A heat-resistant hydroxycinnamate decarboxylase derived from an unusual wickerham sp.

Abstract

The application discloses a heat-resistant hydroxycinnamic acid decarboxylase derived from abnormal Wickerham sp., and belongs to the technical field of enzyme engineering. The optimal reaction temperature of the hydroxycinnamic acid decarboxylase is 40 DEG C, and the activity is stable in the range of 40 DEG C to 70 DEG C. This provides certain theoretical basis and practical significance for industrialization of high-efficiency preparation of 4-vinyl derivatives and other high-grade perfume materials by using the hydroxycinnamic acid decarboxylase.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a heat-resistant hydroxycinnamic acid decarboxylase derived from *Saccharomyces aberrantis*. Background Technology

[0002] Hydroxycinnamate decarboxylase (HCDC) catalyzes the non-oxidative decarboxylation of hydroxycinnamic acid to generate corresponding 4-vinyl derivatives. These 4-vinyl derivatives are not only high-grade fragrances themselves, but also important precursors for the synthesis of other fragrances such as vanillin. Utilizing HCDC for the biosynthesis of 4-vinyl derivatives is an efficient and environmentally friendly preparation route. Therefore, exploring multiple sources of HCDC is of great significance for the industrial biosynthesis of 4-vinyl derivatives.

[0003] The vast majority of HCDCs originate from microorganisms, with a few originating from plants. HCDCs can be obtained from Pseudomonas fluorescens, Bacillus pumilus, Candida guilliermondii, Saccharomyces cerevisiae, Aspergillus luchuensis, and Bacillus licheniformis. Currently, only one hydroxycinnamate decarboxylase from Wickerhamomyces anomalus exists: XM_019186392.1 (Uniport protein sequence number A0A1E3P002).

[0004] Industrial applications often require high-temperature and strongly alkaline environments; therefore, the heat resistance of HCDC is a key issue for its widespread application in industrial production. Regarding heat resistance, the optimal temperature range for PAD derived from *Lactobacillus* is 20–30°C, and enzyme activity significantly decreases above 37°C. LvPAD derived from *Lactobacillus vermolensis* retains approximately 50% of its relative enzyme activity after incubation at 65°C for 90 minutes. Most HCDC derived from yeast are not heat-resistant; hydroxycinnamic acid decarboxylase derived from *Candida guilliermondii* completely loses its activity after incubation at 50°C for 20 minutes. Therefore, exploring hydroxycinnamic acid decarboxylases with high thermal stability will provide a theoretical basis for broadening the industrial application conditions of hydroxycinnamic acid decarboxylases and improving the efficiency of vinylphenol preparation. Summary of the Invention

[0005] The first objective of this invention is to provide a hydroxycinnamic acid decarboxylase (named WaHCDC) as a solid theoretical basis for the efficient expression and utilization of subsequent enzymes, thereby developing new enzyme resources.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The present invention provides a hydroxycinnamic acid decarboxylase (WaHCDC), the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] In one embodiment, the optimal reaction temperature of the hydroxycinnamic acid decarboxylase (WaHCDC) is 40°C, and it can still maintain relatively high activity at 40°C-70°C.

[0009] In one embodiment, the hydroxycinnamic acid decarboxylase (WaHCDC) can catalyze the decarboxylation reaction of hydroxycinnamic acid (ferulic acid, caffeic acid, sinapic acid, p-coumaric acid) to generate the corresponding 4-vinyl derivatives.

[0010] This invention provides a gene encoding the hydroxycinnamic acid decarboxylase.

[0011] The present invention provides a recombinant vector expressing the hydroxycinnamic acid decarboxylase or carrying the gene thereon.

[0012] The present invention provides recombinant cells that express the hydroxycinnamic acid decarboxylase, or contain the gene, or contain the recombinant vector.

[0013] The present invention provides a recombinant Pichia pastoris, using Pichia pastoris GS115 as a host, to express the hydroxycinnamic acid decarboxylase.

