A tyrosine decarboxylase mutant and its preparation method and application

Abstract

The present invention discloses a tyrosine decarboxylase mutant and its preparation method and application. The present invention also discloses a method for synthesizing 3-amino-1-propanol by decarboxylating L-homoserine as a substrate by the tyrosine decarboxylase mutant. The present invention proves through experiments that the tyrosine decarboxylase mutant of the present invention can efficiently catalyze the reaction of the substrate L-homoserine to produce 3-amino-1-propanol. Compared with the wild-type tyrosine decarboxylase, the catalytic activity of the tyrosine decarboxylase mutant is greatly improved, the maximum yield of 3-amino-1-propanol can reach 0.161 mM, and the conversion rate can reach up to 1.61%. Compared with existing chemical synthesis methods, the 3-amino-1-propanol synthesis method provided by the present invention has the advantages of being green, environmentally friendly, and low cost, laying a good foundation for the large-scale industrial production of 3-amino-1-propanol.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme catalysis, and in particular to a tyrosine decarboxylase mutant and a preparation method and application thereof. Background Art

[0002] 3-Amino-1-propanol has a wide range of applications in personal care products, food products, and pharmaceutical intermediates. In the pharmaceutical field, it can be used in the synthesis of anticancer drugs such as cyclophosphamide and cyclopentane. In personal care products and food, 3-amino-1-propanol is primarily used in the preparation of D-panthenol. Panthenol shares the same metabolic process with vitamin B5 in the body, hence its name, provitamin B5. In the food industry and daily chemical products, it is used as a nutritional supplement and enhancer, promoting the metabolism of proteins, fats, and carbohydrates, maintaining skin and mucous membranes, improving hair luster, boosting immunity, and preventing disease. It can also be used as a skin care agent, acting as a deeply penetrating moisturizer, stimulating epithelial cell growth, promoting wound healing, and having anti-inflammatory properties. In recent years, with the improvement of people's living standards, the application of panthenol in personal care products has continued to increase, especially in hair care products and other topical cosmetics, which has significantly driven market demand for 3-amino-1-propanol.

[0003] Currently, the preparation method of 3-amino-1-propanol is relatively extensive. Most of them use 3-hydroxypropionitrile as a raw material to prepare 3-amino-1-propanol. 3-hydroxypropionitrile reacts with hydrogen under ammonia conditions to produce 3-amino-1-propanol. This method can directly synthesize 3-amino-1-propanol in one step, but there is a problem of low selectivity of the target product. There is also a method that uses Raney cobalt catalyst to catalyze the synthesis of 3-amino-1-propanol from 3-hydroxypropionitrile. However, this method has the disadvantages of low acrylonitrile conversion and the generation of a large amount of by-product dicyanoethyl ether, which is highly dangerous and not easy to achieve industrial application. Another part is a method of indirectly synthesizing 3-amino-1-propanol by other processes, such as first preparing 3-benzyloxypropionitrile by adding acrylonitrile and benzyl alcohol, and then further hydrogenating it to obtain 3-amino-1-propanol. However, toluene is also produced as a by-product, resulting in a decrease in product yield. In addition, the by-product is difficult to remove, and the post-processing cost increases. Another example is first preparing 3-chloropropanol by using 1,3-propylene glycol and hydrochloric acid, and then further reacting it with ammonia water to prepare 3-amino-1-propanol. The raw material hydrochloric acid required for this invention will cause serious pollution and corrosion, and it needs to go through complex steps such as extraction, and the preparation process is quite cumbersome. Another example is using 1,4-butyrolactone as a raw material to add hydrazine hydrate to prepare acyl azide, and then further rearrange it to generate 3-amino-1-propanol. The disadvantage of this method is that the raw materials are expensive and extremely toxic, and there is a risk of flammability and explosion. From the above analysis, it can be seen that the existing preparation methods of 3-amino-1-propanol have many problems such as low selectivity, expensive raw materials, complex processes, poor safety, low product yield and conversion rate. Therefore, it is necessary to develop an efficient and simple preparation method to meet the market demand for 3-amino-1-propanol.

[0004] Enzymatic synthesis is currently the mainstream method for synthesizing many compounds due to its environmental friendliness, efficiency, and sustainability. Currently, the commonly used decarboxylases are derived from Corynebacterium glutamicum and Bacillus subtilis. However, the bottleneck in synthesizing 3-amino-1-propanol with these decarboxylases from different sources is low enzyme activity. Summary of the Invention

[0005] The purpose of the present invention is to provide a tyrosine decarboxylase mutant and a preparation method and application thereof.

