTTTGATAAAG (SEQ ID NO:9);

[0091] The nucleotide sequence of the downstream primer for constructing the StGR-R204P mutant: CTTTATCAAAGGTAGGCAGCGGACGATC (SEQ ID NO:10);

[0092] The nucleotide sequence of the upstream primer for constructing the LbFDH-Q222D mutant: TGAAACTGGTTTATAATGACCGTCATCAGCTGCCGG (SEQ ID NO:11);

[0093] The nucleotide sequence of the downstream primer for constructing the LbFDH-Q222D mutant: TCCGGCAGCTGATGACGGTCATTATAAACCAGTTTC (SEQ ID NO:12);

[0094] Amino acid sequence of glutathione reductase StGR-V197E_R198M_K199F_R204P mutant: MVKEYDYIVIGGGSGGIASANRAAMHGAKVILFEGKEVGGTCVNVGCVPKKVMWYGAQVAETLHRYAGEYGFDVTINNFDFATLKANRQAYIDRIHGSFERGFDSNGVERVYEYARFVDPHTVEVAGELYTAPHILIATGGHPLYPNIPGSEYGITSDGFFELDEVPKRTAVIGAGYIAVEVAGVLNALGSDTHLFEMFDRPLPTFDKDIIDVLVDEMAKSGPTLHMHANATEVVKNADDSLTISFDNEETITVDCLIWAVGRAANTSGFGLEKTGVELTERGNIYSDEFENTSVPGIYALGDVTGKLDLTPVAVKAGRQLSERLFNNKVDAKLDYTDVATVVFSHPAIGAIGLTEEKAIAKYGAENIKVYKSSFTPMYTALGDNRQLSTMKLVTLGEDEKIIGLHGIGYGVDEMIQGFSVAIKMGATKADFDNTVAIHPTGSEEFVTMR (SEQ ID NO:13);

[0095]

[0096] Amino acid sequence of formate dehydrogenase LbFDH-Q222D mutant: MTKVLAVLYPDPVDGFPPKYVRDDIPKITHYPDGSTVPTPEGIDFKPGELLGSVSGGLGLKKYLESKGVEFVVTSDKEGPDSVFEKELPTADVVISQPFWPAYLTADLIDKAKKLKLAITAGIGSDHVDLNAANEHNITVAEVTYSNSVSVAEAEVMQLLALVRNFIPAHDIVKAGGWNIADAVSRAYDLEGMTVGVIGAGRIGRAVLERLKPFGVKLVYNDRHQLPDEVENELGLTYFPDVHEMVKVVDAVVLAAPLHAQTYHLFNDEVLATMKRGAYIVNNSRGEEVDRDAIVRALNSGQIGGYSGDVWYPQPAPKDHPWRTMPNEAMTPHMSGTTLSAQARYAAGAREILEDFLEDKPIRPEYLIAQGGSLAGTGAKSYTVKKGEETPGSGEAEK (SEQ ID NO:15);

[0097]

[0098] Amino acid sequence of glutathione bifunctional synthetase StGshF derived from Streptococcus thermophilus: MTLNQLLQKLEATSPILQANFGIERESLRVDRQGQLVHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRFLGAITDVAGRSIATDEVLWPLSMPPRLKAEEIQVAQLENDFERHYRNYLAEKYGTKLQAISGIHYNMELGKDLVEALFQESDQTDMIAFKNALYLKLAQNYLRYRWVITYLFGASPIAEQGFFDQEVPEPMRSFRNSDHGYVNKEEIQVSFVSLEDYVSAIETYIEQGDLIAEKEFYSAVRFRGQKVNRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDQALAQGHALNEKIALSHPLEPLPSEAKTQDIVTALDQLVQHFGLGDYHQDLVKQVKAAFADPNQTLSAQLLPYIKDKSLAEFALNKALAYHDYDWTAHYALKGYEEMELSTQMLLFDAIQKGIHFEILDEQDQFLKLWHQDHVEYVKNGNMTSKDNYVVPLAMANKTVTKKILADAGFPVPSGDEFTSLEEGLAYYPLIKDKQIVVKPKSTNFGLGISIFQEPASLDNYQKALEIAFAEDTSVLVEEFIPGTEYRFFILDGRCEAVLLRVAANVIGDGKHTIRELVAQKNANPLRGRDHRSPLEIIELGDIEQLMLAQQGYTPDDILPEGKKVNLRRNSNISTGGDSIDVTETMDSSYQELAAAMATSMGAWACGVDLIIPDETQIATKENPHCTCIELNFNPSMYMHTYCAEGPGQAITTKILDKLFPEIVAGQT (SEQ ID NO:17);

