Mutants of bsbacd enzyme, enzyme compositions, and methods of making tyrosine dipeptides

Abstract

The present application relates to the technical field of enzyme, in particular to a mutant of BsBacD enzyme, an enzyme composition and a preparation method of tyrosine dipeptide. The present application firstly rationally designs the BsBacD enzyme, then replaces ADP (adenosine diphosphate or its salt) with relatively cheap ATP (adenosine triphosphate or its salt) or even cheaper AMP (adenosine monophosphate or its salt), and introduces polyphosphate kinase ChPPK2 which can regenerate ATP with AMP as a substrate to synthesize tyrosine dipeptide, and optimizes reaction pH, magnesium chloride concentration and substrate tyrosine concentration and other parameters, and develops a low-cost and high-efficiency enzyme synthesis process of three kinds of tyrosine dipeptides, such as propyl tyrosine dipeptide, glycosyl tyrosine dipeptide and silk tyrosine dipeptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of enzyme technology, in particular to a mutant of BsBacD enzyme, an enzyme composition and a preparation method of tyrosine dipeptide. BACKGROUND

[0002] Tyrosine (Tyr) is one of the three aromatic amino acids, which is synthesized from phenylalanine (Phe) by phenylalanine hydroxylase (PheOH) in vivo (Erlandsen et al. 1997). Supplementing tyrosine in the cell culture medium can effectively eliminate the sequence variation of Tyr→Phe, Tyr→His (histidine) and other sequence variations that are prone to occur during the production of monoclonal antibodies using Chinese hamster ovary cells (CHO). However, tyrosine has low solubility in water and is unstable, and the mother liquor needs to be prepared at pH 11, and then accurately added during cell culture (Feeney et al. 2013). Replacing tyrosine with tyrosine dipeptides such as Met-Tyr (methionyl tyrosine), Tyr-His (tyrosyl histidine), and Tyr-Lys (tyrosyl lysine) can significantly reduce the complexity of process control, while improving the yield and activity of monoclonal antibodies (Erdmann et al. 2006, Kang et al. 2012). Ala-Tyr (alanyl tyrosine) and Gly-Tyr (glycyl tyrosine) are the two most widely promoted tyrosine dipeptides on the market, with representatives including Germany's Winkel, Chengdu Bixing, Wuhan Jipei / Hubei Hongpeide, Wuxi Jingyao, and Anhui Hexun.

[0003] The first L-amino acid ligase (BsBacD) was screened from domain set PS 50975 by Japanese Kyowa Hakko Kogyo, and its N-terminal amino acid substrate tends to be Ala (alanine), Gly (glycine), Ser (serine), and its C-terminal amino acid tends to be Phe, Met (methionine) (Tabata, Ikeda, and Hashimoto 2005). The team of Song Wenlu from Jining University coupled BsBacD enzyme with SlPPK2, which regenerates ATP (adenosine triphosphate) using ADP (adenosine diphosphate) as a substrate, and generated 40.1 mM Ala-Tyr with a conversion rate of 89.1% using 45 mM Ala, 45 mM Tyr, 6 mM ADP, and 20 mM PolyP6 (hexametaphosphoric acid) as substrates for 3 h (Cui et al. 2023). However, this work is only a laboratory result, and the residual tyrosine after the reaction cannot be removed by simple membrane separation during purification, which affects the crystallization of the product Ala-Tyr. At the same time, the product concentration is too low at 10.1 g / L (40.1 mM), and the substrate ADP is expensive, resulting in high production cost, which makes it difficult to apply enzyme synthesis of Ala-Tyr to industrialization. SUMMARY

[0004] Therefore, the application provides a mutant of BsBacD enzyme, an enzyme composition and a preparation method of tyrosine dipeptides. Through a large number of design screening and experimental verification, a low-cost and efficient enzyme synthesis process of three kinds of tyrosine dipeptides, propyl tyrosine dipeptide, glycosyl tyrosine dipeptide and silk tyrosine dipeptide, is developed.

[0005] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0006] In a first aspect, the application provides a mutant of BsBacD enzyme, wherein the mutation site of the mutant comprises N108S and L110S.

[0007] The BsBacD enzyme has:

[0008] (I) an amino acid sequence as shown in SEQ ID No. 1;

[0009] (II) a sequence in which one or more amino acids in the amino acid sequence as shown in (I) are substituted, deleted, added and / or replaced; or

[0010] (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence as shown in any one of (I) or (II).

[0011] In a second aspect, the application further provides a nucleic acid molecule encoding the mutant.

[0012] The nucleic acid molecule has:

[0013] (1) a nucleotide sequence as shown in SEQ ID No. 2; or

[0014] (2) a nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence as shown in (1), and having the same or similar function as (1); or

[0015] (3) a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence as shown in (1) or (2).

[0016] In a third aspect, the application further provides an enzyme composition comprising the mutant and a polyphosphate kinase.

[0017] In some specific embodiments of the application, the polyphosphate kinase has:

[0018] (a) an amino acid sequence as shown in SEQ ID No. 11;

[0019] (b) a sequence of one or more amino acids substituted, deleted, added and / or replaced based on the amino acid sequence as shown in (a); or

[0020] (c) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence as shown in any one of (a) or (b).

[0021] In a fourth aspect, the present application further provides a nucleic acid molecule encoding the enzyme composition.

[0022] The nucleic acid molecule encoding the polyphosphatase has:

[0023] (1) a nucleotide sequence as shown in SEQ ID No. 12; or

[0024] (2) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (1), and having the same or similar function as (1); or

[0025] (3) a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence as shown in (1) or (2).

[0026] In a fifth aspect, the present application further provides an expression frame comprising the nucleic acid molecule.

[0027] In a sixth aspect, the present application further provides a plasmid comprising the expression frame.

