Hemoprotein mutant and application thereof in preparation of 5-hydroxy-l-tryptophan

Abstract

This invention discloses a heme protein mutant and its application in the preparation of 5-hydroxy-L-tryptophan, belonging to the fields of enzyme engineering and biocatalysis. This invention uses heme protein A2 as the starting protein and provides a method for direct catalysis with the participation of hydrogen peroxide. L A mutant of heme protein with β-tryptophan hydroxylation. The mutant constructed in this invention can be used in a solution containing 3 mmol / L... L In a reaction system containing 5-hydroxy-L-tryptophan, 15 mmol / L hydrogen peroxide, and 20 μmol / L purification enzyme, a 5-hydroxy-L-tryptophan conversion rate of 24.95% was achieved, compared to the single-mutant A2. M1 The percentage of 5-hydroxy-L-tryptophan was increased by approximately 8.63 times from 2.89%; and the area ratio of 5-hydroxy-L-tryptophan in the main monohydroxylation product peak was increased from approximately 49.8% to 97.1%, while the non-target product peak was reduced.

Description

Technical Field

[0001] This invention relates to a heme protein mutant and its application in the preparation of 5-hydroxy-L-tryptophan, belonging to the fields of enzyme engineering, biocatalysis and microbial engineering technology. Background Technology

[0002] 5-Hydroxy-L-tryptophan (5-HTP) is an important precursor for serotonin synthesis and has significant applications in pharmaceuticals, nutritional supplements, and functional foods. Current methods for producing 5-HTP mainly include plant extraction, chemical synthesis, and biocatalysis.

[0003] Plant extraction methods primarily rely on natural resources such as Ghana seeds, which suffer from limitations in raw material availability, significant batch-to-batch variability, high extraction costs, and insufficient stability for large-scale production. Chemical synthesis methods typically require multiple reaction steps and protection / deprotection processes, and because... L The high electron cloud density and numerous reactive sites of the tryptophan indole ring make it prone to generating regioisomeric byproducts, which limits the selectivity and purification efficiency of the target product.

[0004] Biocatalysis can achieve L-tryptophan hydroxylation under mild conditions, representing a potential green route for 5-HTP production. Existing biological methods mainly rely on natural tryptophan hydroxylases or their mutants. These enzymes typically require tetrahydrobiopterin, metal ions, oxygen, and related cofactor regeneration systems, making the reaction system and cellular metabolic regulation complex. Existing technologies have improved the fermentation production capacity of engineered bacteria by expressing tryptophan hydroxylase mutants and introducing NAD(P)H regeneration modules. For example, CN115948311A discloses a genetically engineered bacterium for 5-HTP production, which introduces the human TPH2-derived tryptophan hydroxylase mutant TM2 and integrates... Exiguobacterium sibiricum The glucose dehydrogenase GDHesi enhances NAD(P)H regeneration and intracellular tryptophan hydroxylation, thereby increasing fermentation yield and reducing [the risk of adverse reactions]. L -Trp accumulation.

[0005] However, the core of the above scheme remains the combination of natural tryptophan hydroxylase mutants and engineered bacterial metabolic modules, with the reaction process relying on cofactor regeneration, intracellular metabolic supply, and fermentation regulation. For in vitro biocatalytic systems that directly prepare 5-HTP using L-tryptophan as a substrate, there is still a need to develop enzyme catalytic systems with simpler catalytic elements, more readily available oxidants, and effective control over product distribution.

[0006] In recent years, heme-dependent bacterial tryptophan hydroxylases have been found to catalyze the release of free heme using oxygen or hydrogen peroxide. Lhydroxylation of the indole ring of tryptophan yields products such as 5-HTP, 4-hydroxy-L-tryptophan, or 6-hydroxy-L-tryptophan, indicating that heme protein can act as... L A potential catalytic element for L-tryptophan hydroxylation. Related studies also indicate that the regioselectivity of different heme-dependent tryptophan hydroxylases may originate from subtle differences in the L-tryptophan binding pattern at the active site, allowing different sites on the indole ring to approach high-valence ferro-oxidative species and undergo oxygen insertion.

