Tyrosine decarboxylase mutant as well as preparation method and application thereof

Abstract

The invention discloses a tyrosine decarboxylase mutant as well as a preparation method and application thereof. The invention also discloses a method for synthesizing 3-amino-1-propanol by taking L-homoserine as a substrate and carrying out decarboxylation on the L-homoserine by using the tyrosine decarboxylase mutant. Experiments prove that the tyrosine decarboxylase mutant disclosed by the invention can efficiently catalyze a reaction of a substrate L-homoserine to generate 3-amino-1-propanol, and compared with wild tyrosine decarboxylase, the tyrosine decarboxylase mutant disclosed by the invention has the advantages that the catalytic activity of the tyrosine decarboxylase mutant is greatly improved, the highest yield of the 3-amino-1-propanol can reach 0.161 mM, and the highest conversion rate can reach 1.61%. Compared with the existing chemical synthesis method, the synthesis method of 3-amino-1-propanol provided by the invention has the advantages of greenness, environmental protection, low cost and the like, and lays a good foundation for large-scale industrial production of 3-amino-1-propanol.

Description

Technical Field

[0001] The 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 is widely used in the fields of daily chemical care products, food and pharmaceutical intermediates. In the pharmaceutical field, it can be used for the synthesis of anticancer drugs such as cyclophosphamide and cyclohexan. In the field of daily chemical care products and food, 3-amino-1-propanol is mainly used for the preparation of D-panthenol. Panthenol has the same metabolic process as vitamin B5 in the body, so it is also called provitamin B5. In the food industry and the field of daily chemicals, it is used as a nutritional supplement and enhancer to promote the metabolism of human protein, fat and sugar, maintain the skin and mucous membranes, improve hair gloss, enhance immunity, and prevent the occurrence of diseases. It can also be used as a skin care agent, which is a deeply penetrating moisturizer, stimulates the growth of epithelial cells, promotes wound healing, and has an anti-inflammatory effect. In recent years, with the improvement of people's living standards, the application of panthenol in daily chemical products has been continuously improved, especially in hair care products and other topical cosmetics. The application is becoming more and more extensive, which has greatly driven the market demand for 3-amino-1-propanol.

[0003] At present, the preparation method of 3-amino-1-propanol is relatively extensive, and most of them adopt 3-hydroxypropionitrile as raw material to prepare 3-amino-1-propanol, and 3-hydroxypropionitrile reacts with hydrogen under ammonia condition to generate 3-amino-1-propanol, and this method can directly synthesize 3-amino-1-propanol in one step, but there is the problem of low selectivity of target product. There is also a part of the method that adopts Raney cobalt catalyst to catalyze 3-hydroxypropionitrile to synthesize 3-amino-1-propanol, but the disadvantage of this method is that the conversion rate of acrylonitrile is low, and a large amount of by-product dicyanoethyl ether will be generated, which is dangerous and difficult to realize industrial application. Another part is a method of indirectly synthesizing 3-amino-1-propanol by other processes, such as first preparing 3-benzylpropionitrile by addition reaction of acrylonitrile and benzyl alcohol, and then further hydrogenating to prepare 3-amino-1-propanol. However, toluene is also produced as a by-product, resulting in a decrease in product yield. Moreover, the by-product is difficult to remove, and the post-processing cost is increased. Another example is first preparing 3-chloropropanol with 1,3-propylene glycol and hydrochloric acid, and then further reacting 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 taking 1,4-butyrolactone as a raw material and adding hydrazine hydrate to prepare acyl azide, and then further rearranging 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 process, 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 the mainstream method for synthesizing many compounds because it is greener, more efficient and sustainable. The commonly used decarboxylases are derived from Corynebacterium glutamicum and Bacillus subtilis, but the bottleneck of decarboxylases from different sources in synthesizing 3-amino-1-propanol is the 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] In order to achieve the above object, the present invention first provides a protein, wherein the protein is obtained by mutating the 99th methionine (Met) in the amino acid sequence of tyrosine decarboxylase to glutamic acid (Glu), which is named V583ADC (M99E) in the present invention.

