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

Abstract

The present invention relates to the field of enzyme engineering, and more particularly to enzyme mutants, enzyme compositions or their immobilized enzymes and their applications and methods for preparing L-carnitine. The present invention provides a simple L-carnitine holoenzyme preparation process, the overall process of which utilizes bulk betaine (N,N,N-trimethylglycine) and acetic acid as raw materials. L-carnitine is directly obtained by two-step continuous conversion by carboxyl reductase (CAR) and aldolase (DERA). This route can be completed in one step using a crude enzyme solution, or by utilizing a recyclable immobilized enzyme, and 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 in process, inexpensive to produce, and easy to scale up.

Description

Technical Field

[0001] The present 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 for preparing L-carnitine. Background Art

[0002] L-carnitine, also known as L-carnitine or vitamin BT, has the chemical formula C7H15NO3 and is chemically known as (R)-3-carboxy-2-hydroxy-N,N,N-trimethylpropylammonium hydroxide inner salt. It is an amino acid derivative widely present in human cells. It plays a vital role in fat metabolism, facilitating the conversion of fat into energy, and is non-toxic to the body. Red meat is the primary source of L-carnitine, and the human body can also synthesize it to meet physiological needs. The body primarily produces L-carnitine in the liver and kidneys, where it is primarily stored in skeletal muscle, heart, brain, and semen. L-carnitine deficiency can hinder fat metabolism, leading to obesity. L-carnitine is not a true vitamin, but rather a vitamin-like substance. It has multiple physiological functions, including fat oxidation and decomposition, weight loss, and fatigue relief. As a food additive, it is widely used in infant and toddler foods, diet foods, athlete foods, nutritional supplements for the elderly, a nutrient fortifier for vegetarians, and an animal feed additive.

[0003] L-carnitine is produced by a variety of methods, including chemical synthesis, biosynthesis, and direct extraction. Chemical synthesis is the most commonly used method, typically starting with epichlorohydrin or ethyl 4-chloroacetoacetate. Through a series of chemical reactions, such as ring-opening, nucleophilic substitution, and hydrolysis, L-carnitine is ultimately produced. This production route is cumbersome, yields low, and poses significant environmental risks. Biosynthesis, which involves microbial fermentation and enzymatic conversion, is well established. L-carnitine is obtained by converting N-trimethyllysine through a four-step enzymatic process. Because L-carnitine is relatively abundant in animal muscle, early methods for producing L-carnitine involved extraction from these meat products. However, this method still suffers from low yields, multiple purification steps, and high costs, making it unsuitable 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 light of this, the present invention provides enzyme mutants, enzyme compositions, or immobilized enzymes thereof, and their applications and methods for preparing L-carnitine. This method utilizes commercially available, inexpensive betaine (trimethylglycine, a widely used feed additive, ¥90 / kg, available from Shaanxi Zelang Biological) and acetic acid as raw materials to produce L-carnitine via a simple, two-step enzymatic process, offering 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 comprising one or more of betaine reductase (SlCAR), L-carnitine aldolase (BaDERA) and / or NADPH regenerating enzyme (ScFDH);

[0008] The mutation sites of the betaine reductase (S1CAR) include 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 (UniprotID: Q08911).

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

[0015] Wherein, the mutant of betaine reductase (S1CAR) 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 that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of (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 that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of (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 that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of (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 differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0039] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions 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 the nucleotide sequence of 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 differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0044] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions 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 the nucleotide sequence of 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 differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0049] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions 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 the nucleotide sequence of 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 differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0054] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions 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 the nucleotide sequence of any one of (I) to (III).

[0056] In a fourth aspect, the present invention further provides an expression vector or 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 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, which is then condensed with acetic acid by the mutant of L-carnitine aldolase (BaDERA) to produce 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 NADPH regenerating enzyme (ScFDH) mutant has an enzymatic activity of 800 to 1200 U; or

[0073] The enzymatic activity of the immobilized enzyme is 4000-8000 U; among the immobilized enzymes, the activity units of the mutant of the betaine reductase (SlCAR), the mutant of the L-carnitine aldolase (BaDERA), the polyphosphate kinase (TvPPK) and the mutant of the NADPH regenerating enzyme (ScFDH) are mixed and immobilized according to (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 holoenzymatic preparation process, the overall process of which utilizes bulk betaine (N,N,N-trimethylglycine) and acetic acid as raw materials. L-carnitine is directly obtained through two-step continuous conversion by carboxyl reductase (CAR) and aldolase (DERA). This route can be completed in one step using a crude enzyme solution, or it can utilize 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 in process, inexpensive to produce, and easy to scale up.