[0007] While the aforementioned studies provide important evidence for the catalytic L-tryptophan hydroxylation of heme proteins, they disclose the regioselectivity of the natural bacterial tryptophan hydroxylase family and its different members. They do not address how to modify specific heme proteins into catalysts with high 5-HTP generation capacity and a high proportion of the target product. For non-natural or unmodified heme proteins, it is still necessary to regulate substrate binding and reaction pathways through specific site mutations. L -Tryptophan enters the active site in a manner that facilitates 5-HTP formation and reduces the formation of competitive byproducts.

[0008] Therefore, there is an urgent need to develop a heme protein mutant that can directly catalyze the hydroxylation of L-tryptophan under hydrogen peroxide-driven conditions, so as to improve the 5-HTP generation capacity, increase the proportion of target products, and reduce the tendency of by-product formation without relying on natural tryptophan hydroxylase and complex cofactor regeneration system. Summary of the Invention

[0009] To address the problems of existing 5-HTP bio-preparation systems that typically rely on natural tryptophan hydroxylase, tetrahydrobiopterin and related cofactor regeneration systems, or on engineered bacterial metabolic modules and produce numerous byproducts, this invention provides a heme protein mutant capable of directly catalyzing the L-tryptophan hydroxylation reaction with the participation of hydrogen peroxide.

[0010] The technical problem to be solved by the present invention is to provide a catalyst that can catalyze L - Tryptophan hydroxylation produces a heme protein mutant of 5-hydroxy-L-tryptophan.

[0011] To solve the above-mentioned technical problems, the present invention provides a method having L -Tryptophan hydroxylation activity of heme protein mutants, using heme protein A2 with the amino acid sequence shown in SEQ ID NO.1 as the parent, has any of the following mutations (a) to (f): (a) Mutate the 279th amino acid from threonine to isoleucine; (b) Mutate the 279th amino acid from threonine to isoleucine, the 287th cysteine ​​to serine, and the 388th tyrosine to alanine; (c) Mutate the 279th amino acid from threonine to isoleucine, the 287th cysteine ​​to serine, the 388th tyrosine to alanine, and the 261st phenylalanine to alanine. (d) Mutate the 279th amino acid from threonine to isoleucine, the 287th cysteine ​​to serine, the 388th tyrosine to alanine, the 261st phenylalanine to alanine, and the 374th tyrosine to phenylalanine. (e) Mutate amino acid 279 from threonine to isoleucine, cysteine ​​from position 287 to serine, tyrosine from position 388 to alanine, phenylalanine from position 261 to alanine, tyrosine from position 374 to phenylalanine, and glycine from position 280 to isoleucine. (f) Mutate amino acid position 279 from threonine to isoleucine, cysteine ​​position 287 from serine, tyrosine position 388 from alanine, phenylalanine position 261 from alanine, tyrosine position 374 from phenylalanine, glycine position 280 from isoleucine, and valine position 285 from isoleucine.

[0012] The present invention also provides an enzyme preparation containing the heme protein mutant.

[0013] The present invention also provides a gene encoding the above-mentioned heme protein mutant.

[0014] The present invention also provides recombinant plasmids carrying the above-mentioned genes.

[0015] In one embodiment of the present invention, the vector for the recombinant plasmid is the pET-28a(+) plasmid.

[0016] The present invention also provides a host cell carrying the above-mentioned gene or the above-mentioned recombinant plasmid.

[0017] In one embodiment of the present invention, the host cell is a bacterium.

[0018] In one embodiment of the present invention, the host cell is Escherichia coli.

[0019] In one embodiment of the present invention, the host cell is Escherichia coli. E. coli BL21(DE3).

[0020] The present invention also provides a method for preparing the above-mentioned heme protein mutant, the method comprising the following steps: Host cells carrying the gene encoding the heme protein mutant were inoculated into a culture medium and cultured. After induction of expression, fermentation broth was obtained. The fermentation broth was centrifuged and the cells were collected. The cells were broken up and centrifuged to obtain the cell lysis supernatant. The cell lysis supernatant was subjected to affinity purification to obtain the heme protein mutant.

