Histidine decarboxylase and its applications, methods for biosynthesizing histamine

Abstract

This invention relates to the field of biosynthesis technology, and particularly to histidine decarboxylases and their applications, as well as methods for biosynthesizing histamine. Through enzyme mining tools such as cluster analysis and phylogenetic tree construction, this invention unexpectedly screened a previously unreported highly active histidine decarboxylase, which catalyzes the synthesis of histamine at concentrations above 55 g / L, enabling a pilot-scale production line with an annual capacity exceeding 13 tons. Furthermore, this invention increases cell mass and accelerates the reaction through cell immobilization, while reducing residual cells, proteins, and nucleic acids in the reaction solution. It is also reusable, significantly reducing production costs.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and in particular to histidine decarboxylase and its application, and methods for biosynthesizing histamine. Background Technology

[0002] Decarboxycarnosine (β-alanylhistamine), also known as decarboxycarnosine, possesses anti-glycation, free radical scavenging, and antioxidant properties, and can be used in cosmetics and health products. As shown below, histamine is a key raw material in the synthesis of decarboxycarnosine.

[0003]

[0004] Histamine is also an important neurotransmitter that regulates a variety of pathophysiological responses by activating four different receptors (H1R, H2R, H3R, H4R), such as allergies, gastric acid secretion, drowsiness, asthma, psoriasis, atopic dermatitis, inflammatory bowel disease, and arthritis.

[0005] Commercially available histamine is in dihydrochloride form and is mostly chemically synthesized. It uses L-histidine as a starting material, adding catalysts such as acetophenone or p-methylacetophenone to undergo a decarboxylation reaction at high temperatures of 110-175°C, as illustrated in patents WO / 2000 / 039098, CN102477014B, CN112266360B, and CN113045500A. Patent CN106432089B reports the oxidation of L-histidine to nitrile methylimidazolium via strong chlorohydrin, followed by reduction with hydrazine hydrate and Raney nickel to produce histamine. The high temperature and pressure required for chemical synthesis pose safety hazards, and Raney nickel is highly dangerous.

[0006] In recent years, biosynthetic histamine technologies, represented by biocatalysis and microbial metabolism, have been increasingly reported. Patent CN112368387A reports the modification of *Yarrowia lipolytica* to express histidine decarboxylase from *Acinetobacter baumannii* AB0057 (UniProt accession number B7I459), yielding 505 mg / L of histamine through fermentation, but the product concentration was low and did not meet commercial requirements. Patent CN117448367A reports a method for preparing histamine using whole-cell catalysis of *Escherichia coli* expressing histidine decarboxylase. The histidine decarboxylase from *Photobacterium aquimaris* (UniProt accession number A0A1A6TPG4) was expressed in *E. coli* as a low-activity inclusion body form. With 15 g / L wet cells catalyzing the reaction of 60 mM L-histidine for 48 h, the maximum histamine yield was only 15.28 mM.

[0007] Biocatalysis methods such as in vitro enzyme catalysis or whole-cell catalysis have the advantages of high reaction concentration, fast speed, high specificity, low impurities, and green safety, which are beneficial for subsequent product separation and purification. Compared with traditional chemical synthesis and microbial metabolism, they often have a cost advantage.

[0008] Histidine decarboxylases (HDCs) that can decarboxylate histidine to histamine exist in nature. They are divided into two categories: HDCs derived from Gram-positive bacteria use pyruvyl as a cofactor, while HDCs derived from Gram-negative bacteria and mammals use PLP as a cofactor. Among them, Gram-negative bacteria exhibit two types: the shorter HDC1 (370-390 aa) and the longer HDC2 (760-800 aa). Existing literature on histidine decarboxylases is mostly limited to HDC1 (Kamath, AV et al., 1991; Bjornsdottir-Butler, K. et al., 2016; Bjornsdottir-Butler, K. et al., 2018; Oda Yuki et al., 2022), while reports on HDC2 are relatively few (Bjornsdottir-Butler, K. et al., 2020). The histamine concentration detected in Photobacterium phosphoreum FS 3.1, which expresses only HDC2, was higher than that in Photobacterium kishitanii, which expresses only HDC1.

