Mutant of BsBacD enzyme, enzyme composition and preparation method of tyrosine dipeptide

Abstract

The invention relates to the technical field of enzymes, in particular to a BsBacD enzyme mutant, an enzyme composition and a preparation method of tyrosine dipeptide. The method comprises the following steps: rationally designing BsBacD enzyme, replacing ADP (adenosine diphosphate or salt thereof) with relatively cheap ATP (adenosine triphosphate or salt thereof) or relatively cheap AMP (adenosine monophosphate or salt thereof), and introducing polyphosphate kinase ChPPK2 capable of regenerating ATP by taking AMP as a substrate to synthesize tyrosine dipeptide; parameters such as reaction pH, magnesium chloride concentration, substrate tyrosine concentration and the like are optimized, and a low-cost and efficient enzymatic synthesis process of three tyrosine dipeptides such as propyl tyrosine dipeptide, glycyl tyrosine dipeptide and sericin tyrosine dipeptide is developed.

Description

Technical Field

[0001] The invention relates to the technical field of enzymes, and in particular to a mutant of a BsBacD enzyme, an enzyme composition and a preparation method of a tyrosine dipeptide. Background Art

[0002] Tyrosine (Tyr) is one of the three aromatic amino acids and is synthesized in vivo from phenylalanine (Phe) by phenylalanine hydroxylase (PheOH) (Erlandsen et al. 1997). Supplementing tyrosine in cell culture medium can effectively eliminate sequence variations such as Tyr→Phe and Tyr→His (histidine) 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. The mother solution 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 greatly reduce the complexity of process control and improve the yield and activity of monoclonal antibodies (Erdmann et al. 2006, Kang et al. 2012). Ala-Tyr (acryloyl-tyrosine dipeptide) and Gly-Tyr (glycolyl-tyrosine dipeptide) are the two most widely promoted tyrosine dipeptides on the market. Representative manufacturers include Germany's Evonik, Chengdu Baishixing, Wuhan Jitide / Hubei Hongpeptide, Wuxi Jingyao and Anhui Herun.

[0003] Kyowa Hakko of Japan screened the first L-amino acid ligase (BsBacD) from the domain set PS 50975. Its substrate N-terminal amino acids tend to be Ala (alanine), Gly (glycine), and Ser (serine), and its C-terminal amino acids tend to be Phe and Met (methionine) (Tabata, Ikeda, and Hashimoto 2005). Song Wenlu's team at Jining University coupled BsBacD enzyme with polyphosphate kinase SlPPK2, which regenerates ATP (adenosine triphosphate) using ADP (adenosine diphosphate) as substrate, and used 45mM Ala, 45mM Tyr, 6mM ADP and 20mM PolyP 6 (Hexametaphosphate) as the substrate, 40.1mM Ala-Tyr was generated in 3h with a conversion rate of 89.1% (Cui et al. 2023). However, this work is only a laboratory result. The residual tyrosine at the end of the reaction cannot be removed by simple membrane separation during the purification process, thus affecting the crystallization of the product Ala-Tyr. At the same time, the product concentration of 10.1g / L (40.1mM) is too low and the substrate ADP is expensive, resulting in high production costs, which makes the enzymatic synthesis of Ala-Tyr difficult to industrialize. Summary of the invention

[0004] In view of this, the present invention provides a mutant of BsBacD enzyme, an enzyme composition and a preparation method of tyrosine dipeptide. After a large number of design screening and experimental verification, a low-cost and efficient enzymatic synthesis process of three tyrosine dipeptides, namely, propyl tyrosine dipeptide, sugar tyrosine dipeptide and serine tyrosine dipeptide, was developed.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a mutant of the BsBacD enzyme, wherein the mutation sites of the mutant include N108S and L110S;

[0007] The BsBacD enzyme has:

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

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

[0011] In a second aspect, the present invention also provides a nucleic acid molecule encoding the mutant;

[0012] The nucleic acid molecule has:

[0013] (1), the nucleotide sequence shown in SEQ ID NO.2; or

[0014] (2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence 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 shown in (1) or (2).

[0016] In a third aspect, the present invention also provides an enzyme composition, comprising the mutant and polyphosphate kinase.