[0007] The tyrosine decarboxylase is a protein described in any one of the following (a1)-(a3): (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (a2) A protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues other than position 99 of the amino acid sequence shown in (a1); (a3) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the amino acid sequence shown in (a1) or (a2).

[0008] 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.

[0009] In (a3) ​​above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0010] The protein in (a2) or (a3) ​​above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0011] 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).

[0012] In order to achieve the above object, the present invention further provides a nucleic acid molecule encoding the above protein.

[0013] 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): (b1) The nucleotide sequence is the DNA molecule shown in Sequence 3; (b2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in (b1) and encoding the above protein.

[0014] 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.

[0015] Those skilled in the art can easily mutate the nucleotide sequence encoding the above protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence encoding the above protein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above protein and have the same function.

[0016] In (b2) above, the identity refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence encoding the V583ADC (M99E) protein of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0017] The 75% or more identity mentioned above may be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or 100% identity.

[0018] In order to achieve the above object, the present invention further provides a biomaterial as described in any one of the following (c1) to (c3): (c1) an expression cassette containing the above nucleic acid molecule; (c2) a recombinant vector containing the above nucleic acid molecule; (c3) A recombinant bacterium containing the above nucleic acid molecule.

[0019] In the above (c1), the expression cassette refers to a DNA capable of expressing the above protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the above protein encoding gene sequence, but also a terminator for terminating transcription of the above protein encoding gene sequence. Furthermore, the expression cassette may also include an enhancer sequence.

[0020] In the above (c2), the recombinant vector refers to a recombinant DNA molecule constructed by connecting the above nucleic acid molecule and the vector in vitro. The vector may be a plasmid, a bacteriophage (such as lambda phage or M13 filamentous phage, etc.), a cosmid (i.e., a cosmid), a Ti plasmid, a viral vector (such as a retrovirus (including a lentivirus), adenovirus, adeno-associated virus, etc.).

[0021] In a specific embodiment of the present invention, the recombinant vector is a vector 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 Sequence 3, while keeping the other sequences of the vector pET-28a(+) unchanged.

[0022] In the above (c3), the recombinant bacteria refers to bacteria obtained by introducing a nucleic acid molecule encoding the above protein into bacteria or fungi. The bacteria may be Escherichia coli (such as Escherichia coli BL21 (DE3)).

[0023] In a specific embodiment of the present invention, the recombinant bacteria is Escherichia coli BL21 (DE3) containing the above-mentioned recombinant vector.

[0024] In order to achieve the above object, the present invention also provides a method for preparing the above protein.

[0025] 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 a biological cell to obtain the above protein.

[0026] Furthermore, the method comprises the following steps: fermenting and culturing the above-mentioned recombinant bacteria to obtain the protein.

[0027] 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 culture to obtain a seed solution; inoculating the seed solution into a fermentation culture medium for culture to obtain a culture solution; adding IPTG to the culture solution for induction culture to obtain a fermentation solution, wherein the fermentation solution contains the above-mentioned protein.

[0028] 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).

[0029] The seed liquid is inoculated into the fermentation medium at a ratio of 1%.

[0030] The conditions for culturing in the seed medium may be overnight culturing at 37° C. and 220 rpm.

[0031] The conditions for culturing in the fermentation medium can be 37°C, 220 rpm, and culturing until the OD 600nm It is 0.6-0.8.

[0032] The final concentration of the IPTG in the fermentation culture system may be 0.5 mM.

[0033] The induction culture condition may be overnight culture at 25°C.

[0034] In order to achieve the above object, the present invention further provides any application described in the following (d1)-(d4): (d1) Use of the above protein as tyrosine decarboxylase; (d2) Use of the above nucleic acid molecules or the above biological materials in the preparation of tyrosine decarboxylase; (d3) Use of the above protein, nucleic acid molecule or biological material in the production of 3-amino-1-propanol; (d4) Use of the above protein, the above nucleic acid molecule or the above biological material in increasing the production of 3-amino-1-propanol.