[0006] To achieve the above objectives, the present invention first provides a protein obtained by mutating the methionine (Met) at position 99 in the amino acid sequence of tyrosine decarboxylase to glutamic acid (Glu). This protein is designated V583ADC (M99E) in the present invention.

[0007] The tyrosine decarboxylase is a protein described in any one of the following (a1)-(a3):

[0008] (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0009] (a2) A protein having the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues other than position 99 in the amino acid sequence of (a1);

[0010] (a3) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in (a1) or (a2).

[0011] In the above (a2), the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0012] In (a3) ​​above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0013] The protein in (a2) or (a3) ​​above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0014] In any of the above proteins, the tyrosine decarboxylase activity of the protein is higher than that of the tyrosine decarboxylase (SEQ ID NO: 1).

[0015] In order to achieve the above object, the present invention further provides a nucleic acid molecule encoding the above protein.

[0016] The nucleic acid molecule encoding the above protein provided by the present invention is a DNA molecule described in any one of the following (b1)-(b2):

[0017] (b1) The nucleotide sequence is the DNA molecule shown in Sequence 3;

[0018] (b2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in (b1) and encodes the above-mentioned protein.

[0019] The nucleic acid molecule may be DNA, such as recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA. The nucleic acid molecule may be a nucleic acid molecule formed by a gene encoding the protein and its regulatory sequence.

[0020] Those skilled in the art can readily mutate the nucleotide sequences encoding the proteins of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequences encoding the proteins are derived from and are equivalent to the nucleotide sequences of the present invention, as long as they encode the proteins and have the same function.

[0021] In (b2) above, identity refers to sequence similarity with a naturally occurring nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater identical to the nucleotide sequence encoding the V583ADC (M99E) protein of the present invention. Identity can be assessed visually or with computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess identity between related sequences.

[0022] The aforementioned 75% or greater identity may be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or 100% identity.

[0023] In order to achieve the above-mentioned object, the present invention further provides a biomaterial as described in any one of the following (c1) to (c3):

[0024] (c1) an expression cassette containing the above nucleic acid molecule;

[0025] (c2) a recombinant vector containing the above nucleic acid molecule;

[0026] (c3) A recombinant bacterium containing the above nucleic acid molecule.

[0027] In (c1) above, the expression cassette refers to DNA capable of expressing the protein in a host cell. This DNA may include not only a promoter for initiating transcription of the protein-encoding gene sequence, but also a terminator for terminating transcription of the protein-encoding gene sequence. Furthermore, the expression cassette may also include an enhancer sequence.

[0028] In (c2) above, the recombinant vector refers to a recombinant DNA molecule constructed by ligating the aforementioned nucleic acid molecule and a vector in vitro. The vector may be a plasmid, a bacteriophage (such as lambda phage or M13 filamentous phage), a cosmid (i.e., a cosmid), a Ti plasmid, or a viral vector (such as a retrovirus (including a lentivirus), an adenovirus, or an adeno-associated virus).

[0029] In a specific embodiment of the present invention, the recombinant vector is obtained by replacing the DNA fragment between the EcoR I and Hind III restriction sites of the vector pET-28a(+) with the V583ADC gene fragment shown in SEQ ID NO: 3, while keeping the other sequences of the vector pET-28a(+) unchanged.

[0030] In (c3) above, the recombinant bacteria refers to bacteria obtained by introducing a nucleic acid molecule encoding the protein into bacteria or fungi. The bacteria may be Escherichia coli (eg, Escherichia coli BL21 (DE3)).

[0031] In a specific embodiment of the present invention, the recombinant bacteria is Escherichia coli BL21 (DE3) containing the above-mentioned recombinant vector.

[0032] In order to achieve the above object, the present invention also provides a method for preparing the above protein.

[0033] The method for preparing the above protein provided by the present invention comprises the following steps: expressing the above nucleic acid molecule in an organism or biological cell to obtain the above protein.

[0034] Furthermore, the method includes the following steps: fermenting and culturing the above-mentioned recombinant bacteria to obtain the protein.

[0035] Furthermore, the fermentation culture method can be carried out according to the following steps: inoculating the above-mentioned recombinant bacteria into a seed culture medium for cultivation to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for cultivation to obtain a culture liquid; adding IPTG to the culture liquid for induction culture to obtain a fermentation liquid, wherein the fermentation liquid contains the above-mentioned protein.

