A method for preparing decarboxylcarnosine using the nonribosomal peptide synthase DpEbony

Abstract

This invention discloses a method for preparing decarboxylated carnosine using the nonribosomal peptide synthase DpEbony, belonging to the fields of functional enzyme technology and biosynthesis technology. The method employs the nonribosomal peptide synthase DpEbony, phosphotransferase Sfp, and histidine decarboxylase HDC to catalyze the synthesis of decarboxylated carnosine from the substrates L-histidine and β-alanine. The amino acid sequence of DpEbony is shown in SEQ ID NO.1. This invention first converts L-histidine to histamine under the action of HDC, and then condenses it with β-alanine under the combined action of DpEbony and Sfp to generate decarboxylated carnosine. This method uses inexpensive L-histidine instead of expensive histamine as the starting substrate, significantly reducing production costs. Furthermore, DpEbony can be efficiently and solublely expressed in E. coli, exhibiting high enzyme activity and good stability.

Description

Technical Field

[0001] This invention relates to a method for preparing decarboxylated carnosine using the nonribosomal peptide synthase DpEbony, and the application of the nonribosomal peptide synthase DpEbony in catalyzing the synthesis of decarboxylated carnosine from histamine and β-alanine, belonging to the fields of functional enzyme technology and biosynthesis technology. Background Technology

[0002] Carcinine, also known as β-alanyl-L-histamine, is a molecular structure composed of β-alanine and histamine linked by an amide bond. The imidazole ring in the molecule is a key functional group that exerts various biological activities. It was first discovered in the neural tissue of crustaceans in 1975, and subsequent studies confirmed its widespread distribution in various tissues of mammals, including the heart, muscles, and brain. Its commercial form is usually dihydrochloride (C8H2O). 14 (N4O·2HCl), with a relative molecular mass of 255.14, is a white or off-white crystalline powder at room temperature, odorless, and with a slightly bitter taste. Decarboxylated carnosine has good solubility in water (solubility greater than 15 g / L at 25℃), but is almost insoluble in organic solvents such as ethanol and ether.

[0003] Compared to carnosine (β-alanyl-L-histidine), which is widely used in the cosmetics industry and has a similar structure, decarboxylated carnosine lacks the carboxyl group on the α-carbon atom of histidine. However, the retention of the imidazole group and free amino active group in decarboxylated carnosine allows it to retain its antioxidant and anti-glycation properties. Because it lacks a carboxyl group, decarboxylated carnosine has a stronger positive charge, does not form intramolecular salts, and exhibits better surface adsorption, permeability, and metal ion chelation ability in the skin. It is also more effective in antioxidation and inhibiting protein cross-linking, and has lower sensitivity to temperature and pH. Furthermore, the human body contains carnosinase, an enzyme that can break down carnosine; decarboxylation or acetylation increases the molecule's resistance to enzymatic hydrolysis. Since decarboxylated carnosine cannot be recognized and broken down by carnosinase, it is more stable and has a longer duration of action. Studies have also verified the metabolic dynamics of carnosine and decarboxylated carnosine on skin microsomes, demonstrating that decarboxylated carnosine does maintain good stability on the skin. All of these findings indicate the broad prospects for the application of decarboxylated carnosine in the cosmetics industry.

[0004] In 1994, EXSYMOL Monaco pioneered the application of decarboxylated carnosine in cosmetics. Thanks to its superior anti-glycation, antioxidant, and anti-inflammatory effects, it quickly became a core active ingredient in anti-aging skincare products. In recent years, with ongoing research, the application value of decarboxylated carnosine in pharmaceutical fields such as neuroprotection, cardiovascular protection, and prevention of diabetic complications has been gradually discovered. However, the limitations of traditional preparation methods severely restrict the large-scale production and application of decarboxylated carnosine. Developing efficient, green, and low-cost preparation technologies has become a key scientific problem urgently needing to be solved in this field.

[0005] Currently, the synthesis methods for decarboxylated carnosine are mainly divided into two categories: chemical synthesis and enzymatic synthesis. Chemical synthesis, which focuses on constructing amide bonds through acyl chloride, suffers from long synthetic routes and cumbersome steps. It also requires peptide coupling reagents and acyl chloride reagents, which can easily cause environmental pollution. Furthermore, the numerous byproducts lead to low yields and high purification costs. The high price of the starting substrate, histamine, further increases the preparation cost. Biosynthetic methods mainly include microbial fermentation and enzymatic catalysis. (Zhao et al., Man Zhao, Xiangting Song, Wei Liu, Fengjie Qi, Tingting Zhao, Keke Xia, Zhiqiang Liu, Yuguo Zheng). The paper "Whole-cell biotransformation for large-scale production of carcininein Escherichia coli" (Journal of Biotechnology, 2022, 354, 45-52) first reported in 2022 a method for producing decarboxycarcinopeptide using recombinant Escherichia coli whole-cell catalysis. They co-expressed the Ebony gene from Drosophila melanogaster and the Sfp gene from Bacillus subtilis to construct a recombinant engineered bacterium, achieving a decarboxycarcinopeptide yield of 1.2 g / L under optimized conditions. Subsequently, Suzhou Huasai Biotechnology Co., Ltd. improved the method by knocking out the pepD and dpp genes (encoding decarboxycarcinopeptide degrading enzyme and uptake enzyme, respectively) in E. coli, reducing product degradation and consumption. They also used the HiEbony gene from black soldier fly larvae, which has higher catalytic activity, further increasing the yield to 3.5 g / L in a 5 L fermenter. While the above whole-cell method avoids the use of chemical reagents, it still suffers from problems such as product degradation, high cost of histamine substrate, and a long fermentation cycle.

