A laccase and its preparation method, and a method for preparing magnolol.

Abstract

This application discloses a laccase and its preparation method, as well as a method for preparing magnolol. The amino acid sequence of the laccase is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, or is an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to any one of SEQ ID NO: 1 to SEQ ID NO: 6. The laccase is obtained by constructing a genetically engineered *E. coli* bacterium, inducing expression in the engineered bacterium, and purifying the laccase. The laccase catalyzes the production of magnolol from 4-allylphenol. This application identifies the key enzyme in magnolol synthesis and achieves heterologous production of magnolol from *E. coli*, which has the advantages of high selectivity, no pollution, and a short production cycle.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, specifically to a laccase and its preparation method, and a method for preparing magnolol. Background Technology

[0002] Magnoliosol (5',5-diallyl-2,2'-biphenyl, C 18 H 18 O2 (266.32) belongs to phenylalanine derivatives and is an important antioxidant in organisms (CAS number 528-43-8). Magnolol is a brown to white fine powder extract of Magnolia officinalis. Magnolol has significant and long-lasting central muscle relaxant and central nervous system depressant effects. It also possesses anti-inflammatory, antibacterial, antimicrobial, anti-ulcer, antioxidant, antitumor, and platelet aggregation inhibitory pharmacological effects. Magnolol can be used to treat acute enteritis, bacterial or amoebic dysentery, and chronic gastritis.

[0003] The production of magnolol primarily relies on plant extraction and synthesis, derived from the dried bark, root bark, and branch bark of Magnolia officinalis. However, natural Magnolia officinalis grows slowly, taking 10 to 20 years to develop medicinal properties, which significantly limits the widespread application of magnolol. Furthermore, harvesting the bark and root bark causes substantial damage to the plant. The plant extraction process is also extremely complex, with cumbersome purification steps, making it difficult to scale up for industrial production.

[0004] Magnolol can also be synthesized chemically. However, the reagents used in chemical synthesis are expensive, the experimental procedures are complex, and it is not conducive to large-scale production. In addition, a large number of byproducts are generated during the production process.

[0005] Therefore, researching the synthesis of magnolol and developing a simple, efficient, and green process route is particularly important. Biosynthesis using enzymes as catalysts offers mild reaction conditions and produces fewer byproducts. Therefore, enzyme-catalyzed synthesis of magnolol is a suitable new research direction. Summary of the Invention

[0006] This application provides a laccase and its preparation method, as well as a method for preparing magnolol, which can achieve heterologous production of magnolol from Escherichia coli.

[0007] In a first aspect, this application provides a laccase whose amino acid sequence is as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar to any one of SEQ ID NO: 1 to SEQ ID NO: 6.

[0008] Secondly, this application provides a nucleic acid molecule comprising a nucleotide sequence encoding the laccase of the first aspect.

[0009] Furthermore, the nucleotide sequence is any of the following:

[0010] (a) The nucleotide sequence is shown in any of the nucleotide sequences in SEQ ID NO: 7 to SEQ ID NO: 12;

[0011] (b) The nucleotide sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to any nucleotide sequence in SEQ ID NO: 7 to SEQ ID NO: 12; or

[0012] (c) The nucleotide sequence is the nucleotide sequence described in (a) or (b) after codon optimization.

[0013] Thirdly, this application provides a genetically engineered bacterium containing the nucleic acid molecules described in the second aspect.

[0014] Furthermore, the genetically engineered bacteria also contain one or more of the following tags: MBP tag, RFP tag, or GFP tag.

[0015] Furthermore, the genetically engineered bacteria is Escherichia coli.

[0016] Fourthly, this application provides a method for constructing genetically engineered bacteria, comprising: constructing a nucleic acid molecule from the second aspect onto a vector to obtain a recombinant vector; and transforming the recombinant vector into an Escherichia coli recipient strain to obtain genetically engineered bacteria.

[0017] Furthermore, the carrier includes pET28a.

