Enzyme mutant, enzyme composition or immobilized enzyme thereof, and application and method for preparing L-L-carnitine

Abstract

The invention relates to the field of enzyme engineering, in particular to an enzyme mutant, an enzyme composition or an immobilized enzyme thereof, and application and a method for preparing L-L-carnitine by using the enzyme mutant and the enzyme composition or the immobilized enzyme thereof. The invention provides a simple process for preparing L-carnitine by a whole enzyme method, which is characterized in that bulk betaine (N, N, N-trimethylglycine) and acetic acid are used as raw materials in the whole process. L-carnitine is directly obtained through two-step continuous conversion of carboxyl reductase (CAR) and aldolase (DERA). The route can be completed in one step by utilizing crude enzyme liquid, recyclable immobilized enzyme can also be utilized, and reduced coenzyme II (NADPH) and adenosine triphosphate (ATP) which are used in the first step of reaction can also be recycled by utilizing corresponding enzyme, so that the production cost is further reduced. Therefore, the process for preparing the L-carnitine has the advantages of being completely green, simple in process, low in production cost, easy for large-scale production and the like.

Description

Technical Field

[0001] The invention relates to the field of enzyme engineering, and in particular to an enzyme mutant, an enzyme composition or an immobilized enzyme thereof, and an application and method of preparing L-carnitine. Background Art

[0002] L-carnitine, also known as L-carnitine and vitamin BT, has a chemical formula of C7H15NO3 and a chemical name of (R)-3-carboxyl-2-hydroxy-N,N,N-trimethylpropylammonium hydroxide inner salt. It is an amino acid derivative widely found in human cells. It plays an important role in the process of fat metabolism, can promote the conversion of fat into energy, and has no toxic side effects on the human body. Red meat is the main source of L-carnitine, and the human body can also synthesize it to meet physiological needs. The human body mainly produces L-carnitine in the liver and kidneys, and it is mainly stored in skeletal muscles, heart, brain and semen. Lack of L-carnitine may lead to obstructed fat metabolism, thereby causing obesity. L-carnitine is not a vitamin in the true sense, but a substance similar to vitamins; it has many physiological functions such as fat oxidation and decomposition, weight loss, and anti-fatigue. As a food additive, it is widely used in infant food, weight loss food, athlete food, nutritional supplements for the middle-aged and elderly, nutritional enhancers for vegetarians, and animal feed additives.

[0003] There are various methods for preparing L-carnitine, mainly including chemical synthesis, biosynthesis and direct extraction. Chemical synthesis is one of the most commonly used methods. It usually uses epichlorohydrin or ethyl 4-chloroacetoacetate as the starting material, and finally produces L-carnitine after a series of chemical reactions, such as ring-opening reaction, nucleophilic substitution reaction, hydrolysis reaction, etc. The preparation route is cumbersome, the final yield is low, and the environmental pollution is also large. The biosynthesis method includes microbial fermentation and enzymatic conversion. The biosynthesis method of L-carnitine in organisms is already very clear. N-trimethyllysine is converted into L-carnitine through a four-step enzymatic conversion. Since L-carnitine is relatively abundant in animal muscles, the early method of L-carnitine is to extract these meat products. However, this method still has low yield, many purification steps, and high cost, which is not suitable for large-scale production.

[0004] Therefore, providing a method for preparing L-carnitine by a simple enzymatic method has important practical significance. Summary of the invention

[0005] In view of this, the present invention provides an enzyme mutant, an enzyme composition or an immobilized enzyme thereof and an application and method of preparing L-carnitine. The present invention uses cheap betaine (trimethylglycine, a widely used feed additive, ￥90 / kg, Shaanxi Zelang Biology) and acetic acid on the market as its raw materials, and prepares L-carnitine through a simple two-step enzymatic method, which has significant advantages.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a mutant of an enzyme, wherein the enzyme comprises one or more of betaine reductase (SlCAR), L-carnitine aldolase (BaDERA) and / or NADPH regenerating enzyme (ScFDH);

[0008] The mutation site of the betaine reductase (S1CAR) includes one or more of V183F, Y186T, Q340M, M366A, N505Q, F509T, K646V, L647S, Q658L, N734Q or P1026G;

[0009] The mutation site of the L-carnitine aldolase (BaDERA) includes one or more of Y71S, S72D, D137V, V152A, D225S, T251M, E252T, T289F, K191D or P292I;

[0010] The mutation sites of the NADPH regenerating enzyme (ScFDH) include one or more of S29I, N121Q, D197A, Y198R, G288V or K289T.

[0011] In some specific embodiments of the present invention, in the mutant of the enzyme, the betaine reductase (SlCAR) is derived from Serpula lacrymans (Uniprot ID: F8P2C8);

[0012] The L-carnitine aldolase (BaDERA) is derived from Bordetella ansorpii (Uniprot ID: A0A157S7Y1);

[0013] The NADPH regenerating enzyme (ScFDH) is derived from Saccharomyces cerevisiae (Uniprot ID: Q08911).