[0009] In order to overcome the shortcomings of slow reaction rate and low conversion rate in the aforementioned biosynthesis patent and make the biocatalytic synthesis of histamine commercially feasible, it is worthwhile to try screening and modifying HDC2 to obtain histidine decarboxylase with higher catalytic efficiency. Summary of the Invention

[0010] In view of this, the present invention provides a histidine decarboxylase and its application, as well as a method for the biosynthesis of histamine. This histidine decarboxylase exhibits high histamine conversion rate and short conversion time, significantly increasing histamine yield and making it suitable for the industrial production of histamine.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0012] Histidine decarboxylase, having any one of the sequences shown in 1) to 3):

[0013] 1) An amino acid sequence as shown in SEQ ID NO: 3, 9 or 10;

[0014] 2) An amino acid sequence in which one or more amino acids are substituted, deleted, or added, and which have the same or similar functions;

[0015] 3) An amino acid sequence that has at least 80% homology with the amino acid sequence shown in 1) or 2).

[0016] This invention marks the first discovery of three novel, previously unreported histidine decarboxylases: Ha2, Ha8, and Ha9. Experiments show that all three enzymes can efficiently catalyze the synthesis of histidine from histidine. Among them, Ha2 exhibits the highest activity, catalyzing the synthesis of histamine at concentrations exceeding 55 g / L, enabling a pilot-scale production line with an annual capacity of over 13 tons.

[0017] In this invention, the substitution includes:

[0018] At least one Cys is replaced by Val, Ser, Phe, Met, His, Ile, Thr, or Ala.

[0019] In the histidine decarboxylase provided by the present invention, the plurality of enzymes is 2 to 155.

[0020] In some embodiments, the present invention also provides a mutant of histidine decarboxylase Ha2, which includes at least one mutation of C525V, C547S, and C558V in the amino acid sequence of the histidine decarboxylase as shown in SEQ ID NO: 3.

[0021] In some specific embodiments, the mutants of the histidine decarboxylase Ha2 are Ha2-Cys525Val, Ha2-Cys547Ser, or Ha2-Cys558Val. Specifically, the mutant Ha2-Cys525Val is obtained by mutating Cys at position 525 of the Ha2 enzyme shown in SEQ ID NO: 3 to Val, with the amino acid sequence SEQ ID NO: 21 and the nucleotide sequence SEQ ID NO: 24. The mutant Ha2-Cys547Ser is obtained by mutating Cys at position 547 of the Ha2 enzyme shown in SEQ ID NO: 3 to Ser, with the amino acid sequence SEQ ID NO: 22 and the nucleotide sequence SEQ ID NO: 25. The mutant Ha2-Cys558Val is obtained by mutating Cys at position 558 of the Ha2 enzyme shown in SEQ ID NO: 3 to Val, with the amino acid sequence SEQ ID NO: 23 and the nucleotide sequence SEQ ID NO: 26.

[0022] Experiments showed that the mutants Ha2-Cys547Ser and Ha2-Cys558Val achieved a conversion rate of 100% after 6 hours of reaction, which is higher than that of Ha2, and the reaction rate is faster. The catalytic activity of Ha2-Cys525Val is comparable to that of Ha2 enzyme.

[0023] The present invention also provides a nucleic acid encoding the histidine decarboxylase or the mutants thereof (i.e., Ha2, Ha8 and Ha9, Ha2-Cys525Val, Ha2-Cys547Ser, Ha2-Cys558Va), which has the following characteristics:

[0024] (I) Nucleotide sequences as shown in SEQ ID No. 13, 19, 20, 24-26; or

[0025] (II) Nucleotide sequences obtained by substitution, deletion, or addition of one or more nucleotides in the nucleotide sequence shown in (I), and which have the same or similar functions; or

[0026] (V) A nucleotide sequence that has at least 80% homology with the nucleotide sequence described in (I) or (II).

[0027] The present invention also provides biomaterials comprising at least one of the following:

[0028] (A) The expression cassette of the nucleic acid described in this invention;

[0029] (B) An expression carrier containing the expression frame described in (A);

[0030] (C) Host cells containing the expression vector described in (B)

[0031] (D) Cultures or metabolites obtained by culturing the host cells described in (C).

[0032] The present invention also provides the use of at least one of the following (I) to (III) in the synthesis of histamine:

[0033] I) The histidine decarboxylases (including Ha2 enzyme and its mutants, Ha8 and Ha9 enzymes) described in this invention;

[0034] II) The nucleic acid encoding the histidine decarboxylase described in I);

[0035] III) The biomaterials described in this invention.