[0028] In a seventh aspect, the present application further provides a bacterium, which:

[0029] (A) expresses the mutant; and / or

[0030] (B) is transformed or transfected with the expression frame; and / or

[0031] (C) is transformed or transfected with the plasmid.

[0032] In an eighth aspect, the present application further provides a composition for synthesizing tyrosine dipeptide, which comprises:

[0033] the enzyme composition; and

[0034] adenosine triphosphate or a salt thereof, or adenosine monophosphate or a salt thereof, but not adenosine diphosphate or a salt thereof; and

[0035] alkaline mother liquor of tyrosine;

[0036] As preferred, the pH of the composition comprises 6.5-9.5; more preferably, the pH of the composition is 8.5;

[0037] As preferred, the concentration of the alkaline mother liquor of tyrosine comprises Tyr 400mM, 500mM, 600mM, 700mM; more preferably, the concentration of the alkaline mother liquor of tyrosine is 500mM;

[0038] As preferred, the pH of the alkaline mother liquor of tyrosine comprises 11.5 or 12.5; more preferably, the pH of the alkaline mother liquor of tyrosine is 11.5;

[0039] As preferred, the composition further comprises magnesium chloride; preferably, the concentration of the magnesium chloride comprises 40mM-100mM; more preferably, the concentration of the magnesium chloride is 60mM.

[0040] In a ninth aspect, the present application further provides the use of any one of the following in the preparation of tyrosine dipeptide:

[0041] (i) the mutant; and / or

[0042] (ii) the nucleic acid molecule; and / or

[0043] (iii) the enzyme combination; and / or

[0044] (iv) the expression cassette; and / or

[0045] (v) the plasmid; and / or

[0046] (vi) the bacterial cell;

[0047] (vii) the composition;

[0048] The tyrosine dipeptide comprises one or more of propyl tyrosine dipeptide, glycol tyrosine dipeptide and / or silk tyrosine dipeptide.

[0049] In a tenth aspect, the present application further provides a method for preparing tyrosine dipeptide, comprising:

[0050] (a) mixing amino acid with the mutant or the enzyme combination to obtain dipeptide; or

[0051] (b) expressing the nucleic acid molecule to obtain protein product, and mixing the protein product with amino acid to obtain dipeptide; or

[0052] (c) expressing the expression cassette or the plasmid to obtain protein product, and mixing the protein product with amino acid to obtain dipeptide; or

[0053] (d) culturing the bacterial cells to obtain a protein product mixed with amino acids to obtain a dipeptide; or

[0054] (e) mixing the composition with amino acids to obtain a dipeptide;

[0055] The tyrosine dipeptides include one or more of propyltyrosine dipeptide, glycoltyrosine dipeptide, and / or serinetyrosine dipeptide.

[0056] The amino acids include one or more of tyrosine, alanine, glycine, or serine.

[0057] In some embodiments of the present application, the mixing includes feeding the alkaline mother liquor of the tyrosine; preferably, the feeding speed includes 0.5 mL / min to 26 mL / min.

[0058] In some embodiments of the present application, the mixing is preceded by a preheating step, and the preheating temperature includes 38°C.

[0059] In some embodiments of the present application, the reaction time after the mixing includes 0.5 h, 1 h, 1.5 h, 2 h, 3 h, or 4 h.

[0060] In some embodiments of the present application, the culturing is followed by a step of breaking the bacterial cells using a breaking buffer; preferably, the breaking buffer includes 20 mM K2HPO4 / KH2PO4 and 100 mM KCl; preferably, the pH of the breaking buffer includes 7.6.

[0061] In some embodiments of the present application, the ratio of the wet weight of the bacterial cells to the volume of the breaking buffer includes 1:4.

[0062] In some embodiments of the present application, the concentration of the bacterial cells added includes 7.7 g / L, 7.9 g / L, 13.2 g / L, or 11 g / L.

[0063] In some embodiments of the present application, the enzyme activity of the mutant of the BsBacD enzyme includes 7 U / mL, 5 U / mL, 9 U / mL, or 10 U / mL.

[0064] In some embodiments of the present application, the enzyme activity of the polyphosphate kinase includes 3 U / mL.

[0065] In some specific embodiments of the present invention, the concentration of the alkaline mother solution of tyrosine includes 400-700 mM, the final concentration of tyrosine includes 100-175 mM, and the pH of the alkaline mother solution of tyrosine includes 11.5 or 12.5; the rate of the flow addition includes 1 mL / min, 8 mL / min, 0.5 mL / min, 26 mL / min, 7.5 mL / min or 13 mL / min; preferably, the concentration of the alkaline mother solution of tyrosine includes 400 mM, 500 mM, 600 mM or 700 mM, the final concentration of tyrosine includes 100 mM, 125 mM, 150 mM, 175 mM, and the pH of the alkaline mother solution of tyrosine includes 11.5; more preferably, the concentration of the alkaline mother solution of tyrosine includes 500 mM, and the concentration of tyrosine in the reaction solution includes 125 mM.

[0066] In some specific embodiments of the present invention, the concentration of AMP is 2 mM or 1 mM; the concentration of ATP-Na2 is 2 mM or 1 mM.

[0067] In some embodiments of the present invention, the concentration of sodium hexametaphosphate includes 40 mM;

[0068] In some specific embodiments of the present invention, the concentration of alanine includes 110 to 262.5 mM; preferably, the concentration of alanine includes 110 mM, 130 mM, 170 mM, 187.5 mM, 150 mM, 225 mM or 262.5 mM;

[0069] In some specific embodiments of the present invention, the equivalent ratio of the alanine to the tyrosine is 1.1 to 1.7; preferably, the equivalent ratio of the alanine to the tyrosine is 1.1, 1.3, 1.5 or 1.7; more preferably, the equivalent ratio of the alanine to the tyrosine is 1.5;

[0070] In some specific embodiments of the present invention, the concentration of glycine is 130-170 mM; preferably, the concentration of glycine is 130 mM, 150 mM or 170 mM; more preferably, the concentration of glycine is 150 mM.