[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] This invention is highly original. L-carnitine has a wide range of applications. The method described in this patent differs from existing commercial preparation processes by effectively converting two inexpensive, bulk products, betaine and sodium acetate, into L-carnitine. There are no similar preparation methods on the market. Therefore, this invention protects the enzyme preparation method and the use of SlCAR and BaDERA enzymes in the industrial production 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 following briefly introduces the drawings required for describing the embodiments or the prior art.

[0085] Figure 1 1 is an SDS-PAGE gel assay of the enzymes prepared in 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 enzyme mutants, enzyme compositions or their immobilized enzymes and their applications and methods for preparing L-carnitine. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It is particularly important to note that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0088] The present invention utilizes commercially inexpensive betaine (trimethylglycine, a widely used feed additive, ￥90 / kg, Shaanxi Zelang Biological) and acetic acid as its raw materials to prepare 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.-). The aldehyde is then condensed with acetic acid under the action of aldolase (DERA, EC 4.1.2.42) to produce L-carnitine. Through directed evolution of the above two enzymes, their activity, stability and chiral selectivity can be greatly improved, thereby achieving higher conversion rates.

[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) obtains a relatively high betaine carboxyl reduction function. Its 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 weak L-carnitine decomposition and condensation function. After systematic modification, (BaDERA) 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 (UniprotID: Q8DI82), this enzyme can naturally use polyphosphate to regenerate AMP and ADP to ATP.

[0097] NADPH regeneration enzyme (ScFDH): Saccharomyces cerevisiae (Uniprot ID: Q08911), the natural enzyme (WTScFDH) can regenerate NADH, but its activity towards NADPH is very weak; by mutation (ScFDH), its ability to regenerate NADPH was improved, and its expression was 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] This invention is highly original. L-carnitine has a wide range of applications. The method described in this patent differs from existing commercial preparation processes by effectively converting two inexpensive, bulk products, betaine and sodium acetate, into L-carnitine. There are no similar preparation methods on the market. Therefore, this invention protects the enzyme preparation method and the use of SlCAR and BaDERA enzymes in the industrial production 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 the embodiments:

[0110] Preparation Example 1 Fermentation production of enzyme:

[0111] The enzymes used in this patent are all produced by in-house fermentation in the laboratory. The following is the basic process for preparing the enzyme. First, the gene sequence corresponding to the enzyme is synthesized by a gene company (Anhui General Biotechnology). Then, it is subcloned into the pET28a plasmid using the NdeI / XhoI restriction sites. The plasmid is then transformed into E. coli (BL21) (Qingke Biotechnology) cells for plate culture. Finally, a single clone is selected for liquid amplification culture. The following is the basic process for cell amplification culture: First, a single colony on the plate is transferred to 5 ml of LB culture medium containing 50 μM kanamycin (37°C) for culture. When the cells reach the logarithmic phase, they are inoculated into 250 ml of LB culture medium containing the same antibiotic and finally transferred to a 5L culture fermenter for culture. When the cell OD reaches ~30, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added at 25°C to induce protein expression for 8 hours. Then, 30-45 g of wet cells are collected by centrifugation (4000 rpm, 15 minutes). To verify enzyme expression, a small amount of cells was first mixed with 50 mM Tris-HCl buffer (pH 8.0). Cells were then disrupted by freeze-thaw, centrifuged at high speed, and the supernatant was run on an SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel) to confirm soluble protein expression. The remaining cells, confirmed to be correct, were then mixed with buffer (10 g of wet cells in approximately 200 ml of commercially available buffer), followed by high-pressure cell disruption and high-speed centrifugation (16,000 rpm, 45 min) to remove cell walls. The resulting enzyme-containing supernatant was either used directly (the liquid enzyme activity ranged from 400 to 560 U / ml, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature) or further purified and immobilized (for solid enzyme reactions). LB medium consisted of 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate, and 5% glycerol.