[0006] Enzymatic catalysis utilizes purified enzymes or crude enzyme solutions as catalysts to convert substrates into decarboxylated carnosine in vitro. It offers advantages such as rapid reaction speed, high selectivity, high product concentration, and simple separation and purification. Currently, the enzymes used for decarboxylated carnosine synthesis mainly include carboxylate reductase (CAR) and the non-ribosomal peptide synthase (Ebony). In 2022, Shenzhen Ruidelin Biotechnology Co., Ltd. reported a method for synthesizing decarboxylated carnosine using a CAR mutant enzyme. This method boasts high yield and good product purity, but requires the use of protected β-alanine as a substrate, increasing production costs and generating waste during deprotection. In contrast, the non-ribosomal peptide synthase Ebony, combined with phosphotransferase Sfp, can directly catalyze the synthesis of decarboxylated carnosine from β-alanine and histamine without requiring substrate protection or deprotection. The reaction steps are simple, making it a highly promising method for preparing decarboxylated carnosine. Currently, research on enzymatic catalysis using this enzyme is limited, and further exploration is needed.

[0007] Despite the advantages of the Ebony enzymatic method, such as mild reaction, environmental friendliness, and few byproducts, several technical bottlenecks remain: First, the number of non-ribosomal peptide synthases discovered is limited, the activity and yield of natural enzymes are low, and some enzymes, when expressed heterologously, easily form inclusion bodies that cannot be obtained from bacterial cell precipitates; second, traditional methods for detecting decarboxycarnitine require derivatization with dansyl chloride, which is complex, costly, and does not allow for direct separation of substrate and product; third, existing enzymatic methods mostly use expensive histamine as the initial substrate, failing to achieve direct utilization of the inexpensive substrate L-histidine, and the high preparation cost limits its application.

[0008] Non-ribosomal peptide synthases are the core enzymes catalyzing the synthesis of decarboxylated carnosine. Ebony enzymes from different sources exhibit significant differences in enzyme activity, expression stability, and substrate specificity. Discovering and screening Ebony homologs with high activity and easy expression, introducing the inexpensive substrate L-histidine, and constructing a multi-enzyme reaction system by combining it with the histidine decarboxylase HDC to achieve the enzymatic synthesis of decarboxylated carnosine is key to solving the high cost of decarboxylated carnosine preparation and realizing its green and efficient production. This invention is formally proposed based on this principle. Summary of the Invention

[0009] To address the shortcomings of the existing technologies, this invention provides a method for preparing decarboxylated carnosine using the non-ribosomal peptide synthase DpEbony. By screening for high-activity non-ribosomal peptide synthase DpEbony, and combining it with phosphotransferase Sfp and histidine decarboxylase HDC to construct a multi-enzyme reaction system, using inexpensive L-histidine as the starting substrate, a green, efficient, and low-cost preparation of decarboxylated carnosine is achieved, providing a new enzyme resource for the biosynthesis of decarboxylated carnosine. Simultaneously, this invention also establishes a novel method for detecting decarboxylated carnosine, which eliminates the need for derivatization reactions, reducing detection steps and lowering production costs.

[0010] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a method for preparing decarboxylated carnosine using a nonribosomal peptide synthase DpEbony. The method involves using the nonribosomal peptide synthase DpEbony, a conjugated phosphotransferase Sfp, and a histidine decarboxylase HDC to catalyze the synthesis of decarboxylated carnosine from substrates L-histidine and β-alanine. The amino acid sequence of the nonribosomal peptide synthase DpEbony is shown in SEQ ID NO.1.

[0011] Furthermore, the specific method of catalytic synthesis is as follows: First, HDC is added to a solution containing L-histidine, and the reaction is carried out at 20-60℃ and pH 6.0-8.0 for 30 min-12 h to convert L-histidine into histamine; then, the reaction temperature is adjusted to 20-50℃ using staged temperature control, and β-alanine, DpEbony and Sfp, adenosine triphosphate (ATP) and Mg²⁺ are directly added, and the reaction is carried out for 3 h-24 h to jointly catalyze the generation of decarboxylated carnosine.