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

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

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

[0021] In a fourth aspect, the present invention also provides a nucleic acid molecule encoding the enzyme composition;

[0022] Wherein, the nucleic acid molecule encoding the polyphosphate kinase has:

[0023] (1), the nucleotide sequence shown in SEQ ID NO.12; or

[0024] (2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence 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 shown in (1) or (2).

[0026] In a fifth aspect, the present invention also provides an expression frame, including the nucleic acid molecule.

[0027] In a sixth aspect, the present invention also provides a plasmid comprising the expression cassette.

[0028] In a seventh aspect, the present invention further provides a bacterial cell, wherein:

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

[0030] (B), transformed or transfected with the expression cassette; and / or

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

[0032] In an eighth aspect, the present invention further provides a composition for synthesizing tyrosine dipeptide, comprising:

[0033] The enzyme composition; and

[0034] including adenosine triphosphate or its salt, or adenosine monophosphate or its salt, but excluding adenosine diphosphate or its salt; and

[0035] Alkaline stock solution of tyrosine;

[0036] Preferably, the pH of the composition is 6.5 to 9.5; more preferably, the pH of the composition is 8.5;

[0037] Preferably, the concentration of the alkaline mother solution of tyrosine includes Tyr 400mM, 500mM, 600mM, and 700mM; more preferably, the concentration of the alkaline mother solution of tyrosine is 500mM;

[0038] Preferably, the pH of the alkaline mother solution of tyrosine is 11.5 or 12.5; more preferably, the pH of the alkaline mother solution of tyrosine is 11.5;

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

[0040] In a ninth aspect, the present invention 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 frame; and / or

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

[0046] (vi), the bacterial cell;

[0047] (vii), the composition described;

[0048] The tyrosine dipeptide includes one or more of acyl tyrosine dipeptide, glycol tyrosine dipeptide and / or serine tyrosine dipeptide.

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

[0050] (a), mixing amino acids with the mutant or the enzyme composition to obtain a dipeptide; or

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

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

[0053] (d), culturing the bacteria, mixing the obtained protein product with amino acids to obtain a dipeptide; or

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

[0055] The tyrosine dipeptide includes one or more of acyl tyrosine dipeptide, glycol tyrosine dipeptide and / or serine tyrosine dipeptide;

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

[0057] In some specific embodiments of the present invention, the mixing comprises adding the alkaline mother solution of tyrosine; preferably, the adding speed comprises 0.5 mL / min to 26 mL / min.

[0058] In some specific embodiments of the present invention, a preheating step is included before the mixing, and the preheating temperature includes 38°C.

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

[0060] In some specific embodiments of the present invention, the culturing comprises a step of using a disruption buffer to disrupt the bacterial cells; preferably, the disruption buffer comprises 20 mM K 2 HPO 4 / KH 2 PO 4 and 100 mM KCl; preferably, the pH of the disruption buffer comprises 7.6;

[0061] In some specific embodiments of the present invention, the volume ratio of the wet weight of the bacterial cells to the disruption buffer is 1:4.

[0062] In some specific embodiments of the present invention, the concentration of added bacteria includes 7.7 g / L, 7.9 g / L, 13.2 g / L, and 11 g / L.

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

[0064] In some specific embodiments of the present invention, the enzyme activity of the polyphosphate kinase comprises 3 U / mL.

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

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

[0067] In some specific 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 110-262.5 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 alanine to tyrosine includes 1.1 to 1.7; preferably, the equivalent ratio of alanine to tyrosine includes 1.1, 1.3, 1.5 or 1.7; more preferably, the equivalent ratio of alanine to tyrosine includes 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 includes 1.3 to 1.7; preferably, the equivalent ratio of the glycine to the tyrosine includes 1.3, 1.5 or 1.7; more preferably, the equivalent ratio of the glycine to the tyrosine includes 1.5 or 1.7.

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

[0073] The present invention firstly rationally designs the BsBacD enzyme, replaces ADP with relatively cheap ATP or even cheaper AMP, introduces polyphosphate kinase ChPPK2 which can regenerate ATP with AMP as substrate to synthesize tyrosine dipeptide, optimizes parameters such as reaction pH, magnesium chloride concentration and substrate tyrosine concentration, and develops a low-cost and efficient enzymatic synthesis process of three tyrosine dipeptides, namely, propyltyrosine dipeptide, glytyrosine dipeptide and serinetyrosine dipeptide.