[0036] Furthermore, both the seed culture medium and the fermentation culture medium can be 2YT liquid culture medium containing kanamycin (the final concentration can be 50 μg / mL).

[0037] The seed liquid is inoculated into the fermentation medium at a ratio of 1%.

[0038] The culture conditions in the seed culture medium may be overnight culture at 37°C and 220 rpm.

[0039] The culture conditions in the fermentation medium can be 37 ° C, 220 rpm and cultured until the OD600nm It is 0.6-0.8.

[0040] The final concentration of IPTG in the fermentation culture system can be 0.5 mM.

[0041] The induction culture condition may be overnight culture at 25°C.

[0042] In order to achieve the above-mentioned object, the present invention further provides any one of the following applications (d1) to (d4):

[0043] (d1) Use of the above protein as a tyrosine decarboxylase;

[0044] (d2) Use of the aforementioned nucleic acid molecules or biological materials in the preparation of tyrosine decarboxylase;

[0045] (d3) Use of the above protein, nucleic acid molecule or biological material in the production of 3-amino-1-propanol;

[0046] (d4) Use of the above protein, nucleic acid molecule or biological material in increasing the production of 3-amino-1-propanol.

[0047] In order to achieve the above object, the present invention finally provides a method for producing 3-amino-1-propanol.

[0048] The method for producing 3-amino-1-propanol provided by the present invention comprises the following steps: using the above protein as a biological enzyme to catalyze L-homoserine to generate 3-amino-1-propanol.

[0049] Furthermore, the protein (ie, tyrosine decarboxylase mutant V583ADC (M99E)) can perform catalysis in the form of a crude enzyme solution.

[0050] The crude enzyme solution can be prepared as follows: the fermentation solution is centrifuged to collect bacterial cells, and then the bacterial cells are resuspended in a buffer solution and ultrasonically disrupted to obtain the crude enzyme solution.

[0051] Furthermore, the centrifugation condition may be 4° C., 6000 rpm, 10 min.

[0052] The buffer may be 50 mM phosphate (dipotassium hydrogen phosphate and potassium dihydrogen phosphate) buffer (pH 7.0).

[0053] The catalytic reaction system is composed of the above crude enzyme solution and L-homoserine. The concentration of the crude enzyme solution can be 10OD 600nm / ml, the final concentration of L-homoserine in the reaction system can be 10mM.

[0054] The catalytic reaction conditions may be 37° C., 200 rpm, and 12 h.

[0055] The present invention provides a tyrosine decarboxylase mutant and a method for synthesizing 3-amino-1-propanol using L-homoserine as a substrate and subsequent decarboxylation by the tyrosine decarboxylase mutant. Experiments have demonstrated that the tyrosine decarboxylase mutant of the present invention can efficiently catalyze the reaction of the substrate L-homoserine to produce 3-amino-1-propanol. Compared with wild-type tyrosine decarboxylase, the catalytic activity of the tyrosine decarboxylase mutant is greatly improved, with a 3-amino-1-propanol yield of up to 0.161 mM and a conversion rate of up to 1.61%. Compared with existing chemical synthesis methods, the 3-amino-1-propanol synthesis method provided by the present invention has advantages such as environmental friendliness and low cost, laying a good foundation for the large-scale industrial production of 3-amino-1-propanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of the synthesis of 3-amino-1-propanol.

[0057] Figure 2 This is the HPLC detection spectrum of L-homoserine standard.

[0058] Figure 3 This is the HPLC detection spectrum of 3-amino-1-propanol standard.

[0059] Figure 4 HPLC detection spectrum of the conversion product. DETAILED DESCRIPTION

[0060] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0061] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0062] The experimental materials and their sources involved in the following examples are as follows:

[0063] The vector pET-28a(+) in the following examples is a product of Invitrogen.

[0064] The Escherichia coli BL21 (DE3) in the following examples is a product of Qingke Biotechnology Co., Ltd.

[0065] The L-homoserine standard in the following examples is a product of Aladdin, with the product number H105430.

[0066] The 3-amino-1-propanol standard in the following examples is a product of ACMEC, with the product number A41420.

[0067] The solvent of the 2YT liquid culture medium in the following examples is water, and the solutes and their concentrations are respectively 0.5% (mass fraction) of NaCl, 1% (mass fraction) of yeast extract, and 1.6% (mass fraction) of tryptone.