[0036] This invention also provides a method for synthesizing histamine, comprising:

[0037] Histamine is synthesized using histidine as a substrate under the catalysis of the histidine decarboxylase described in this invention.

[0038] This may include: using histidine as a substrate, adding bacterial cells, bacterial cell lysate, immobilized cells or immobilized enzymes, reacting to obtain histamine;

[0039] The method for preparing the bacterial cells includes: collecting the bacterial cells after inducing host cells in the biomaterials of the present invention through shake flask or fermentation culture;

[0040] The method for preparing the bacterial cell lysate includes: culturing the host in the biomaterial of the present invention in a shake flask or fermentation culture, collecting the bacterial cells, and lysing them to obtain the bacterial cell lysate;

[0041] The method for preparing the immobilized cells includes: culturing the host cells in the biomaterial of the present invention in a shake flask or by fermentation, collecting the bacterial cells, and using the bacterial cells to prepare immobilized cells;

[0042] The method for preparing the immobilized enzyme includes: culturing the host in the biomaterial of the present invention in a shake flask or fermentation culture, collecting the cells, breaking them, and preparing the immobilized enzyme using the lysate.

[0043] In this invention, there are no special restrictions on the specific method for preparing immobilized enzymes using the lysis solution; any method commonly used in the art is acceptable.

[0044] In this invention, the substrate is a 100mM to 600mM histidine aqueous solution with a pH of 5-8.

[0045] In this invention, the reaction is carried out at 20℃~30℃ and pH 5-8 with stirring; the specific temperature of the reaction can be 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, or more; the specific pH of the reaction can be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.

[0046] In this invention, the mass ratio of immobilized cells to histidine is 30–120:62–93; in some specific embodiments of this invention, the mass ratio is specifically 40:77.5, and can also be 30:62, 60:93, or more. In this reaction, increasing the amount of immobilized cells can accelerate the reaction; for example, a mass ratio of immobilized cells to histidine of 60:62, 80:77.5, or 120:93 will increase the reaction rate.

[0047] The synthesis method provided by this invention includes the following steps for preparing the immobilized cells:

[0048] The bacterial culture of the host cells described in this invention is mixed with an aqueous sodium alginate solution and then hardened in a 1-6 wt% calcium chloride solution for 2-6 hours. The hardened microsphere-like immobilized cells are then collected. Specifically, the mass percentage concentration of the calcium chloride solution can be 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, or 6 wt%. The concentration of the sodium alginate aqueous solution is 10-40 g / L, specifically 10 g / L, 20 g / L, 30 g / L, or 40 g / L; the concentration of the bacterial culture of the host cells is 50-140 g / L, specifically 50 g / L, 60 g / L, 80 g / L, 100 g / L, 120 g / L, or 140 g / L. The mixing is preferably done in equal volumes.

[0049] In this invention, when histidine is added as a substrate, the mass ratio of the bacterial cells to histidine is 15-30:62-93.

[0050] In this invention, the conditions for shake flask or fermentation culture are: adding an inducer and culturing at 16-37℃ for 4-20 hours; in some specific embodiments, the conditions for shake flask or fermentation culture are: culturing at 16℃ for 16-20 hours, culturing at 25℃ for 8-12 hours, and culturing at 37℃ for 4-6 hours.

[0051] In this invention, the culture medium for shake flask culture is LB medium; the culture medium for fermentation culture comprises the following components:

[0052]

[0053]

[0054] In some specific embodiments, the culture medium for fermentation includes the following components:

[0055]

[0056] This invention marks the first discovery of previously unreported highly active histidine decarboxylases (Ha2, Ha8, and Ha9 enzymes). The Ha2 enzyme alone catalyzes histamine synthesis at concentrations exceeding 55 g / L, enabling a pilot-scale production line with an annual capacity exceeding 13 tons. Furthermore, this invention mutates the Ha2 enzyme, obtaining a Ha2 enzyme mutant with an even higher reaction rate. Even further, this invention increases cell mass through cell immobilization, accelerating the reaction and reducing residual cells, proteins, and nucleic acids in the reaction solution, while also allowing for reuse and significantly reducing production costs. Attached Figure Description

[0057] Figure 1 The results of enzyme cluster analysis are shown.

[0058] Figure 2 Show the phylogenetic tree results;

[0059] Figure 3 Showing the results of multiple sequence alignments;

[0060] Figure 4 The structural prediction results for Ha2_A0A7J4PLZ2 are shown.