[0014] In one implementation, pPIC9K is used as the expression vector.

[0015] The present invention provides a method for synthesizing 4-vinyl derivatives, using the hydroxycinnamic acid decarboxylase, or the recombinant cells, or the recombinant Pichia pastoris as a catalyst, and ferulic acid, caffeic acid, sinapic acid, or p-coumaric acid as substrates for the reaction.

[0016] In one embodiment, the reaction is carried out at 10-80°C for at least 15 minutes.

[0017] In one embodiment, the initial concentration of the substrate is at least 1 mM.

[0018] In one embodiment, the initial concentration of the substrate is 1-8 mM.

[0019] The present invention provides the use of the hydroxycinnamic acid decarboxylase, or the recombinant cells, or the recombinant Pichia pastoris in the preparation of 4-vinyl derivatives.

[0020] The present invention provides the use of the hydroxycinnamic acid decarboxylase, or the recombinant cells, or the recombinant Pichia pastoris in the degradation of ferulic acid, caffeic acid, sinapic acid, or p-coumaric acid.

[0021] Beneficial effects:

[0022] This invention screened a hydroxycinnamic acid decarboxylase from *Wickerhamomyces anomalus*. The predicted isoelectric point (pI) of the protein is 5.95, the theoretical molecular weight (MW) is 41.9 kDa, the optimal temperature is 40°C, and the activity is stable within the temperature range of 40°C-70°C. It can catalyze the production of ferulic acid, caffeic acid, sinapic acid, and p-coumaric acid. This provides a theoretical basis and practical significance for the industrialization of the efficient preparation of 4-vinyl derivatives and other high-grade flavoring substances using hydroxycinnamic acid decarboxylase. Attached Figure Description

[0023] Figure 1 This is an electrophoresis diagram of the PCR amplification verification of the hydroxycinnamic acid decarboxylase gene described in this invention.

[0024] Figure 2 This is an amino acid sequence comparison between the hydroxycinnamic acid decarboxylase described in this invention and the hydroxycinnamic acid decarboxylase (XM_019186392.1) derived from *Saccharomyces aberrantus* in GenBank.

[0025] Figure 3 This is a nucleotide sequence comparison between the hydroxycinnamic acid decarboxylase described in this invention and the hydroxycinnamic acid decarboxylase (XM_019186392.1) from *Saccharomyces aberrantus* in GenBank.

[0026] Figure 4 This is the decarboxylation and colorimetric result of the reaction between WaHCDC and hydroxycinnamic acid described in this invention.

[0027] Figure 5 The results of SDS-PAGE gel electrophoresis of heterologous expression of WaHCDC (lane 2) and purified WaHCDC (lane 1) in this invention are shown.

[0028] Figure 6 This is the substrate-specific result of WaHCDC described in this invention.

[0029] Figure 7 The results are the optimal reaction temperature, temperature tolerance, optimal reaction pH, and pH tolerance of WaHCDC as described in this invention.

[0030] Figure 8 The results show the optimal reaction temperature and heat resistance of the hydroxycinnamic acid decarboxylase XM_019186392.1 derived from *Saccharomyces aberrantus*. Detailed Implementation

[0031] The technical solution of the invention will be described in detail below with reference to the accompanying drawings:

[0032] The culture media involved in the following examples are as follows:

[0033] Each liter of YPD liquid culture medium consists of: 2% peptone, 1% yeast extract, and 2% glucose.

[0034] Each liter of BMMY liquid culture medium contains: 2% peptone, 1% yeast extract, 1% potassium phosphate buffer, 1.34% YNB, and 5 × 10⁻⁶ biotin. -5 % , methanol 0.5%.

[0035] Each liter of BMGY liquid culture medium contains: 2% peptone, 1% yeast extract, 1% potassium phosphate buffer, 1.34% YNB, and 5 × 10⁻⁶ biotin. -5 % glycerol 1%.