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

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

[0076] 2. Introduce low-cost ATP regeneration system into the enzymatic synthesis of propyl tyrosine dipeptide, glyceryl tyrosine dipeptide and serine tyrosine 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 drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[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 The protein electrophoresis results of the small-tank fermentation of BsBacD-N108S_L110S are shown; among them, "whole" represents the sample after disruption;

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

[0086] Figure 8 The results of pH optimization of Ala-Tyr reaction are shown;

[0087] Fig. 9 The figure shows the optimization results of magnesium chloride concentration in Ala-Tyr reaction;

[0088] Fig.10 The figure shows the optimization results of the tyrosine concentration of the Ala-Tyr reaction substrate;

[0089] Fig.11 The reaction formula for the enzymatic synthesis of Gly-Tyr is shown;

[0090] Fig.12 The optimization results of glycine equivalent in Gly-Tyr reaction are shown;

[0091] Fig.13 The reaction formula for enzymatic synthesis of Ser-Tyr is shown. DETAILED DESCRIPTION

[0092] The present invention discloses a mutant of BsBacD enzyme, an enzyme composition and a method for preparing tyrosine dipeptide. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the above. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0093] The present invention provides an enzyme and its application, and a method for synthesizing a dipeptide. Firstly, the present invention changes the substrate specificity of the BsBacD enzyme through rational design at the structural level, improves the catalytic performance, and thus improves the conversion rate of the reaction.

[0094] Secondly, ADP is replaced by cheaper ATP or even cheaper AMP, and the polyphosphate kinase ChPPK2 that can regenerate ATP using AMP as a substrate is introduced. Compared with the ADP→ATP regeneration system, it can also pull AMP degraded by miscellaneous protein phosphatases back into the regeneration system, thereby reducing the total amount of adenosine phosphate (AMP+ADP+AMP) used and thus reducing material costs.

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

[0096] The relevant sequences involved in the present invention are as follows:

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

[0098] MERKTVLVIADLGGCPPHMFYKSAAEKYNLVSFIPRPFAITASHAALIEKYSVAVIKDKDYFKSLADFEHPDSIYWAHEDHNKPEEEVVEQIVKVAEMFGADAITTNNELFIAPMAKA CERLGLRGAGVQAAENARDKNKMRDAFNKAGVKSIKNKRVTTLEDFRAALEEIGTPLILKPTYLASSIGVTLITDTETAEDEFNRVNDYLKSINVPKAVTFEAPFIAEEFLQGEYGDW YQTEGYSDYISIEGIMADGEYFPIAIHDKTPQIGFTETSHITPSILDEEAKKKIVEAAKKANEGLGLQNCATHTEIKLMKNREPGLIESAARFAGWNMIPNIKKVFGLDMAQLLLDVL CFGKDADLPDGLLDQEPYYVADCHLYPQHFKQNGQIPETAEDLVIEAIDIPDGLLKGDTEIVSFSAAAPGTSVDLTLFEAFNSIAAFELKGSNSQDVAESIRQIQQHAKLTAKYVLPV

[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. The 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] ATGGCCACCGATTTCTCCAAATTGAGCAAGTACGTGGAAACCCTGCGCGTTAAACCCAAACAGAGCATTGATTTAAAAAAGGACTTCGACACGGATTACGATCACAAAATGCTGACTAAGGAGGAAGGCGAGGAACTGTTGAACTTAGGTATTAGTAAGCTGAGCGAGATCCAGGAAAAGTTATACGCTAGCGGCACTAAATCGGTGCTGATTGTGTTCCAGGCAATG GACGCAGCTGGCAAAGATGGCACGGTAAAGCATATCATGACGGGCCTTAACCCGCAGGGCGTCAAGGTCACTTCCTTTTAAGGTTCCGTCCAAGATCGAGTTAAGTCATGATTACCTGTGGCGTCATTATGTTGCCTTGCCTGCTACCGGTGAAATCGGCATCTTTAATCGCTCACATTATGAGAATGTTCTTGTAACTCGCGTCCACCCCGAGTATCTGTTATCGGAAC AAACGAGCGGTGTGACAGCCATCGAGCAGGTCAATCAGAAGTTTTGGGACAAGCGCTTTCAGCAAATTAATAACTTCGAACAGCACATCAGTGAGAACGGGACGATCGTTCTTAAATTCTTTCTTCACGTCAGCAAAAAAGAGCAAAAAAAGCGCTTTATTGAGCGCATTGAACTTGACACTAAAAACTGGAAGTTCTCTACGGGTGACCTGAAGGAGCGTGCTCATTG GAAGGACTATCGCAACGCATATGAAGATATGCTGGCTAACACTTCGACAAAGCAAGCGCCCTGGTTCGTGATTCCGGCAGATGATAAGTGGTTTACGCGTTTATTAATTGCAGAAATTATCTGTACAGAACTGGAGAAACTTAATCTTACATTTTCCAACTGTCTCTTTGGAGCAAAAGGCAGAACTTGAAAAAGCCAAGGCCGAGTTAGTGGCCGAAAAGAGTTCCGAC