[0018] Furthermore, the recipient strains of Escherichia coli include Escherichia coli DH5α, Escherichia coli BL21(DE3) or Escherichia coli TOPO.

[0019] Furthermore, one or more of the following tags can be added when constructing the recombinant vector: MBP (maltose-binding protein) tag, RFP (red fluorescent protein) tag, or GFP (green fluorescent protein) tag.

[0020] Fifthly, this application provides a method for preparing laccase, comprising: inducing expression of genetically engineered bacteria according to the third aspect using IPTG, disrupting the bacterial cells, and purifying the laccase using nickel column affinity chromatography.

[0021] Furthermore, the concentration of IPTG can be 0.1–1 mM, 0.3–0.7 mM, or 0.5 mM.

[0022] Furthermore, the induction time can be 13–18 h, 14–17 h, or 15–16 h.

[0023] Sixthly, this application provides the application of laccase prepared by the method of the first aspect or the fifth aspect in the preparation of magnolol using 4-allylphenol as a substrate.

[0024] Seventhly, this application provides a method for preparing magnolol, comprising: using 4-allylphenol as a substrate, and catalyzing it with laccase from the first aspect, laccase induced and expressed by genetically engineered bacteria from the third aspect, or laccase prepared by the fifth aspect, to obtain magnolol.

[0025] Furthermore, the mass ratio of 4-allylphenol to laccase can be 1:(1-5), 1:(1.5-3), or 1:2.

[0026] Furthermore, the catalytic reaction temperature can be 50–70℃, 55–65℃, or 60℃.

[0027] Furthermore, the catalytic reaction time can be 3 to 7 hours, 4 to 6 hours, or 5 hours.

[0028] This application provides a biosynthetic method for producing magnolol, which has the following beneficial effects:

[0029] Traditional magnolol production processes involve plant extraction and organic chemical synthesis, with plant extraction being the primary method. Organic chemical production is cumbersome, produces excessive amounts of residual organic solvents, and generates significant chemical waste, resulting in severe pollution. Furthermore, the extraction of magnolol from plants is often limited by climate, season, and human factors. This application identifies magnolol-related proteins in Magnolia officinalis and overexpresses the laccase gene in Escherichia coli, constructing a recombinant E. coli strain capable of efficiently synthesizing laccase, and then efficiently synthesizing magnolol via laccase. Compared to plant extraction and chemical synthesis, the biosynthesis of magnolol in this application exhibits milder reaction conditions and higher synthesis efficiency. Therefore, this application is of great significance for the industrial production and large-scale application of magnolol. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of a protein sequence tree;

[0032] Figure 2 This is a schematic diagram of the 28a-mbp-PROTEIN1 recombinant vector;

[0033] Figure 3A This is an SDS-PAGE image of the 28A-PROTEIN1 protein. Figure 3B This is an SDS-PAGE image of the 28a-mbp-PROTEIN1 protein. Figure 3C This is an SDS-PAGE image of the 28a-mbp-PROTEIN1-rfp protein;

[0034] Figure 4 This is the standard curve of magnolol in liquid chromatography;

[0035] Figure 5 This is the mass spectrometry standard curve for the detection of magnolol;

[0036] Figure 6 This is a liquid chromatography-mass spectrometry (LC-MS) image of magnolol.

[0037] Figure 7 This is a comparison of the effects of whole-cell catalysis in producing magnolol.

[0038] Figure 8 This study compares the effects of different crude enzyme solutions on the production of magnolol. Detailed Implementation

[0039] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0040] This application provides a laccase and its preparation method, as well as a method for preparing magnolol. These are described in detail below. It should be noted that the order of description in the following examples is not intended to limit the preferred order of the examples.

[0041] Experimental methods not specifically described in the following examples are generally performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this application. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0043] Unless otherwise stated, all reagents and materials mentioned herein are commercially available or can be prepared by those skilled in the art based on common knowledge.