[0021] In one embodiment of the present invention, IPTG is added during the induction of expression.

[0022] In one embodiment of the present invention, FeSO4 and 5-aminolevulinic acid are added during the induction of expression to promote the assembly of heme cofactor.

[0023] In one embodiment of the present invention, the heme protein mutant is purified by nickel column affinity chromatography.

[0024] This invention also provides the above-mentioned heme protein mutant in catalysis L Application in the preparation of 5-hydroxy-L-tryptophan by hydroxylation of 5-tryptophan.

[0025] In one embodiment of the present invention, the reaction system contains L-tryptophan, hydrogen peroxide, and a heme protein mutant.

[0026] In one embodiment of the present invention, the L -Tryptophan concentration is 1~20 mmol / L.

[0027] In one embodiment of the present invention, the L -Tryptophan concentration was 3 mmol / L.

[0028] In one embodiment of the present invention, the concentration of hydrogen peroxide is 1~50 mmol / L.

[0029] In one embodiment of the present invention, the hydrogen peroxide concentration is 15 mmol / L.

[0030] In one embodiment of the present invention, the concentration of the heme protein mutant is 1~100 μM.

[0031] In one embodiment of the present invention, the concentration of the heme protein mutant is 20 μM.

[0032] In one embodiment of the present invention, the reaction temperature is 10~40°C.

[0033] In one embodiment of the present invention, the reaction temperature is 20°C.

[0034] In one embodiment of the present invention, the reaction time is 5 to 120 minutes.

[0035] In one embodiment of the present invention, the reaction time is 30 min.

[0036] In one embodiment of the present invention, the reaction buffer is HEPES buffer.

[0037] In one embodiment of the present invention, the pH of the reaction buffer is 6.0 to 9.0.

[0038] In one embodiment of the present invention, the pH of the reaction buffer is 8.0.

[0039] The present invention also provides the use of the mutant in the preparation of 5-hydroxy-L-tryptophan or products containing 5-hydroxy-L-tryptophan.

[0040] Beneficial effects: (1) The heme protein mutant provided by the present invention can catalyze with the participation of hydrogen peroxide. L -Tryptophan is hydroxylated to generate 5-hydroxy-L-tryptophan. Compared with the original heme protein A2, the A2 of the present invention... M1 The mutant activates previously undetectable mutations through the T279I mutation. L -Tryptophan hydroxylation activity, with the target product ratio reaching 49.8%.

[0041] (2) The present invention constructs A2 M1 Based on the mutant, A2 was obtained by combining the T279I, C287S, and Y388A mutations. M3 This reduces the amount of 5-HTP generated from A2. M1 The percentage of 5-HTP increased from 2.89% to 5.24%, and the proportion of the target product increased from approximately 49.8% to approximately 75.2%, indicating that the above combined mutations can simultaneously improve the 5-HTP generation capacity and improve product distribution.

[0042] (3) The mutant A2 obtained by the present invention through active pocket iterative mutation M7 This can reduce the amount of 5-HTP generated from A2 M1 The percentage of the target product increased from 2.89% to 24.95%, and the proportion of the target product increased from approximately 49.8% to 97.1%. This result indicates that A2 M7 Not only improved L The 5-tryptophan hydroxylation activity also significantly enhanced the target product selectivity of 5-HTP and reduced the tendency for byproduct formation. Attached Figure Description

[0043] Figure 1 The results show the expression, heme assembly, and purification of A2 heme protein.

[0044] Figure 2 Catalysis by heme protein mutant LHPLC analysis results of the α-tryptophan hydroxylation reaction.

[0045] Figure 3 Catalysis of representative combined mutants L HPLC analysis results showing a reduction in byproducts during tryptophan hydroxylation.