[0121] The preparation materials and reagents used in the mutant of BsBacD enzyme, enzyme composition and tyrosine dipeptide preparation method provided by the present invention can all be purchased from the market.

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

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

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

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

[0126] The present invention will be further described below in conjunction with embodiments:

[0127] Example 1 Modification of substrate selectivity of BsBacD enzyme

[0128] The BsBacD-ADP-P0D (PDB: 3VMM) complex structure ( Figure 1 ) analysis, Leu12, Trp76, Asn108, and Leu110 formed a local substrate pocket ( Figure 2 ), interacting with the imidazole ring of the reaction intermediate analog P0D, simulating the binding with the real substrate Phe, it can be seen that the terminal NH 2 Groups that would restrict 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, molecular docking was performed using AutoDockVina with BsBacD-ADP-Ala (PDB: 3WO0) as the target and Phe as the ligand ( Figure 3 ), among the 20 models generated, one of the models ( Figure 4 ) showed that the distance between Leu110 and Phe was reduced from 4.7 Å in the BsBacD-ADP-P0D complex structure to 3.6 Å, 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 template, and the plasmids were transformed into BL21 (DE3) competent cells, and then expanded and cultured in LB liquid medium (Kan), and induced to express under the conditions of 20°C and IPTG 0.1mM. The cells were collected by centrifugation and the wet cells: disruption buffer (20mM K 2 HPO 4 / KH 2 PO 4 ,100mM KCl,pH7.6)=1:9(w / v),resuspended in the disruption buffer, and then ultrasonically disrupted and centrifuged at high speed. The supernatant and precipitate were sampled for protein electrophoresis. The wild type and different mutants of BsBacD enzyme were expressed in the supernatant ( Figure 5The crude enzyme solution after ultrasonic disruption was diluted 50 times with disruption buffer, and 100 μL was added to 10 mL of Ala-Tyr activity test solution (Tyr 10 mM, Ala 30 mM, ATP-Na2 15 mM, MgCl 2 20 mM, pH 9), react at 38°C for 10 min, and detect the amount of product Ala-Tyr by high performance liquid chromatography (HPLC) to calculate the enzyme activity.

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

[0132] Table 1

[0133]

[0134] Example 2 Enzymatic Synthesis of Acetoacetyl Dipeptide

[0135] 1. Optimization of reaction pH

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

[0137] Table 2

[0138]

[0139]

[0140] When the biomass OD 600 =20-30, start cooling to 20°C, then add 0.1mM IPTG to induce expression for 20-24h, and place the tank in a jar. 600 =80-120. Collect the cells by centrifugation and store at -20°C for later use. The supernatant of BsBacD-N108S_L110S has high expression level ( Figure 6 ).

[0141] Weigh 19.58 g (40 mM) of sodium hexametaphosphate (reaction solution volume, a total of 800 mL (ignore the volume effect of crude enzyme solution)), 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) and dissolve in 500 mL of pure water, divide into 4 portions, adjust pH to 6.5, 7.5, 8.5, and 9.5 respectively, and make up to 150 mL, preheat at 38 ° C, add wet bacteria of BsBacD-N108S_L110S or ChPPK2: disruption buffer = 1:4 (w / v), resuspend and homogenize to obtain 7.6 mL (i.e., ~7 U / mL in the reaction solution) of BsBacD-N108S_L110S crude enzyme solution, 2 mL (~3 U / mL) of ChPPK2 crude enzyme solution, and Tyr 50 mL of 400 mM pH 12.5 mother solution was added at a rate of 1 mL / min. The fermentation bacteria used had an enzyme activity of 900 U / g for BsBacD-N108S_L110S and 1700 U / g for ChPPK2. The wet weight of the bacteria used was obtained by centrifugation after shaking flask or fermentation culture of BsBacD-N108S_L110S or ChPPK2, and weighing the wet weight.