[0036] The components of each liter of MD solid medium include: 2% agarose, 2% glucose, 1.34% YNB, and 5 × 10⁻⁶ biotin. -5 .

[0037] The main instruments involved in the following examples are: High Performance Liquid Chromatography (Agilent-Lc1260 Infinity I, Agilent Technologies), Electroporation Unit (BIO-RAD-MicroPulser, Fuyue Biotechnology Co., Ltd.), PCR System (GE4852T, Hangzhou Baiheng Technology Co., Ltd.), and Electrophoresis Unit (BEP-600, Beijing Berlant Instrument Equipment Co., Ltd.).

[0038] The main reagents involved in the following examples are: plasmid extraction kit (B518191-0100, Sangon Biotech (Shanghai) Technology Co., Ltd.), enzyme digestion product purification kit (B518141-0100, Sangon Biotech (Shanghai) Co., Ltd.), EcoRI (Baori Biotech Co., Ltd.), NotRI (Baori Biotech Co., Ltd.), T4 ligase (Baori Biotech Co., Ltd.), ferulic acid (Shanghai Yien Chemical Technology Co., Ltd.), caffeic acid (Shanghai Yien Chemical Technology Co., Ltd.), sinapic acid (Shanghai Yien Chemical Technology Co., Ltd.), and p-coumaric acid (Shanghai Yien Chemical Technology Co., Ltd.).

[0039] Example 1: Obtaining the gene encoding hydroxycinnamic acid decarboxylase

[0040] 1. Design and synthesis of specific primers

[0041] Yeast strains were isolated from Daqu (a type of starter culture), and strains exhibiting hydroxycinnamic acid decarboxylase activity and vinylphenol reductase activity were screened. These strains were identified as *Saccharomyces cerevisiae*.

[0042] Based on the gene sequence of hydroxycinnamic acid decarboxylase (accession number: XM_019186392.1) in *Saccharomyces cerevisiae* from NCBI (National Center of Biotechnology Information) GenBank, upstream and downstream primers were designed using PrimerBlast, as shown in Table 1.

[0043] Table 1 Specific primers

[0044]

[0045] 2. Extraction of yeast RNA

[0046] RNA was extracted from the abnormal Wickham yeast according to the instructions of the RNA extraction kit.

[0047] 3. RT-PCR

[0048] Add the RNA sample, primers, and reagents according to the RT-CR reagent instructions, and then perform PCR amplification.

[0049] 4. Gel electrophoresis

[0050] The above PCR products were subjected to gel electrophoresis. Figure 1 The PCR product was sent to Shanghai Sangon Biotech for sequencing to obtain the gene encoding hydroxycinnamic acid decarboxylase, as shown in positions 660 to 1787 of SEQ ID NO. 2. The amino acid sequence of the hydroxycinnamic acid decarboxylase is shown in SEQ ID NO. 1. Comparison with the amino acid sequence of WaHCDC enzyme from *Saccharomyces cerevisiae* in GenBank revealed differences in the amino acid and nucleotide sequences of the WaHCDC enzyme in this patent compared to existing sequences. Figure 2 , Figure 3 ).

[0051] Example 2: Recombinant and Expression of Hydroxycinnamic Acid Decarboxylase

[0052] 1. Plasmid construction

[0053] NotⅠ was added before position 660 of WaHCDC (SEQ ID NO.2). The sequence from position 660 to 1787 was excised by double digestion with EcoRlⅠ and NotⅠ. At the same time, the vector pPIC9K was double digested, purified and recovered, and ligated overnight at 4°C with T4 ligase to construct the recombinant plasmid pPIC9K-WaHCDC. The plasmid was chemically transformed into E. coli DH5α, and colony PCR and sequencing were used for verification. The results were correct.