[0112] Preparation Example 2 Mixed Immobilization of Enzymes:

[0113] To the crude enzyme solutions of betaine reductase (SlCAR), L-carnitine aldolase (BaDERA), polyphosphate kinase (TvPPK), and NADPH regenerating enzyme (ScFDH) collected above, solid ammonium sulfate was gradually added until the enzymes precipitated (40%-60%, w / v ammonium sulfate / buffer). The enzyme solids were then collected by centrifugation (10,000 rpm, 12 min) and slowly dissolved in 25 mM Tris buffer (pH 8.0). Finally, the enzymes were desalted using a G25 size exclusion chromatography column (purchased from Sigma) and separated using a DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain pre-purified liquid enzymes of SlCAR, BaDERA, TvPPK, and ScFDH. This enzyme solution was directly used for subsequent enzyme immobilization. For the mixed immobilization of SlCAR / BaDERA / TvPPK / ScFDH, the pre-purified enzymes were immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) in a ratio of 2:2:3:1 activity units. The basic immobilization method is as follows: 6000 U of the enzyme mixture, mixed according to the above activity unit ratio, is dissolved in 2 L of 50 mM potassium phosphate solution, pH 8.0. 40 mM phenoxyacetic acid and 600 g of LX-1000EP epoxy resin are then added to the buffer. After stirring at room temperature for 6 hours, the immobilized enzyme is filtered out and washed three times with water and three times with 25 mM phosphate buffer, pH 8.0, before being dried at low temperature for use. The immobilized SlCAR / BaDERA / TvPPK / ScFDH enzyme mixture exhibits 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 of 100 mM Tris-HCl (pH 8.0), 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). The pH of the reaction solution was then 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 initiate the reaction. The reaction was stirred gently at 37°C, and the pH was maintained at approximately 7.0-8.5 throughout the reaction. After 6 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 the pH was adjusted to 8.0. After 7.0, phosphoric acid-containing impurities were removed using D201 anion exchange resin, and the crude product was then purified 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 9.9 g of a 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 of 100 mM Tris-HCl solution (pH 8.0), 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). The pH of the reaction solution was then 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 initiate the reaction. The reaction was stirred gently at 37°C, and the pH was maintained at around 7.0-8.5 throughout the reaction. After 6 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 8. After 7.0, phosphoric acid-containing impurities were removed using D201 anion exchange resin, and the crude product was then purified 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 9.1 g of a 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 of 100 mM Tris-HCl solution (pH 8.0), 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). The pH of the reaction solution was then 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 initiate the reaction. The reaction was stirred gently at 37°C, and the pH was maintained at around 7.0-8.5 throughout the reaction. After 6 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 the pH was adjusted to 8. After 7.0, phosphoric acid-containing impurities were removed using D201 anion exchange resin, and the crude product was then purified 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 11.4 g of a 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 was added to the reaction solution to recycle ATP.