[0061] Figure 5 This shows a locally magnified view of the Ha2_A0A7J4PLZ2 structure.

[0062] Figure 6 The results of activity tests for different enzymes are shown.

[0063] Figure 7 The expression of Ha2 enzyme at different induction temperatures is shown;

[0064] Figure 8 The results of whole-cell catalysis test are shown.

[0065] Figure 9 The conversion efficiency of different cell masses at a substrate concentration of 600 mM is shown.

[0066] Figure 10 The conversion efficiency of different cell masses at a substrate concentration of 500 mM is shown.

[0067] Figure 11 The conversion rates of different Ha2 enzyme mutants are shown.

[0068] Figure 12 The conversion rate of different cell masses in whole cells is shown;

[0069] Figure 13 The appearance of the microsphere-immobilized cells shown in Example 7;

[0070] Figure 14 The conversion rate of the immobilized cells is shown. Detailed Implementation

[0071] This invention provides histidine decarboxylase and its applications, as well as a method for the biosynthesis of histamine. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0072] The sequence information involved in this invention is as follows:

[0073] The amino acid sequence (SEQ ID No:1) of the PpHDC2 enzyme is as follows:

[0074]

[0075] The amino acid sequence of the Ha1 enzyme (SEQ ID No:2) is as follows:

[0076]

[0077] The amino acid sequence of the Ha2 enzyme (SEQ ID No:3) is as follows:

[0078]

[0079]

[0080] The amino acid sequence of the Ha3 enzyme (SEQ ID No:4) is as follows:

[0081]

[0082] The amino acid sequence of the Ha4 enzyme (SEQ ID No:5) is as follows:

[0083]

[0084] The amino acid sequence of the Ha5 enzyme (SEQ ID No:6) is as follows:

[0085]

[0086]

[0087] The amino acid sequence of the Ha6 enzyme (SEQ ID No:7) is as follows:

[0088]

[0089] The amino acid sequence of the Ha7 enzyme (SEQ ID No:8) is as follows:

[0090]

[0091] The amino acid sequence of the Ha8 enzyme (SEQ ID No:9) is as follows:

[0092]

[0093]

[0094] The amino acid sequence of the Ha9 enzyme (SEQ ID No:10) is as follows:

[0095]

[0096] The nucleic acid sequence (SEQ ID No:11) of the PpHDC2 enzyme is as follows:

[0097]

[0098]

[0099] The nucleic acid sequence of the Ha1 enzyme (SEQ ID No:12) is as follows:

[0100]

[0101]

[0102] The nucleic acid sequence of the Ha2 enzyme (SEQ ID No:13) is as follows:

[0103]

[0104]

[0105] The nucleic acid sequence of the Ha3 enzyme (SEQ ID No:14) is as follows:

[0106]

[0107]

[0108] The nucleic acid sequence of the Ha4 enzyme (SEQ ID No:15) is as follows:

[0109]

[0110]

[0111] The nucleic acid sequence of the Ha5 enzyme (SEQ ID No:16) is as follows:

[0112]

[0113]

[0114] The nucleic acid sequence of the Ha6 enzyme (SEQ ID No:17) is as follows:

[0115]

[0116]

[0117] The nucleic acid sequence of the Ha7 enzyme (SEQ ID No:18) is as follows:

[0118]

[0119]

[0120] The nucleic acid sequence of the Ha8 enzyme (SEQ ID No:19) is as follows:

[0121]

[0122]

[0123] The nucleic acid sequence of the Ha9 enzyme (SEQ ID No:20) is as follows:

[0124]

[0125] The amino acid sequence (SEQ ID No:21) of the Ha2-Cys525Val enzyme is as follows:

[0126]

[0127] The amino acid sequence (SEQ ID No:22) of the Ha2-Cys547Ser enzyme is as follows:

[0128]

[0129] The amino acid sequence (SEQ ID No:23) of the Ha2-Cys558Val enzyme is as follows:

[0130]

[0131]

[0132] In the amino acid sequences of Ha2-Cys525Val, Ha2-Cys547Ser, and Ha2-Cys558Val, the underlined amino acids are those at mutation sites.