[0014] In some specific embodiments of the present invention, the mutant of the enzyme includes a mutant of betaine reductase (SlCAR), a mutant of L-carnitine aldolase (BaDERA) and / or a mutant of NADPH regenerating enzyme (ScFDH);

[0015] Wherein, the mutant of betaine reductase (SlCAR) has:

[0016] (I), the amino acid sequence shown in SEQ ID No.1;

[0017] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0018] (III), an amino acid sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identity with the amino acid sequence described in (I) or (II); or

[0019] The mutant of L-carnitine aldolase (BaDERA) has:

[0020] (I), the amino acid sequence shown in SEQ ID No.2;

[0021] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0022] (III), an amino acid sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identity with the amino acid sequence described in (I) or (II); or

[0023] The mutant of the NADPH regenerating enzyme (ScFDH) has:

[0024] (I), the amino acid sequence shown in SEQ ID No.4;

[0025] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0026] (III) an amino acid sequence that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to the amino acid sequence described in (I) or (II).

[0027] In a second aspect, the present invention also provides an enzyme composition or an immobilized enzyme thereof, including a mutant of the enzyme;

[0028] As a preference, it also includes polyphosphate kinase (TvPPK);

[0029] Preferably, the polyphosphate kinase (TvPPK) is derived from Thermosynechococcus vestitus (Uniprot ID: Q8DI82);

[0030] Preferably, the polyphosphate kinase (TvPPK) has:

[0031] (I), the amino acid sequence shown in SEQ ID No.3;

[0032] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0033] (III), an amino acid sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identity with the amino acid sequence described in (I) or (II); or

[0034] In a third aspect, the present invention also provides a nucleic acid molecule encoding a mutant of the enzyme or the enzyme composition or the immobilized enzyme thereof.

[0035] In some specific embodiments of the present invention, the nucleic acid molecule includes a nucleic acid molecule encoding a mutant of the betaine reductase (SlCAR), a nucleic acid molecule encoding a mutant of the L-carnitine aldolase (BaDERA), a nucleic acid molecule encoding the polyphosphate kinase (TvPPK) and / or a nucleic acid molecule encoding a mutant of the NADPH regenerating enzyme (ScFDH);

[0036] The nucleic acid molecule encoding the mutant of betaine reductase (S1CAR) has:

[0037] (I), the nucleotide sequence shown in SEQ ID NO.5; or

[0038] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0039] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0040] (IV), a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of (I) to (III); or

[0041] The nucleic acid molecule encoding the mutant of L-carnitine aldolase (BaDERA) has:

[0042] (I), the nucleotide sequence shown in SEQ ID NO.6; or

[0043] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0044] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0045] (IV), a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of (I) to (III); or

[0046] The nucleic acid molecule encoding the polyphosphate kinase (TvPPK) has:

[0047] (I), the nucleotide sequence shown in SEQ ID NO.7; or

[0048] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0049] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0050] (IV), a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of (I) to (III); or

[0051] The nucleic acid molecule encoding the mutant of the NADPH regenerating enzyme (ScFDH) has:

[0052] (I), the nucleotide sequence shown in SEQ ID NO.8; or

[0053] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0054] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0055] (IV) A nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of the nucleotide sequences of (I) to (III).

[0056] In a fourth aspect, the present invention further provides an expression vector or a host, wherein the expression vector comprises the nucleic acid molecule;

[0057] The host is transfected or transformed with the expression vector.

[0058] In a fifth aspect, the present invention further provides the use of any of the following items in the preparation of L-carnitine:

[0059] (I), a mutant of the enzyme;

[0060] (II), the enzyme composition or the immobilized enzyme thereof;

[0061] (III), the nucleic acid molecule; or

[0062] (IV), the expression vector or host.

[0063] In a sixth aspect, the present invention also provides a method for preparing L-carnitine, using acetic acid or its salt and betaine as raw materials, and converting any of the following items to obtain L-carnitine:

[0064] (I), a mutant of the enzyme;

[0065] (II), the enzyme composition or the immobilized enzyme thereof;

[0066] (III), the nucleic acid molecule; or

[0067] (IV), the expression vector or host.

[0068] In some specific embodiments of the present invention, the betaine is catalyzed by the mutant of betaine reductase (SlCAR) to generate the corresponding aldehyde, and then condensed with acetic acid by the mutant of L-carnitine aldolase (BaDERA) to obtain L-carnitine;

[0069] The enzyme activity of the mutant of betaine reductase (SlCAR) is 1800-2200 U; or

[0070] The enzyme activity of the mutant of L-carnitine aldolase (BaDERA) is 1500-2400 U; or

[0071] The enzymatic activity of the polyphosphate kinase (TvPPK) is 2800-3400 U; or

[0072] The enzyme activity of the mutant of the NADPH regenerating enzyme (ScFDH) is 800-1200 U; or

[0073] The enzyme activity of the immobilized enzyme is 4000-8000U; among the immobilized enzymes, the mutants of the betaine reductase (SlCAR), the mutants of the L-carnitine aldolase (BaDERA), the polyphosphate kinase (TvPPK) and the mutants of the NADPH regenerating enzyme (ScFDH) are mixed and immobilized according to the activity units (1.8-2.4):(1.5-2.2):(2.4-3.4):(0.8-1.2).