[0099]

[0100] Amino acid sequence of polyphosphate kinase SlPPK derived from Sulfurovum lithotrophicum: MKKNIYKKELYKLQVELVKFQKYVIEENVAVCLVFEGRDTAGKDGTIKRFTEHLSPREARTVALGVPSDKEKKSWYFQRYVPHLPSAGEIVFFNRSWYNRAGVEKVMGFCTKKQYKAFMEEVGSFEQMLTHSNIRFFKYYLDISKKEQKKRLEARKTDPLKQWKLSPIDAKAQKMWDAYSKARDDMFNKTSFIYAPWYVVHTDDKKEARINIMKHFLSLNDYPDKDKALLVYDHDVICKFDPVCYEKEMIAP (SEQ ID NO:19);

[0101] Nucleotide sequence (codon-optimized) of polyphosphate kinase SlPPK derived from Sulfurovum lithotrophicum: ATGAAGAAGAACATCTACAAGAAAGAGCTGTACAAACTGCAGGTTGAACTGGTGAAATTCCAGAAATATGTGATCGAAGAAAATGTTGCCGTTTGCCTGGTTTTTGAAGGTCGTGATACCGCAGGTAAAGATGGCACCATTAAACGTTTTACCGAACATCTGAGTCCGCGTGAAGCACGTACCGTTGCACTGGGTGTTCCGAGCGATAAAGAAAAGAAAAGCTGGTATTTTCAGCGCTATGTTCCGCATCTGCCGAGTGCCGGTGAAATTGTTTTCTTTAATCGTAGCTGGTATAATCGTGCCGGTGTTGAAAAAGTTATGGGTTTTTGTACCAAAAAGCAGTATAAAGCCTTCATGGAAGAAGTGGGTAGCTTTGAGCAGATGCTGACCCATAGCAATATTCGCTTTTTCAAATACTACCTGGACATCAGCAAGAAAGAACAGAAAAAGCGTCTGGAAGCCCGTAAAACCGATCCGCTGAAACAGTGGAAACTGAGCCCGATTGATGCAAAAGCACAGAAAATGTGGGATGCATATAGCAAAGCACGTGATGACATGTTTAACAAGACCAGCTTTATCTATGCACCGTGGTATGTTGTTCATACCGATGATAAGAAAGAAGCGCGTATCAACATCATGAAACACTTTCTGAGCCTGAACGATTATCCGGACAAAGATAAAGCACTGCTGGTGTATGATCATGACGTGATCTGTAAATTTGATCCGGTGTGCTATGAGAAAGAAATGATTGCACCGTAA (SEQ ID NO:20).

[0102] In Comparative Example 1 and Examples 1 to 3 of the present invention, the raw materials and reagents used are all commercially available.

[0103] The present invention is further illustrated below in conjunction with examples:

[0104] Comparative Example 1 Glutathione reductase StGR, glucose dehydrogenase BcGDH reduces GSSG

[0105] Glutathione reductase StGR from Streptococcus thermophilus and glucose dehydrogenase BcGDH from Burkholderia cenocepacia were selected, codon-optimized for the expression host Escherichia coli using the online tool GeneOptimizer. After obtaining the corresponding nucleotide sequences, they were submitted to GenScript Nanjing for whole gene synthesis and constructed on the pET-28a vector (Novagen).

[0106] 0.5 μg of the obtained plasmid dry powder was centrifuged briefly, then resuspended in 50 μL of ddH2O. 1 μL was taken and transferred into the expression-competent cells BL21(DE3) (Kangti Biotech) thawed on ice, and incubated in an ice bath for 20 min. After heat shock in a 42 °C water bath for 30 s, it was placed back on ice and allowed to stand for 2 min. Then 700 μL of antibiotic-free LB medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride) was added and cultured at 37 °C with shaking at 220 rpm for 1 h. 100 μL was taken and streaked on a Kana-resistant LB plate (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 12 g / L agar). After placing it in a 37 °C incubator and allowing it to stand for 15 min, it was inverted and cultured overnight.