[0133] The nucleotide sequence (SEQ ID No:24) encoding the Ha2-Cys525Val enzyme is as follows:

[0134]

[0135]

[0136] The nucleotide sequence encoding the Ha2-Cys547Ser enzyme (SEQ ID No:25) is as follows:

[0137]

[0138]

[0139] The nucleotide sequence (SEQ ID No:26) encoding the Ha2-Cys558Val enzyme is as follows:

[0140]

[0141]

[0142] This invention, through enzyme mining tools such as cluster analysis and phylogenetic tree construction, unexpectedly screened previously unreported highly active histidine decarboxylases (such as Ha2 enzymes), catalyzing the synthesis of histamine at concentrations above 55 g / L, enabling a pilot-scale production line with an annual capacity exceeding 13 tons. Furthermore, this invention increases cell mass and accelerates the reaction through cell immobilization, while reducing residual cells, proteins, and nucleic acids in the reaction solution. Simultaneously, the cells can be reused, significantly reducing production costs.

[0143] The specific analytical process and experimental steps involved in this invention are as follows:

[0144] (I) Enzyme Clustering Analysis

[0145] The HDC2 enzyme is incorrectly labeled as arginine decarboxylase (ADC) in databases such as UniProt and NCBI. To effectively isolate the histidine decarboxylase, the PpHDC2 enzyme sequence was input into BLAST to search for homologous sequences. The first 1000 results were used to generate a sequence similarity network (SSN) using an enzyme similarity analysis tool (EFI-EST). The threshold was set to 10. -240 At that time, the Blast sequence library was classified into 7 clusters of different sizes (see...). Figure 1 PpHDC2, located in the third largest cluster, is more likely to be primarily histidine decarboxylase.

[0146] (II) Constructing a phylogenetic tree

[0147] 116 results from the third major category were extracted using Cytoscape software, and sequences were extracted using UniProt's ID mapping tool. These sequences were then imported into MEGA software for alignment and a Neighbor-Joining phylogenetic tree was constructed (see...). Figure 2 ) Sequences 1, 4, and 5 were selected from Group I (12 sequences), Group II (43 sequences), and Group III (61 sequences) respectively for subsequent verification.

[0148] (III) Gene Synthesis

[0149] The predicted secondary and tertiary structures of the above candidate sequences (see Table 1) were analyzed. After removing the disordered regions at the N-terminus and C-terminus, the sequences shown in SEQ ID No:1-10 were submitted to Universal Biotech (Anhui) for codon optimization and whole-gene synthesis (the corresponding nucleotide sequences are SEQ ID No:11-20 in sequence). The pET-28a vector was constructed using NdeI and XhoI restriction endonucleases and then transformed into the BL21(DE3) expression host to produce stab bacteria.

[0150] Table 1

[0151]

[0152] (iv) Expression detection

[0153] First, streak the stab-collected bacteria onto LB agar plates (Kan) and incubate overnight at 37°C. Then, pick a single colony and incubate in 5 ml of LB liquid medium (Kan) at 37°C and 200 rpm for 6-8 hours until turbidity appears. Take 1 ml to prepare glycerol culture medium for storage, and add the remaining 5 ml of LB liquid medium and 0.1 mM IPTG to induce expression at 16°C and 200 rpm for 16-20 hours. Collect the bacterial cells by centrifugation, add Lysis buffer (20 mM K2HPO4 / KH2PO4, 100 mM NaCl, pH 7.6), sonicate, and centrifuge at high speed. Collect samples of the lysate and supernatant for protein electrophoresis to detect expression.

[0154] (v) Enzyme activity screening

[0155] Take 0.1 ml of the lysate and add it to 0.9 ml of reaction solution (100 mM histidine, pH 6.5). Incubate in a 25°C water bath and take samples at 0.5 h and 1 h respectively. HPLC is used to detect the concentration of substrate and product.

[0156] (vi) Shake flask culture

[0157] Transfer the glycerol bacteria to 20 ml of LB liquid medium (Kan) and incubate overnight at 37°C and 200 rpm. Then transfer to 400 ml x 3 LB liquid medium (Kan) and incubate at 37°C and 200 rpm for 2-3 hours until OD (Organic Depth). 600 =0.6-1.0, add 0.1mM IPTG and incubate at 16℃ for 16-20h, 25℃ for 8-12h, and 37℃ for 4-6h respectively. Collect bacterial cells by centrifugation, add Lysis buffer, sonicate and centrifuge at high speed, and sample the lysate and supernatant for protein electrophoresis to detect expression and determine the optimal induction temperature.

