Method for preparing D-lysine hydrochloride through enzyme catalysis cascade reaction

Abstract

The invention discloses a method for preparing D-lysine hydrochloride through enzyme catalysis cascade reaction, and belongs to the technical field of biochemistry. The method comprises the following steps: by taking L-lysine hydrochloride as a substrate, carrying out racemization reaction through racemase to prepare DL-lysine, then carrying out decarboxylation on the L-lysine in a reaction solution under the action of decarboxylase to generate pentamethylene diamine, and finally extracting to obtain the D-lysine hydrochloride. The reaction liquid is extracted and purified to obtain a high-purity D-lysine hydrochloride product, and the conversion rate of the decarboxylase to the L-lysine reaches 99% or above. After decoloration, rotary evaporation, alcohol washing and drying, the purity of the D-lysine hydrochloride product is detected to be more than 99%, the enantiomeric excess value ee is 100%, and the yield reaches the theoretical value 85% or above of the D-lysine hydrochloride. The method provided by the invention has the characteristics of high conversion rate and high yield, and is easy to industrialize.

Description

Technical Field

[0001] The invention relates to a method for preparing D-lysine hydrochloride through enzyme-catalyzed cascade reaction, belonging to the technical field of biochemistry. Background Art

[0002] D-lysine hydrochloride, English name D-lysine hydrochloride, white powder, chemical formula C6H 15 ClN2O2. CAS: 7274-88-6. Molecular weight: 182.65. It is one of the most important amino acids, primarily used in food and medicine.

[0003] D-lysine is a non-proteinogenic amino acid. Replacing one or several L-amino acids in a short peptide with D-amino acids can produce new peptides with significantly different biological functions or properties than the original peptides. Amino acid antibiotics doped with D-amino acids are less susceptible to bacterial enzyme degradation and thus do not contribute to drug resistance. Due to the chiral nature of D-lysine, the preparation of D-lysine hydrochloride is difficult to achieve by microbial fermentation or extraction. Instead, it is primarily obtained by racemization and resolution of L-lysine hydrochloride.

[0004] The most economical method for the optical resolution of racemates is the preferential crystallization method, because this method does not require the use of other substances or the conversion of amino acid lipids into other forms. However, the product yield of this method is low and the chiral content does not meet the quality requirements of chiral compounds.

[0005] The chemical chiral acid resolution method uses a chiral acid as a resolving agent to react with DL-lysine to form diastereomeric salts. Based on the different solubilities of the two diastereomeric salts, the D-lysine diastereomeric salts and the L-lysine diastereomeric salts are separated. The D-lysine diastereomeric salts are then decomposed and D-lysine is purified and extracted. However, the production yield of D-lysine is lower than that of the biological method.

[0006] A prior art document (Modern Chemical Industry, 2007, 27, 32) reports a method for preparing D-lysine using a chemical-biological coupling method. L-lysine is chemically racemized to DL-lysine, and the L-enantiomer of DL-lysine is converted to pentamethylenediamine using lysine decarboxylase from Hafnia alvei AS1.1009. The D-lysine yield is 56.6%, which remains to be improved.

[0007] In summary, it is necessary to continue to conduct in-depth research on the enzymatic preparation of D-lysine hydrochloride in order to obtain a process route with more competitive advantages. Summary of the Invention

[0008] To overcome the above-mentioned technical deficiencies, the present invention provides a method for preparing D-lysine hydrochloride via an enzyme-catalyzed cascade reaction. Using L-lysine hydrochloride as a substrate, a racemization reaction is first performed with a racemase to produce DL-lysine. Subsequently, the L-lysine in the reaction solution is decarboxylated to form pentamethylenediamine by a decarboxylase, and finally, D-lysine hydrochloride is extracted. Extraction and purification of the reaction solution yield a high-purity D-lysine hydrochloride product, with the decarboxylase converting L-lysine to over 99%. After decolorization, rotary evaporation, alcohol washing, and drying, the D-lysine hydrochloride product exhibits a purity exceeding 99%, an enantiomeric excess (EE) of 100%, and a yield exceeding 85% of the theoretical value for D-lysine hydrochloride. The present method exhibits high conversion and yield, making it readily commercializable.

[0009] The invention discloses a method for preparing D-lysine hydrochloride by enzyme-catalyzed cascade reaction, comprising the following steps: using L-lysine hydrochloride as a raw material, treating it with racemase and deacidase respectively to obtain D-lysine hydrochloride.