[0012] Furthermore, the amount of non-ribosomal peptide synthase DpEbony added is 400-600 μg / mL, and DpEbony and Sfp are added at an enzyme ratio of 1:1. The amount of HDC added is 250-350 μg / mL.

[0013] Furthermore, the concentration of L-histidine in the solution containing L-histidine is 5–15 mg / mL, the molar ratio of β-alanine to L-histidine is 1:8–8:1 (preferably 1:1–1:8 or 2:1–8:1), and all substrates are dissolved in phosphate-buffered saline (PBS) at pH 6.0.

[0014] Furthermore, ATP and Mg are added to the reaction system. 2+ The final concentration of ATP was 2.5 mmol / L, and Mg... 2+ The final concentration was 10 mmol / L, providing a cofactor for the activation of the nonribosomal peptide synthase DpEbony, wherein the Mg 2+ It was added in the form of MgCl2.

[0015] Furthermore, the stage temperature control is as follows: after converting L-histidine to histamine at 50°C, the reaction temperature is adjusted to 35°C.

[0016] Furthermore, the reaction time is 1-3 h (preferably 2 h) after L-histidine is converted into histamine, and other substances are added and the reaction is carried out for 12-16 h.

[0017] Furthermore, the reaction was carried out in phosphate-buffered saline (PBS) at pH 6.0.

[0018] Furthermore, high-performance liquid chromatography-evaporative light scattering (HPLC-ELSD) was used to detect the decarboxylated carnosine product. The HPLC-ELSD method established in this invention abandons the traditional dansyl chloride derivatization detection method, achieving non-derivative, specific, and accurate detection of decarboxylated carnosine. It can directly separate the substrate and the product decarboxylated carnosine, and is suitable for rapid detection and system optimization in enzymatic synthesis processes.

[0019] Secondly, the present invention provides the application of the non-ribosomal peptide synthase DpEbony in the preparation of decarboxylated carnosine, wherein the amino acid sequence of the non-ribosomal peptide synthase DpEbony is shown in SEQ ID NO.1.

[0020] Thirdly, the present invention provides a recombinant expression vector carrying a coding gene encoding the non-ribosomal peptide synthase DpEbony, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0021] Furthermore, the recombinant expression vector uses pET-28a as the base plasmid and inserts the coding gene of DpEbony into the multiple cloning site of pET-28a to construct the recombinant plasmid pET28a-DpEbony.

[0022] Fourthly, a recombinant engineered bacterium is provided, comprising the recombinant expression vector, with Escherichia coli as the host.

[0023] Fifthly, the present invention provides an enzyme preparation comprising the aforementioned non-ribosomal peptide synthase DpEbony, and further comprising histidine decarboxylase HDC and phosphotransferase Sfp; the enzyme preparation is in the form of a pure enzyme or crude enzyme solution, and can be directly used for the biosynthesis of decarboxylated carnosine.

[0024] The present invention also provides the application of the recombinant expression vector and recombinant engineered bacteria in the preparation of nonribosomal peptide synthase DpEbony; the application of the enzyme preparation in the catalytic synthesis of decarboxylated carnosine; and the application in the industrial production of decarboxylated carnosine.

[0025] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: The nonribosomal peptide synthase DpEbony of this invention is derived from fruit flies. Drosophila persimilis It is a non-ribosomal peptide synthase that can be expressed heterologously in Escherichia coli without inclusion body formation. The optimal reaction temperature is 35℃, and the optimal reaction pH is 6.0 (PBS buffer). It has a relative enzyme activity of more than 80% at 25-40℃.

[0026] Furthermore, stability tests revealed that the enzyme exhibits excellent stability, retaining over 50% of its activity after incubation at 30°C for 120 hours; and retaining over 60% of its activity after storage at pH 6.0 / pH 7.0 and 4°C for 120 hours, making it an enzyme with outstanding stability.

[0027] This invention obtains an enzyme preparation through gene cloning, heterologous expression, purification, and lyophilization. It can be used in combination with histidine decarboxylase HDC and phosphotransferase Sfp. The ability to prepare decarboxylated carnosine from L-histidine was verified in vitro. The product was identified by high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) and mass spectrometry (MS). The optimal temperature, pH, and substrate concentration for decarboxylated carnosine preparation were optimized, thus establishing the core conditions for its production. The histidine decarboxylase HDC is derived from Lactobacillus sp. 30A, with an optimal catalytic temperature of 50°C and an optimal catalytic pH of 5.0 (citrate-phosphate buffer, CPBS). The phosphotransferase Sfp is derived from... Bacillus subtilis subsp. subtilis str. 168 provides an auxiliary role in the activation of the nonribosomal peptide synthase DpEbony. The method for preparing decarboxylated carnosine in this invention achieves multi-enzyme catalytic conversion from inexpensive L-histidine to decarboxylated carnosine, which is of great significance for realizing the green, low-cost, and large-scale preparation of decarboxylated carnosine.