[0107] Three colonies of uniform size were picked from the plate into 10 mL of Kana-resistant LB medium and cultured at 37 °C with shaking at 220 rpm until turbid. 0.5 mL was taken and mixed with 0.5 mL of 40% glycerol in a 1.5 mL EP tube and stored at -40 °C. The remaining medium was transferred to 500 mL of Kana-resistant LB medium and cultured at 37 °C with shaking at 220 rpm until OD 600 = 0.6 - 1.0. After cooling to 20 °C, 0.1 mM IPTG was added and the culture was continued overnight at 37 °C with shaking at 220 rpm.

[0108] The cells were collected by centrifugation at 8000 rpm for 5 min at 4 °C, resuspended in the lysis buffer (20 mM KH2PO4 / K2HPO4, 100 mM KCl, pH 7.6) at a ratio of 1 g of wet cells : 9 mL, and then sonicated to obtain the crude enzyme solution. After centrifugation at 20000 rpm for 25 min at 4 °C, samples of the supernatant and the resuspended pellet were taken and analyzed by electrophoresis to detect the expression ( Figure 1 ). Both StGR and BcGDH were expressed in the supernatant.

[0109] Weigh 12.25 g of oxidized glutathione GSSG (100 mM, with a total reaction solution volume of 200 mL), 3.96 g of glucose monohydrate (100 mM), and 0.31 g of nicotinamide adenine dinucleotide phosphate disodium salt NADP-Na2 (2 mM), dissolve them in 160 mL of pure water, divide them into 4 portions, adjust the pH to 7.0, 8.0, 9.0, and 10.0 respectively, and make up the volume to 48 mL. After preheating at 25 °C, add 1.5 mL of the crude StGR enzyme solution (enzyme activity 550 U / mL) and 0.5 mL of the crude BcGDH enzyme solution (enzyme activity 2778 U / mL) to each portion to start the reaction. The reaction formula is shown in Figure 2 . During the reaction process, the production of the product GSH is detected by high performance liquid chromatography (HPLC).

[0110] As shown in Table 1, the highest amount of GSH generated in the pH 9.0 group after 3 h of reaction was 71.46 mM, but the conversion rate was only 36%. This may be due to the large difference between the optimal pH of StGR (7.0 - 7.6) and that of BcGDH (9.3 - 9.7).

[0111] Table 1

[0112]

[0113] Example 1 Reduction of GSSG by glutathione reductase StGR and formate dehydrogenase LbFDH

[0114] To solve the problem of the low conversion rate that may be caused by the large difference in the optimal pH between StGR and BcGDH in Comparative Example 1, formate dehydrogenase LbFDH from Lentilactobacillus buchneri was selected. The optimized nucleotide sequence was submitted to Nanjing Genscript for whole gene synthesis and constructed on the pET-28a vector (Novagen). Referring to the procedure in Comparative Example 1, the obtained plasmid was transformed into the expression competent cell BL21(DE3), and after streaking, enlarged culture, and disruption, the crude LbFDH enzyme solution was obtained. Centrifugation was carried out, samples of the supernatant and precipitate were taken, and the expression was detected by electrophoresis ( Figure 3 ), the expression of LbFDH was low, but mainly in the supernatant.

[0115] Weigh 6.13 g of GSSG (100 mM, reaction solution volume 100 mL), 0.88 g of sodium formate (130 mM), and 0.16 g of NADP-Na2 (2 mM), dissolve them in 80 mL of pure water, adjust the pH to 7.0, and make up the volume to 94 mL. After preheating at 25 °C, add 3 mL of the crude StGR enzyme solution (enzyme activity 550 U / mL) and 3 mL of the crude LbFDH enzyme solution (enzyme activity 428 U / mL) to start the reaction. The reaction formula is shown in Figure 4 .

[0116] As Figure 5As shown, 162.45 mM of GSH was generated after 3 h of reaction, with a conversion rate of 81%, which was significantly higher than 36% in the comparative example.

[0117] Example 2 Coenzyme Specificity Modification of Glutathione Reductase StGR

[0118] The enzyme StGR can only utilize the expensive NADP as a coenzyme and cannot utilize the relatively inexpensive nicotinamide adenine dinucleotide NAD. To reduce the material cost of the reaction, it is necessary to change the coenzyme specificity of the enzyme StGR from NADP to NAD. Based on the predicted protein complex structure and catalytic mechanism analysis, four mutations, namely V197E, R198M, K199F, and R204P, were introduced (this quadruple mutant was named M4). At the same time, to improve the reduction efficiency of LbFDH for NAD, based on sequence alignment, one mutation, Q222D (M1), was introduced. Using pET28a-StGR and pET28a-LbFDH as templates respectively, the primers in Table 2 were designed and the plasmids of the mutants StGR-M4 and LbFDH-M1 were constructed using the QuickChange site-directed mutagenesis kit (Agilent).