[0071] In some specific embodiments of the present invention, the equivalent ratio of the glycine to the tyrosine is 1.3 to 1.7; preferably, the equivalent ratio of the glycine to the tyrosine is 1.3, 1.5 or 1.7; more preferably, the equivalent ratio of the glycine to the tyrosine is 1.5 or 1.7.

[0072] In some embodiments of the present invention, the concentration of serine comprises 150 mM.

[0073] The present invention first rationally designs the BsBacD enzyme, replaces ADP with relatively cheap ATP or even cheaper AMP, and introduces the polyphosphate kinase ChPPK2 that can regenerate ATP using AMP as a substrate to synthesize tyrosine dipeptides. Parameters such as the reaction pH, magnesium chloride concentration, and substrate tyrosine concentration are optimized to develop a low-cost, efficient enzymatic synthesis process for three tyrosine dipeptides: propyltyrosine dipeptide, glytyrosine dipeptide, and serinetyrosine dipeptide.

[0074] The beneficial effects of the present invention include:

[0075] 1. Through rational design and modification at the structural level, high-performance catalytic enzymes of propyl-tyrosine dipeptide, glyceryl-tyrosine dipeptide, and serine-tyrosine dipeptide were obtained;

[0076] 2. Introducing a low-cost ATP regeneration system into the enzymatic synthesis of acylglycerol dipeptide, glycosylglycerol dipeptide, and serine dipeptide to reduce material costs;

[0077] 3. Add the insoluble tyrosine into the reaction system in the form of mother liquor flow addition, which greatly increases the concentration of substrate and product of the reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0079] Figure 1 Shown is the BsBacD-ADP-POD complex structure;

[0080] Figure 2 Shows the local substrate pocket formed by Leu12, Trp76, Asn108, and Leu110;

[0081] Figure 3 Shown is the molecular docking model of BsBacD-ADP-Ala and Phe;

[0082] Figure 4 A partial enlarged view of the BsBacD-ADP-Ala and Phe molecular docking model #2 is shown;

[0083] Figure 5 Shows the protein electrophoresis results of BsBacD mutants;

[0084] Figure 6 Figure 2 shows the protein electrophoresis results of BsBacD-N108S_L110S small-tank fermentation; “whole” represents the sample after disruption;

[0085] Figure 7 The reaction formula for the enzymatic synthesis of Ala-Tyr is shown;

[0086] Figure 8 Results of optimization of Ala-Tyr reaction pH;

[0087] Figure 9 Results of optimization of MgCl2 concentration in Ala-Tyr reaction;

[0088] Figure 10 Results of optimization of substrate tyrosine concentration in Ala-Tyr reaction;

[0089] Figure 11 Reaction formula of enzymatic synthesis of Gly-Tyr;

[0090] Figure 12 Results of optimization of glycine equivalent in Gly-Tyr reaction;

[0091] Figure 13 Reaction formula of enzymatic synthesis of Ser-Tyr. DETAILED DESCRIPTION

[0092] The present application discloses mutants of BsBacD enzyme, enzyme compositions and methods for preparing tyrosine dipeptides. Those skilled in the art can refer to the content herein and make appropriate improvements to the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0093] The present application provides enzymes and their applications, and methods for synthesizing dipeptides. First, the present application changes the substrate specificity of BsBacD enzyme and improves the catalytic performance at the structural level through rational design, thereby improving the conversion rate of the reaction.

[0094] Secondly, ADP is replaced by relatively inexpensive ATP or even cheaper AMP, and polyphosphate kinase ChPPK2 capable of regenerating ATP using AMP as a substrate is introduced. Compared with the regeneration system of ADP→ATP, the introduced ChPPK2 can also pull the AMP degraded by the heterologous phosphatase back into the regeneration system, thereby reducing the total consumption of adenosine phosphate (AMP+ADP+AMP) and further reducing the material cost.

[0095] Finally, the poorly soluble tyrosine is configured into a high-concentration alkaline mother liquor and added to the reaction system in a flow addition manner, which greatly improves the substrate and product concentrations in the reaction, thereby improving the production efficiency and reducing the labor and manufacturing costs.

[0096] The related sequences involved in the present application are as follows:

[0097] 1. Amino acid sequence of BsBacD derived from Bacillus subtilis (as shown in SEQ ID No. 1):

[0098] MERKTVLVIADLGGCPPHMFYKSAAEKYNLVSFIPRPFAITASHAALIEKYSVAVIKDKDYFKSLADFEHPDSIYWAHEDHNKPEEEVVEQIVKVAEMFGADAITTNNELFIAPMAKACERLGLRGAGVQAAENARDKNKMRDAFNKAGVKSIKNKRVTTLEDFRAALEEIGTPLILKPTYLASSIGVTLITDTETAEDEFNRVNDYLKSINVPKAVTFEAPFIAEEFLQGEYGDWYQTEGYSDYISIEGIMADGEYFPIAIHDKTPQIGFTETSHITPSILDEEAKKKIVEAAKKANEGLGLQNCATHTEIKLMKNREPGLIESAARFAGWNMIPNIKKVFGLDMAQLLLDVLCFGKDADLPDGLLDQEPYYVADCHLYPQHFKQNGQIPETAEDLVIEAIDIPDGLLKGDTEIVSFSAAAPGTSVDLTLFEAFNSIAAFELKGSNSQDVAESIRQIQQHAKLTAKYVLPV

[0099] 2. Nucleotide sequence encoding BsBacD (as shown in SEQ ID No. 2):

[0100]