[0035] In order to achieve the above object, the present invention finally provides a method for producing 3-amino-1-propanol.

[0036] The method for producing 3-amino-1-propanol provided by the invention comprises the following steps: using the above protein as a biological enzyme to catalyze L-homoserine to generate 3-amino-1-propanol.

[0037] Furthermore, the protein (ie, tyrosine decarboxylase mutant V583ADC (M99E)) can catalyze in the form of a crude enzyme solution.

[0038] The preparation method of the crude enzyme solution can be prepared according to the following method: centrifuging the fermentation solution, collecting bacterial cells, and then resuspending the bacterial cells in a buffer solution and performing ultrasonic disruption to obtain the crude enzyme solution.

[0039] Furthermore, the centrifugation condition may be 4° C. and 6000 rpm for 10 min.

[0040] The buffer may be 50 mM phosphate (dipotassium hydrogen phosphate and potassium dihydrogen phosphate) buffer (pH 7.0).

[0041] 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.

[0042] The catalytic reaction conditions may be 37° C., 200 rpm, and 12 h.

[0043] The present invention provides a tyrosine decarboxylase mutant, and also provides a method for synthesizing 3-amino-1-propanol by decarboxylating 3-amino-1-propanol using L-homoserine as a substrate by the tyrosine decarboxylase mutant. Experiments have shown that the tyrosine decarboxylase mutant of the present invention can efficiently catalyze the reaction of the substrate L-homoserine to generate 3-amino-1-propanol, and compared with the wild-type tyrosine decarboxylase, the catalytic activity of the tyrosine decarboxylase mutant is greatly improved, the 3-amino-1-propanol yield can reach up to 0.161 mM, and the conversion rate can reach up to 1.61%. Compared with the existing chemical synthesis method, the 3-amino-1-propanol synthesis method provided by the present invention has the advantages of green environmental protection and low cost, and has laid a good foundation for the large-scale industrial production of 3-amino-1-propanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the synthesis of 3-amino-1-propanol.

[0045] Figure 2 This is the HPLC detection spectrum of L-homoserine standard.

[0046] Figure 3 This is the HPLC detection spectrum of 3-amino-1-propanol standard.

[0047] Figure 4 The HPLC detection spectrum of the conversion product. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0049] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0050] The experimental materials and their sources involved in the following embodiments are as follows: The vector pET-28a(+) in the following examples is a product of Invitrogen.

[0051] The Escherichia coli BL21 (DE3) in the following examples is a product of Qingke Biotechnology Co., Ltd.

[0052] The L-homoserine standard in the following examples is a product of Aladdin, with a product number of H105430.

[0053] The 3-amino-1-propanol standard in the following examples is a product of ACMEC, with the product number A41420.

[0054] 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.

[0055] The tyrosine decarboxylase ADC in the following examples is derived from Enterococcus faecalis V583, abbreviated as V583ADC. The amino acid sequence of V583ADC is shown in Sequence 1, and the coding gene sequence of V583ADC is shown in Sequence 2.

[0056] Example 1. Preparation of V583 ADC mutants 1. Construction of recombinant bacteria 1. Construction of wild-type recombinant plasmid The DNA fragment between the EcoR I and Hind III restriction sites of the vector pET-28a(+) was replaced with the gene fragment shown in sequence 2, and the other sequences of the vector pET28a(+) were kept unchanged to obtain the wild-type recombinant plasmid pET28a(+)-V583ADC.

[0057] 2. Construction of mutant recombinant plasmid Using the wild-type recombinant plasmid in step 1 as a template, 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), pET28a(+)-V58 3ADC (M99E), pET28a(+)-V583ADC (M99S), pET28a(+)-V583ADC (M99A), pET28a(+)-V583ADC (M99W), pET28a(+)-V583ADC (M99R), pET28a(+)-V583ADC (M99H), pET28a(+)-V583ADC (M99Y). The primer sequences are shown in Table 1.