[0044] Definitions and Explanations:

[0045] As used in this application, "recombinant vector" refers to a DNA construct containing a nucleic acid molecule operatively linked to a suitable control sequence that enables the expression of the nucleic acid molecule in a suitable expression system. In the embodiments of this application, a recombinant vector refers to a DNA construct formed by inserting a foreign gene into a vector using molecular biology techniques; Example 28a-mbp-PROTEIN 1 is a recombinant vector.

[0046] As used in this application, "nucleotide sequence" means a sequence of nucleotides from the 5' to 3' ends of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded.

[0047] As used in this application, "codon" refers to a trinucleotide sequence that specifies a particular amino acid.

[0048] This application provides a laccase whose amino acid sequence is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to any one of SEQ ID NO: 1 to SEQ ID NO: 6. Through transcriptome data analysis of differential gene expression and comparison of laccase with homologous sequences, six protein sequences (SEQ ID NO: 1 to SEQ ID NO: 6) from *Magnolia officinalis* Rehd. et Wils. were screened. The proteins with amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 6 were named MoSKU5F, MoLAC7B, MoLAC4A, MoLAC4B, MoLAC17F, and MoLAC14, respectively.

[0049] This application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the laccase described above.

[0050] In some embodiments of this application, the nucleotide sequence is any one of the following:

[0051] (a) The nucleotide sequence is shown in any of the nucleotide sequences in SEQ ID NO: 7 to SEQ ID NO: 12; respectively encoding the protein sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6.

[0052] (b) The nucleotide sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to any nucleotide sequence in SEQ ID NO: 7 to SEQ ID NO: 12; or

[0053] (c) The nucleotide sequence is the nucleotide sequence described in (a) or (b) after codon optimization.

[0054] This application provides a genetically engineered bacterium containing the aforementioned nucleic acid molecules. The bacterium also contains a vector plasmid for expressing laccase protein. Since laccase itself has low specific activity, it typically forms inclusion bodies or is expressed at very low levels. Therefore, using an *E. coli* recombinant expression system to produce laccase can significantly increase yield, meeting the requirements for industrial applications.

[0055] In some embodiments of this application, the genetically engineered bacteria also contain one or more of the following tags: MBP tag, RFP tag, or GFP tag.

[0056] In some embodiments of this application, the genetically engineered bacteria is *Escherichia coli*. Because *E. coli* has a small genome, a mature and well-developed expression system, rapid bacterial reproduction, simple culture, convenient operation, and genetic stability, the *E. coli* expression system is currently the most widely used prokaryotic expression system.

[0057] This application also provides a method for constructing genetically engineered bacteria, comprising: constructing the above-mentioned nucleic acid molecules on a vector to obtain a recombinant vector; and transforming the recombinant vector into an Escherichia coli recipient strain to obtain genetically engineered bacteria.

[0058] In some embodiments of this application, the vector includes an expression vector or a transformation vector.

[0059] In some embodiments of this application, the carrier includes pET28a.

[0060] In some embodiments of this application, the Escherichia coli recipient strains include Escherichia coli DH5α, Escherichia coli BL21(DE3) or Escherichia coli TOPO.

[0061] In some embodiments of this application, one or more of the following tags—MBP (maltose-binding protein), RFP (red fluorescent protein), or GFP (green fluorescent protein)—are added when constructing the recombinant vector. Laccase often forms inclusion bodies or exhibits very low expression levels when expressed in *E. coli*, making it difficult to purify the protein using nickel column adsorption. This application, by adding one or more of the following tags—MBP soluble protein, RFP, or GFP—to the recombinant vector, enhances the solubility and stability of laccase, while simultaneously making the protein expression and purification process visible, optimizing the laccase expression process, and significantly improving the purification efficiency of laccase.

[0062] Compared with the recipient strain of Escherichia coli, the genetically engineered bacteria showed increased expression of the laccase gene and / or increased content of laccase protein and / or increased activity of laccase.