[0101] 3. The nucleotide sequence of the upstream primer for constructing the BsBacD-N108S mutant plasmid (as shown in SEQ ID No. 3):

[0102] ATGCGATCACAACTAACAGTGAACTGTTTATCGCTCCG

[0103] 4. The nucleotide sequence of the downstream primer for constructing the BsBacD-N108S mutant plasmid (as shown in SEQ ID No. 4):

[0104] GGAGCGATAAACAGTTCACTGTTAGTTGTGATCGCATC

[0105] 5. The nucleotide sequence of the upstream primer for constructing the BsBacD-L110N mutant plasmid (as shown in SEQ ID No. 5):

[0106] TCACAACTAACAATGAAAATTTTATCGCTCCGATGG

[0107] 6. The nucleotide sequence of the downstream primer for constructing the BsBacD-L110N mutant plasmid (as shown in SEQ ID No. 6):

[0108] GCCATCGGAGCGATAAAATTTTTCATTGTTAGTTGTG

[0109] 7. The nucleotide sequence of the upstream primer for constructing the BsBacD-L110S mutant plasmid (as shown in SEQ ID No. 7):

[0110] TCACAACTAACAATGAAAGTTTTATCGCTCCGATGG

[0111] 8. The nucleotide sequence of the downstream primer for constructing the BsBacD-L110S mutant plasmid (as shown in SEQ ID No. 8):

[0112] GCCATCGGAGCGATAAAACTTTCATTGTTAGTTGTG

[0113] 9. The nucleotide sequence of the upstream primer for constructing the BsBacD-N108S_L110S mutant plasmid (as shown in SEQ ID No. 9):

[0114] TGCGATCACAACTAACAGTGAAAGTTTTATCGCTCCGATGG

[0115] 10. The nucleotide sequence of the downstream primer for constructing the BsBacD-N108S_L110S mutant plasmid (as shown in SEQ ID No. 10):

[0116] GCCATCGGAGCGATAAAACTTTCACTGTTAGTTGTGATCGC

[0117] 11. Amino acid sequence of ChPPK2 from Cytophaga hutchinsonii (as shown in SEQ ID No. 11):

[0118] MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAMDAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPATGEIGIFNRSHYENVLVTRVHPEYLLSE QTSGVTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKERAHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSLEQKAELEKAKAELVAEKSSD

[0119] 12. Nucleotide sequence encoding ChPPK2 (as shown in SEQ ID No. 12):

[0120] ATGGCCACCGATTTCTCCAAATTGAGCAAGTACGTGGAAACCCTGCGCGTTAAACCCAAACAGAGCATTGATTTAAAAAAGGACTTCGACACGGATTACGATCACAAAATGCTGACTAAGGAGGAAGGCGAGGAACTGTTGAACTTAGGTATTAGTAAGCTGAGCGAGATCCAGGAAAAGTTATACGCTAGCGGCACTAAATCGGTGCTGATTGTGTTCCAGGCAATGGACGCAGCTGGCAAAGATGGCACGGTAAAGCATATCATGACGGGCCTTAACCCGCAGGGCGTCAAGGTCACTTCCTTTAAGGTTCCGTCCAAGATCGAGTTAAGTCATGATTACCTGTGGCGTCATTATGTTGCCTTGCCTGCTACCGGTGAAATCGGCATCTTTAATCGCTCACATTATGAGAATGTTCTTGTAACTCGCGTCCACCCCGAGTATCTGTTATCGGAACAAACGAGCGGTGTGACAGCCATCGAGCAGGTCAATCAGAAGTTTTGGGACAAGCGCTTTCAGCAAATTAATAACTTCGAACAGCACATCAGTGAGAACGGGACGATCGTTCTTAAATTCTTTCTTCACGTCAGCAAAAAAGAGCAAAAAAAGCGCTTTATTGAGCGCATTGAACTTGACACTAAAAACTGGAAGTTCTCTACGGGTGACCTGAAGGAGCGTGCTCATTGGAAGGACTATCGCAACGCATATGAAGATATGCTGGCTAACACTTCGACAAAGCAAGCGCCCTGGTTCGTGATTCCGGCAGATGATAAGTGGTTTACGCGTTTATTAATTGCAGAAATTATCTGTACAGAACTGGAGAAACTTAATCTTACATTTCCAACTGTCTCTTTGGAGCAAAAGGCAGAACTTGAAAAAGCCAAGGCCGAGTTAGTGGCCGAAAAGAGTTCCGAC

[0121] The mutant of BsBacD enzyme, enzyme composition and preparation method of tyrosine dipeptide provided by the application can use the market-purchased anticipation and reagent.

[0122] The amino acid raw materials were purchased from Hebei Huayang Biology;

[0123] Sodium hexametaphosphate was purchased from Hubei Xingfa Chemical;

[0124] ATP-Na2 and AMP were purchased from Anhui CuiNiao Biology;

[0125] Magnesium chloride hexahydrate was purchased from Jiangsu Longtaiwei Food.

[0126] The application will be further described below in combination with examples:

[0127] Example 1 Modification of BsBacD enzyme substrate selectivity

[0128] By analyzing the BsBacD-ADP-P0D (PDB:3VMM) complex structure, Figure 1 Leu12, Trp76, Asn108 and Leu110 form a local substrate pocket Figure 2 which interacts with the imidazole ring of the reaction intermediate analog P0D, and simulates the binding with the real substrate Phe. It can be seen that the terminal NH2 group of Asn108 will limit larger substrates such as Tyr.

[0129] In order to observe the more extensive residues involved in the process of substrate Phe entering the enzyme active pocket, the BsBacD-ADP-Ala (PDB:3WO0) was used as the target and Phe as the ligand, and AutoDockVina was used for molecular docking Figure 3 Among the 20 generated models, one model Figure 4 shows that the distance between Leu110 and Phe is reduced from 4.7 angstroms in the BsBacD-ADP-P0D complex structure to 3.6 angstroms, which may also be directly involved in the substrate specificity of the enzyme.