[0058] Table 1

[0059] Note: The bases in bold are the introduced mutations.

[0060] The PCR amplification reaction system was as follows (total volume was 50 μL): ApexHFHS DNA 2x25 μL, forward primer (10 pm) 1.5 μL, reverse primer (10 pm) 1.5 μL, wild-type recombinant plasmid (100 ng / μL) 1 μL, and water 22 μL.

[0061] 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, 4°C forever.

[0062] After the PCR amplification reaction was completed, 1.5 μL Dpn I (20 U / μL) was added to the PCR reaction solution and reacted at 37°C for 3 h.

[0063] 3. Construction of recombinant bacteria 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). 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).

[0064] 2. Preparation of crude enzyme solution of V583ADC mutant 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).

[0065] Experimental method: The test strain was added to 5 mL 2YT liquid medium with a final concentration of 50 μg / ml kanamycin, and cultured at 37°C, 220 rpm overnight to obtain seed solution; the seed solution was inoculated into 100 mL 2YT liquid medium containing a final concentration of 50 μg / ml kanamycin at a ratio of 1% (V / V), and cultured at 37°C, 220 rpm to obtain culture solution; when the OD value of the culture solution reached 600nm When the pH value was between 0.6 and 0.8, an inducer IPTG with a final concentration of 0.5 mM was added for induction culture, and the induction was carried out at 25°C overnight to obtain a fermentation broth. The fermentation broth was centrifuged at 4°C and 6000 rpm for 10 min, the bacteria were collected, suspended in 50 mM phosphate (dipotassium hydrogen phosphate and potassium dihydrogen phosphate) buffer (pH 7.0), and ultrasonically disrupted (200W, 5s / 7s, 20min) to obtain a crude enzyme solution.

[0066] The crude enzyme solution prepared by the recombinant bacteria pET28a (+) -V583ADC-BL21 (DE3) contains V583ADC protein, and the amino acid sequence of the V583ADC protein is shown in Sequence 1.

[0067] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(H98A)-BL21(DE3) contains V583ADC protein mutant V583ADC(H98A), and the amino acid sequence of V583ADC protein mutant V583ADC(H98A) is the sequence obtained by mutating the histidine at position 98 of sequence 1 to alanine.

[0068] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(H98G)-BL21(DE3) contains V583ADC protein mutant V583ADC(H98G), and 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.

[0069] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99T)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99T), and the amino acid sequence of the V583ADC protein mutant V583ADC(M99T) is the sequence obtained by mutating methionine at position 99 of sequence 1 to threonine.

[0070] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99L)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99L), and the amino acid sequence of V583ADC protein mutant V583ADC(M99L) is the sequence obtained by mutating methionine at position 99 of sequence 1 to leucine.

[0071] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99E)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99E), and the amino acid sequence of V583ADC protein mutant V583ADC(M99E) is the sequence obtained by mutating methionine at position 99 of sequence 1 to glutamic acid.

[0072] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99S)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99S), and 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.

[0073] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99A)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99A), and the amino acid sequence of V583ADC protein mutant V583ADC(M99A) is the sequence obtained by mutating methionine at position 99 of sequence 1 to alanine.

[0074] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99W)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99W), and the amino acid sequence of V583ADC protein mutant V583ADC(M99W) is the sequence obtained by mutating methionine at position 99 of sequence 1 to tryptophan.

[0075] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99R)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99R), and the amino acid sequence of V583ADC protein mutant V583ADC(M99R) is the sequence obtained by mutating methionine at position 99 of sequence 1 to arginine.

[0076] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99H)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99H). The amino acid sequence of the V583ADC protein mutant V583ADC(M99H) is the sequence obtained by mutating methionine at position 99 of sequence 1 to histidine.

[0077] The crude enzyme solution prepared by the recombinant bacteria pET28a(+)-V583ADC(M99Y)-BL21(DE3) contains V583ADC protein mutant V583ADC(M99Y). The amino acid sequence of V583ADC protein mutant V583ADC(M99Y) is the sequence obtained by mutating methionine at position 99 of sequence 1 to tyrosine.