[0074] The present invention provides a simple L-carnitine holoenzyme preparation process, the overall process of which is to use bulk betaine (N, N, N-trimethylglycine) and acetic acid as raw materials. L-carnitine is directly obtained by two-step continuous conversion of carboxyl reductase (CAR) and aldolase (DERA). This route can be completed in one step using crude enzyme solution, or by using recyclable immobilized enzymes. The reduced coenzyme II (NADPH) and adenosine triphosphate (ATP) used in the first step of the reaction can also be recycled using corresponding enzymes to further reduce production costs. Therefore, the patented L-carnitine preparation process has many advantages such as being fully green, simple process, cheap production, and easy large-scale production.

[0075] In some specific embodiments of the present invention, the ratio of the raw material to the enzyme mutant or immobilized enzyme is:

[0076] Betaine: sodium acetate: adenosine triphosphate disodium salt ATP: coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH: SlCAR mutant: BaDERA mutant = 100mM: (250~350mM): (90~120mM): (80~120mM): (1800~2200U): (1500~2400U).

[0077] In some specific embodiments of the present invention, the ratio of the raw material to the enzyme mutant or immobilized enzyme is:

[0078] Betaine: sodium acetate: adenosine triphosphate disodium salt ATP: coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH: SlCAR mutant: BaDERA mutant: TvPPK=100mM: (250~350mM): (5~20mM): (80~120mM): (1800~2200U): (1800~2400U): (2800~3400U).

[0079] In some specific embodiments of the present invention, the ratio of the raw material to the enzyme mutant or immobilized enzyme is:

[0080] Betaine: sodium acetate: adenosine triphosphate disodium salt ATP: coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH: sodium formate: SlCAR mutant: BaDERA mutant: TvPPK: ScFDH = 100mM: (250~350mM): (8~12mM): (3~6mM): (100~150mM): (1800~2200U): (1800~2200U): (2700~3500U): (800~1200U).

[0081] In some specific embodiments of the present invention, the ratio of the raw material to the enzyme mutant or immobilized enzyme is:

[0082] Betaine: sodium acetate: adenosine triphosphate disodium salt ATP: coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH: immobilized enzyme = 100mM: (250-350mM): (8-12mM): (2-6mM): (4000-8000U).

[0083] The invention has a relatively high originality. L-carnitine has a wide range of applications. The method introduced in this patent is different from the current preparation process on the market. It uses two cheap bulk products, betaine and sodium acetate, to effectively convert into L-carnitine. There is no similar preparation method on the market. Therefore, the invention protects the enzyme preparation method and the application of SlCAR enzyme and BaDERA enzyme in the industrial preparation of L-carnitine. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0085] Figure 1 2 shows an SDS-PAGE gel detection diagram of the enzyme prepared by the present invention, wherein lane 1 is SlCAR, lane 2 is ScFDH, lane 3 is BaDERA, and lane 4 is TvPPK;

[0086] Figure 21H-NMR of L-carnitine prepared in Example 10 of the present invention is shown, D2O is used as solvent, and Varian 600 MHz NMR is used. DETAILED DESCRIPTION

[0087] The present invention discloses an enzyme mutant, an enzyme composition or an immobilized enzyme thereof and an application and method for preparing L-carnitine. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0088] The present invention utilizes cheap betaine (trimethylglycine, a widely used feed additive, ￥90 / kg, Shaanxi Zelang Biology) and acetic acid on the market as its raw materials, and prepares L-carnitine through a simple two-step enzymatic method, which has significant advantages.

[0089] Betaine can be converted into the corresponding aldehyde under the catalysis of carboxyl acid reductase (EC 1.2.1.-), and then the aldehyde can be condensed with acetic acid under the action of aldolase (DERA, EC 4.1.2.42) to prepare L-carnitine. The above two enzymes can be greatly improved in their activity, stability and chiral selectivity through directed evolution in the laboratory, thereby achieving a higher conversion rate.

[0090]

[0091] Information about the enzymes used above:

[0092] Table 1 Meanings of special terms and English abbreviations

[0093]

[0094] Betaine reductase (SlCAR): Derived from Serpula lacrymans (Uniprot ID: F8P2C8), its natural enzyme (WTSlCAR) has a certain carboxyl reduction function for non-polar amino acids such as L-alanine and L-glycine. After comprehensive modification of its structure (SlCAR), a relatively high betaine carboxyl reduction function is obtained. The specific mutation sites are: V183F, Y186T, Q340M, M366A, N505Q, F509T, K646V, L647S, Q658L, N734Q, P1026G.

[0095] L-carnitine aldolase (BaDERA): derived from Bordetella ansorpii (UniprotID: A0A157S7Y1), its natural enzyme (WTBaDERA) has a weak L-carnitine decomposition and condensation function. After systematic modification (BaDERA), it has high activity and stability. Its specific mutation sites are: Y71S, S72D, D137V, V152A, D225S, T251M, E252T, T289F, K191D, P292I.

[0096] Polyphosphate kinase (TvPPK): Thermosynechococcus vestitus (Uniprot ID: Q8DI82), this enzyme can naturally use polyphosphate to regenerate AMP and ADP to ATP.