[0046] Figure 4 The results show the color changes of the reaction solution and the formation of byproducts. Detailed Implementation

[0047] Unless otherwise specified, heme protein A2 and its mutants catalyze L The total volume of the system for the β-tryptophan hydroxylation reaction was 400 μL, containing L The reaction mixture consisted of 3 mmol / L tryptophan, 15 mmol / L hydrogen peroxide, and 20 μmol / L purified enzyme, in HEPES buffer at pH 8.0. The reaction was carried out at 20°C for 30 min. After the reaction was complete, three volumes of methanol were added to the reaction solution to terminate the reaction. The mixture was then centrifuged to remove the precipitate, and the supernatant was filtered and analyzed by HPLC.

[0048] Product detection was performed using an Agilent 1260 HPLC system equipped with a UV detector. The chromatographic column was a Diamonsil C18 column, 250 mm × 4.6 mm, 5 μm. Mobile phase A was water containing 0.05% formic acid, and mobile phase B was acetonitrile containing 0.05% formic acid. The flow rate was 0.6 mL / min, the column temperature was 30℃, the detection wavelength was 280 nm, and the injection volume was 10 μL.

[0049] The gradient elution program was as follows: 0 min, 5% B; 12 min, 59% B; 13 min, 95% B; 14 min, 5% B; 19 min, 5% B.

[0050] The target product, 5-hydroxy-L-tryptophan, was confirmed by comparing its retention time with that of a 5-hydroxy-L-tryptophan standard, and its production amount was calculated based on the 5-hydroxy-L-tryptophan standard curve. In this invention, the production amount of 5-HTP refers to the molar amount of 5-HTP calculated based on the 5-HTP standard curve as a percentage of the initial product. L - Percentage of molar amount of tryptophan.

[0051] The target product ratio is used to represent the relative proportion of the target product 5-hydroxy-L-tryptophan in the total peak area of ​​the detected products, and is calculated according to the following formula: 5-HTP ratio = S5-HTP / (S5-HTP + S 副产物 ) × 100% Wherein, S5-HTP represents the HPLC peak area corresponding to 5-hydroxy-L-tryptophan, and Sbyproduct represents the HPLC peak area of ​​the corresponding byproduct. Unless otherwise specified, the reaction data are the average of three independent parallel experiments.

[0052] Example 1: Construction, expression and purification of heme protein A2 and its mutants The gene encoding heme protein A2 with the amino acid sequence shown in SEQ ID NO.1 (nucleotide sequence shown in SEQ ID NO.2) was chemically synthesized and cloned into the EcoRI and HindIII restriction sites of the pET-28a(+) vector to obtain the recombinant plasmid pET28a-A2.

[0053] Using recombinant plasmid pET28a-A2 as a template, site-directed mutagenesis was performed using whole-plasmid PCR to construct plasmids containing T279I (abbreviated as A2). M1 T279I / C287S / Y388A (abbreviated as A2) M3 T279I / C287S / Y388A / F261A (abbreviated as A2) M4 T279I / C287S / Y388A / F261A / Y374F (abbreviated as A2) M5 ), T279I / C287S / Y388A / F261A / Y374F / G280I (abbreviated as A2) M6 ) and T279I / C287S / Y388A / F261A / Y374F / G280I / V285I (abbreviated as A2) M7 The mutated recombinant plasmids were used in the study. The primers used in the study are shown in Table 1.

[0054] Table 1 Primer Table

[0055] PCR amplification products were digested with DpnI and transformed into Escherichia coli. E. coli BL21(DE3) was used to select positive single clones for sequencing verification, yielding the heme protein mutant A2. M1 A2 M3 A2 M4 A2 M5 A2M6 and A2 M7 Recombinant strains.

[0056] The recombinant bacteria were inoculated into LB medium containing kanamycin and cultured at 37°C until OD500. 600 When the concentration reaches 0.6–0.8, IPTG at a final concentration of 0.2 mM is added for induction of expression. FeSO4 and 5-aminolevulinic acid are also added to promote heme assembly. After induction culture, the bacterial cells are collected by centrifugation.