[0010] Furthermore, in the above technical solution, the method for preparing D-lysine hydrochloride by enzyme-catalyzed cascade reaction of the present invention is represented by the following chemical reaction equation:

[0011]

[0012] Furthermore, in the above technical solution, the process is as follows: L-lysine hydrochloride is freed by adding alkali, racemase is used to generate DL-lysine, and then reacted in the presence of decarboxylase, and hydrochloric acid is added to obtain D-lysine hydrochloride.

[0013] Furthermore, in the above technical solution, the amino acid sequence of the racemase is SEQ ID NO.2.

[0014] Furthermore, in the above technical solution, the amino acid sequence of the decarboxylase is SEQ ID NO.4.

[0015] Furthermore, in the above technical solution, the racemase-expressing engineered bacteria are constructed by codon-optimizing the racemase (lysine racemase / LR) amino acid sequence to obtain a gene sequence. The racemase gene is amplified by PCR using primers LR-F / LR-R and inserted into the pET-28a(+) expression vector between the BamHI and EcoRI restriction sites to obtain the racemase expression vector pET28a-LR. The recombinant plasmid is then heat-shock transformed into Escherichia coli BL21(DE3) competent cells to obtain the racemase-expressing engineered bacteria EcLR.

[0016] Furthermore, in the above technical solution, the method for constructing the decarboxylase-expressing engineered bacteria includes: codon-optimizing the decarboxylase (lysine decarboxylase / LDC) amino acid sequence to obtain a gene sequence; PCR amplifying the racemase gene using primers LDC-F / LDC-R and inserting it into the pET-28a(+) expression vector between the BamHI and EcoRI restriction sites to obtain the racemase expression vector pET28a-LDC; and heat-shock-transforming the recombinant plasmid into Escherichia coli BL21(DE3) competent cells to obtain the decarboxylase-expressing engineered bacteria EcLDC.