[0097] NADPH regenerating enzyme (ScFDH): Saccharomyces cerevisiae (Uniprot ID: Q08911), the natural enzyme (WTScFDH) can regenerate NADH, but its activity on NADPH is very weak; by mutation (ScFDH), its ability to regenerate NADPH is improved, and its expression is also partially improved. The final mutation sites are: S29I, N121Q, D197A, Y198R, G288V, K289T.

[0098] Table 2

[0099]

[0100]

[0101] Table 3

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] The invention has a relatively high originality. L-carnitine has a wide range of applications. The method introduced in this patent is different from the current preparation process on the market. It uses two cheap bulk products, betaine and sodium acetate, to effectively convert into L-carnitine. There is no similar preparation method on the market. Therefore, the invention protects the enzyme preparation method and the application of SlCAR enzyme and BaDERA enzyme in the industrial preparation of L-carnitine.

[0108] In the enzyme mutant, enzyme composition or immobilized enzyme thereof and the application and method for preparing L-carnitine provided by the present invention, the raw materials and reagents used can be purchased from the market.

[0109] The present invention will be further described below in conjunction with embodiments:

[0110] Preparation Example 1 Fermentation production of enzyme:

[0111] The enzymes used in this patent are all produced by laboratory fermentation. The following is the basic operation process for preparing the enzyme. First, the gene sequence corresponding to the enzyme is synthesized by a gene company (Anhui General Biotechnology), and then subcloned into the pET28a plasmid through the NdeI / XhoI restriction site, and the plasmid is transferred into E. coli (BL21) (Qingke Biology) cells for plate culture, and finally a single clone is selected for liquid step-by-step amplification culture. The following is the basic process of step-by-step cell amplification culture. First, the single colony on the plate is transferred into 5ml LB culture medium containing 50μM kanamycin (37℃) for culture. When the cells grow to the logarithmic phase, they are inoculated into 250ml LB culture medium containing the same antibiotics, and finally transferred to a 5L culture fermenter for culture; when the cell OD is ~ 30, 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added at 25℃ to induce protein expression for 8 hours, and then centrifuged (4000rpm, 15min) to collect 30-45g of wet cells. To verify the expression of the enzyme, first take a small amount of cells and mix them evenly with Tris.HCl buffer (50mM, pH 8.0), then use the freeze-thaw method to break the cells, take the supernatant after high-speed centrifugation and run SDS-PAGE protein gel (sodium dodecyl sulfate-polyacrylamide gel) to determine the soluble expression of the protein; confirm that the remaining cells are correct and mix them with buffer (10 grams of wet cells are mixed with about 200ml of marketed buffer), then high-pressure cell breaking and high-speed centrifugation (16000rpm, 45min) are performed to remove the cell wall, and the enzyme-containing clear liquid obtained is directly used in the subsequent use (the liquid enzyme activity is 400-560U / ml, U is the amount of enzyme required to convert 1μmol of substrate in one minute at room temperature) or further purified and immobilized for use (for solid enzyme reaction). LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

[0112] Preparation Example 2 Mixed immobilization of enzymes:

[0113] Ammonium sulfate solid was gradually added to the crude enzyme solution of betaine reductase (SlCAR), L-carnitine aldolase (BaDERA), polyphosphate kinase (TvPPK), and NADPH regeneration enzyme (ScFDH) collected above until the enzyme precipitated (40%-60%, w / v ammonium sulfate / buffer). The enzyme solid was then collected by centrifugation (10000rpm, 12min) and slowly dissolved in 25mM pH 8.0 Tris buffer, and finally desalted by G25 size exclusion chromatography column (purchased from Sigma) and separated by DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain the initially purified liquid enzyme SlCAR, BaDERA, TvPPK, ScFDH, and the enzyme solution can be directly used for subsequent enzyme fixation. In the mixed fixation of SlCAR / BaDERA / TvPPK / ScFDH enzyme, the initially purified enzyme was mixed and fixed using LX-1000EP epoxy resin (Xi'an Lanxiao Company) according to the activity unit 2:2:3:1. The basic method of fixation is: 6000U mixed enzyme mixed according to the above activity unit ratio is dissolved in 2L 50mM pH 8.0 potassium phosphate solution, then 40mM phenoxyacetic acid and 600g LX-1000EP epoxy resin are added to the buffer solution, and the immobilized enzyme is filtered out after stirring at room temperature for 6 hours, and finally washed three times with clean water and 25mM pH 8.0 phosphate buffer solution, and then dried at low temperature for use; SlCAR / BaDERA / TvPPK / ScFDH immobilized mixed enzyme has 70-85% of the activity of the corresponding liquid enzyme.