[0057] The bacterial cells were resuspended in a buffer solution containing 300 mM NaCl, 50 mM phosphate, and 20 mM imidazole. After sonication and centrifugation, the supernatant was collected. The supernatant was filtered and loaded onto a nickel column, and purified heme protein and its mutants were obtained by imidazole gradient elution. SDS-PAGE analysis of the purified protein showed that the target protein band matched the theoretical molecular weight, indicating that a purified enzyme suitable for catalytic reactions had been obtained.

[0058] Example 2: Heme protein mutant catalyzes L-tryptophan hydroxylation reaction Using L-tryptophan as a substrate, the ability of the heme protein mutant obtained in Example 1 to catalyze the generation of 5-HTP was tested.

[0059] The total volume of the reaction system was 400 μL, containing 3 mM L-tryptophan, 15 mM hydrogen peroxide, and 20 μM of the purified enzyme prepared in Example 1. The reaction buffer was HEPES buffer, pH 8.0. The reaction was carried out at 20 °C for 30 min. After the reaction was completed, three volumes of methanol were added to terminate the reaction. The precipitate was removed by centrifugation, and the supernatant was filtered and analyzed by HPLC.

[0060] HPLC results showed that no significant 5-HTP formation was detected in the initial heme protein A2; however, the T279I mutant A2M1 showed a 5-HTP product peak. According to the initial... L Calculate A2 based on a tryptophan concentration of 3 mmol / L. M1 The 5-HTP conversion rate was 2.89%, corresponding to a 5-HTP concentration of 19.1 mg / L; A2 M1 The target product proportion was approximately 49.8%. These results indicate that the T279I mutation can activate heme protein A2. L -Tryptophan hydroxylation activity endows the starting protein, which previously showed no obvious 5-HTP generation, with the ability to generate 5-HTP. However, A2... M1 The 5-HTP conversion rate was still low, and the target product ratio was only about 49.8%, indicating that the single-point activation mutant still had problems with insufficient catalytic ability and byproduct competition. Further combinatorial mutations are needed to improve the 5-HTP conversion rate and improve product distribution.

[0061] Example 3: Effect of conserved site mutations on product distribution In A2 M1 Based on this, the conserved residues near the substrate were mutated according to the method of Example 1 to obtain mutants such as E86P, L273A, D281G, C287S and Y388A. The purified enzyme protein was prepared and its ability to catalyze the L-tryptophan hydroxylation reaction was tested according to the method of Example 2.

[0062] The purpose of this round of mutations is not only to increase the production of 5-HTP, but also to improve the product distribution of the hydroxylation reaction. Since there are multiple potential hydroxylation sites in the L-tryptophan molecule, insufficient control of the enzyme over the substrate binding conformation and reaction sites may lead to the simultaneous generation of multiple monohydroxylation byproducts, resulting in a decrease in the area ratio of 5-HTP in the main product. Therefore, the 5-HTP area ratio is used to evaluate the relative proportion of the target product in the main monohydroxylation products; a higher ratio indicates that the reaction is more inclined to generate the target product 5-HTP, rather than hydroxylation byproducts from other regions.

[0063] The results showed that, compared with A2 M1 In comparison, the 5-HTP conversion rates of E86P and L273A decreased to 2.74% and 2.29%, respectively; the 5-HTP conversion rate of D281G increased to 3.34%, but the 5-HTP area ratio did not improve significantly; the 5-HTP conversion rate of C287S was 2.85%, and the 5-HTP area ratio decreased to approximately 36.4%, indicating that this single-point mutation failed to improve the distribution of the target product; Y388A simultaneously increased both the 5-HTP production and the target product ratio, increasing the 5-HTP ratio to approximately 68.1%.

[0064] Further combining T279I, C287S, and Y388A yielded mutant A2. M3 With A2 M1 Compared to A2 M3 The 5-HTP conversion rate increased from 2.89% to 5.24%, an increase of approximately 1.81 times; the 5-HTP area ratio increased from approximately 49.8% to approximately 75.2%. These results indicate that the combined mutations of T279I, C287S, and Y388A not only improved the substrate's ability to convert to 5-HTP but also reduced the relative proportion of byproducts in the main monohydroxylation products.