[0130] The primers shown in Table 1 were used to construct different mutant plasmids with pET28a-BsBacD as the template, which were respectively transformed into BL21(DE3) competent cells, then expanded in LB liquid medium (Kan), and induced to express under the condition of 20℃ and IPTG 0.1mM. The bacterial cells were collected by centrifugation, resuspended with the addition of crushing buffer (20mM K2HPO4 / KH2PO4, 100mM KCl, pH7.6) = 1:9 (w / v), and then ultrasonically crushed and high-speed centrifuged. The protein electrophoresis detection was performed on the supernatant and precipitate samples, and the wild type and different mutants of BsBacD enzyme were expressed in the supernatant Figure 5). The crude enzyme solution was diluted 50 times with the lysis buffer, and 100 μL of the diluted solution was added to 10 mL of Ala-Tyr assay solution (Tyr 10 mM, Ala 30 mM, ATP-Na2 15 mM, MgCl2 20 mM, pH 9) and reacted at 38°C for 10 min. The amount of the product Ala-Tyr was determined by high performance liquid chromatography (HPLC), and the enzyme activity was calculated.

[0131] As shown in Table 1, the enzyme activity of the BsBacD mutant N108S_L110S was increased by 404% compared to the wild type (WT).

[0132] Table 1

[0133]

[0134] Example 2 Enzymatic synthesis of propyl tyrosine dipeptide

[0135] 1. Optimization of reaction pH

[0136] BsBacD-N108S_L110S BL21(DE3) and ChPPK2 BL21(DE3) stored in glycerol tubes were inoculated into LB liquid medium (Kan) for expansion and then inoculated into small tanks for fermentation. The fermentation medium formula was as follows:

[0137] Table 2

[0138]

[0139]

[0140] When the biomass OD 600 was 20-30, the temperature was lowered to 20°C, and then 0.1 mM IPTG was added to induce expression for 20-24 h, and the tank OD 600 was 80-120. The bacterial cells were collected by centrifugation and stored at -20°C for later use. The supernatant of BsBacD-N108S_L110S had a high expression level Figure 6 ).

[0141] Take 19.58 g (40 mM, (reaction volume, a total of 800 mL (ignore the volume impact of the crude enzyme solution))) of sodium hexametaphosphate, 12.12 g (170 mM) of Ala, 6.51 g (40 mM) of magnesium chloride hexahydrate, and 0.88 g (2 mM) of adenosine triphosphate disodium (ATP-Na2) in 500 mL of pure water, divide into 4 portions, adjust the pH to 6.5, 7.5, 8.5, and 9.5 respectively, and make up to 150 mL, preheat at 38°C, then add the wet bacteria according to BsBacD-N108S_L110S or ChPPK2:crushing buffer = 1:4 (w / v) resuspended after homogenization crushing to obtain BsBacD-N108S_L110S crude enzyme solution 7.6 mL (i.e. ~7 U / mL in the reaction solution), ChPPK2 crude enzyme solution 2 mL (~3 U / mL), Tyr 400 mM pH 12.5 stock solution 50 mL at a rate of 1 mL / min. The enzyme activity of the fermentation bacteria used is 900 U / g for BsBacD-N108S_L110S and 1700 U / g for ChPPK2. The wet weight of the bacteria used is the wet weight of the bacteria after shake flask or fermentation culture, centrifugal collection of the bacteria, and weighing of the wet weight.

[0142] Figure 7 The reaction formula for the enzymatic synthesis of Ala-Tyr is shown. As shown in Figure 8 The Tyr→Ala-Tyr conversion rate of the pH 8.5 group is the highest, reaching 91.9% after 2 h of reaction, while the conversion rates of the pH 7.5 group and the pH 9.5 group are only 69.0% and 40.4% respectively after 2 h of reaction, indicating that the suitable pH of BsBacD-N108S_L110S enzyme is relatively narrow.

[0143] 2. Optimization of magnesium chloride concentration

[0144] Take 19.58 g (40 mM) of sodium hexametaphosphate, 12.12 g (170 mM) of Ala, and 0.88 g (2 mM) of adenosine triphosphate disodium (ATP-Na2) in 500 mL of pure water, divide into 4 portions, dissolve 1.63 g (40 mM), 2.44 g (60 mM), 3.25 g (80 mM), and 4.07 g (100 mM) of magnesium chloride hexahydrate respectively, adjust the pH to 8.5, and make up to 150 mL, preheat at 38°C, then add the wet bacteria according to BsBacD-N108S_L110S or ChPPK2:crushing buffer = 1:4 (w / v) resuspended after homogenization crushing to obtain BsBacD-N108S_L110S crude enzyme solution 8 mL (~7 U / mL), ChPPK2 crude enzyme solution 2 mL (~3 U / mL), Tyr 400 mM pH 12.5 stock solution 50 mL at a rate of 1 mL / min. The enzyme activity of the fermentation bacteria used is 900 U / g for BsBacD-N108S_L110S and 1700 U / g for ChPPK2.

[0145] As shown in Figure 9 Figure 6, the conversion rate of Tyr→Ala-Tyr was the highest in the 60 mM group of magnesium chloride 60 mM, and the conversion rate reached 95.0% after 2 h of reaction. In comparison, the conversion rates of the 40 mM group and the 80 mM group were 91.9% and 89.0% respectively after 2 h of reaction. The Tyr residue was lower in the 60 mM group, which was easier to purify.