[0114] Table 4: Summary of enzyme properties covered by this patent

[0115]

[0116] Example 1 Using betaine and sodium acetate as raw materials, two liquid enzymes (SICAR and BaDERA) were used to prepare L-carnitine at one time

[0117]

[0118] 11.8 g of betaine (100 mM) was added to 1 L 100 mM pH 8.0 Tris.HCl solution, followed by 24.6 g of sodium acetate (300 mM), 60.8 g of adenosine triphosphate disodium salt ATP (110 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), and 76.6 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 100 mM), and then the pH value of the reaction solution was adjusted back to 8.0, and finally 2000 U of SlCAR crude enzyme solution and 2000 U of BaDERA crude enzyme solution were added at once to start the reaction; the reaction was stirred lightly at 37 ° C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. After 6 hours, the reaction was completely adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the reaction, and then adjusted to pH 7.0, D201 anion exchange resin was used to remove phosphoric acid impurities, and then D101 non-polar resin was used to purify and collect the crude product, and finally a reverse osmosis membrane was used to desalinate, concentrate, and crystallize (ethanol:H2O=4:1, V:V) to obtain 9.9 g of white solid (final yield 61%).

[0119] Example 2 Using betaine and sodium acetate as raw materials, two liquid enzymes (SICAR and BaDERA) were used to prepare L-carnitine at one time

[0120]

[0121] 11.8 g of betaine (100 mM) was added to 1 L 100 mM pH 8.0 Tris.HCl solution, followed by 20.5 g of sodium acetate (250 mM), 49.7 g of adenosine triphosphate disodium salt ATP (90 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), and 61.3 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 80 mM), and then the pH value of the reaction solution was adjusted back to 8.0, and finally 1800 U of SlCAR crude enzyme solution and 1500 U of BaDERA crude enzyme solution were added at once to start the reaction; the reaction was stirred lightly at 37 ° C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. After 6 hours, the reaction was completely adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the reaction, and then adjusted to pH 7.0, D201 anion exchange resin was used to remove phosphoric acid impurities, and then D101 non-polar resin was used to purify and collect the crude product, and finally a reverse osmosis membrane was used to desalinate, concentrate, and crystallize (ethanol:H2O=4:1, V:V) to obtain 9.1 g of white solid (final yield 56%).

[0122] Example 3 Using betaine and sodium acetate as raw materials, two liquid enzymes (SICAR and BaDERA) were used to prepare L-carnitine at one time

[0123]

[0124] 11.8 g of betaine (100 mM) was added to 1 L 100 mM pH 8.0 Tris.HCl solution, followed by 28.7 g of sodium acetate (350 mM), 66.3 g of adenosine triphosphate disodium salt ATP (120 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), and 92.0 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 120 mM), and then the pH value of the reaction solution was adjusted back to 8.0, and finally 2200 U of SlCAR crude enzyme solution and 2400 U of BaDERA crude enzyme solution were added at once to start the reaction; the reaction was stirred lightly at 37 ° C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. After 6 hours, the reaction was completely adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the reaction, and then adjusted to pH 7.0, D201 anion exchange resin was used to remove phosphoric acid impurities, and then D101 non-polar resin was used to purify and collect the crude product, and finally a reverse osmosis membrane was used to desalinate, concentrate, and crystallize (ethanol: H2O = 4:1, V:V) to obtain 11.4 g of white solid (final yield 70%).

[0125] Example 4 Using betaine and sodium acetate as raw materials, three liquid enzymes (SICAR, BaDERA, TvPPK) were used to prepare L-carnitine at one time

[0126]

[0127] Similar to Example 1, but TvPPK enzyme is added to the reaction solution to regenerate ATP.

[0128] In 1L 100mM pH 8.0 Tris.HCl solution, add 11.8g betaine (100mM), then add 24.6g sodium acetate (300mM), 5.5g adenosine triphosphate disodium salt ATP (10mM), 40.7g sodium hexaphosphate (66.7mM), 4.2g magnesium chloride hexahydrate (20mM), 76.6g coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 100mM), then adjust the solution pH back to 8.0, and finally add 2000U SlCAR crude enzyme solution, 2000U BaDERA crude enzyme solution, 3000U The reaction was started with TvPPK crude enzyme solution; the reaction was lightly stirred at 37°C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. After 4 hours, the reaction was complete and the reaction solution was adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the reaction, and then the pH was adjusted to 7.0 and D201 anion exchange resin was used to remove phosphoric acid impurities. Thereafter, the crude product was purified and collected using D101 non-polar resin, and finally desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=4:1, V:V) to obtain 11.2 grams of white solid (final yield 69%).

[0129] Example 5 Using betaine and sodium acetate as raw materials, three liquid enzymes (SICAR, BaDERA, TvPPK) were used to prepare L-carnitine at one time

[0130]

[0131] Similar to Example 2, but TvPPK enzyme was added to the reaction solution to recycle ATP. 11.8 g of betaine (100 mM) was added to 1L 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 20.5 g of sodium acetate (250 mM), 2.8 g of adenosine triphosphate disodium salt ATP (5 mM), 40.7 g of sodium hexaphosphate (66.7 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), and 61.3 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 80 mM), and then the pH value of the solution was adjusted back to 8.0, and finally 1800 U of SlCAR crude enzyme solution, 1800 U of BaDERA crude enzyme solution, and 2800 U of TvPPK crude enzyme solution were added at once to start the reaction; the reaction was stirred lightly at 37°C, and the reaction pH was maintained during the entire reaction. The pH of the reaction mixture was adjusted to about 7.0-8.5. After 4 hours, the reaction was complete. The reaction mixture was adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the reaction. The pH was then adjusted to 7.0 and phosphoric acid-containing impurities were removed using D201 anion exchange resin. The crude product was then purified and collected using D101 non-polar resin. Finally, the product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: H2O = 4:1, V:V) to obtain 10.4 g of a white solid (final yield 64%).