[0065] Example 4: Mutation of the active pocket improves 5-HTP conversion rate and target product ratio In A2 M3Based on this, analysis of the active pocket residues revealed that mutations in F261, Y374, G280, and V285 can improve catalytic performance. These were further used for iterative mutation. In A2 M3 Based on this, the F261A mutation was introduced to obtain A2. M4 ; in A2 M4 Based on this, the Y374F mutation was introduced to obtain A2. M5 ; in A2 M5 Based on this, the G280I mutation was introduced to obtain A2. M6 ; in A2 M6 Based on this, the V285I mutation was introduced to obtain A2. M7 .

[0066] The test results show that from A2 M1 To A2 M7 The 5-HTP formation increased from 2.89% to 24.95%, an increase of approximately 8.63 times. The 5-HTP proportion increased from approximately 49.8% to 97.1%. Simultaneously, HPLC analysis showed that with the gradual introduction of combinatorial mutations, the peak area of ​​non-target products gradually decreased. These results indicate that A2 M7 While improving the conversion rate of 5-HTP, it also increased the proportion of the target product and reduced the tendency for the formation of non-target oxidation products.

[0067] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Possesses L A heme protein mutant with β-tryptophan hydroxylation activity, characterized in that... The heme protein, relative to the amino acid sequence shown in SEQ ID NO.1, has any of the following mutations (a) to (f): (a) Mutate the 279th amino acid from threonine to isoleucine; (b) Mutate the 279th amino acid from threonine to isoleucine, the 287th cysteine ​​to serine, and the 388th tyrosine to alanine; (c) Mutate the 279th amino acid from threonine to isoleucine, the 287th cysteine ​​to serine, the 388th tyrosine to alanine, and the 261st phenylalanine to alanine. (d) Mutate the 279th amino acid from threonine to isoleucine, the 287th cysteine ​​to serine, the 388th tyrosine to alanine, the 261st phenylalanine to alanine, and the 374th tyrosine to phenylalanine. (e) Mutate amino acid 279 from threonine to isoleucine, cysteine ​​from position 287 to serine, tyrosine from position 388 to alanine, phenylalanine from position 261 to alanine, tyrosine from position 374 to phenylalanine, and glycine from position 280 to isoleucine. (f) Mutate amino acid position 279 from threonine to isoleucine, cysteine ​​position 287 from serine, tyrosine position 388 from alanine, phenylalanine position 261 from alanine, tyrosine position 374 from phenylalanine, glycine position 280 from isoleucine, and valine position 285 from isoleucine.

2. An enzyme preparation containing the heme protein mutant of claim 1.

3. The gene encoding the heme protein mutant of claim 1.

4. A recombinant plasmid carrying the gene of claim 3.

5. A host cell carrying the gene of claim 3 or the recombinant plasmid of claim 4.

6. Recombinant Escherichia coli, characterized in that, With Escherichia coli E. coli Using BL21(DE3) as the host and pET28(a) as the vector, the heme protein mutant described in claim 1 is expressed.

7. A method for preparing the heme protein mutant of claim 1, characterized in that, Includes the following steps: The recombinant Escherichia coli of claim 5 is cultured in a culture medium for a period of time, and fermentation broth is obtained after induction expression; the fermentation broth is centrifuged and the bacterial cells are collected; the bacterial cells are broken and centrifuged to obtain cell lysis supernatant; the cell lysis supernatant is subjected to affinity purification to obtain heme protein mutant; optionally, IPTG, FeSO4 and 5-aminolevulinic acid are added during the induction expression process.

8. A method for preparing 5-hydroxy-L-tryptophan, characterized in that, The heme protein mutant described in claim 1 is used as a catalyst; the reaction system contains L-tryptophan and hydrogen peroxide.

9. The method according to claim 8, characterized in that, The reaction temperature is 10~40℃; the reaction time is 5~120min.

10. The use of the heme protein mutant of claim 1 in the preparation of 5-hydroxy-L-tryptophan or products containing 5-hydroxy-L-tryptophan.

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