[0146] 3. Optimization of the substrate alanine equivalent

[0147] Sodium hexametaphosphate 19.58 g (40 mM), magnesium chloride hexahydrate 9.76 g (60 mM), adenosine triphosphate disodium (ATP-Na2) 0.88 g (2 mM) were weighed into 500 mL of pure water, divided into 4 portions, and Ala 1.96 g (110 mM), 2.32 g (130 mM), 2.67 g (150 mM), and 3.03 g (170 mM) were weighed and dissolved, respectively, adjusted to 8.5 and diluted to 150 mL. After preheating at 38°C, 8 mL of crude enzyme solution of BsBacD-N108S_L110S (~ 7 U / mL) and 2 mL of crude enzyme solution of ChPPK2 (~ 3 U / mL) obtained by resuspending the wet bacterial cells in the lysis buffer at a ratio of 1:4 (w / v) and homogenizing were added, and 50 mL of Tyr 400 mM pH 12.5 stock solution was added at a rate of 1 mL / min. The enzyme activity of the fermentation bacterial cells used was 870 U / g for BsBacD-N108S_L110S and 1500 U / g for ChPPK2.

[0148] As shown in Table 3, when the Ala:Tyr equivalent was greater than or equal to 1.5, it could better promote the conversion of Tyr→Ala-Tyr, but excessive Ala would lead to excessive organic nitrogen in the wastewater. In summary, the Ala:Tyr equivalent of 1.5 was more appropriate.

[0149] Table 3

[0150] Ala:Tyr equivalent 1h conversion rate 2h conversion rate 1.1 80.7％ 85.8％ 1.3 83.1％ 91.3％ 1.5 85.4％ 93.6％ 1.7 86.0％ 95.0％

[0151] 4. Optimization of the substrate tyrosine concentration

[0152] Dissolve 19.58 g (40 mM) of sodium hexametaphosphate, 9.76 g (60 mM) of magnesium chloride hexahydrate, and 0.88 g (2 mM) of adenosine triphosphate disodium (ATP-Na2) in 500 mL of pure water, divide into 4 portions, and respectively take 2.67 g (150 mM) of Ala, 3.34 g (187.5 mM) of Ala, 4.01 g (225 mM) of Ala, and 4.68 g (262.7 mM) of Ala. Adjust the pH to 8.5 and make up to 150 mL. After preheating at 38°C, add 8 mL of crude enzyme solution of BsBacD-N108S_L110S (about 7 U / mL) and 2 mL of crude enzyme solution of ChPPK2 (about 3 U / mL) obtained by resuspending the wet bacterial cells in the lysis buffer at a ratio of 1:4 (w / v) and then ultrasonic lysis. Add 50 mL of Tyr 400 mM, 500 mM, 600 mM, and 700 mM pH 11.5 stock solution to the Ala 150 mM, 187.5 mM, 225 mM, and 262.5 mM reaction solutions at a rate of 1 mL / min, respectively. The fermentation bacterial cells used are BsBacD-N108S_L110S with an enzyme activity of 900 U / g and ChPPK2 with an enzyme activity of 1500 U / g.

[0153] As shown in Figure 10 When the concentration of tyrosine in the reaction solution does not exceed 125 mM, the conversion rate of Tyr to Ala-Tyr is higher than 99% after 2 hours of reaction. However, when the concentration of Tyr is 150 mM and 175 mM, the conversion rates after 2 hours of reaction are 88.7% and 77.9%, respectively. When the reaction time is extended to 3 hours, the conversion rate of Tyr 150 mM group is only 92.4%, indicating that the concentration of substrate Tyr 150 mM is too high, and 125 mM is more appropriate. At the same time, when the pH of Tyr stock solution is reduced from 12.5 to 11.5, the conversion rate after 2 hours of reaction is increased from 93.6% in the alanine equivalent optimization experiment to 99.6%, which may be due to the reduction of the influence of pH change in the reaction solution on the enzyme.

[0154] 5. Laboratory 3L scale-up

[0155] Take 73.41 g (40 mM) of sodium hexametaphosphate, 50.11 g (187.5 mM) of Ala, 36.59 g (60 mM) of magnesium chloride hexahydrate, 1.65 g (1 mM) of adenosine triphosphate disodium (ATP-Na2) in 1800 mL of pure water, adjust the pH to 8.5 and make up to 2250 mL, preheat to 38°C, then add 86 mL of crude enzyme solution of BsBacD-N108S_L110S (~5 U / mL, cell concentration 5.7 g / L) and 30 mL of crude enzyme solution of ChPPK2 (~3 U / mL, cell concentration 2 g / L) obtained by resuspending the wet cells in 4 times (w / v) of the breaking buffer and then homogenizing, and add 750 mL of Tyr 500 mM pH 11.5 stock solution at a rate of 13 mL / min. The fermentation cells used are BsBacD-N108S_L110S with an enzyme activity of 900 U / g and ChPPK2 with an enzyme activity of 1500 U / g.

[0156] The Tyr→Ala-Tyr conversion rates at 0.5 h, 1 h, 1.5 h and 2 h are 96.9%, 90.5%, 98.8% and 100%, respectively. The substrate concentration, conversion rate and reaction time are all superior to the reported laboratory results.

[0157] 6. Laboratory 6L scale-up

[0158] Take 73.41 g (40 mM) of sodium hexametaphosphate, 50.11 g (187.5 mM) of Ala, 36.59 g (60 mM) of magnesium chloride hexahydrate, 1.65 g (1 mM) of adenosine triphosphate disodium (ATP-Na2) in 1800 mL of pure water, adjust the pH to 8.5 and make up to 2250 mL, preheat to 38°C, then add 86 mL of crude enzyme solution of BsBacD-N108S_L110S (~5 U / mL, cell concentration 5.7 g / L) and 30 mL of crude enzyme solution of ChPPK2 (~3 U / mL, cell concentration 2 g / L) obtained by resuspending the wet cells in 4 times (w / v) of the breaking buffer and then homogenizing, and add 750 mL of Tyr 500 mM pH 11.5 stock solution at a rate of 13 mL / min. The fermentation cells used are BsBacD-N108S_L110S with an enzyme activity of 900 U / g and ChPPK2 with an enzyme activity of 1500 U / g.