[0132] Example 6 Using betaine and sodium acetate as raw materials, three liquid enzymes (SICAR, BaDERA, TvPPK) were used to prepare L-carnitine at one time

[0133]

[0134] Similar to Example 3, but TvPPK enzyme was added to the reaction solution to regenerate ATP. 11.8 g of betaine (100 mM) was added to 1 L 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 20.5-28.7 g of sodium acetate (350 mM), 2.8-11 g of adenosine triphosphate disodium salt ATP (20 mM), 40.7 g of sodium metabisphosphate (66.7 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), 91.9 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 120 mM), and then the pH value of the solution was adjusted back to 8.0, and finally 2200 U SlCAR crude enzyme solution, 2200 U BaDERA crude enzyme solution, and 3400 U The reaction was started with TvPPK crude enzyme solution; the reaction was lightly stirred at 37°C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. After 4 hours, the reaction was complete and the reaction solution was adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the reaction, and then the pH was adjusted to 7.0 and D201 anion exchange resin was used to remove phosphoric acid impurities. Thereafter, the crude product was purified and collected using D101 non-polar resin, and finally desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=4:1, V:V) to obtain 11.8 grams of white solid (final yield 73%).

[0135] Example 7 Using betaine and sodium acetate as raw materials, four liquid enzymes (SICAR, BaDERA, TvPPK, ScFDH) were used to prepare L-carnitine at one time

[0136] Similar to Example 4, but TvPPK enzyme and ScCAR enzyme were added to the reaction solution to recycle ATP and NADPH.

[0137] In 1L 100mM pH 8.0 Tris.HCl solution, add 11.8g betaine (100mM), then add 24.6g sodium acetate (300mM), 5.5g adenosine triphosphate disodium salt ATP (10mM), 40.7g sodium hexaphosphate (66.7mM), 4.2g magnesium chloride hexahydrate (20mM), 3.9g coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 5mM) and 9.8g sodium formate (120mM), then adjust the solution pH back to 8.0, and finally add 2000U SlCAR crude enzyme solution, 2000U BaDERA crude enzyme solution, 3000U TvPPK crude enzyme solution and 1000U ScFDH started the reaction; the reaction was gently stirred at 37°C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. After 3 hours, the reaction was complete and the reaction solution was adjusted to acidity with dilute hydrochloric acid aqueous solution to inactivate the reaction, and then adjusted to pH 7.0 and used D201 anion exchange resin to remove phosphoric acid impurities, and then used D101 non-polar resin to purify and collect the crude product, and finally used a reverse osmosis membrane to desalinate, concentrate, and crystallize (ethanol:H2O=4:1, V:V) to obtain 13.6 grams of white solid (final yield 84%).

[0138] Example 8 Using betaine and sodium acetate as raw materials, four liquid enzymes (SICAR, BaDERA, TvPPK, ScFDH) were used to prepare L-carnitine at one time

[0139] Similar to Example 5, but TvPPK enzyme and ScCAR enzyme were added to the reaction solution to recycle ATP and NADPH.

[0140] 11.8 g betaine (100 mM) was added to 1 L 100 mM pH 8.0 Tris.HCl solution, followed by 20.5 g sodium acetate (250 mM), 4.4 g adenosine triphosphate disodium salt ATP (8 mM), 40.7 g sodium hexaphosphate (66.7 mM), 4.2 g magnesium chloride hexahydrate (20 mM), 2.4 g coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 3 mM) and 8.2 g sodium formate (100 mM), then the pH value of the solution was adjusted back to 8.0, and finally 1800 U SlCAR crude enzyme solution, 1800 U BaDERA crude enzyme solution, 2700 U TvPPK crude enzyme solution and 800 U ScFDH were added at once to start the reaction; the reaction was stirred lightly at 37 °C, and the reaction pH was maintained during the entire reaction. The pH of the reaction mixture was adjusted to about 7.0-8.5. After 3 hours, the reaction was complete. The reaction mixture was adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the reaction. The pH was then adjusted to 7.0 and phosphoric acid-containing impurities were removed using D201 anion exchange resin. The crude product was then purified and collected using D101 non-polar resin. Finally, the product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: H2O = 4:1, V:V) to obtain 13.0 g of a white solid (final yield 80%).

[0141] Example 9 Using betaine and sodium acetate as raw materials, four liquid enzymes (SICAR, BaDERA, TvPPK, ScFDH) were used to prepare L-carnitine at one time

[0142] Similar to Example 6, but TvPPK enzyme and ScCAR enzyme were added to the reaction solution to recycle ATP and NADPH.