[0063] In some embodiments of this application, a total of 6 gene sequences, SEQ ID NO: 7 to SEQ ID NO: 12, were constructed on the vector pET28a plasmid to obtain recombinant vectors 28a-mbp-PROTEIN1 / 2 / 3 / 4 / 5 / 6 or 28a-mbp-PROTEIN1 / 2 / 3 / 4 / 5 / 6-rfp. The constructed recombinant vectors were then transformed into Escherichia coli DH5α strain. After correct sequencing, the plasmid was extracted and transformed into BL21(DE3) strain.

[0064] This application provides a method for preparing laccase, comprising: inducing expression of the above-mentioned genetically engineered bacteria using IPTG, disrupting the bacterial cells, and purifying the laccase using nickel column affinity chromatography.

[0065] In some embodiments of this application, the concentration of IPTG can be 0.1–1 mM, 0.3–0.7 mM, or 0.5 mM.

[0066] In some embodiments of this application, the induction time can be 13-18 hours, 14-17 hours, or 15-16 hours.

[0067] In some embodiments of this application, the constructed BL21DE3 strains of 28a-PROTEIN1 / 2 / 3 / 4 / 5 / 6, 28a-mbp-PROTEIN1 / 2 / 3 / 4 / 5 / 6, or 28a-mbp-PROTEIN1 / 2 / 3 / 4 / 5 / 6-rfp were streaked. Single colonies were picked and cultured in LB medium with kanamycin resistance at 37°C for 8-10 h, then transferred to 50 mL of LB medium and cultured for about 3 h, followed by transfer to 800 mL of 2×YT medium. After about 3 h of culture, when the OD value reached 0.6, 0.5 mM IPTG was added, and filtered sterilized CuSO4 solution was added to a final concentration of 0.5 mM. After expression at 16°C for 16 h, the bacteria were harvested at 6000 rpm. The bacterial pellet was resuspended in 50 mM PBS solution at pH 7.0. The cells were then disrupted using an ultra-high pressure sterilizer at 700-1000 psi for 3 min. The sample was then centrifuged at 8000 rpm for 60 min in a large centrifuge at low temperature. The supernatant was passed through a nickel column and then purified and eluted using imidazole containing 0 mM, 20 mM, 50 mM, 100 mM, 200 mM, and 300 mM.

[0068] The protein elution buffer was then concentrated using an Ultracel-30 centrifuge filter, and the protein storage system was finally replaced with 50 mM PBS solution at pH 7.0. Protein concentration was tested using Thermo's BCA protein assay kit. The purified protein was then flash-frozen in liquid nitrogen and finally stored at -80°C. This yielded purified laccase.

[0069] Sixthly, this application provides the application of the above-described laccase or the laccase prepared by the above-described method in the preparation of magnolol using 4-allylphenol as a substrate.

[0070] Seventhly, this application provides a method for preparing magnolol, comprising: using 4-allylphenol (also known as piperine, p-allylphenol, p-allylphenol, CAS No.: 501-92-8) as a substrate, and catalyzing it with the above-mentioned laccase, the laccase induced and expressed by the above-mentioned genetically engineered bacteria, or the laccase prepared by the above-mentioned method, to obtain magnolol.

[0071]

[0072] Furthermore, the mass ratio of 4-allylphenol to laccase can be 1:(1-5), 1:(1.5-3), or 1:2.

[0073] Furthermore, the catalytic reaction temperature can be 50–70℃, 55–65℃, or 60℃.

[0074] Furthermore, the catalytic reaction time can be 3 to 7 hours, 4 to 6 hours, or 5 hours.

[0075] Furthermore, adding copper ions to the reaction as a catalyst for laccase can increase its activity; for example, copper sulfate can be added to the reaction to improve its efficiency.

[0076] Description of culture media in the embodiments of this application

[0077] (1) LB liquid culture medium

[0078] Contains 1% NaCl, 1% tryptone, and 0.5% yeast extract. After thorough mixing, adjust the pH to 7.0 and autoclave at 121°C for 30 minutes. Add kanamycin before use.