[0159] The Tyr→Ala-Tyr conversion rates at 0.5 h, 1 h, 1.5 h and 2 h are 96.9%, 90.5%, 98.8% and 100%, respectively. The substrate concentration, conversion rate and reaction time are all superior to the reported laboratory results.

[0160] Example 3 Enzymatic synthesis of glycyl-tryptophan dipeptide

[0161] 1. Optimization of the substrate glycine equivalent

[0162] Take 14.68 g (40 mM) of sodium hexametaphosphate, 7.32 g (60 mM) of magnesium chloride hexahydrate, 0.66 g (2 mM) of adenosine triphosphate disodium (ATP-Na2) in 360 mL of pure water, divide into 3 parts, and take Gly 1.95 g (130 mM), Gly 2.25 g (150 mM), and Gly 2.55 g (170 mM) respectively, adjust pH to 8.5 and dilute to 150 mL, preheat at 38°C, then add 10 mL of the crude enzyme solution of BsBacD-N108S_L110S obtained by resuspending the wet cells according to the ratio of wet cells to breaking buffer = 1:4 (w / v) and homogenizing, 2 mL of the crude enzyme solution of ChPPK2, 50 mL of Tyr 400 mM mother liquor at a rate of 0.5 mL / min. The enzyme activity of the fermentation cells used is 900 U / g for BsBacD-N108S_L110S and 1500 U / g for ChPPK2. The wet weight of the cells used is the weight of the cells collected by centrifugation after shake flask or fermentation culture of BsBacD-N108S_L110S or ChPPK2 cells.

[0163] Figure 11 The reaction formula for the enzymatic synthesis of Gly-Tyr is shown as follows. Figure 12 As shown in the following table, when the Gly:Tyr equivalent is greater than or equal to 1.5, the conversion rate of Tyr to Gly-Tyr is higher than 97% after 3 hours of reaction. Compared with the optimization experiment results of the substrate alanine equivalent in Example 2, the conversion rate of 50.0% of the Gly:Tyr equivalent 1.3 group after 1 hour of reaction is much lower than that of the Gly:Tyr 1.5 group of 90.5%, which indicates that the low excess substrate has a slower positive driving effect on the reaction of Gly-Tyr compared with Ala-Tyr.

[0164] 2. Laboratory 3L scale-up

[0165] Take 14.68 g (40 mM) of sodium hexametaphosphate, 7.32 g (60 mM) of magnesium chloride hexahydrate, 0.66 g (2 mM) of adenosine triphosphate disodium (ATP-Na2) in 360 mL of pure water, divide into 3 parts, and take Gly 1.95 g (130 mM), Gly 2.25 g (150 mM), and Gly 2.55 g (170 mM) respectively, adjust pH to 8.5 and dilute to 150 mL, preheat at 38°C, then add 10 mL of the crude enzyme solution of BsBacD-N108S_L110S obtained by resuspending the wet cells according to the ratio of wet cells to breaking buffer = 1:4 (w / v) and homogenizing, 2 mL of the crude enzyme solution of ChPPK2, 50 mL of Tyr 400 mM mother liquor at a rate of 0.5 mL / min. The enzyme activity of the fermentation cells used is 900 U / g for BsBacD-N108S_L110S and 1500 U / g for ChPPK2. The wet weight of the cells used is the weight of the cells collected by centrifugation after shake flask or fermentation culture of BsBacD-N108S_L110S or ChPPK2 cells.

[0166] Reaction 1h, 2h, 3h, Tyr→Gly-Tyr conversion rate was 94.9%, 94.9%, 95.7%, respectively.

[0167] 3. Laboratory 3L scale-up

[0168] Sodium hexametaphosphate 73.41 g (40 mM), Gly 33.78 g (150 mM), magnesium chloride hexahydrate 36.59 g (60 mM), adenosine monophosphate (AMP) 1.04 g (1 mM) were weighed into 1800 mL of pure water, dissolved, adjusted to pH 8.5 and made up to 2250 mL. After preheating at 38°C, 168 mL of crude enzyme solution of BsBacD-N108S_L110S (~ 10 U / mL, cell concentration 11.2 g / L) and 30 mL of crude enzyme solution of ChPPK2 (~ 3 U / mL, cell concentration 2 g / L) obtained by resuspending the wet cells in the ratio of 1:4 (w / v) and homogenizing were added. The Tyr 400 mM stock solution was added at a rate of 7.5 mL / min. The fermentation cells used were BsBacD-N108S_L110S with an enzyme activity of 900 U / g and ChPPK2 with an enzyme activity of 1700 U / g.

[0169] Reaction 1h, 2h, 3h, Tyr→Gly-Tyr conversion rate was 95.9%, 94.3%, 96.2%, respectively.

[0170] Example 4 Enzymatic synthesis of serine tyrosyl dipeptide

[0171] 1. Laboratory 3L scale-up

[0172] Sodium hexametaphosphate 73.41 g (40 mM), Ser 47.29 g (150 mM), magnesium chloride hexahydrate 36.59 g (60 mM), adenosine triphosphate disodium (ATP-Na2) 1.65 g (1 mM) were weighed into 1800 mL of pure water, dissolved, adjusted to pH 8.5 and made up to 2250 mL. After preheating at 38°C, 135 mL of crude enzyme solution of BsBacD-N108S_L110S (~ 9 U / mL, cell concentration 9 g / L) and 30 mL of crude enzyme solution of ChPPK2 (~ 3 U / mL, cell concentration 2 g / L) obtained by resuspending the wet cells in the ratio of 1:4 (w / v) and homogenizing were added. The Tyr 400 mM stock solution was added at a rate of 8 mL / min. The fermentation cells used were BsBacD-N108S_L110S with an enzyme activity of 1000 U / g and ChPPK2 with an enzyme activity of 1500 U / g. The wet weight of the cells used was obtained by centrifuging the cells after shake flask or fermentation culture, and weighing the wet weight of the cells.