[0158] (vii) Small-scale fermentation

[0159] Spread a glycerol bacterium onto an LB agar plate (Kan) and incubate overnight at 37°C. Then scrape off all colonies and incubate in 400ml LB liquid medium (Kan) at 37°C and 200rpm for 6-8 hours until OD reaches 0.5. 600 =2-3 is the seed culture, which is then transferred to a 15L fermenter containing 7.5L of initial fermentation medium by flame inoculation and cultured at 37℃. The medium formula is as follows:

[0160] Table 2

[0161]

[0162] When biomass OD 600 When the temperature reaches 20-30°C, begin cooling to the induction temperature, then add 0.1 mM IPTG to induce expression for 6-24 hours, and then remove the container from the OD chamber. 600 =80-120. Centrifuge to collect bacterial cells and store at -20℃ for later use.

[0163] (viii) Whole-cell catalysis test

[0164] Weigh 31g of L-histidine and dissolve it in 900ml of pure water. Adjust the pH to 6.5 with 4M HCl and bring the volume to 1L. Add 12g of bacterial cells and stir the reaction at room temperature, maintaining the pH between 6.5 and 7.0 during the reaction. Take samples at 1h, 4h, and 6h, and determine the substrate and product concentrations by HPLC to calculate the conversion rate.

[0165] (ix) Comparative analysis of different enzymes

[0166] Multiple sequence alignment was performed on the aforementioned candidate sequences using the online tools ClustalOmega and ESPript, and the more conserved Cys sequence was annotated (see...). Figure 3 ).

[0167] Observe the positions and pairings of the labeled Cys333, Cys339, Cys525, Cys547, and Cys558 in the prediction structure of Ha2_A0A7J4PLZ2 (see...). Figure 4 ～5 Among them, Cys333 and Cys339 may form disulfide bonds to stabilize the internal structure of the protein.

[0168] The online tool PrimerX was used to design Cys525Val, Cys547Ser, and Cys558Val mutant primers, which were then submitted to Qingke Biotechnology (Beijing) for synthesis. The corresponding plasmids were then constructed using the Quick-Change site-directed mutagenesis kit (Agilent) with pET28-ha2 as a template. After successful sequencing, the plasmids were transformed into the BL21(DE3) expression host.

[0169] The results showed that mutating the Cys of the HDC2 enzyme to the amino acids Val, Ser, and Ala significantly improved the stability and activity of histidine decarboxylase.

[0170] (ix) Cell immobilization

[0171] Weigh 2g of sodium alginate and dissolve it in 100ml of pure water by heating. After cooling to room temperature, mix it with 100ml of 120g / l bacterial solution and add it dropwise into 300ml of 2% calcium chloride solution to harden for 4 hours. Collect the hardened microsphere immobilized cells, rinse them with pure water, and store them in the refrigerator.

[0172] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0173] The present invention will be further illustrated below with reference to the embodiments:

[0174] Example 1: Activity Test of Different Enzymes

[0175] Take 0.1 ml of the lysate from the shake-flask culture of PpHDC2 and Ha1-9 mentioned in "(IV) Expression Detection" above, add 0.9 ml of reaction solution (100 mM histidine, pH 6.5), incubate at 25°C, and incubate for 0.5 h and 1 h respectively. The reaction results are as follows. Figure 6 As shown.

[0176] The results showed that the histamine concentration synthesized by PpHDC2, Ha2, Ha8 and Ha9 was significantly higher than that of other enzymes, with Ha2 producing the highest concentration of the product.

[0177] Example 2: Shaking Flask Expression Temperature Optimization

[0178] Referring to the previous section "(VI) Shake Flask Culture", the pET28a-Ha2 BL21(DE3) glycerol bacteria were expanded to 400 ml shake flasks. The expression results at different induction temperatures are as follows: Figure 7 . Figure 7 In this context, "16℃ complete, 25℃ complete, and 37℃ complete" refer to bacterial cell lysate samples (containing supernatant and precipitate) collected from induction cultures at 16℃, 25℃, and 37℃, respectively. "16℃ supernatant, 25℃ supernatant, and 37℃ supernatant" refer to the supernatant samples after high-speed centrifugation of the bacterial cell lysate collected from induction cultures at 16℃, 25℃, and 37℃, respectively.

[0179] The results showed that 37℃ was the optimal induction temperature, so 37℃ was used as the induction temperature for the subsequent "(VII) Small-scale fermentation".