[0068] The tyrosine decarboxylase ADC in the following examples is derived from Enterococcus faecalis V583 and is abbreviated as V583ADC. The amino acid sequence of V583ADC is shown in SEQ ID NO: 1, and the encoding gene sequence of V583ADC is shown in SEQ ID NO: 2.

[0069] Example 1. Preparation of V583 ADC mutants

[0070] 1. Construction of recombinant bacteria

[0071] 1. Construction of wild-type recombinant plasmid

[0072] The DNA fragment between the EcoRI and HindIII restriction sites of the vector pET-28a(+) was replaced with the gene fragment shown in Sequence 2, while keeping the other sequences of the vector pET28a(+) unchanged to obtain the wild-type recombinant plasmid pET28a(+)-V583ADC.

[0073] 2. Construction of mutant recombinant plasmid

[0074] The wild-type recombinant plasmid in step 1 was used as a template, and PCR amplification was performed using the primers in Table 1 to obtain mutant recombinant plasmids pET28a(+)-V583ADC(H98A), pET28a(+)-V583ADC(H98G), pET28a(+)-V583ADC(M99T), pET28a(+)-V583ADC(M99L), and pET28a(+)-V583ADC(H98A). 3ADC (M99E), pET28a(+)-V583ADC (M99S), pET28a(+)-V583ADC (M99A), pET28a(+)-V583ADC (M99W), pET28a(+)-V583ADC (M99R), pET28a(+)-V583ADC (M99H), and pET28a(+)-V583ADC (M99Y). The primer sequences are shown in Table 1.

[0075] Table 1

[0076]

[0077] Note: The bases in bold are the introduced mutations.

[0078] The PCR amplification reaction system was as follows (total volume 50 μL): 2x25 μL of ApexHFHS DNA, 1.5 μL of forward primer (10 pM), 1.5 μL of reverse primer (10 pM), 1 μL of wild-type recombinant plasmid (100 ng / μL), and 22 μL of water.

[0079] The PCR amplification reaction conditions were as follows: 98°C for 10 min, 98°C for 10 s, 55°C for 30 s, 72°C for 4 min, 35 cycles, 72°C for 10 mins, and 4°C forever.

[0080] After the PCR amplification reaction was completed, 1.5 μL of Dpn I (20 U / μL) was added to the PCR reaction solution and reacted at 37°C for 3 h.

[0081] 3. Construction of recombinant bacteria

[0082] The wild-type recombinant plasmid pET28a-V583ADC, mutant recombinant plasmids pET28a(+)-V583ADC (H98A), pET28a(+)-V583ADC (H98G), pET28a(+)-V583ADC (M99T), pET28a(+)-V583ADC (M99L), pET28a(+)-V583ADC (M99E), pET28a(+)-V583ADC (M99S), and pET28a(+)-V583ADC (M99T) were respectively used to analyze the effect of different plasmids. 3ADC (M99A), pET28a(+)-V583ADC (M99W), pET28a(+)-V583ADC (M99R), pET28a(+)-V583ADC (M99H), and pET28a(+)-V583ADC (M99Y) were transformed into Escherichia coli BL21 (DE3) to obtain the recombinant bacteria pET28a-V583ADC-BL21 (DE3) containing the wild-type recombinant plasmid pET28a-V583ADC and the recombinant plasmids containing different mutants. The recombinant bacteria pET28a(+)-V583ADC(H98A)-BL21(DE3), pET28a(+)-V583ADC(H98G)-BL21(DE3), pET28a(+)-V583ADC(M99T)-BL21(DE3), pET28a(+)-V583ADC(M99L)-BL21(DE3), pET28a(+)-V583ADC(M99E)-BL21(DE3), pET28a(+)-V583A DC(M99S)-BL21(DE3), pET28a(+)-V583ADC(M99A)-BL21(DE3), pET28a(+)-V583ADC(M99W)-BL21(DE3), pET28a( +)-V583ADC(M99R)-BL21(DE3), pET28a(+)-V583ADC(M99H)-BL21(DE3), pET28a(+)-V583ADC(M99Y)-BL21(DE3).