[0054] 2. Construction of expression carrier

[0055] A Pichia pastoris expression system was constructed using electroporation. 10 μL of pPIC9K-WaHCDC plasmid was added to 80 μL of competent yeast GS115 cells and gently mixed. The mixture was transferred to a pre-chilled electroporation cuvette and electroporated once. Immediately after electroporation, 1 mL of sorbitol was added and mixed in the cuvette, then transferred to a new centrifuge tube. The mixture was incubated in a metal bath at 28°C for 1-2 hours. The bacterial culture was then plated onto MD plates containing ampicillin and incubated at 30°C for approximately 48 hours.

[0056] 3. Recombinant protein induced expression

[0057] Positive clones selected from MD plates were inoculated into BMGY medium and cultured for 24 hours (OD). 600 After centrifuging (approximately 1), the entire bacterial cell was transferred to BMMY medium for further culture. Methanol at a final concentration of 1% (v / v) was added every 24 hours to induce the incubation. After 3 days of induction, the bacterial solution was centrifuged to obtain the supernatant, which yielded the crude enzyme solution.

[0058] Add 10 μL of the obtained crude enzyme solution to 200 μL of hydroxycinnamic acid-bromocresol purple solution (hydroxycinnamic acid content is 50 mM). The substrate solution changes from yellow to purple. Figure 4 The crude enzyme solution was passed through a Ni-agarose gel 6FF gravity column and washed with high-concentration imidazole to obtain the purified protein. The crude enzyme solution and the purified protein were analyzed by SDS-PAGE gel electrophoresis. Figure 5 ).

[0059] Example 3 Enzymatic properties of hydroxycinnamic acid decarboxylase

[0060] 1. Substrate specificity

[0061] Different concentrations (10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM) of ferulic acid, caffeic acid, sinapic acid, and p-coumaric acid were prepared as substrates. 0.1 mL of each substrate solution at different concentrations, 0.1 mL of enzyme solution filtered through a 0.22 μm membrane, and 0.8 mL of disodium hydrogen phosphate-citrate buffer (pH 6) were added to a total reaction volume of 1 mL. The reaction was terminated by adding 2 mL of methanol after incubating in a water bath at 40 °C for 15 min. Three replicates were performed for each substrate concentration. The residues of the hydroxycinnamic acid substrate were determined by HPLC after filtration through a 0.22 μm membrane.

[0062] 2. Optimal reaction pH

[0063] Prepare disodium hydrogen phosphate-citric acid buffer solutions with different pH values ​​(3.0, 4.0, 5.0, 6.0, 7.0, 8.0). Take 0.1 mL of ferulic acid solution (50 mM), 0.1 mL of enzyme solution filtered through a 0.22 μm filter membrane, and 0.8 mL of disodium hydrogen phosphate-citric acid buffer solutions with different pH values, for a total reaction system of 1 mL. After reacting in a water bath at 40 °C for 15 min, add 2 mL of methanol to terminate the reaction. Perform three replicates at different pH values. After filtration through a 0.22 μm filter membrane, analyze the residual content of the substrate hydroxycinnamic acid by HPLC.

[0064] 3. pH tolerance

[0065] Prepared disodium hydrogen phosphate-citric acid buffer solutions with different pH values ​​(3.0, 4.0, 5.0, 6.0, 7.0, 8.0). Take 0.1 mL of ferulic acid solution (50 mM), 0.1 mL of enzyme solution filtered through a 0.22 μm membrane, and 0.8 mL of disodium hydrogen phosphate-citric acid buffer solutions with different pH values, totaling 1 mL of reaction system. After reacting in a water bath at 40℃ for 2 h, add 2 mL of methanol to terminate the reaction. Perform three replicates at different pH values. After filtration through a 0.22 μm membrane, analyze the residual content of the substrate hydroxycinnamic acid by HPLC.

[0066] 4. Optimal reaction temperature

[0067] Take 0.1 mL of ferulic acid solution (50 mM), 0.1 mL of enzyme solution filtered through a 0.22 μm filter membrane, and 0.8 mL of disodium hydrogen phosphate-citric acid buffer solution at pH 6.0, for a total reaction system of 1 mL. React in a water bath at 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 15 min, respectively, and then add 2 mL of methanol to terminate the reaction. Perform three replicates at different temperatures. After filtration through a 0.22 μm filter membrane, the residual content of the substrate hydroxycinnamic acid is detected by HPLC.