[0119] Table 2

[0120]

[0121] Referring to the procedure in Comparative Example 1, the obtained plasmids were respectively transformed into the expression competent cell BL21(DE3), and after streaking, enlarged culture, and disruption, the crude enzyme solutions of StGR-M4 and LbFDH-M1 were obtained.

[0122] Weigh 12.25 g of GSSG (100 mM, total reaction solution volume of 200 mL), 1.77 g of sodium formate (130 mM), and 0.27 g of NAD (2 mM), dissolve them in 160 mL of pure water, adjust the pH to 7.0, and make up the volume to 188 mL. After preheating 2 aliquots at 25 °C, add 3 mL of the crude enzyme solution of StGR (550 U / mL) and 3 mL of the crude enzyme solution of LbFDH (428 U / mL) to one aliquot, and add 3 mL of the crude enzyme solution of StGR-M4 (enzyme activity 468 U / mL) and 3 mL of the crude enzyme solution of LbFDH-M1 (695 U / mL) to the other aliquot, and start the reaction.

[0123] As Figure 6 shown, in the StGR-M4 & LbFDH-M1 group, 158.20 mM of GSH was generated after 3 h of reaction, 168.40 mM was generated after 4 h, and the conversion rate was 84.2%. While in the StGR & LbFDH group, only 42.88 mM of GSH was generated after 4 h of reaction. It shows that through rational design and modification, the coenzyme specificity of the enzyme StGR was successfully changed from NADP to NAD.

[0124] Example 3 Synthesis of GSH by Glutathione Bifunctional Synthase StGshF and Polyphosphate Kinase SlPPK

[0125] Select the glutathione bifunctional synthase StGshF from Streptococcus thermophilus and the polyphosphate kinase SlPPK from Sulfurovum lithotrophicum. Submit the optimized nucleotide sequence to GenScript Nanjing for whole gene synthesis and construct it on the pET-28a vector (Novagen). Referring to the procedure in Comparative Example 1, the obtained plasmids were respectively transformed into the expression competent cell BL21(DE3). After streaking, scale-up culture, and disruption, crude enzyme solutions of StGshF and SlPPK were obtained. Centrifuge, take samples of the supernatant and precipitate, and detect the expression by electrophoresis ( Figure 7 ), and both were expressed in the supernatant.

[0126] Weigh 5.89 g of L-glutamic acid (200 mM, total reaction volume of 200 mL), 3.30 g of glycine (220 mM), 7.34 g of sodium hexametaphosphate (60 mM), 2.44 g of magnesium chloride hexahydrate (60 mM), 0.27 g of sodium formate (20 mM), 0.27 g of NAD (2 mM), and 0.14 g of adenosine monophosphate AMP (2 mM) and dissolve them in 130 mL of pure water. Adjust the pH to 7.0 and make up the volume to 144 mL. After preheating 2 aliquots at 25 °C, add 5 mL of crude enzyme solution of StGshF (600 U / mL), 1 mL of crude enzyme solution of SlPPK (enzyme activity 360 U / mL, the same below), 1 mL of crude enzyme solution of StGR-M4 (enzyme activity 468 U / mL), and 1 mL of crude enzyme solution of LbFDH-M1 (enzyme activity 695 U / mL) to one aliquot, and add only 5 mL of crude enzyme solution of StGshF and 1 mL of crude enzyme solution of SlPPK to the other aliquot and make up to 80 mL with pure water. Insert an air tube and pass industrial nitrogen. Weigh 7.38 g of L-cysteine hydrochloride monohydrate and dissolve it in 40 ml of pure water, that is, a 1.05 M mother liquor is divided into 2 aliquots and added dropwise to the reaction solutions of StGshF_SlPPK_StGR-M4_LbFDH-M1 and StGshF_SlPPK in the time range of 0 - 1 h respectively to start the reaction.