[0083] 2. Preparation of V583ADC mutant crude enzyme solution

[0084] Test strains: pET28a(+)-V583ADC-BL21(DE3), pET28a(+)-V583ADC(H98A)-BL21(DE3), pET28a(+)-V583ADC(H98G)-BL21(DE3), pET28a(+)-V583ADC(M99T)-BL21(DE3), pET28a(+)-V583ADC(M99L)-BL21(DE3), pET28a(+)-V583ADC(M99E)-BL21(DE3), pET28a(+)-V583ADC(M99S)-BL21(DE3), pET28a(+)-V583ADC(M99A)-BL21(DE3), pET28a(+)-V583ADC(M99W)-BL21(DE3), pET28a(+)-V583ADC(M99R)-BL21(DE3), pET28a(+)-V583ADC(M99H)-BL21(DE3), pET28a(+)-V583ADC(M99Y)-BL21(DE3).

[0085] Experimental method: The test strain was cultured in 5 mL 2YT liquid medium supplemented with kanamycin at a final concentration of 50 μg / ml at 37°C and 220 rpm overnight to obtain seed solution; the seed solution was inoculated into 100 mL 2YT liquid medium supplemented with kanamycin at a final concentration of 50 μg / ml at 1% (V / V) and cultured at 37°C and 220 rpm to obtain culture solution; the culture solution OD 600nm When the pH value is between 0.6 and 0.8, inducer IPTG is added to a final concentration of 0.5 mM for induction. Induction is carried out at 25°C overnight to obtain a fermentation broth. The fermentation broth is centrifuged at 6000 rpm for 10 minutes at 4°C. The cells are harvested and suspended in 50 mM phosphate (dipotassium hydrogen phosphate and potassium dihydrogen phosphate) buffer (pH 7.0). Ultrasonic disruption is performed (200W, 5s / 7s, 20min) to obtain a crude enzyme solution.

[0086] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC-BL21(DE3) contains V583ADC protein, and the amino acid sequence of the V583ADC protein is shown in SEQ ID NO: 1.

[0087] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(H98A)-BL21(DE3) contains the V583ADC protein mutant V583ADC(H98A). The amino acid sequence of the V583ADC protein mutant V583ADC(H98A) is the sequence obtained by mutating the histidine at position 98 of sequence 1 to alanine.

[0088] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(H98G)-BL21(DE3) contains the V583ADC protein mutant V583ADC(H98G). The amino acid sequence of the V583ADC protein mutant V583ADC(H98G) is the sequence obtained by mutating the histidine at position 98 of sequence 1 to glycine.

[0089] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99T)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99T). The amino acid sequence of the V583ADC protein mutant V583ADC(M99T) is the sequence obtained by mutating the methionine at position 99 of sequence 1 to threonine.

[0090] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99L)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99L). The amino acid sequence of the V583ADC protein mutant V583ADC(M99L) is the sequence obtained by mutating the methionine at position 99 of sequence 1 to leucine.

[0091] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99E)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99E). The amino acid sequence of the V583ADC protein mutant V583ADC(M99E) is the sequence obtained by mutating the methionine at position 99 of sequence 1 to glutamic acid.

[0092] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99S)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99S). The amino acid sequence of the V583ADC protein mutant V583ADC(M99S) is the sequence obtained by mutating methionine at position 99 of sequence 1 to serine.

[0093] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99A)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99A). The amino acid sequence of the V583ADC protein mutant V583ADC(M99A) is the sequence obtained by mutating the methionine at position 99 of sequence 1 to alanine.

[0094] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99W)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99W). The amino acid sequence of the V583ADC protein mutant V583ADC(M99W) is the sequence obtained by mutating the methionine at position 99 of sequence 1 to tryptophan.

[0095] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99R)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99R). The amino acid sequence of the V583ADC protein mutant V583ADC(M99R) is the sequence obtained by mutating the methionine at position 99 of sequence 1 to arginine.

[0096] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99H)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99H). The amino acid sequence of the V583ADC protein mutant V583ADC(M99H) is the sequence obtained by mutating the methionine at position 99 of sequence 1 to histidine.

[0097] The crude enzyme solution prepared by the recombinant bacterium pET28a(+)-V583ADC(M99Y)-BL21(DE3) contains the V583ADC protein mutant V583ADC(M99Y). The amino acid sequence of the V583ADC protein mutant V583ADC(M99Y) is the sequence obtained by mutating the methionine at position 99 of sequence 1 to tyrosine.