[0143] 11.8 g betaine (100 mM) was added to 1 L 100 mM pH 8.0 Tris.HCl solution, followed by 28.7 g sodium acetate (350 mM), 6.6 g adenosine triphosphate disodium salt ATP (12 mM), 40.7 g sodium hexaphosphate (66.7 mM), 4.2 g magnesium chloride hexahydrate (20 mM), 24.7 g coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 6 mM) and 12.3 g sodium formate (150 mM), then the pH value of the solution was adjusted back to 8.0, and finally 2200 U SlCAR crude enzyme solution, 2200 U BaDERA crude enzyme solution, 3500 U TvPPK crude enzyme solution and 1200 U ScFDH were added at once to start the reaction; the reaction was stirred lightly at 37 °C, and the reaction pH was maintained during the entire reaction. The pH of the reaction mixture was adjusted to about 7.0-8.5. After 3 hours, the reaction was complete and the reaction solution was adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the reaction. The pH was then adjusted to 7.0 and phosphoric acid-containing impurities were removed using D201 anion exchange resin. The crude product was then purified and collected using D101 non-polar resin. Finally, the product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: H2O = 4:1, V:V) to obtain 14.2 g of a white solid (final yield 88%).

[0144] Example 10: Using betaine and sodium acetate as raw materials, immobilized mixed enzymes were used to prepare L-carnitine at one time

[0145]

[0146] The reaction is similar to the above embodiment, but the immobilized enzyme can be recovered and reused later.

[0147] 11.8 g of betaine (100 mM) was added to 1 L 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 24.6 g of sodium acetate (300 mM), 5.5 g of adenosine triphosphate disodium salt ATP (10 mM), 40.7 g of sodium hexaphosphate (66.7 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), 3.9 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 5 mM) and 9.8 g of sodium formate (120 mM), then the pH value of the solution was adjusted back to 8.0, and 6000 U of immobilized mixed enzyme was added at one time to start the reaction; the reaction was stirred lightly at 37°C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. The reaction was complete after 6 hours, and then the immobilized mixed enzyme was collected by filtration (the mixed enzyme still retained 70% of its initial activity after being used 8 times). Similar to the above treatment, the reaction supernatant was treated with D201 anion exchange resin to remove phosphoric acid impurities, and then purified with D101 non-polar resin to collect the crude product. Finally, it was desalted with a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=4:1, V:V) to obtain 14.7 g of white solid (final yield 91%).

[0148] Example 11: Using betaine and sodium acetate as raw materials, immobilized mixed enzymes were used to prepare L-carnitine at one time

[0149]

[0150] The reaction is similar to the above embodiment, but the immobilized enzyme can be recovered and reused later.

[0151] 11.8 g of betaine (100 mM) was added to 1 L 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 20.5 g of sodium acetate (250 mM), 4.4 g of adenosine triphosphate disodium salt ATP (8 mM), 40.7 g of sodium hexaphosphate (66.7 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), 1.6 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 2 mM) and 9.8 g of sodium formate (120 mM), then the pH value of the solution was adjusted back to 8.0, and finally 4000 U of immobilized mixed enzyme was added at once to start the reaction; the reaction was stirred lightly at 37°C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. The reaction was complete after 6 hours, and then the immobilized mixed enzyme was collected by filtration (the mixed enzyme still retained 70% of its initial activity after being used 8 times). Similar to the above treatment, the reaction supernatant was treated with D201 anion exchange resin to remove phosphoric acid impurities, and then purified with D101 non-polar resin to collect the crude product. Finally, it was desalted with a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=4:1, V:V) to obtain 14.2 g of white solid (final yield 88%).

[0152] Example 12: Using betaine and sodium acetate as raw materials, immobilized mixed enzymes were used to prepare L-carnitine at one time

[0153]

[0154] The reaction is similar to the above embodiment, but the immobilized enzyme can be recovered and reused later.

[0155] 11.8 g of betaine (100 mM) was added to 1 L 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 28.7 g of sodium acetate (350 mM), 6.6 g of adenosine triphosphate disodium salt ATP (12 mM), 40.7 g of sodium hexaphosphate (66.7 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), 4.7 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 6 mM) and 9.8 g of sodium formate (120 mM), then the pH value of the solution was adjusted back to 8.0, and finally 8000 U of immobilized mixed enzyme was added at once to start the reaction; the reaction was stirred lightly at 37°C, and the reaction pH was maintained at about 7.0-8.5 during the entire reaction process. The reaction was complete after 6 hours, and then the immobilized mixed enzyme was collected by filtration (the mixed enzyme still retained 70% of its initial activity after being used 8 times). Similar to the above treatment, the reaction supernatant was treated with D201 anion exchange resin to remove phosphoric acid impurities, and then purified with D101 non-polar resin to collect the crude product. Finally, it was desalted with a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=4:1, V:V) to obtain 15.2 g of white solid (final yield 94%).

[0156] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An enzyme mutant, characterized in that The enzyme comprises one or more of betaine reductase (SlCAR), L-carnitine aldolase (BaDERA) and / or NADPH regenerating enzyme (ScFDH); The mutation site of the betaine reductase (S1CAR) includes one or more of V183F, Y186T, Q340M, M366A, N505Q, F509T, K646V, L647S, Q658L, N734Q or P1026G; The mutation site of the L-carnitine aldolase (BaDERA) includes one or more of Y71S, S72D, D137V, V152A, D225S, T251M, E252T, T289F, K191D or P292I; The mutation sites of the NADPH regenerating enzyme (ScFDH) include one or more of S29I, N121Q, D197A, Y198R, G288V or K289T.