[0028] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0029] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 Image 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) images of DpEbony, Sfp, and HDC proteins; Image 2: DpEbony and Sfp proteins, where M: 180 kDa protein molecular weight marker, lane 2: empty pET-28a cell lysis supernatant, lane 3: Sfp crude enzyme solution, lane 4: Sfp purified enzyme, lane 5: DpEbony crude enzyme solution, lane 6: DpEbony purified enzyme, lane 7: DpEbony precipitate; Image 3: HDC protein electrophoresis image of HDC proteins, where M: 180 kDa protein molecular weight marker, lane 2: empty pET-28a cell lysis supernatant, lane 3: HDC crude enzyme solution, lane 4: HDC purified enzyme.

[0031] Figure 2MS analysis chromatogram of decarboxylated carnosine, the product catalyzed by DpEbony ([Carcinine+H)). + Characteristic peaks).

[0032] Figure 3 HPLC chromatogram of decarboxylated carnosine, the product of DpEbony catalysis.

[0033] Figure 4 Effect of pH on DpEbony enzyme activity.

[0034] Figure 5 pH stability of DpEbony.

[0035] Figure 6 The effect of temperature on DpEbony enzyme activity.

[0036] Figure 7 Temperature stability of DpEbony.

[0037] Figure 8 Effect of pH on HDC enzyme activity.

[0038] Figure 9 pH stability of HDC.

[0039] Figure 10 The effect of temperature on HDC enzyme activity.

[0040] Figure 11 Temperature stability of HDC.

[0041] Figure 12 Optimization of substrate concentration in HDC reaction system.

[0042] Figure 13 The effect of substrate concentration in the HDC reaction system on enzyme activity.

[0043] Figure 14 Optimization of enzyme dosage in HDC reaction system.

[0044] Figure 15 Optimization of substrate concentration in the DpEbony reaction system.

[0045] Figure 16 Optimization of enzyme dosage in the DpEbony reaction system. Detailed Implementation

[0046] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0047] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0049] Example 1: Construction of the recombinant expression vector pET28a-DpEbony The encoding gene of the non-ribosomal peptide synthase DpEbony in this invention was synthesized from the whole genome and is a homologous gene of Ebony from *Drosophila persimilis*, mined from the NCBI database. Its NCBI code is XP_002023902.1. Currently, there are no literature reports on the expression, purification, and catalytic function of this enzyme. Histidine decarboxylase HDC and phosphotransferase Sfp are reported enzymes with activity, with NCBI codes J02613.1 and X63158.1, respectively. The expression vector was synthesized from the whole genome by a gene synthesis company (Shanghai Sangon Biotech). The target gene fragment was inserted into the multiple cloning site of the pET-28a plasmid. After codon optimization using *E. coli*, the sequence was confirmed to be correct by DNA sequencing, and recombinant plasmids were obtained, named pET28a-DpEbony, pET28a-HDC, and pET28a-Sfp, respectively.

[0050] The application of the nonribosomal peptide synthase DpEbony in the preparation of decarboxylated carnosine, wherein the amino acid sequence of the nonribosomal peptide synthase DpEbony is shown in SEQ ID NO.1, and codon optimization only changes the nucleotide sequence but not the amino acid sequence.

[0051] The nucleotide sequence of the gene encoding the non-ribosomal peptide synthase DpEbony is shown in SEQ ID NO.2, which is a codon-optimized sequence.

[0052] Example 2: Construction of recombinant engineered bacteria expressing DpEbony The recombinant plasmid pET28a-DpEbony constructed in Example 1 was transformed into E. coli BL21(DE3) competent cells using a heat shock transformation method: 1 μL of plasmid (approximately 50 ng) was added to 50 μL of competent cells, heat-shocked at 42°C for 90 seconds, followed by an ice bath for 2 minutes, and then 500 μL of LB liquid medium was added. The cells were then incubated at 37°C and 200 rpm for 45 minutes. After incubation, the cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 12 h. Single colonies grown on the plates were picked, and the plasmid was extracted and digested with enzymes to confirm the presence of the recombinant plasmid. The correctly verified strain was the constructed recombinant engineered bacterium E. coli BL21-pET28a-DpEbony. Using the same method, the recombinant plasmids pET28a-HDC and pET28a-Sfp were transformed into E. coli BL21(DE3) competent cells to construct recombinant engineered bacteria E. coli BL21-pET28a-HDC and E. coli BL21-pET28a-Sfp.