[0079] (2) 2×YT culture medium

[0080] Mix 1.6% tryptone, 1% yeast extract, and 0.5% NaCl thoroughly, adjust the pH to 7.0, and autoclave at 121°C for 30 minutes. Add kanamycin before use.

[0081] The details of plasmids and competent cells involved in the embodiments of this application are shown in Table 1 below:

[0082] Table 1

[0083]

[0084]

[0085] Description of gene fragments and reagents involved in the embodiments of this application:

[0086] The gene fragments involved in the embodiments of this application, including primers and nucleotide sequences such as those shown in SEQ ID NO: 7-12, were synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.

[0087] The technical solution and beneficial effects of this application are further illustrated below with reference to the embodiments.

[0088] Example 1: Screening of Laccase

[0089] The laccase screening method in this embodiment is as follows:

[0090] The *Magnolia officinalis* genome used in this application was provided by Professor Gao Jihai of Chengdu University of Traditional Chinese Medicine. Its total genome length is 1,685,228,614, the average length is 582,720.80, and the N50 is 76,619,249. The genome assembly and data are normal. N50 is the weighted average of the assembled contigs or backbone. Protein sequences from 100 laccase genes obtained from genome annotation were extracted and aligned using clustawl. The sequences were filtered using gblocks alignment, and then a phylogenetic tree was constructed using raxml-ng.

[0091] After annotating the transcriptome, and combining transcriptome analysis with previous genome tree construction results, six key genes with effective laccase activity were identified. The protein sequence tree is shown below. Figure 1 As shown. We screened MoSKU5F, MoLAC7B, MoLAC4A, MoLAC4B, MoLAC17F, and MoLAC14 from the genome of Magnolia officinalis Rehd. et Wils., which encode a total of 6 protein sequences, namely PROTEIN SEQ ID No. 1 / 2 / 3 / 4 / 5 / 6, and synthesized them at Wuhan Jinkairui Biotechnology Co., Ltd.

[0092] Example 2: Construction of genetically engineered bacteria containing the laccase gene

[0093] The method for constructing the genetically engineered bacteria containing the laccase gene in this embodiment is as follows:

[0094] The six gene sequences, represented by the nucleotide sequences of SEQ ID NO: 7–12, were constructed into the pET28a plasmid to obtain the recombinant vectors 28a-mbp-PROTEIN1 / 2 / 3 / 4 / 5 / 6 or 28a-mbp-PROTEIN1 / 2 / 3 / 4 / 5 / 6-rfp. The vector map of the 28a-mbp-PROTEIN1 recombinant vector is shown below. Figure 2 As shown, the constructed recombinant vector was transformed into Escherichia coli DH5α strain. After correct sequencing, the plasmid was extracted and transformed into BL21(DE3) strain.

[0095] Example 3: Induced expression of genetically engineered bacteria containing the laccase gene

[0096] This embodiment provides purified laccase, which is obtained through the following steps:

[0097] 1) Expression was performed using the BL21(DE3) strain. Taking the 28a-mbp-PROTEIN1 plasmid as an example, the 28a-mbp-PROTEIN1 plasmid was transformed into the BL21DE3 strain and plated on kanamycin (kana) resistant plates. Single colonies were picked and cultured in LB medium with kanamycin resistance of 100ug / mL at 37℃ for 8-10 h, then transferred to 50 mL of LB medium and cultured for about 3 h, and then transferred to 400 mL of 2×YT medium with 100ug / mL kana resistance.

[0098] 2) After incubating for approximately 3 hours until the OD value reaches 0.6, add 0.5 mM IPTG and then add filtered sterile CuSO4 solution to a final concentration of 0.5 mM. After expressing at 16°C for 16 hours, harvest the bacteria at 6000 rpm for 10 minutes. Resuspend the bacteria in 50 mM PBS solution (pH 7.0).