[0127] Figure 8 is the production amount of GSH during the reaction of the two groups, Figure 9 is the liquid phase chromatogram of the reaction for 4 h, Figure 10It is the first-order mass spectrometry map of the GSH product peak. The GSH produced by the StGshF_SlPPK group after 4 hours of reaction is 183.76 mM, and the conversion rate of Cys→GSH is 87.5%. After introducing the GSSG reduction system composed of StGR-M4 and LbFDH-M1 in the StGshF_SlPPK_StGR-M4_LbFDH-M1 group, 196.82 mM of GSH is produced after 4 hours of reaction, and the conversion rate is 93.7%. Moreover, the production of GSSG cannot be observed in the liquid phase spectrum, showing obvious advantages in terms of conversion rate and GSSG residue.

[0128] The above are only the preferred embodiments of the present invention. It should be pointed out 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, characterized in that, Comprising: A mutant of glutathione reductase and / or a mutant of formate dehydrogenase; The mutation sites of the mutant of glutathione reductase include: one or more of the 197th, 198th, 199th, and 204th positions of the wild-type amino acid sequence of glutathione reductase; The mutation site of the mutant of formate dehydrogenase includes: the 222nd position of the wild-type amino acid sequence of formate dehydrogenase.

2. The mutant according to claim 1, wherein The mutant of glutathione reductase includes: the 197th position mutated from V to E, the 198th position mutated from R to M, the 199th position mutated from K to F, and the 204th position mutated from R to P; The mutant of formate dehydrogenase includes: the 222nd position mutated from Q to D.

3. The mutant according to claim 1 or 2, characterized in that, The mutant of glutathione reductase has: (1), an amino acid sequence as shown in SEQ ID NO:13; or (2), an amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence as shown in (1), and having the same or similar function as the amino acid sequence as shown in (1); or (3), an amino acid sequence having at least 80% identity with the amino acid sequence as shown in (1) or (2); and / or The mutant of formate dehydrogenase has: (4), an amino acid sequence as shown in SEQ ID NO:15; or (5), an amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence as shown in (4), and having the same or similar function as the amino acid sequence as shown in (4); or (6), an amino acid sequence having at least 80% identity with the amino acid sequence as shown in (4) or (5).

4. An enzyme composition, characterized in that, Comprising: The mutant, glutathione bifunctional synthase, and polyphosphate kinase as described in any one of claims 1 to 3.

5. A nucleic acid molecule encoding the mutant according to any one of claims 1 to 3 or the enzyme composition according to claim 4, characterized in that, The nucleic acid molecule encoding the mutant of glutathione reductase has: (7), a nucleotide sequence as shown in SEQ ID NO:14; or (8), a nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence as shown in (7), and having the same or similar function as the nucleotide sequence as shown in (7); or (9), a nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (7) or (8); and / or The nucleic acid molecule encoding the mutant of formate dehydrogenase has: (10), a nucleotide sequence as shown in SEQ ID NO:16; or (11), a nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence as shown in (10), and having the same or similar function as the nucleotide sequence as shown in (10); or (12), a nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (10) or (11); and / or The nucleic acid molecule encoding the glutathione bifunctional synthase has: (13), a nucleotide sequence as shown in SEQ ID NO:18; or (14) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (13), and having the same or similar function as the nucleotide sequence as shown in (13); or (15) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (13) or (14); and / or The nucleic acid molecule encoding the polyphosphate kinase has: (16) A nucleotide sequence as shown in SEQ ID NO:20; or (17) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (13), and having the same or similar function as the nucleotide sequence as shown in (13); or (18) A nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (13) or (14).

6. Recombinant vector, characterized in that, Comprising: The nucleic acid molecule according to claim 5.

7. Host, characterized in that, Transforming and / or transfecting the recombinant vector according to claim 6.

8. Use of the mutant according to any one of claims 1 to 3, the enzyme composition according to claim 4, the nucleic acid molecule according to claim 5, the recombinant vector according to claim 6 and / or the host according to claim 7 in the preparation of glutathione.

9. A method for preparing glutathione, characterized in that, Glutathione is prepared by transforming the raw material by any of the following: (I) The mutant according to any one of claims 1 to 3; or (II) The enzyme composition according to claim 4; or (III) The nucleic acid molecule according to claim 5; or (IV) The recombinant vector according to claim 6; or (V) The host according to claim 7; The raw material includes: glutamic acid, glycine, cysteine, sodium hexametaphosphate and AMP.

10. Product, characterized in that, Comprising: The mutant according to any one of claims 1 to 3, the enzyme composition according to claim 4, the nucleic acid molecule according to claim 5, the recombinant vector according to claim 6 and / or the host according to claim 7.

Images