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

[0143] 2. Optimization of magnesium chloride concentration

[0144] Weigh 19.58g (40mM) of sodium hexametaphosphate, 12.12g (170mM) of Ala, and 0.88g (2mM) of adenosine triphosphate disodium (ATP-Na2) and dissolve them in 500mL of pure water, divide them into 4 portions, weigh 1.63g (40mM), 2.44g (60mM), 3.25g (80mM), and 4.07g (100mM) of magnesium chloride hexahydrate and dissolve them respectively, adjust to 8.5 and make the volume to 150mL, preheat at 38°C, add 8mL (~7U / mL) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to wet bacteria: disruption buffer = 1:4 (w / v), 2mL (~3U / mL) of ChPPK2 crude enzyme solution, and 50mL of Tyr 400mM pH 12.5 mother solution at a rate of 1mL / min. The enzyme activity of the fermentation bacteria BsBacD-N108S_L110S used was 900U / g, and the enzyme activity of ChPPK2 was 1500U / g.

[0145] like Fig. 9 As shown, the conversion rate of Tyr→Ala-Tyr in the 60 mM magnesium chloride group was the highest, reaching 95.0% after 2 hours 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 hours. The residual Tyr in the 60 mM group was lower and easier to purify.

[0146] 3. Optimization of substrate alanine equivalent

[0147] Weigh 19.58g (40mM) of sodium hexametaphosphate, 9.76g (60mM) of magnesium chloride hexahydrate, and 0.88g (2mM) of adenosine triphosphate disodium (ATP-Na2) and dissolve them in 500mL of pure water, divide them into 4 portions, weigh 1.96g (110mM) of Ala, 2.32g (130mM), 2.67g (150mM), and 3.03g (170mM) and dissolve them respectively, adjust 8.5 and make the volume to 150mL, preheat at 38°C, add 8mL (~7U / mL) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to wet bacteria: disruption buffer = 1:4 (w / v), 2mL (~3U / mL) of ChPPK2 crude enzyme solution, and 50mL of Tyr 400mM pH 12.5 mother solution at a rate of 1mL / min. The enzyme activity of the fermentation bacteria BsBacD-N108S_L110S used was 870U / g, and the enzyme activity of ChPPK2 was 1500U / g.

[0148] As shown in Table 3, when the Ala:Tyr equivalent is greater than or equal to 1.5, the conversion of Tyr→Ala-Tyr can be promoted well, but excessive Ala will cause the organic nitrogen in the wastewater to exceed the standard. In summary, the Ala:Tyr equivalent of 1.5 is more suitable.

[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 substrate tyrosine concentration

[0152] Weigh 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) and dissolve them in 500 mL of pure water. Divide them into 4 portions, weigh 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, respectively, adjust the pH to 8.5 and make the volume to 150 mL. After preheating at 38°C, add 8 mL (~7 U / mL) of crude enzyme solution of BsBacD-N108S_L110S and 2 mL (~3 U / mL) of crude enzyme solution of ChPPK2 obtained by resuspending and ultrasonically disrupting wet cells: disruption buffer = 1:4 (w / v), Tyr 50 mL of 400 mM, 500 mM, 600 mM, 700 mM pH 11.5 mother liquors were added to the Ala 150 mM, 187.5 mM, 225 mM, and 262.5 mM reaction solutions at a rate of 1 mL / min. The fermentation bacteria used had an enzyme activity of 900 U / g for BsBacD-N108S_L110S and 1500 U / g for ChPPK2.

[0153] like Fig.10 As shown in the figure, when the tyrosine concentration in the reaction solution does not exceed 125mM, the conversion rate of Tyr→Ala-Tyr in 2h reaction is higher than 99%, while the conversion rates of Tyr 150mM and 175mM groups in 2h are 88.7% and 77.9% respectively. When extended to 3h, the conversion rate of Tyr 150mM group is only 92.4%, indicating that the concentration of substrate Tyr 150mM is too high, and 125mM is more suitable. At the same time, after the pH of Tyr mother solution was reduced from 12.5 to pH 11.5, the conversion rate in 2h increased from 93.6% in the alanine equivalent optimization experiment to 99.6%, which may be because the effect of pH change in the reaction solution on the enzyme was reduced.