[0180] Example 3 Whole-cell catalysis test

[0181] In a 1L 200mM histamine reaction solution, 12g of batch 20240123 Ha2 cells, fermented according to the method described in "(VII) Small Tank Fermentation" above, were added. The reaction results are as follows. Figure 8 As shown in the figure. The results indicate that the conversion rate was 96.5% after 5 hours.

[0182] Example 4: Optimization of cell mass at high substrate concentrations

[0183] Weigh 93g of L-histidine into 900ml of pure water and stir. Adjust the pH to 6.0 with 4M HCl, and bring the volume to 1L (L-histidine concentration is 600mM). Continue stirring for half an hour until the substrate is completely dissolved. Divide the reaction solution into four portions and add Ha2 cells obtained according to the "(VII) Small Tank Fermentation" method described above at cell concentrations of 25g / L, 35g / L, 45g / L, and 55g / L. Stir the reaction at room temperature. During the reaction, the pH gradually rises to 6.5 and is then maintained at 6.5-7.0. The reaction results are as follows. Figure 9 As shown.

[0184] The results showed that the transformation rate was 95.8% with a substrate concentration of 600 mM and a cell concentration of 55 g / L after 7 h of reaction, but the cell quantity was too high and the cells were difficult to separate after the reaction.

[0185] Weigh 77.5g of L-histidine into 900ml of pure water and stir. Adjust the pH to 6.0 with 4M HCl, and bring the volume to 1L (L-histidine concentration is 500mM). Continue stirring for 15 minutes until the substrate is completely dissolved. Divide the reaction solution into four portions and add Ha2 cells obtained according to the "(VII) Small Tank Fermentation" method described above at cell concentrations of 40g / L, 45g / L, 50g / L, and 55g / L. Stir the reaction at room temperature. During the reaction, the pH gradually rises to 6.5 and is then maintained at 6.5-7.0. The reaction results are as follows. Figure 10 As shown.

[0186] The results showed that the transformation rate was 49.9% after 3 hours of reaction with 500 mM substrate and 40 g / L cells, and 100% overnight.

[0187] Example 5: Screening of Ha2 enzyme mutants

[0188] Fermentation cells of Ha2, Ha2-Cys525Val (SEQ ID NO: 21), Ha2-Cys547Ser (SEQ ID NO: 22), and Ha2-Cys558Val (SEQ ID NO: 23) were prepared according to the process described in “(VII) Small Tank Fermentation” above.

[0189] A 500 mM L-histidine reaction solution was prepared and divided into four equal portions. 40 g / L of the above-mentioned bacterial cells were added to each portion, and the mixture was stirred at room temperature. During the reaction, the pH was gradually increased to 6.5 and then maintained at 6.5-7.0. The reaction results are as follows: Figure 11 .

[0190] The results showed that the mutants Ha2-Cys547Ser and Ha2-Cys558Val exhibited higher reaction rates compared to the Ha2 enzyme.

[0191] Example 6: Whole-cell cell mass optimization experiment

[0192] A 500 mM L-histidine reaction solution was prepared and divided into three equal portions. Ha2-Cys547Ser fermentation cells were added to the reaction solution at cell concentrations of 20 g / L, 30 g / L, and 40 g / L, respectively. The mixture was stirred at room temperature, and the pH was gradually increased to 6.5 and then maintained at 6.5-7.0. The Ha2-Cys547Ser fermentation cells were prepared according to the "(VII) Small-Scale Fermentation" process described above. The reaction results are as follows: Figure 12 As shown.

[0193] The results showed that the transformation rate of Ha2-Cys547Ser 20g / L cells after 3 hours of reaction was 70.9%, which was comparable to that of 55g / L Ha2 cells, thus achieving the effect of reducing the amount of cells used.

[0194] Example 7: Effect of Immobilized Cells on Synthetic Reactions

[0195] Cell immobilization: Dissolve 2g of sodium alginate in 100ml of pure water by heating. After cooling to room temperature, mix with 100ml of 120g / L bacterial solution, then add dropwise to 300ml of 2% calcium chloride solution for hardening for 4 hours. Collect the hardened microsphere-like immobilized cells (see...). Figure 13 Rinse with pure water and store in the refrigerator.

[0196] Weigh Figure 13 40g of immobilized cells were added to 1L of 500mM L-histidine reaction solution and stirred at low speed at room temperature. After the reaction, the immobilized cells were separated, recovered, and reused. The reaction results are shown below. Figure 14 .