[0068] 5. Temperature tolerance

[0069] Take 0.1 mL of ferulic acid solution (50 mM), 0.1 mL of enzyme solution filtered through a 0.22 μm filter membrane, and 0.8 mL of disodium hydrogen phosphate-citric acid buffer solution at pH 6.0, for a total reaction system of 1 mL. React in a water bath at 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 2 h, respectively. Then, add 2 mL of methanol to terminate the reaction. Perform three replicates at different temperatures. After filtration through a 0.22 μm filter membrane, analyze the residual content of the substrate hydroxycinnamic acid by HPLC.

[0070] All the above results are expressed as relative enzyme activity (relative enzyme activity under other conditions is calculated with the highest enzyme activity as 100%).

[0071]

[0072] In the formula A r A represents relative enzyme activity, A represents absolute enzyme activity, and A represents absolute enzyme activity. max denoted as 'maximum enzyme activity' and 'm' as 'mass'.

[0073] The results showed that WaHCDC had a better ability to decompose ferulic acid than to decompose caffeic acid, sinapic acid, and p-coumaric acid, and the catalytic effect was optimal at a concentration of 5 mM. Figure 6 The optimal reaction pH is 6.0, and the optimal reaction temperature is 40℃. Figure 7 To demonstrate the high heat resistance of the WaHCDC, the heat resistance of hydroxycinnamic acid decarboxylase XM derived from *Wickhamia lanceolata* was determined in the same manner. The results showed that the optimal temperature for hydroxycinnamic acid decarboxylase XM was 40°C, and the relative enzyme activity decreased to approximately 40% upon incubation at 60°C. Figure 8 The WaHCDC described in this patent can still maintain 80% relative activity when the temperature reaches 70℃, until the relative enzyme activity decreases to about 58% when the temperature reaches 80℃. Figure 7 This indicates that the WaHCDC described in this patent has better heat resistance than XM.

[0074] 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. A hydroxycinnamic acid decarboxylase, characterized in that, The amino acid sequence of the hydroxycinnamic acid decarboxylase is shown in SEQ ID NO.

1.

2. The gene encoding the hydroxycinnamic acid decarboxylase of claim 1.

3. Expressing the hydroxycinnamic acid decarboxylase of claim 1, or a recombinant vector carrying the gene of claim 2.

4. Recombinant cells expressing the hydroxycinnamic acid decarboxylase of claim 1, or containing the gene of claim 2, or containing the recombinant vector of claim 3.

5. A recombinant Pichia pastoris, characterized in that, Using Pichia pastoris GS115 as a host, the hydroxycinnamic acid decarboxylase described in claim 1 is expressed.

6. A method for synthesizing 4-vinyl derivatives, characterized in that, The reaction is carried out using the hydroxycinnamic acid decarboxylase of claim 1, the recombinant cell of claim 4, or the recombinant Pichia pastoris of claim 5 as a catalyst, and ferulic acid, caffeic acid, sinapic acid, or p-coumaric acid as a substrate.

7. The method as described in claim 6, characterized in that, React at 10-80℃ for at least 15 minutes.

8. The method as described in claim 6 or 7, characterized in that, The initial concentration of the substrate is at least 1 mM.

9. The use of the hydroxycinnamic acid decarboxylase of claim 1, or the recombinant cell of claim 4, or the recombinant Pichia pastoris of claim 5 in the preparation of 4-vinyl derivatives.

10. The use of the hydroxycinnamic acid decarboxylase of claim 1, or the recombinant cell of claim 4, or the recombinant Pichia pastoris of claim 5 in the degradation of ferulic acid, caffeic acid, sinapic acid, or p-coumaric acid.

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