[0154] 5. Laboratory 3L Amplification

[0155] Weigh 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, and 1.65 g (1 mM) of adenosine triphosphate disodium (ATP-Na2) and dissolve them in 1800 mL of pure water, adjust the pH to 8.5 and make the volume to 2250 mL, preheat at 38°C, add 86 mL (~5 U / mL, bacterial concentration 5.7 g / L) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to wet bacteria: disruption buffer = 1:4 (w / v), 30 mL (~3 U / mL, bacterial concentration 2 g / L) of ChPPK2 crude enzyme solution, and 750 mL of Tyr 500 mM pH 11.5 mother solution at a rate of 13 mL / min. The enzyme activity of the fermentation bacteria BsBacD-N108S_L110S used was 900U / g, and the enzyme activity of ChPPK2 was 1500U / g.

[0156] The conversion rates of Tyr→Ala-Tyr were 96.9%, 90.5%, 98.8% and 100% at reaction times of 0.5h, 1h, 1.5h and 2h, respectively. The substrate concentration, conversion rate and reaction time were better than those reported in the laboratory.

[0157] 6. Laboratory 6L Amplification

[0158] Weigh 146.82 g (40 mM) of sodium hexametaphosphate, 100.23 g (187.5 mM) of Ala, 73.19 g (60 mM) of magnesium chloride hexahydrate, and 2.08 g (1 mM) of adenosine monophosphate (AMP) and dissolve them in 3600 mL of pure water, adjust the pH to 8.5 and make the volume to 4500 mL. After preheating at 38°C, add 178 mL (5 U / mL, bacterial concentration 5.9 g / L) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to wet bacteria: disruption buffer = 1:4 (w / v), 60 mL (3 U / mL, bacterial concentration 2 g / L) of ChPPK2 crude enzyme solution, and 1500 mL of Tyr 500 mM pH 11.5 mother solution at a rate of 26 mL / min. The enzyme activity of the fermentation bacteria BsBacD-N108S_L110S used was 900U / g, and the enzyme activity of ChPPK2 was 1500U / g.

[0159] The conversion rates of Tyr→Ala-Tyr were 91.6%, 91.0%, 100% and 100% after reaction for 0.5h, 1h, 1.5h and 2h, respectively.

[0160] Example 3 Enzymatic Synthesis of Glycine Dipeptide

[0161] 1. Optimization of substrate glycine equivalents

[0162] Weigh 14.68 g (40 mM) of sodium hexametaphosphate, 7.32 g (60 mM) of magnesium chloride hexahydrate, and 0.66 g (2 mM) of adenosine triphosphate disodium (ATP-Na2) and dissolve them in 360 mL of pure water. Divide them into 3 portions, weigh 1.95 g (130 mM) of Gly, 2.25 g (150 mM) of Gly, and 2.55 g (170 mM) of Gly respectively, adjust the pH to 8.5 and make the volume to 150 mL, preheat at 38 ° C, add 10 mL (~9 U / mL) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to wet bacteria: disruption buffer = 1:4 (w / v), 2 mL (~3 U / mL) of ChPPK2 crude enzyme solution, and 50 mL of Tyr400 mM stock solution, all at a rate of 0.5 mL / min. The fermentation bacteria used had an enzyme activity of 900 U / g for BsBacD-N108S_L110S and 1500 U / g for ChPPK2. The wet weight of the bacteria used was obtained by centrifuging the BsBacD-N108S_L110S or ChPPK2 bacteria after shaking flask or fermentation culture and weighing the wet weight.

[0163] Fig.11 The following is the reaction formula for the enzymatic synthesis of Gly-Tyr. Fig.12 As shown, when the Gly:Tyr equivalent is greater than or equal to 1.5, the conversion rate of Tyr→Gly-Tyr after 3h reaction is higher than 97%. Compared with the optimization experimental results of the substrate alanine equivalent in Example 2, the conversion rate of 50.0% in the Gly:Tyr equivalent 1.3 group after 1h reaction is significantly lower than the 90.5% in the Gly:Tyr 1.5 group, indicating that the low excess substrate of Gly-Tyr promotes the reaction more slowly than that of Ala-Tyr.