[0197] The results showed that the conversion rates for the first three reactions after 9 hours were 100%, 100%, and 95.6%, respectively, indicating good activity retention. Moreover, compared with the whole-cell reaction in Example 3, the immobilized cell reaction method resulted in easier cell separation after the reaction, less foaming of the reaction solution, and higher subsequent purification yield.

[0198] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of histidine decarboxylase, characterized in that, The amino acid sequence of the histidine decarboxylase is shown in SEQ ID NO: 3; the mutation site of the mutant is one of C525V, C547S, and C558V.

2. The nucleic acid encoding the mutant of claim 1.

3. The nucleic acid according to claim 2, characterized in that, Its nucleotide sequence is shown in any one of SEQ ID NO: 24~26.

4. A biomaterial, characterized in that, It includes at least one of the following: (A) An expression frame containing the nucleic acid of claim 2 or 3; (B) An expression carrier containing the expression frame described in (A); (C) Host cells containing the expression vector described in (B).

5. The use of at least one of the following (I) to (III) in the synthesis of histamine: I) Histidine decarboxylase with an amino acid sequence as shown in SEQ ID NO: 3; II) The nucleic acid as described in claim 2 or 3; III) The biomaterial described in claim 4.

6. A method for synthesizing histamine, characterized in that, include: Histamine was synthesized using histidine as a substrate under the catalysis of the histidine decarboxylase shown in SEQ ID NO: 3 or the mutant described in claim 1.

7. The synthesis method according to claim 6, characterized in that, The substrate is a 100mM~600mM aqueous solution of histidine at pH 5-8.

8. A method for synthesizing histamine, characterized in that, include: Histamine is obtained by adding bacterial cells, bacterial cell lysate, immobilized cells or immobilized enzymes to histidine as a substrate. The method for preparing the bacterial cells includes: collecting the bacterial cells after inducing the host cells in the biomaterial of claim 4 through shake flask or fermentation culture; The method for preparing the bacterial cell lysate includes: culturing the host cells in the biomaterial described in claim 4 in a shake flask or by fermentation, collecting the bacterial cells, lysing them, and obtaining the bacterial cell lysate; The method for preparing the immobilized cells includes: inducing host cells in the biomaterial of claim 4 by shaking flask or fermentation culture, collecting the bacterial cells, and using the bacterial cells to prepare immobilized cells; The method for preparing the immobilized enzyme includes: inducing host cell culture in the biomaterial described in claim 4 by shaking flask or fermentation, collecting the cells, breaking them, and preparing the immobilized enzyme using the lysate.

9. The synthesis method according to claim 8, characterized in that, The substrate is a 100mM~600mM aqueous solution of histidine at pH 5-8.

10. The synthesis method according to claim 8, characterized in that, The mass ratio of immobilized cells to histidine is (30~120):(62~93); the reaction is carried out under stirring conditions at 20℃~30℃ and pH 5-8.

11. The synthesis method according to claim 8, characterized in that, The method for preparing the immobilized cells includes: After mixing the bacterial culture containing the host cells described in claim 4 with an aqueous sodium alginate solution, the mixture was hardened in a 1-6% calcium chloride solution for 2-6 hours, and the hardened microsphere-shaped immobilized cells were collected.

12. The synthesis method according to claim 11, characterized in that, The concentration of the sodium alginate aqueous solution is 10~40 g / L; the concentration of the bacterial solution is 50~140 g / L.

13. The synthesis method according to claim 11, characterized in that, The mixing is an equal-volume mixing.

14. The synthesis method according to claim 8, characterized in that, When histidine is added to the bacterial cells or bacterial cell lysate, the mass ratio of bacterial cells to histidine is (15~30):(62~93).

15. The synthesis method according to any one of claims 8 to 14, characterized in that, The conditions for the shake flask or fermentation culture are: add an inducing agent and culture at 16~37℃ for 4-20h; The culture medium used for the shake flask culture is LB medium; the culture medium for the fermentation culture includes the following components: Yeast powder 12~24g / L Peptone 4~12g / L Glycerin 3~6g / L Sodium chloride 0.5~1g / L Magnesium sulfate 1~2g / L Dipotassium hydrogen phosphate trihydrate 3~5g / L Potassium dihydrogen phosphate 3~5g / L Disodium hydrogen phosphate dodecahydrate 1~2.5g / L Defoamer 0.4~1g / L.

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