[0078] Example 2: Detection of catalytic activity of V583 ADC and its mutants In this example, L-homoserine was used as a substrate to synthesize 3-amino-1-propanol ( Figure 1 As shown in Figure 2, the catalytic activity of V583ADC and its mutants was detected. The specific steps are as follows: 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.

[0079] 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.

[0080] 3. Heat-treat 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 to collect the supernatant.

[0081] 4. The supernatant obtained in step 3 was derivatized with DNFB (2,4-dinitrofluorobenzene) and then the content of 3-amino-1-propanol was detected by HPLC.

[0082] The DNFB derivatization method is as follows: take 50 μl of sample, 20 μl of 1M sodium bicarbonate, and 80 μl of DNFB derivatization agent, mix well, and place at 60°C for 30 minutes. After 30 minutes, take out and add 50 μl of 1M hydrochloric acid, mix well, centrifuge at 12000 rpm for 5 minutes, take the supernatant and filter it through a 0.22 μM filter membrane, collect the filtrate, and detect the 3-amino-1-propanol content in the filtrate by HPLC. HPLC uses an RP-C18 column, mobile phase: 35% acetonitrile + 65% 0.1% formic acid solution, flow rate is 0.7 ml / min, column temperature is 25°C, injection is 10 μl, and detection wavelength is 360 nm.

[0083] The experiment was repeated three times and the results were averaged.

[0084] 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 is 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 3-amino-1-propanol and conversion rate of V583ADC (M99E) were significantly improved compared with those of V583ADC (Table 2).

[0085] Table 2

[0086] Note: Conversion rate = 3-amino-1-propanol (mM) / L-homoserine (mM)*100%.

[0087] 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A protein obtained by mutating the 99th methionine in the amino acid sequence of tyrosine decarboxylase to glutamic acid.

2. The protein according to claim 1, characterized in that: The tyrosine decarboxylase is a protein described in any one of the following (a1)-(a3): (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (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 of the amino acid sequence shown in (a1); (a3) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the amino acid sequence shown in (a1) or (a2).

3. A nucleic acid molecule encoding the protein according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleic acid molecule is a DNA molecule as described in any one of the following (b1)-(b2): (b1) The nucleotide sequence is the DNA molecule shown in Sequence 3; (b2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in (b1) and encoding the protein of claim 1 or 2.

5. The biological material described in any one of the following (c1) to (c3): (c1) an expression cassette containing the nucleic acid molecule according to claim 3 or 4; (c2) a recombinant vector containing the nucleic acid molecule according to claim 3 or 4; (c3) A recombinant bacterium containing the nucleic acid molecule according to claim 3 or 4.

6. The biomaterial according to claim 5, characterized in that: The recombinant bacteria are obtained by introducing a nucleic acid molecule encoding the protein according to claim 1 or 2 into Escherichia coli.

7. A method for preparing the protein of claim 1 or 2, comprising the following steps: expressing the nucleic acid molecule of claim 3 or 4 in an organism or biological cell to obtain the protein of claim 1 or 2.

8. The method according to claim 7, characterized in that: The method comprises the following steps: fermenting and culturing the recombinant bacteria according to claim 5 or 6 to obtain the protein according to claim 1 or 2.

9. Any application described in (d1) to (d4) below: (d1) Use of the protein according to claim 1 or 2 as tyrosine decarboxylase; (d2) Use of the nucleic acid molecule according to claim 3 or 4 or the biological material according to claim 5 in the preparation of L-aspartate α-decarboxylase; (d3) Use of the protein according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, or the biological material according to claim 5 in the production of 3-amino-1-propanol; (d4) Use of the protein according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, or the biomaterial according to claim 5 for increasing the production of 3-amino-1-propanol.

10. A method for producing 3-amino-1-propanol, comprising the following steps: using the protein according to claim 1 or 2 as a biological enzyme to catalyze L-homoserine to produce 3-amino-1-propanol.

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