[0164] 2. Laboratory 3L Amplification

[0165] Weigh 73.41 g (40 mM) of sodium hexametaphosphate, 33.78 g (150 mM) of Gly, 36.59 g (60 mM) of magnesium chloride hexahydrate, and 1.65 g (1 mM) of adenosine triphosphate disodium (ATP-Na2) and dissolve them in 1800 mL of pure water, adjust the pH to 8.5 and make the volume to 2250 mL. After preheating at 38°C, add 168 mL (~10 U / mL, bacterial concentration 11.2 g / L) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to wet bacteria: disruption buffer = 1:4 (w / v), 30 mL (~3 U / mL, bacterial concentration 2 g / L) of ChPPK2 crude enzyme solution, and 750 mL of Tyr 400 mM mother solution at a rate of 8 mL / min. The enzyme activity of the fermentation bacteria BsBacD-N108S_L110S used was 900U / g, and the enzyme activity of ChPPK2 was 1700U / g.

[0166] After 1 h, 2 h, and 3 h of reaction, the conversion rates of Tyr→Gly-Tyr were 94.9%, 94.9%, and 95.7%, respectively.

[0167] 3. Laboratory 3L Amplification

[0168] Weigh 73.41 g (40 mM) of sodium hexametaphosphate, 33.78 g (150 mM) of Gly, 36.59 g (60 mM) of magnesium chloride hexahydrate, and 1.04 g (1 mM) of adenosine monophosphate (AMP) and dissolve them in 1800 mL of pure water, adjust the pH to 8.5 and make the volume to 2250 mL. After preheating at 38°C, add 168 mL (~10 U / mL, bacterial concentration 11.2 g / L) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to wet bacteria: disruption buffer = 1:4 (w / v), 30 mL (~3 U / mL, bacterial concentration 2 g / L) of ChPPK2 crude enzyme solution, and 750 mL of Tyr 400 mM mother solution at a rate of 7.5 mL / min. The enzyme activity of the fermentation bacteria BsBacD-N108S_L110S used was 900U / g, and the enzyme activity of ChPPK2 was 1700U / g.

[0169] After 1 h, 2 h, and 3 h of reaction, the conversion rates of Tyr→Gly-Tyr were 95.9%, 94.3%, and 96.2%, respectively.

[0170] Example 4 Enzymatic Synthesis of Serine Dipeptide

[0171] 1. Laboratory 3L scale-up

[0172] Weigh 73.41 g (40 mM) of sodium hexametaphosphate, 47.29 g (150 mM) of Ser, 36.59 g (60 mM) of magnesium chloride hexahydrate, and 1.65 g (1 mM) of adenosine triphosphate disodium (ATP-Na2) and dissolve them in 1800 mL of pure water, adjust the pH to 8.5 and make the volume to 2250 mL. After preheating at 38°C, add 135 mL (~9 U / ml, bacterial concentration 9 g / L) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to the ratio of wet bacteria: disruption buffer = 1:4 (w / v), 30 mL (~3 U / mL, bacterial concentration 2 g / L) of ChPPK2 crude enzyme solution, and 750 mL of Tyr 400 mM mother solution at a rate of 8 mL / min. The fermentation bacteria used had an enzyme activity of 1000 U / g for BsBacD-N108S_L110S and 1500 U / g for ChPPK2. The wet weight of the bacteria used was obtained by centrifuging BsBacD-N108S_L110S or ChPPK2 bacteria after shaking flask or fermentation culture and weighing the wet weight.

[0173] Fig.13The following is the reaction formula for the enzymatic synthesis of Ser-Tyr. The conversion rates of Tyr→Ser-Tyr were 62.3%, 80.7%, 86.0%, and 88.4% after 1h, 2h, 3h, and 4h of reaction, respectively.

[0174] 2. Laboratory 3L Amplification

[0175] Weigh 73.41 g (40 mM) of sodium hexametaphosphate, 47.29 g (150 mM) of Ser, 36.59 g (60 mM) of magnesium chloride hexahydrate, and 1.04 g (1 mM) of adenosine monophosphate (AMP) and dissolve them in 1800 mL of pure water, adjust the pH to 8.5 and make the volume to 2250 mL. After preheating at 38°C, add 135 mL (~9 U / ml, bacterial concentration 9 g / L) of BsBacD-N108S_L110S crude enzyme solution obtained by resuspending and homogenizing according to the ratio of wet bacteria: disruption buffer = 1:4 (w / v), 30 mL (~3 U / mL, bacterial concentration 2 g / L) of ChPPK2 crude enzyme solution, and 750 mL of Tyr400 mM mother solution at a rate of 8 mL / min. The fermentation bacteria BsBacD-N108S_L110S had an enzyme activity of 1000U / g, and ChPPK2 had an enzyme activity of 1500U / g.