[0017] Racemase gene sequence SEQ ID NO.1

[0018] ATGAAAAAAGCAACCTGTGGGCTTGCCTGGGCGCTGGTCTGCTGCATATCCAGAGCTCTT

[0019] TCGCAGCACCGCTGCTGACCCCACACCTGTCTGCTACTCAAGTGGCAGCTCCGCAGGCAA

[0020] ACGCTTGGGTGGAGATTAACGTGCAGACTTTCGAACAGAACATTCAAACCCTGCAACAGC

[0021] AGCTGCAAGATAAGAGCCAGTTCTGTGCTGTCATGAAAGCGGATGCGTATGGCCACGGCAT

[0022] CGAACTGCTGATGCCATCCATCATTAAGCTGAATGTGCCGTGTGTAGGCATCACCTCCAAC

[0023] GCGGAAGCTGCACTGGTTCGCAAAAGCGGTTACCAGGGTCGTCTGCTGCGTCTGCGTGCA

[0024] GCGACTGACCAGGAAATTCAAAACGCGGCTTCTCTGAACATGGAGGAACTGCTGGGTAAC

[0025] TATGAACAGGCGCAACGTCTGTCCGAATGGGCGAAACAGCAGGGCCTGATTCTGGAATAC

[0026] CATCTGGGTCTGAACGCAGGTGGTATGGATCGCAACGGCCTGGAACTGAACAGCGCACTG

[0027] GGTAAACAGCAGGCGCTGCGTATGACCAAACTGCCGAACCTGAAACTGGTTGGCATCATG

[0028] ACTCACTACGCCGTAGAAGACGAAAAATATGTGCGTGAACGCCTGGCCGTTTTCAACCAG

[0029] CAAACCGATTGGATCATCAAGAAAGCAAAGCTGAAACGCGAGGCCCTGACCCTGCACACC

[0030] GCAAACTCTTTTGCGACCCTGACCGTGCCGGAAAGCCACCTGGACATGGTTCGTGCTGGT

[0031] GGTCAGATTTATGGCGATTCCCTGCCGGATCGTGTTGAATACAAAAAACTGATGACCTTCA

[0032] AAACCCAGGTTGCTACTGTAAACGCGTATCTGAAAGGCACCACTGTTGGTTACGACCAGA

[0033] CCTATACTCTGAAACGCGACTCTCTGCTGGCAAACCTGCCGCTGGGTTACTCTGATGGCTA

[0034] CCGTCGTGCGTTTTCCAACAAAGCGTATGTGCTGATCCGTGGTCACAAAGTTCCGGTTGTA

[0035] GGTCGTACGTCCATGAACACCACGATGGTCGATATCACCGATTTTCCGGATATTCGCGCGAA

[0036] CGATGAAGTCGTGCTGTTCGGCGAGCAGGGTGGTGCTATTGTGAAACAGTCCGACCTGGA

[0037] GGAATACAACGGTGCACTGCTGGCTGACGTTTACACCATGTGGGGCAACAGCAATCCGAA

[0038] GGTCGTTAAATAA

[0039] 消旋酶氨基酸SEQ ID NO.2

[0040] MKKATLWACLGAGLLHIQSSFAAPLLTPHLSATQVAAPQANAWVEINVQTFEQNIQTLQQQLQ

[0041] DKSQFCAVMKADAYGHGIELLMPSIIKLNVPCVGITSNAEAALVRKSGYQGRLLRLRAATDQE

[0042] IQNAASLNMEELLGNYEQAQRLSEWAKQQGLILEYHLGLNAGGMDRNGLELNSALGKQQAL

[0043] RMTKLPNLKLVGIMTHYAVEDEKYVRERLAVFNQQTDWIIKKAKLKREALTLHTANSFATLTV

[0044] PESHLDMVRAGGQIYGDSLPDRVEYKKLMTFKTQVATVNAYLKGTTVGYDQTYTLKRDSLL

[0045] ANLPLGYSDGYRRAFSNKAYVLIRGHKVPVVGRTSMNTTMVDITDFPDIRANDEVVLFGEQGGAIVKQSDLEEYNGALLADVYTMWGNSNPKVVK*

[0046] 脱羧酶基因序列SEQ ID NO.3

[0047] ATGAACATCATCGCCATTATGAACGACCTGTCCGCTTACTTTAAGGAAGAACCGCTGCGTG

[0048] AACTGCACCAGGAGCTGGAAAAAGAGGGTTTCCGTATTGCGTACCCGAAGGATCGCAACG

[0049] ACCTGCTGAAACTGATCGAAAACAATTCCCGTCTGTGCGGTGTCATCTTCGACTGGGATAA

[0050] ATATAACCTGGAACTGTCTGCAGAAATCAGCGAACTGAACAAGCTGCTGCCGATTTACGCT

[0051] TTCGCAAACACTTACTCTACCCTGGACGTAAACATGAGCGATCTGCGTCTGAACGTTCGTT

[0052] TCTTTGAATATGCTCTGGGTTCCGCCCAGGACATTGCAACTAAAATCCGCCAGTCTACTGAT

[0053] CAGTACATCGACACCATTCTGCCGCCACTGACCAAAGCCCTGTTCAAATACGTTAAGGAAG

[0054] AAAAATACACTGTCTGTACCCCGGGTCACATGGGTGGCACCGCTTTTGACAAAAGCCCGG

[0055] TTGGCTCCCTGTTCTATGACTTCTTCGGCGAAAACACTATGCGCTCCGATATCTCTATCAGC

[0056] GTCTCTGAACTGGGTTCCCTGCTGGATCATTCTGGTCCGCACCGTGACGCAGAAGAATACA