[0053] Example 3: Preparation of nonribosomal peptide synthase DpEbony using engineered Escherichia coli Single colonies of the recombinant engineered E. coli BL21-pET28a-DpEbony constructed in Example 2 were selected and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. After culturing at 37°C and 220 rpm for 12 h, 1% of the culture was inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm until OD600 = 0.6–0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the system to a final concentration of 0.5 mM, and expression was induced at 20°C and 200 rpm for 16 h. After induction, the bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 15 min. The bacterial cells were resuspended in lysis buffer (500 mM NaCl, 50 mM Tris-HCl, pH 8.0) and sonicated for 30 minutes under ice bath conditions. After disruption (power 200W, 3s operation / 5s interval), centrifuge at 4℃ and 8000rpm for 15min, and collect the supernatant as DpEbony crude enzyme solution. The crude enzyme solution is purified using a Ni-NTA affinity chromatography column. First, equilibrate the column with equilibration buffer (500 mM NaCl, 50 mM Tris-HCl, pH 7.5). After loading the sample, wash unbound proteins with equilibration buffer, elute weakly bound proteins with 20 mM imidazole solution (20 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), and finally elute the target protein with 150 mM imidazole solution (150 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). Collect the eluent and concentrate it to 1 mL using an ultrafiltration tube (molecular weight cutoff 30 kDa) to obtain pure enzyme solution. The obtained pure enzyme solution was analyzed by SDS-PAGE to check whether the bands were uniform and whether the size was accurate. The results are as follows: Figure 1 As shown in the figure, a band of 103.3 kDa was obtained, consistent with the prediction, confirming that the target protein is the DpEbony protein shown in SEQ ID NO.1. SDS-PAGE analysis was performed on the supernatant (crude enzyme solution) and precipitate of the fragmented DpEbony protein. The target protein was mainly distributed in the supernatant, indicating that DpEbony is mainly expressed in a soluble form with no obvious inclusion body formation. The protein concentration in the purified enzyme solution was determined using the Bradford method to be 2.37 mg / mL. Histidine decarboxylase HDC purified enzyme solution (target protein eluted with 80 mM imidazole solution) and phosphotransferase Sfp purified enzyme solution (target protein eluted with 150 mM imidazole solution) were prepared using the same method. SDS-PAGE analysis of the obtained purified enzyme solutions yielded the following results: Figure 1 As shown, stripes with sizes of 30.7 kDa and 38.8 kDa were obtained.

[0054] Example 4: Enzyme activity assay of nonribosomal peptide synthase DpEbony Enzyme activity assay system: Histamine and β-alanine were used as substrates, with final substrate concentrations of 20 mM and 10 mM, respectively. ATP (final concentration 2.5 mM), MgCl2 (final concentration 10 mM), Sfp pure enzyme solution (final concentration 125 μg / mL), and DpEbony pure enzyme solution (final concentration 300 μg / mL) were added. The system was then brought to 1 mL with PBS buffer (pH 6.0) and reacted in a 35°C water bath for 6 h. The pure enzyme solution obtained in Example 3 was used as the enzyme index, and the same applies below. A control group of DpEbony was set up by boiling inactivation, in which DpEbony was pre-boiled and inactivated for 10 minutes, and the other components (including Sfp, substrate, ATP, and Mg²⁺) were the same as those in the experimental group. Product detection: After the reaction was completed, an equal volume of acetonitrile was added to terminate the reaction. The mixture was centrifuged at 4°C and 12,000 rpm for 5 min. The supernatant was collected and analyzed by mass spectrometry (MS) to identify the decarboxycarnitine product. At the same time, the amount of decarboxycarnitine generated was detected by the HPLC-ELSD method established in this invention.

[0055] MS analysis conditions: Electrospray positive ion mode (ESI⁺), scan range m / z 50-500.

[0056] HPLC-ELSD detection conditions: Comixsil RP-100 (4.6×250 mm, 5 μm); mobile phase A: water, mobile phase B: acetonitrile, mobile phase C: 100 mM ammonium formate; gradient elution: 0–8 min 90%–67% A, 0%–3% B, 10%–30% C; 8–14 min 67%–0% A, 3%–60% B, 30%–40% C; flow rate 1.0 mL / min; column temperature 25℃; ELSD parameters: drift tube temperature 80℃, nitrogen flow rate 2.5 L / min.

[0057] Test results as follows Figure 2 The MS analysis showed a characteristic peak of [Carcinine+H]+, indicating the formation of decarboxylated carnosine, demonstrating the activity of the non-ribosomal peptide synthase DpEbony discovered in this invention; Meanwhile, the liquid chromatography results showed... Figure 3 As shown, under these conditions, 3.47 mM of decarboxycarnosine can be detected, while no decarboxycarnosine is detected in the control group, proving that DpEbony is the core enzyme catalyzing the synthesis of decarboxycarnosine and requires Sfp for activation.