[0176] After reaction for 1 h, 2 h, 3 h, and 4 h, the conversion rates of Tyr→Ser-Tyr were 76.5%, 81.0%, 87.3%, and 89.2%, respectively.

[0177] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant of the BsBacD enzyme, characterized in that: The mutation sites of the mutant include N108S and L110S; The BsBacD enzyme has: (I), the amino acid sequence shown in SEQ ID No.1; (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence as shown in any one of (I) or (II).

2. A nucleic acid molecule, characterized in that Encoding the mutant according to claim 1; The nucleic acid molecule has: (1), the nucleotide sequence shown in SEQ ID NO.2; or (2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (1), and having the same or similar function as (1); or (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 shown in (1) or (2).

3. An enzyme composition, characterized in that The method comprises the mutant as claimed in claim 1 and polyphosphate kinase.

4. The enzyme composition according to claim 3, characterized in that The polyphosphate kinase has: (a), the amino acid sequence shown in SEQ ID No.11; (b) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (a); or (c) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence as shown in any of (a) or (b).

5. A nucleic acid molecule, characterized in that Encoding the enzyme composition according to claim 3 or 4; Wherein, the nucleic acid molecule encoding the polyphosphate kinase has: (1) the nucleotide sequence shown in SEQ ID NO.12; or (2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (1), and having the same or similar function as (1); or (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 shown in (1) or (2).

6. An expression cassette, characterized in that Comprising the nucleic acid molecule according to claim 2 or 5.

7. A plasmid, characterized in that Comprising the expression frame as claimed in claim 6.

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

9. A composition for synthesizing tyrosine dipeptide, characterized in that: include: The enzyme composition according to claim 3 or 4; and Including adenosine triphosphate or its salt, or adenosine monophosphate or its salt, but excluding adenosine diphosphate or its salt; and Alkaline stock solution of tyrosine; Preferably, the pH of the composition is 6.5 to 9.5; more preferably, the pH of the composition is 8.5; Preferably, the concentration of the alkaline mother solution of tyrosine includes Tyr 400mM, 500mM, 600mM, and 700mM; more preferably, the concentration of the alkaline mother solution of tyrosine is 500mM; Preferably, the composition further comprises magnesium chloride; preferably, the concentration of the magnesium chloride comprises 40 mM to 100 mM; more preferably, the concentration of the magnesium chloride is 60 mM.

10. Use of any of the following in the preparation of tyrosine dipeptide: (i) the mutant according to claim 1; and / or (ii), a nucleic acid molecule as claimed in claim 2 or 5; and / or (iii) the enzyme combination according to claim 3 or 4; and / or (iv) the expression frame as claimed in claim 6; and / or (v) a plasmid as claimed in claim 7; and / or (vi) The bacterial cell according to claim 8; (vii) The composition according to claim 9; The tyrosine dipeptide includes one or more of acyl tyrosine dipeptide, acyl tyrosine dipeptide and / or acyl tyrosine dipeptide.

11. A method for preparing tyrosine dipeptide, characterized in that: include: (a), mixing amino acids with the mutant according to claim 1 or the enzyme composition according to claim 3 or 4 to obtain a dipeptide; or (b), expressing the nucleic acid molecule according to claim 2 or 5, and mixing the obtained protein product with amino acids to obtain a dipeptide; or (c) expressing the expression cassette of claim 6 or the plasmid of claim 7, and mixing the obtained protein product with amino acids to obtain a dipeptide; or (d) culturing the bacterial cell as claimed in claim 8, and mixing the obtained protein product with amino acids to obtain a dipeptide; or (e), mixing the composition according to claim 9 with amino acids to obtain a dipeptide; The tyrosine dipeptide includes one or more of acyl tyrosine dipeptide, acyl tyrosine dipeptide and / or acyl tyrosine dipeptide; The amino acids include one or more of tyrosine, alanine, glycine or serine.

12. The preparation method according to claim 11, characterized in that: The mixing includes adding the alkaline mother solution of tyrosine; Preferably, the rate of the flow addition is 0.5 mL / min to 26 mL / min.

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