[0099] 3) Use a pre-cooled ultra-high pressure sterilizer at 700-1000 psi for 3 minutes to break the mixture. Then centrifuge at 8000 rpm in a large centrifuge at low temperature for 60 minutes.

[0100] 4) Column affinity chromatography: The supernatant was purified using Ni-NTA affinity chromatography, followed by elution with 50mM PBS solution containing 20mM, 50mM, 100mM, 200mM, and 300mM imidazole. The elution buffer was then concentrated using an Ultracel-30 centrifuge filter, and the protein storage system was finally replaced with 50mM PBS solution at pH 7.0. Protein concentration was determined using Thermo's BCA protein assay kit. The purified MoSKU5F protein was flash-frozen in liquid nitrogen and ultimately stored at -80°C.

[0101] The eluent was subjected to SDS-PAGE to obtain the following results: Figures 3A-3C The results show that the target protein was clearly expressed in the elution buffers of 100mM, 200mM, and 300mM imidazole, and the 28a-mbp-PROTEIN1 protein showed the best purification effect.

[0102] Example 4: Laccase-catalyzed production of magnolol

[0103] The steps of laccase catalysis for the production of magnolol from 4-allylphenol in this embodiment are as follows:

[0104] 1) First, prepare a pH 7.0, 50mM potassium phosphate solution and filter it for later use. Use methanol to prepare a 20mg / ml 4-allylphenol solution as a substrate and prepare a 20mM copper sulfate solution and filter it for later use.

[0105] 2) Preparation of reaction system: The reaction system volume is 200uL. Use the prepared pH 7.0, 50mM potassium phosphate solution as buffer, add 10uL of 4-allylphenol solution to make the final concentration 1g / L, add 10uL of 20mM copper sulfate to make the final concentration 1mM, and add laccase from Example 3 to make the final concentration 1mg / mL.

[0106] 3) The reaction system was placed at 60℃ and shaken for 5 hours at a shaking frequency of 1000 rpm;

[0107] 4) After the reaction is complete, add an equal volume of ethyl acetate to extract the reaction sample at room temperature for 3 hours at 1000 rpm / min. Centrifuge the reaction tube at 12000 rpm / min for 2 minutes at room temperature. Transfer 100 μL of the supernatant ethyl acetate solution to a new centrifuge tube and vacuum dry it at 60°C for approximately 5 minutes using a rotary evaporator. Then dissolve the solution in 100 μL of methanol, centrifuge again at 12000 rpm / min for 20 minutes at 4°C, collect the supernatant, and prepare a liquid chromatography vial.

[0108] Example 5: Liquid Chromatography-Mass Spectrometry Detection of Magnolol

[0109] The steps for the liquid chromatography-mass spectrometry detection of magnolol in this embodiment are as follows:

[0110] Liquid chromatography detection conditions: C18 column, mobile phase A:B = methanol:0.1% formic acid water = 80:20, sample injection volume 30 μL, column temperature 37 °C, flow rate 0.6 mL / min, absorbance 290 nm, 277 nm.

[0111] Liquid chromatography-mass spectrometry (LC-MS) conditions: C18 column, mobile phase A:B = methanol:0.1% formic acid water = 80:20, sample injection volume 30 μL, column temperature 37 °C, flow rate 0.6 mL / min, absorbance 290 nm, 277 nm. Mass spectrum 265.28.

[0112] magnolol liquid chromatography standard curve as follows Figure 4 As shown, the mass spectrometry detection standard curve of magnolol is as follows: Figure 5 As shown, the results of magnolol detection by liquid chromatography-mass spectrometry are as follows: Figure 6 As shown, laccase catalyzes the production of magnolol from 4-allylphenol.

[0113] Example 6: Whole-cell catalytic production of magnolol

[0114] The steps for whole-cell catalytic production of magnolol in this embodiment are as follows:

[0115] 1) Expression was performed using the BL21(DE3) strain of 28a-mbp-PROTEIN1. Strawberries were streaked, and single colonies were picked and transferred to LB medium with 100 μg / mL kana resistance. The culture was carried out at 37°C for 8-10 h, and then transferred to 400 mL of 100 μg / mL kana 2×YT medium.