[0058] Example 5: Determination of the effect of pH on the nonribosomal peptide synthase DpEbony Effect of pH on enzyme activity: Following the enzyme activity assay system of Example 4, the following buffers were used: citrate-phosphate buffer (CPBS) at pH 3.0, 4.0, and 5.0; phosphate buffer (PBS) at pH 5.0, 6.0, and 7.0; Tris-HCl buffer at pH 7.0, 8.0, and 9.0; and glycine-NaOH buffer at pH 9.0, 10.0, and 11.0. DpEbony was placed directly in the buffers of different pH values ​​and reacted at 37°C under the conditions of Example 4, and enzyme activity was measured at different pH values. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated.

[0059] The results are as follows Figure 4 As shown, the optimal catalytic pH for DpEbony is 6.0 (PBS buffer).

[0060] pH stability: DpEbony was placed in PBS buffer at pH 5.0, 6.0, and 7.0 and incubated at 4°C for 144 h. The remaining enzyme activity was then measured according to the conditions in Example 4.

[0061] The results are as follows Figure 5 As shown, DpEbony retained more than 50% of its enzyme activity after 144 h in buffer solutions at pH 5.0, 6.0, and 7.0, indicating that the enzyme has good stability.

[0062] Example 6: Determination of the effect of temperature on the nonribosomal peptide synthase DpEbony Effect of temperature on enzyme activity: Following the enzyme activity assay system of Example 4, the reaction was carried out at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃ and 50℃ (pH 6.0 PBS buffer), and the enzyme activity at different temperatures was measured. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated.

[0063] The results are as follows Figure 6 As shown, the optimal catalytic temperature of DpEbony is 35℃, and the relative enzyme activity is above 60% in the range of 30~45℃, indicating good temperature adaptability.

[0064] Temperature stability: DpEbony pure enzyme solution was incubated in water baths at 30℃, 35℃, and 40℃ for 72 h, respectively. Samples were taken at regular intervals, and the remaining enzyme activity was determined according to the conditions in Example 4. The results are as follows: Figure 7 As shown, DpEbony retains more than 50% of its enzyme activity after being placed at 30 and 35°C for 72 hours, indicating good enzyme activity stability.

[0065] Example 7 Enzyme activity assay of histidine decarboxylase HDC HDC enzyme activity was determined colorimetrically: L-histidine was dissolved in PBS (pH 6.0) to a final concentration of 5 mg / mL. HDC purified enzyme solution (final concentration 250 μg / mL) was added, and the volume was brought to 1 mL with PBS buffer (pH 6.0). The reaction was carried out at 50°C for 30 min. After the reaction, the supernatant was collected by centrifugation, and the absorbance was measured at OD420 using a commercial histamine kit (proceded according to instructions, Shanghai Baisun Biotechnology Co., Ltd., catalog number: 12829). The histamine production was calculated based on the histamine standard curve. The results showed that under these conditions, after 30 min of reaction, the histamine production was 2.8 mM, indicating that HDC can efficiently catalyze the conversion of L-histidine to histamine.

[0066] Example 8: Determination of the effect of pH on histidine decarboxylase HDC Effect of pH on enzyme activity: Following the enzyme activity assay system of Example 7, the following buffers were used: CPBS buffer at pH 3.0, 4.0, and 5.0; PBS buffer at pH 5.0, 6.0, and 7.0; Tris-HCl buffer at pH 7.0, 8.0, and 9.0; and glycine-NaOH buffer at pH 9.0, 10.0, and 11.0. HDC was placed directly in buffers of different pH values ​​and reacted at 50°C under the conditions of Example 7. Enzyme activity was measured at different pH values, and the highest enzyme activity was taken as 100% to calculate the relative enzyme activity.

[0067] The results are as follows Figure 8 As shown, the optimal catalytic pH for HDC is 5.0 (CPBS buffer).

[0068] pH stability: HDC was placed in CPBS buffer at pH 3.0, 4.0, and 5.0 (and PBS buffer at pH 6.0 and 7.0) and incubated at 4°C for 240 h (without substrate). Samples were then taken and the remaining enzyme activity was determined under the optimal conditions of Example 7 (pH 5.0 CPBS buffer, 50°C).

[0069] The results are as follows Figure 9 As shown, after incubation in buffer solutions at pH 5.0, 6.0, and 7.0 for 240 h, HDC retained more than 75% of its original enzyme activity, indicating that the enzyme has excellent stability.

[0070] Example 9: Determination of the effect of temperature on histidine decarboxylase HDC Effect of temperature on enzyme activity: Following the enzyme activity assay system of Example 7, CPBS buffer with pH 5.0 was used instead of PBS buffer. Reactions were carried out at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃ and 60℃ respectively. Enzyme activity at different temperatures was measured, and the highest enzyme activity was taken as 100% to calculate the relative enzyme activity.

[0071] The results are as follows Figure 10 As shown, the optimal catalytic temperature of HDC is 50℃, and its relative enzyme activity is above 60% in the range of 45~60℃. It has good temperature adaptability and belongs to the category of relatively heat-resistant enzymes.