[0173] Figure 13The enzyme synthesis of Ser-Tyr is shown. The Tyr→Ser-Tyr conversion rates at 1h, 2h, 3h, 4h are 62.3%, 80.7%, 86.0%, 88.4%, respectively.

[0174] 2. Laboratory 3L scale-up

[0175] Sodium hexametaphosphate 73.41g (40mM), Ser 47.29g (150mM), magnesium chloride hexahydrate 36.59g (60mM), adenosine monophosphate (AMP) 1.04g (1mM) were weighed and dissolved in 1800mL pure water, adjusted to pH 8.5 and constant volume to 2250mL, 38℃ preheating, then added 135mL of BsBacD-N108S_L110S crude enzyme solution (~9U / ml, cell concentration 9g / L) and 30mL of ChPPK2 crude enzyme solution (~3U / mL, cell concentration 2g / L) obtained by resuspending the wet cell according to the ratio of cell:crushing buffer = 1:4 (w / v) and homogenizing, Tyr 400mM stock solution 750mL was added at a rate of 8mL / min. The fermentation cell used was BsBacD-N108S_L110S with an enzyme activity of 1000U / g and ChPPK2 with an enzyme activity of 1500U / g.

[0176] The Tyr→Ser-Tyr conversion rates at 1h, 2h, 3h, 4h are 76.5%, 81.0%, 87.3%, 89.2%, respectively.

[0177] The above only describes the preferred embodiments of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make several improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Mutants of BsBacD enzyme characterized in that: The mutant is simultaneously mutated at N108S and L110S based on the amino acid sequence shown as SEQ ID No.

1.

2. A nucleic acid molecule, characterized in that, The mutant as claimed in claim 1.

3. Enzyme composition, characterized in that, The mutant as claimed in claim 1 and polyphosphate kinase.

4. The enzyme composition of claim 3, wherein, The amino acid sequence of the polyphosphate kinase is shown as SEQ ID No.

11.

5. A nucleic acid molecule, characterized in that, The enzyme composition as claimed in claim 3 or 4; The nucleotide sequence of the nucleic acid molecule encoding the polyphosphate kinase is shown as SEQ ID No.

12.

6. An expression cassette characterized in that, The nucleic acid molecule as claimed in claim 2 or 5.

7. A plasmid characterized in that, The expression cassette as claimed in claim 6.

8. A bacterial cell, characterized in that, The bacterial cell: (A) expressing the mutant as claimed in claim 1; and / or (B) transformed with the expression cassette as claimed in claim 6; and / or (C) transformed with the plasmid as claimed in claim 7.

9. A composition for the synthesis of a tyrosine dipeptide, characterized in that, The composition comprises: The enzyme composition as claimed in claim 3 or 4; and The composition comprises adenosine triphosphate or a salt thereof, or adenosine monophosphate or a salt thereof, but does not comprise adenosine diphosphate or a salt thereof; and The alkaline mother liquor of tyrosine.

10. The composition of claim 9, wherein The pH of the composition is 8.

5.

11. The composition of claim 9, wherein The concentration of the alkaline mother liquor of tyrosine is Tyr 400 mM or 500 mM.

12. The composition of claim 9, wherein The concentration of the alkaline mother liquor of tyrosine is Tyr 500 mM.

13. The composition of claim 9, wherein The composition further comprises magnesium chloride.

14. The composition of claim 9, wherein The concentration of the magnesium chloride is between 40 mM and 100 mM.

15. The composition of claim 9, wherein The concentration of the magnesium chloride is 60 mM.

16. Use of any of the following in the preparation of a tyrosine dipeptide: (i) the mutant as claimed in claim 1; and / or (ii) the nucleic acid molecule as claimed in claim 2 or 5; and / or (iii) the enzyme composition as claimed in claim 3 or 4; and / or (iv) the expression cassette as claimed in claim 6; and / or (v) the plasmid as claimed in claim 7; and / or (vi) the bacterial cell as claimed in claim 8; (vii) the composition as claimed in any one of claims 9 to 15; The tyrosine dipeptide comprises one or more of propyl-tyrosine dipeptide, glycyl-tyrosine dipeptide and / or seryl-tyrosine dipeptide.

17. A method for the preparation of a tyrosine dipeptide, characterized in that, The composition comprises: (a) mixing amino acids with the mutant as claimed in claim 1 or the enzyme composition as claimed in claim 3 or 4 to obtain a dipeptide; or (b) expressing the nucleic acid molecule as claimed in claim 2 or 5, mixing the obtained protein product with amino acids to obtain a dipeptide; or (c) expressing the expression cassette as claimed in claim 6 or the plasmid as claimed in claim 7, mixing the obtained protein product with amino acids to obtain a dipeptide; or (d) culturing the bacterial cell as claimed in claim 8, mixing the obtained protein product with amino acids to obtain a dipeptide; or (e) mixing the composition as claimed in any one of claims 9 to 15 with amino acids to obtain a dipeptide; The tyrosine dipeptide comprises one or more of propyl-tyrosine dipeptide, glycyl-tyrosine dipeptide and / or seryl-tyrosine dipeptide. The amino acids are selected from tyrosine, alanine, glycine or serine.

18. The production method according to claim 17, wherein The mixing comprises feeding the alkaline mother liquor of tyrosine.

19. The production method according to claim 18, wherein The feeding speed is between 0.5 mL / min and 26 mL / min.

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