[0116] 2) After incubating for approximately 3 hours, when the OD value reaches 0.6, add 0.5 mM IPTG and then add filtered, sterilized CuSO4 solution to a final concentration of 0.5 mM. After expressing at 16℃ for 16 hours, centrifuge at 6000 rpm for 10 minutes to collect the bacteria.

[0117] 3) Resuspend the bacterial strain in 30 mL of filtered pH 7.0, 50 mM potassium phosphate solution, mix well, centrifuge again, and then resuspend the bacterial strain in 20 mL of pH 7.0, 50 mM potassium phosphate solution to obtain the genetically engineered bacterial culture.

[0118] 4) The reaction system is prepared as follows: The reaction system is 200 μL, 10 μL of 4-allylphenol solution to make the final concentration 1 g / L, 10 μL of 20 mM copper sulfate to make the final concentration 1 mM, and 20 times concentrated bacterial solution as catalyst to make up to 200 μL.

[0119] 5) Reaction conditions: The reaction system was placed at 60℃ and shaken for 5 hours at a shaking frequency of 1000 rpm / min. After the reaction was complete, an equal volume of ethyl acetate was added to extract the reaction sample at room temperature for 3 hours at 1000 rpm / min. The reaction tube was then centrifuged at 12000 rpm / min for 2 minutes at room temperature. 100 μL of the upper ethyl acetate solution was transferred to a new centrifuge tube and vacuum dried at 60℃ for approximately 5 minutes using a rotary evaporator. Then, 100 μL of methanol was used to dissolve the solution, and the tube was centrifuged again at 4℃ and 12000 rpm / min for 20 minutes. The supernatant was collected, and a liquid chromatography vial was prepared for analysis. The whole-cell catalysis results are shown in the figure below. Figure 7 It is evident that all six engineered bacteria were able to successfully catalyze the synthesis of magnolol from piperine, with the strain containing the MoLAC14 gene exhibiting the highest catalytic efficiency.

[0120] Example 7: Crude enzyme catalysis to produce magnolol

[0121] The steps for crude enzyme-catalyzed production of magnolol in this embodiment are as follows:

[0122] 1) Expression was performed using the BL21(DE3) strain 28a-mbp-PROTEIN1. The strain was streaked, and single colonies were picked. Single colonies were transferred to LB medium with 100 μg / mL kana resistance. The medium was incubated at 37℃ for 8-10 h, then transferred to 400 mL of 2×YT medium containing 100 μg / mL kana.

[0123] 2) After incubating for approximately 3 hours, when the OD value reaches 0.6, add 0.5 mM IPTG and then add filtered, sterilized CuSO4 solution to a final concentration of 0.5 mM. After expressing at 16℃ for 16 hours, centrifuge at 6000 rpm for 10 minutes to collect the bacteria.

[0124] 3) Resuspend the bacteria in 50mM PBS solution at pH 7.0. After mixing, centrifuge again at 6000 rpm / min for 10 min, and then resuspend the bacteria in 20 mL of 50mM PBS solution at pH 7.0. Disrupt the cells using a pre-cooled autoclave at 700-1000 psi for 3 min. Then centrifuge at 8000 rpm / min for 60 min at low temperature. Separate the precipitate from the supernatant to obtain the crude enzyme supernatant.

[0125] 4) Prepare the reaction system: The reaction system is 200 μL. Add 10 μL of 4-allylphenol solution to make the final concentration 1 g / L. Add 10 μL of 20 mM copper sulfate to make the final concentration 1 mM. Add crude enzyme supernatant as a catalyst to make up to 200 μL.