[0072] Temperature stability: HDC pure enzyme solution was incubated in water baths at 45℃, 50℃, and 55℃ respectively. Samples were taken at regular intervals, and the remaining enzyme activity was measured under the optimal conditions of Example 7 (pH 5.0 CPBS buffer, 50℃).

[0073] The results are as follows Figure 11 As shown, HDC incubation at high temperature for 24 h gradually increases enzyme activity, followed by a gradual decrease. After being placed at 45 and 50 °C for 108 h, the enzyme activity can still retain more than 50%, indicating good enzyme activity stability. At the same time, the results also suggest that the enzyme can be incubated at high temperature before participating in the reaction.

[0074] Example 10 Optimization of the reaction system for histidine decarboxylase HDC Following the method in Example 7, the amount of histamine produced under different conditions was determined, and the conversion rate of L-histidine was calculated.

[0075] (a) Substrate concentration optimization With a fixed HDC enzyme dosage of 100 μg / mL, and L-histidine concentrations of 5, 10, and 15 mg / mL, the amount of histamine produced at different time points was measured.

[0076] The results are as follows Figure 12 , 13 As shown, at low enzyme concentrations (100 μg / mL), the conversion rates at substrate concentrations of 10 mg / mL and 15 mg / mL were similar, but both were limited by the enzyme concentration and the conversion rates were not high. To obtain higher histamine yields, the enzyme concentration needs to be increased.

[0077] (II) Optimization of enzyme dosage With the L-histidine concentration fixed at 10 mg / mL, the amount of HDC enzyme added was set at 250, 300, and 350 μg / mL, and the amount of histamine produced at different time points was measured.

[0078] The results are as follows Figure 14As shown, when the enzyme dosage is 300 μg / mL, the conversion rate of L-histidine can reach 100% under the conditions of 50℃ and pH 6.0 (PBS) for 1-2 hours.

[0079] (III) Determination of the optimal reaction system Taking into account both substrate utilization efficiency and reaction time, the optimal reaction conditions for HDC were determined to be: L-histidine concentration 10 mg / mL, HDC enzyme dosage 300 μg / mL, 50℃, pH 6.0 PBS buffer, and reaction time 1–2 h.

[0080] It should be noted that although the optimal pH for HDC is 5.0 ( Figure 8 However, it can still maintain high activity at pH 6.0, and pH 6.0 is the optimal pH for the subsequent DpEbony reaction. Therefore, the HDC reaction was carried out in PBS buffer at pH 6.0.

[0081] Example 11 Optimization of the reaction system for the nonribosomal peptide synthase DpEbony First, L-histidine was completely catalyzed to histamine under the optimal conditions determined in Example 10. After the reaction, the reaction solution was divided into several equal portions and diluted by adding different volumes of reaction buffer (to keep the total volume and composition of the buffer consistent in each group of reaction solutions), or by adding different amounts of histamine standards to set different final histamine concentrations. Then, β-alanine, DpEbony, Sfp, ATP (final concentration 2.5 mM), and MgCl2 (final concentration 10 mM) were added to start the reaction. After the reaction, the amount of decarboxycarboxylated carnosine generated was detected by HPLC-ELSD, and the conversion rate was calculated (conversion rate % = amount of decarboxylated carnosine generated / initial histamine concentration × 100%).

[0082] (a) Optimization of substrate molar ratio The molar ratios of histamine to β-alanine were fixed at 1:1, 1:2, 1:3, 1:4, 1:5, 1:8, 2:1, 4:1, 6:1, and 8:1. Both DpEbony and Sfp were fixed at 300 μg / mL, and the reaction was carried out at 35℃ in pH 6.0 PBS buffer for 12 h.

[0083] The results are as follows Figure 15 As shown, the conversion rate of decarboxylated carnosine is highest when the substrate molar ratio (histamine:β-alanine) is 1:1.

[0084] (II) Optimization of DpEbony enzyme dosage The molar ratio of histamine to β-alanine was fixed at 1:1, and the concentration of both was 40 mM. The Sfp enzyme dosage was fixed at 600 μg / mL, and the DpEbony enzyme dosage was set at 400, 450, 500, 550, and 600 μg / mL. The reaction was carried out at 35℃ in PBS buffer at pH 6.0 for 12 h.

[0085] The results are as follows Figure 16 As shown, the conversion rate gradually increased with the increase of DpEbony enzyme dosage, and reached its highest value when the enzyme dosage was 600 μg / mL.

[0086] (III) Determination of the optimal reaction system Based on the above results, the optimal reaction conditions for DpEbony were determined to be: a histamine to β-alanine molar ratio of 1:1, a DpEbony enzyme dosage of 600 μg / mL, a Sfp enzyme dosage of 600 μg / mL, 35℃, pH 6.0 PBS buffer, and a reaction time of 12 h. Under these conditions, the conversion rate of decarboxylated carnosine reached 99.13%.