[0128] 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), and 76.6 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 100 mM). The pH value of the solution was then adjusted back to 8.0, and finally 2000 U of SlCAR crude enzyme solution, 2000 U of BaDERA crude enzyme solution, and 3000 U of Gluconolactone were added at once. The reaction was initiated with crude TvPPK enzyme solution; the reaction was gently stirred at 37°C, and the pH was maintained at approximately 7.0-8.5 throughout the reaction. After 4 hours, the reaction was complete, and the reaction solution was adjusted to acidity with dilute aqueous hydrochloric acid to inactivate the enzyme. 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 using D101 non-polar resin, collected, and finally desalinated using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O = 4:1, v:v) to obtain 11.2 g of a 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 1 L of 100 mM pH 8.0 tris(hydroxymethylaminomethane) hydrochloric acid (Tris.HCl) solution, followed by 20.5 g of sodium acetate (250 mM), 2.8 g of adenosine triphosphate disodium salt (5 mM), 40.7 g of sodium metabisphosphate (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). The pH of the solution was then 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 initiate the reaction. The reaction was stirred gently at 37°C, and the pH was maintained throughout the reaction. The pH was approximately 7.0-8.5. After 4 hours, the reaction was complete. The reaction solution was acidified with dilute hydrochloric acid 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 using D101 non-polar resin and collected. 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, except that TvPPK enzyme was simultaneously added to the reaction solution to recycle ATP. 11.8 g of betaine (100 mM) was added to 1 L of 100 mM Tris-HCl solution (pH 8.0), 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), and 91.9 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt, NADPH, 120 mM). The pH of the solution was then adjusted back to 8.0, and finally, 2200 U of SlCAR crude enzyme solution, 2200 U of BaDERA crude enzyme solution, and 3400 U of PEG were added all at once. The reaction was initiated with crude TvPPK enzyme solution; the reaction was gently stirred at 37°C, and the pH was maintained at approximately 7.0-8.5 throughout the reaction. After 4 hours, the reaction was complete, and the reaction solution was adjusted to acidity with dilute aqueous hydrochloric acid to inactivate the enzyme. 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 using D101 non-polar resin, collected, and finally desalinated using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O = 4:1, v:v) to obtain 11.8 g of a 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] 11.8 g of betaine (100 mM) was added to 1 L of 100 mM Tris-HCl solution (pH 8.0), 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). The pH of the solution was then adjusted back to 8.0, and finally 2000 U of SlCAR crude enzyme solution, 2000 U of BaDERA crude enzyme solution, 3000 U of TvPPK crude enzyme solution and 1000 U of ELISA were added at once. The reaction was initiated with ScFDH; the reaction was gently stirred at 37°C, and the pH was maintained at approximately 7.0-8.5 throughout the reaction. After 3 hours, the reaction was complete, and the reaction solution was acidified with dilute aqueous hydrochloric acid 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 using D101 non-polar resin, and finally desalinated using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O = 4:1, v:v) to obtain 13.6 g of a 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] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, 11.8 g of betaine (100 mM) was added, 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 metabisphosphate (66.7 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), 2.4 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 3 mM), and 8.2 g of sodium formate (100 mM). The pH of the solution was then adjusted back to 8.0, and finally, 1800 U of SlCAR crude enzyme solution, 1800 U of BaDERA crude enzyme solution, 2700 U of TvPPK crude enzyme solution, and 800 U of ScFDH were added at once to initiate the reaction. The reaction was gently stirred at 37°C, and the pH was maintained throughout the reaction. The pH was approximately 7.0-8.5. After 3 hours, the reaction was complete. The reaction solution was acidified with dilute hydrochloric acid 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 using D101 non-polar resin and collected. 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] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, 11.8 g of betaine (100 mM) was added, 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 metabisphosphate (66.7 mM), 4.2 g of magnesium chloride hexahydrate (20 mM), 24.7 g of coenzyme II (nicotinamide adenine dinucleotide phosphate monosodium salt NADPH, 6 mM), and 12.3 g of sodium formate (150 mM). The pH of the solution was then adjusted back to 8.0, and finally, 2200 U of SlCAR crude enzyme solution, 2200 U of BaDERA crude enzyme solution, 3500 U of TvPPK crude enzyme solution, and 1200 U of ScFDH were added all at once to initiate the reaction. The reaction was gently stirred at 37°C, and the pH was maintained throughout the reaction. The reaction mixture was acidified with dilute hydrochloric acid to inactivate the reaction mixture after 3 hours. 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 using D101 non-polar resin and collected. Finally, the product was desalinated 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 enzyme was used to prepare L-carnitine in one step

[0145]

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

[0147] To 1 L of 100 mM Tris-HCl (pH 8.0) solution was added 11.8 g of betaine (100 mM), 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 metabisphosphate (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). The pH of the solution was then adjusted back to 8.0, and finally, 6000 U of the immobilized enzyme mix was added in one portion to initiate the reaction. The reaction was gently stirred at 37°C, and the pH was maintained at approximately 7.0-8.5 throughout the reaction. The reaction was complete after 6 hours, and the immobilized enzyme mix was collected by filtration (the enzyme mix retained 70% of its initial activity after eight uses). Similar to the above treatment, the reaction supernatant was treated with D201 anion exchange resin to remove phosphoric acid-containing impurities. The crude product was then purified using D101 non-polar resin and collected. Finally, the product was desalinated using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O = 4:1, v:v) to obtain 14.7 g of a white solid (final yield 91%).