2. The enzyme mutant according to claim 1, characterized in that The betaine reductase (SlCAR) is derived from Serpula lacrymans (Uniprot ID: F8P2C8); The L-carnitine aldolase (BaDERA) is derived from Bordetella ansorpii (UniprotID: A0A157S7Y1); The NADPH regenerating enzyme (ScFDH) is derived from Saccharomyces cerevisiae (Uniprot ID: Q08911).

3. The mutant of the enzyme according to claim 1 or 2, characterized in that The mutants of the enzyme include mutants of betaine reductase (SlCAR), mutants of L-carnitine aldolase (BaDERA) and / or mutants of NADPH regenerating enzyme (ScFDH); Wherein, the mutant of betaine reductase (SlCAR) has: (I), the amino acid sequence shown in SEQ ID No.1; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III), an amino acid sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identity with the amino acid sequence described in (I) or (II); or The mutant of L-carnitine aldolase (BaDERA) has: (I), the amino acid sequence shown in SEQ ID No.2; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III), an amino acid sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identity with the amino acid sequence described in (I) or (II); or The mutant of the NADPH regenerating enzyme (ScFDH) has: (I), the amino acid sequence shown in SEQ ID No.4; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III) an amino acid sequence that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to the amino acid sequence described in (I) or (II).

4. An enzyme composition or an immobilized enzyme thereof, characterized in that: A mutant comprising the enzyme according to any one of claims 1 to 3; As a preference, it also includes polyphosphate kinase (TvPPK); Preferably, the polyphosphate kinase (TvPPK) is derived from Thermosynechococcus vestitus (Uniprot ID: Q8DI82); Preferably, the polyphosphate kinase (TvPPK) has: (I), the amino acid sequence shown in SEQ ID No.3; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III) an amino acid sequence that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to the amino acid sequence described in (I) or (II).

5. A nucleic acid molecule encoding a mutant of the enzyme according to any one of claims 1 to 3 or an enzyme composition or an immobilized enzyme thereof according to claim 4.

6. The nucleic acid molecule according to claim 5, characterized in that A nucleic acid molecule comprising a mutant of the betaine reductase (SlCAR), a mutant of the L-carnitine aldolase (BaDERA), a nucleic acid molecule encoding the polyphosphate kinase (TvPPK) and / or a mutant of the NADPH regenerating enzyme (ScFDH); The nucleic acid molecule encoding the mutant of betaine reductase (S1CAR) has: (I), the nucleotide sequence shown in SEQ ID NO.5; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV), a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of (I) to (III); or The nucleic acid molecule encoding the mutant of L-carnitine aldolase (BaDERA) has: (I), the nucleotide sequence shown in SEQ ID NO.6; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV), a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of (I) to (III); or The nucleic acid molecule encoding the polyphosphate kinase (TvPPK) has: (I), the nucleotide sequence shown in SEQ ID NO.7; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV), a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of (I) to (III); or The nucleic acid molecule encoding the mutant of the NADPH regenerating enzyme (ScFDH) has: (I), the nucleotide sequence shown in SEQ ID NO.8; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) A nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of the nucleotide sequences of (I) to (III).

7. An expression vector or host, characterized in that The expression vector comprises the nucleic acid molecule according to claim 5 or 6; The host is transfected or transformed with the expression vector.

8. Use of any of the following in the preparation of L-carnitine; (I) A mutant of the enzyme according to any one of claims 1 to 3; (II), the enzyme composition or the immobilized enzyme thereof as claimed in claim 4; (III), a nucleic acid molecule according to claim 5 or 6; or (IV) The expression vector or host according to claim 7.

9. A method for preparing L-carnitine, characterized in that: Using acetic acid or its salt and betaine as raw materials, L-carnitine is obtained by any of the following conversions: (I) A mutant of the enzyme according to any one of claims 1 to 3; (II), the enzyme composition or the immobilized enzyme thereof as claimed in claim 4; (III), a nucleic acid molecule according to claim 5 or 6; or (IV) The expression vector or host according to claim 7.

10. The preparation method according to claim 9, characterized in that: The betaine is catalyzed by the mutant of betaine reductase (SlCAR) to generate the corresponding aldehyde, and then the mutant of L-carnitine aldolase (BaDERA) is condensed with acetic acid to obtain L-carnitine; The enzyme activity of the mutant of betaine reductase (SlCAR) is 1800-2200 U; or The enzyme activity of the mutant of L-carnitine aldolase (BaDERA) is 1500-2400 U; or The enzymatic activity of the polyphosphate kinase (TvPPK) is 2800-3400 U; or The enzyme activity of the mutant of the NADPH regenerating enzyme (ScFDH) is 800-1200 U; or The enzyme activity of the immobilized enzyme is 4000-8000U; among the immobilized enzymes, the mutants of the betaine reductase (SlCAR), the mutants of the L-carnitine aldolase (BaDERA), the polyphosphate kinase (TvPPK) and the mutants of the NADPH regenerating enzyme (ScFDH) are mixed and immobilized according to the activity units (1.8-2.4):(1.5-2.2):(2.4-3.4):(0.8-1.2).

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