[0087] Example 12: One-pot multi-enzyme reaction for the preparation of decarboxylated carnosine First, L-histidine is converted to histamine under the catalysis of HDC. The reaction system consisted of 10 mg / mL L-histidine, 300 μg / mL HDC, and PBS buffer at pH 6.0. The reaction was carried out in a 50°C water bath for 2 h.

[0088] Tests showed that the L-histidine conversion rate could reach 100%.

[0089] Subsequently, using staged temperature control, the temperature was lowered to 35°C within 5 minutes using a circulating water bath. β-alanine (added in a molar ratio of 1:1 with L-histidine), DpEbony (600 μg / mL), Sfp (600 μg / mL), ATP (final concentration 2.5 mM), and Mg2+ (final concentration 10 mM) were added directly to the same reaction system, and the reaction was continued for 12 hours to co-catalyze the generation of decarboxylated carnosine.

[0090] Product detection: After the reaction was completed, an equal volume of acetonitrile was added to terminate the reaction. The supernatant was collected by centrifugation and detected by HPLC-ELSD and MS (detection conditions were the same as in Example 4). The results showed that decarboxycarnosine was successfully generated in the system, with no obvious substrate residue or byproducts, achieving a one-step conversion from L-histidine to decarboxycarnosine.

[0091] Under optimal reaction conditions, the catalytic yield of decarboxycarnitine can reach 39.651 mM, with a conversion rate of 99.13% and a production intensity of 0.602 g / (L·h) (3.3 mM / h), which is significantly higher than the production intensity of decarboxycarnitine produced by non-ribosomal peptide synthases reported to date.

[0092] Example 13 Preparation of enzyme preparations The DpEbony, HDC, and Sfp pure enzyme solutions prepared in Example 3 were mixed according to the optimal enzyme addition ratio determined in Example 12, and then freeze-dried using a vacuum freeze dryer to obtain a composite enzyme preparation containing DpEbony, HDC, and Sfp. This enzyme preparation is in powder form, can be stored for a long time, and can be directly used for the preparation of decarboxycarnitine after dissolving in buffer solution. The operation is simple and suitable for industrial applications. Industrial Applicability The non-ribosomal peptide synthase DpEbony of this invention can be efficiently and heterologously expressed in Escherichia coli with high enzyme activity and stable expression. The method for preparing decarboxylated carnosine using DpEbony as the core enzyme employs inexpensive L-histidine as the initial substrate and combines it with histidine decarboxylase to construct a multi-enzyme reaction system, which can achieve green, efficient, and low-cost preparation of decarboxylated carnosine. The reaction conditions are mild, the steps are simple, and the product purity is high. At the same time, the newly established detection method can further reduce production costs and has significant industrial application value, which can promote the large-scale production and application of decarboxylated carnosine in cosmetics and other fields.

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing decarboxylated carnosine using the nonribosomal peptide synthase DpEbony, characterized in that: The nonribosomal peptide synthase DpEbony, Sfp, and HDC are used to catalyze the synthesis of decarboxylated carnosine from substrates L-histidine and β-alanine. The amino acid sequence of the nonribosomal peptide synthase DpEbony is shown in SEQ ID NO.

1. The specific method of catalytic synthesis is as follows: First, HDC is added to a solution containing L-histidine, and the reaction is carried out at 20-60℃ and pH 6.0-8.0 for 30 min-12 h to convert L-histidine into histamine; the amount of HDC added is 250-350 μg / mL, and the concentration of L-histidine in the solution containing L-histidine is 5-15 mg / mL; Subsequently, the reaction temperature was adjusted to 35°C using staged temperature control, and β-alanine, DpEbony and Sfp, ATP and Mg were directly added. 2 + The reaction was carried out for 12 hours, and the decarboxylated carnosine was co-catalyzed to generate carnosine. The amount of DpEbony added was 600 μg / mL, the amount of Sfp added was 600 μg / mL, and the molar ratio of histamine to β-alanine was 1:

1. When the reaction is carried out in a one-pot process, β-alanine and L-histidine are added to the reaction system in a molar ratio of 1:

1.

2. The method according to claim 1, characterized in that, The final concentration of ATP added to the reaction system was 2.5 mmol / L, and the Mg content was... 2+ The final concentration was 10 mmol / L, and the Mg... 2+ It is added in the form of MgCl2; The temperature control during the stage is as follows: after converting L-histidine to histamine at 50℃, the reaction temperature is adjusted to 35℃.

3. The application of the non-ribosomal peptide synthase DpEbony in the preparation of decarboxylated carnosine, characterized in that, The amino acid sequence of the nonribosomal peptide synthase DpEbony is shown in SEQ ID NO.1.

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