[0148] Example 11: Using betaine and sodium acetate as raw materials, immobilized mixed enzyme was used to prepare L-carnitine in one step

[0149]

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

[0151] To 1 L of 100 mM Tris-HCl (pH 8.0) solution was added 11.8 g of betaine (100 mM), 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 metabisphosphate (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). The pH of the solution was then adjusted back to 8.0, and finally, 4000 U of the immobilized enzyme mix was added in one portion to initiate the reaction. The reaction was gently stirred at 37°C, and the pH was maintained at approximately 7.0-8.5 throughout the reaction. The reaction was complete after 6 hours, and the immobilized enzyme mix was collected by filtration (the enzyme mix retained 70% of its initial activity after eight uses). Similar to the above treatment, the reaction supernatant was treated with D201 anion exchange resin to remove phosphoric acid-containing impurities. The crude product was then purified using D101 non-polar resin and collected. 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%).

[0152] Example 12: Using betaine and sodium acetate as raw materials, immobilized mixed enzyme was used to prepare L-carnitine in one step

[0153]

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

[0155] To 1 L of 100 mM Tris-HCl (pH 8.0) solution was added 11.8 g of betaine (100 mM), 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 metabisphosphate (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). The pH of the solution was then adjusted back to 8.0, and finally, 8000 U of the immobilized enzyme mix was added in one portion to initiate the reaction. The reaction was gently stirred at 37°C, and the pH was maintained at approximately 7.0-8.5 throughout the reaction. The reaction was complete after 6 hours, and the immobilized enzyme mix was collected by filtration (the enzyme mix retained 70% of its initial activity after eight uses). Similar to the above treatment, the reaction supernatant was treated with D201 anion exchange resin to remove phosphoric acid-containing impurities. The crude product was then purified using D101 non-polar resin and collected. Finally, the product was desalinated using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O = 4:1, v:v) to obtain 15.2 g of a 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An enzyme mutant, characterized in that The enzyme includes betaine reductase, and the amino acid sequence of the mutant of the betaine reductase is shown in SEQ ID No.

1.

2. An enzyme mutant, characterized in that The enzyme includes L-carnitine aldolase, and the mutant of the L-carnitine aldolase is shown as SEQ ID No.

2.

3. An enzyme composition or an immobilized enzyme thereof, characterized in that The invention also includes mutants of the enzymes according to claims 1 and 2.

4. The enzyme composition or immobilized enzyme thereof according to claim 3, wherein The enzyme also includes NADPH regenerating enzyme; the amino acid sequence of the mutant of the NADPH regenerating enzyme is shown in SEQ ID No.

4.

5. The enzyme composition or immobilized enzyme thereof according to claim 4, wherein The enzyme composition or the immobilized enzyme thereof further comprises polyphosphate kinase; the amino acid sequence of the polyphosphate kinase is shown in SEQ ID No.

3.

6. A nucleic acid molecule encoding the enzyme composition or immobilized enzyme thereof according to any one of claims 3 to 5.

7. The nucleic acid molecule according to claim 6, wherein The nucleotide sequence of the nucleic acid molecule encoding the betaine reductase mutant is shown in SEQ ID NO.5; The nucleotide sequence of the nucleic acid molecule encoding the mutant of L-carnitine aldolase is shown in SEQ ID NO.6; The nucleotide sequence of the nucleic acid molecule encoding the polyphosphate kinase is shown in SEQ ID NO.7; The nucleotide sequence of the nucleic acid molecule encoding the mutant of the NADPH regenerating enzyme is shown in SEQ ID NO.

8.

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

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

10. A method for preparing L-carnitine, characterized in that: L-carnitine is prepared by using acetic acid or its salts and betaine as raw materials through any of the following conversions: (I) The enzyme composition or immobilized enzyme thereof according to any one of claims 3 to 5; (II), the nucleic acid molecule according to claim 6 or 7; or (III) The expression vector or host according to claim 8.

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

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