[0126] 5) Reaction conditions: The reaction system was placed at 60℃ and shaken for 5 hours at a shaking frequency of 1000 rpm. After the reaction was complete, an equal volume of ethyl acetate was added to extract the reaction sample at room temperature at 1000 rpm / min for 3 hours. The reaction tube was then centrifuged at 12000 rpm / min for 2 minutes at room temperature. 100 μL of the upper ethyl acetate solution was transferred to a new centrifuge tube and vacuum dried using a rotary evaporator at 60℃ for approximately 5 minutes. Then, 100 μL of methanol was used to dissolve the solution, and the tube was centrifuged again at 4℃ at 12000 rpm / min for 20 minutes. The supernatant was collected and a liquid chromatography vial was prepared for analysis. The crude enzyme catalysis results are shown in [Figure number missing]. Figure 8 It can be seen that the crude enzymes prepared by the six engineered bacteria can all successfully catalyze the synthesis of magnolol from piperine, among which the crude enzyme prepared by the strain containing the MoLAC14 gene has the highest catalytic efficiency.

[0127] This application provides a method for the biosynthesis of magnolol, which successfully catalyzes the synthesis of magnolol from piperine by expressing laccase derived from magnolia bark in Escherichia coli. This method is highly selective, pollution-free, and has a short production cycle.

[0128] The foregoing has provided a detailed description of a laccase and its preparation method, as well as a method for preparing magnolol, provided in the embodiments of this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A laccase, characterized in that, The amino acid sequence of the laccase is shown in any one of SEQ ID NO: 1 to SEQ ID NO:

6.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is a nucleotide sequence encoding the laccase as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence is any one of the following: (a) The nucleotide sequence is shown as any one of SEQ ID NO: 7 to SEQ ID NO: 12; or (b) The nucleotide sequence is the nucleotide sequence obtained by codon optimization of the nucleotide sequence in (a).

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain nucleic acid molecules as described in any one of claims 2 to 3.

5. The genetically engineered bacterium according to claim 4, characterized in that, The genetically engineered bacteria also contain one or more of the following tags: MBP tag, RFP tag, or GFP tag.

6. The genetically engineered bacterium according to claim 4, characterized in that, The genetically engineered bacteria is Escherichia coli.

7. A method for constructing a genetically engineered bacterium, characterized in that, The construction method includes: constructing the nucleic acid molecule of any one of claims 2 to 3 on a vector to obtain a recombinant vector; and transforming the recombinant vector into an Escherichia coli recipient strain to obtain the genetically engineered bacteria.

8. The method for constructing genetically engineered bacteria according to claim 7, characterized in that, The vector includes pET28a; and / or the Escherichia coli recipient strain includes Escherichia coli DH5α, Escherichia coli BL21(DE3) or Escherichia coli TOPO.

9. The method for constructing genetically engineered bacteria according to claim 7, characterized in that, One or more of the following tags are added when constructing the recombinant vector: MBP tag, RFP tag, or GFP tag.

10. A method for preparing laccase, characterized in that, The preparation method includes: inducing expression of the genetically engineered bacteria according to any one of claims 4 to 6 using IPTG, lysing the bacterial cells, and purifying the laccase using nickel column affinity chromatography.

11. The method for preparing laccase according to claim 10, characterized in that, The induction conditions include: the concentration of IPTG is 0.1–1 mM; and / or the induction time is 13–18 h.

12. The use of a laccase as described in claim 1 or a laccase prepared by any one of claims 10-11 in the preparation of magnolol using 4-allylphenol as a substrate.

13. A method for preparing magnolol, characterized in that, The preparation method includes: using 4-allylphenol as a substrate, and catalyzing it with laccase as described in claim 1, laccase induced and expressed by genetically engineered bacteria as described in any one of claims 4 to 6, or laccase prepared by the preparation method as described in any one of claims 10 to 11, to obtain magnolol.

14. The method for preparing magnolol according to claim 13, characterized in that, The mass ratio of 4-allylphenol to laccase is 1:(1-5).

15. The method for preparing magnolol according to claim 13, characterized in that, The conditions for the catalytic reaction include: a reaction temperature of 50–70°C; and / or a reaction time of 3–7 hours.

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