[0098] Example 2: Catalytic activity detection of V583 ADC and its mutants

[0099] In this example, L-homoserine was used as a substrate to synthesize 3-amino-1-propanol ( Figure 1 ) to detect the catalytic activity of V583ADC and its mutants. The specific steps are as follows:

[0100] 1. Add L-homoserine to the crude enzyme solution prepared in step 2 of Example 1 to obtain a mixed solution. The concentration of the crude enzyme solution is 10 OD 600nm / ml, and the concentration of L-homoserine was 10 mM.

[0101] 2. The mixed solution obtained in step 1 was subjected to catalytic reaction at 37° C. and 220 rpm for 12 hours to obtain a conversion solution.

[0102] 3. Heat the conversion solution obtained in step 2 in a boiling water bath at 80°C for 10 min. After the heat treatment, centrifuge at 4°C and 12,000 rpm for 5 min and collect the supernatant.

[0103] 4. The supernatant obtained in step 3 was derivatized with DNFB (2,4-dinitrofluorobenzene) and the content of 3-amino-1-propanol was detected by HPLC.

[0104] The DNFB derivatization method is as follows: 50 μl of sample, 20 μl of 1 M sodium bicarbonate, and 80 μl of DNFB derivatization reagent are mixed and incubated at 60°C for 30 minutes. After 30 minutes, the sample is removed and 50 μl of 1 M hydrochloric acid is added. After mixing, the supernatant is centrifuged at 12,000 rpm for 5 minutes. The supernatant is filtered through a 0.22 μM filter membrane and the filtrate is collected. The 3-amino-1-propanol content in the filtrate is determined by HPLC. HPLC is performed using an RP-C18 column with a mobile phase of 35% acetonitrile and 65% 0.1% formic acid solution at a flow rate of 0.7 ml / min, a column temperature of 25°C, and a 10 μl injection. The detection wavelength is 360 nm.

[0105] The experiment was repeated three times and the results were averaged.

[0106] The results showed that the retention time of L-homoserine standard was 8.28min ( Figure 2 ), the retention time of 3-amino-1-propanol standard was 12.835min ( Figure 3 There are peaks with retention times of 8.216min and 12.795min in the conversion solution ( Figure 4 ), indicating that 3-amino-1-propanol was generated using L-homoserine as a substrate, and the amount of 3-amino-1-propanol and conversion rate of V583ADC (M99E) were significantly improved compared with those of V583ADC (Table 2).

[0107] Table 2

[0108]

[0109] Note: Conversion rate = 3-amino-1-propanol (mM) / L-homoserine (mM) * 100%.

[0110] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A tyrosine decarboxylase mutant, wherein the tyrosine decarboxylase mutant is a protein obtained by mutating the methionine at position 99 of the tyrosine decarboxylase amino acid sequence to glutamic acid; the tyrosine decarboxylase is a protein consisting of the amino acid sequence shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the tyrosine decarboxylase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule whose nucleotide sequence is shown in SEQ ID NO:

3.

4. Any of the following biological materials (c1) to (c3): (c1) an expression cassette containing the nucleic acid molecule according to claim 2 or 3; (c2) a recombinant vector containing the nucleic acid molecule according to claim 2 or 3; (c3) A recombinant bacterium containing the nucleic acid molecule according to claim 2 or 3.

5. The biomaterial according to claim 4, characterized in that: The recombinant bacterium is obtained by introducing a nucleic acid molecule encoding the tyrosine decarboxylase mutant according to claim 1 into Escherichia coli.

6. A method for preparing the tyrosine decarboxylase mutant according to claim 1, comprising the steps of expressing the nucleic acid molecule according to claim 2 or 3 in a biological cell to obtain the tyrosine decarboxylase mutant according to claim 1.

7. The method according to claim 6, characterized in that: The method comprises the following steps: fermenting and culturing the recombinant bacteria according to claim 4 or 5 to obtain the tyrosine decarboxylase mutant according to claim 1.

8. Use of the nucleic acid molecule according to claim 2 or 3 or the biomaterial according to claim 4 or 5 in the preparation of tyrosine decarboxylase.

9. Use of the tyrosine decarboxylase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the biomaterial according to claim 4 or 5 in the production of 3-amino-1-propanol.

10. Use of the tyrosine decarboxylase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the biomaterial according to claim 4 or 5 in increasing the yield of 3-amino-1-propanol.

11. A method for producing 3-amino-1-propanol, comprising the following steps: using the tyrosine decarboxylase mutant according to claim 1 as a biological enzyme to catalyze L-homoserine to produce 3-amino-1-propanol.

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