[0057] TTGCACGTACCTTCAACGCGGACCGTTCTTATATCGTAACGAACGGTACCAGCACCGCAAA

[0058] CAAAATTGTAGGCATGTATTCCTCTCCAGCGGGTGCGACTATCCTGATTGATCGTAACTGCC

[0059] ACAAAAGCCTGACCCACCTGATGATGATGTCTAATGTTGTTCCGGTTTACCTGCGTCCGAC

[0060] TCGCAACGCGTATGGTATCCTGGGTGGCATTCCACAGTCTGAATTTACCCGTGCTTCCATCG

[0061] AAGAAAAAGTTAAAAACACCCCGAACGCTACGTGGCCGGTTCATGCGGTGGTCACCAACT

[0062] CCACGTACGATGGTCTGTTCTACAACACCGAGTACATCAAAAACACCCTGGACGTCAAAA

[0063] GCATCCATTTCGACTCTGCATGGGTTCCTTACACTAACTTCCATCCGATTTATCAGGGTAAG

[0064] GCGGGTATGAGCGGTGAACGTGTTCCAGGTAAAATCATCTACGAAACCCAGAGCACGCAC

[0065] AAACTGCTGGCGGCGTTCTCTCAGGCGAGCATGATCCACGTCAAAGGTGAAATTAACGAG

[0066] GAGACGTTCAACGAGGCGTACATGATGCACACGTCTACCTCCCCGCATTACGGTATTGTTG

[0067] CTTCTACCGAAACCGCTGCGGCGATGATGAAAGGTAACGCTGGTAAACGTCTGATCAACG

[0068] GCTCTATCGAACGCGCAATTCGTTTTCGTAAGGAAATTCGTCGTCTGCGTACCGAAAGCGA

[0069] TGGCTGGTTCTTCGATGTGTGGCAGCCGGACAACATCGATGAGGTAGCTTGCTGGCCGCTG

[0070] AACCCGCGTAATGAATGGCATGGCTTTCCGAACATCGATAACGATCACATGTACCTGGACC

[0071] CGATCAAAGTTACTCTGCTGACGCCGGGTCTGGCACCAAATGGTACTCTGGAGGAGGAAG

[0072] GTATCCCGGCGTCTATCGTGTCTAAATATCTGGACGAACACGGTATCATTGTGGAAAAAACT

[0073] GGCCCGTATAACCTGCTGTTCCTGTTCTCCATCGGCATTGATAAAACGAAAGCACTGTCCCT

[0074] GCTGCGTGCTCTGACGGACTTCAAACGTGTTTACGACCTGAACCTGCGCGTAAAAAACGT

[0075] TCTGCCGTCCCTGTACAACGAAGCTCCGGATTTCTACAAGGAAATGCGCATCCAGGAACTG

[0076] GCTCAGGGTATTCACGCCCTGGTGAAACACCATAATCTGCCGGATCTGATGTATCGTGCCTT

[0077] TGAAGTCCTGCCGAAACTGGTGATGACCCCGCACGATGCATTCCAGGATGAAGTCCGTGG

[0078] CAACATCGAACCGTGCGCACTGGATGACATGCTGGGCAAAGTGTCTGCTAACATGATCCTG

[0079] CCATATCCGCCGGGCGTTCCGGTAGTTATGCCGGGTGAAATGCTGACGAAAGAATCCCGTC

[0080] CGGTGCTGTCCTTCCTGCAGATGCTGTGCGAAATTGGTGCGCACTACCCAGGCTTTGAGAC

[0081] TGATATCCACGGCGTTCACCGTGATGGCGCCACCGGTAAATATATGGTGCCGGTTCTGAAA

[0082] CAGGGCGCAGATCCGGGTGATAAACCATCCGATACTGTTAAAAAGGCGCCGGGCAAAAAA

[0083] CCGTCTGCGGCTAAAAAGTCCTAA

[0084] Decarboxylase amino acid sequence SEQ ID NO. 4

[0085] MNIIAIMNDLSAYFKEEPLRELHQELEKEGFRIAYPKDRNDLLKLIENNSRLCGVIFDWDKYNL

[0086] ELSAEISELNKLLPIYAFANTYSTLDVNMSDLRLNVRFFEYALGSAQDIATKIRQSTDQYIDTILP

[0087] PLTKALFKYVKEEKYTVCTPGHMGGTAFDKSPVGSLFYDFFGENTMRSDISISVSELGSLLDHS

[0088] GPHRDAEEYIARTFNADRSYIVTNGTSTANKIVGMYSSPAGATILIDRNCHKSLTHLMMMSNV

[0089] VPVYLRPTRNAYGILGGIPQSEFTRASIEEKVKNTPNATWPVHAVVTNSTYDGLFYNTEYIKNT

[0090] LDVKSIHFDSAWVPYTNFHPIYQGKAGMSGERVPGKIIYETQSTHKLLAAFSQASMIHVKGEI

[0091] NEETFNEAYMMHTSTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIRFRKEIRRLRTESDG

[0092] WFFDVWQPDNIDEVACWPLNPRNEWHGFPNIDNDHMYLDPIKVTLLTPGLAPNGTLEEEGIPA

[0093] SIVSKYLDEHGIIVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRVYDLNLRVKNVLPSLYNE

[0094] APDFYKEMRIQELAQGIHALVKHHNLPDLMYRAFEVLPKLVMTPHDAFQDEVRGNIEPCALD

[0095] DMLGKVSANMILPYPPGVPVVMPGEMLTKESRPVLSFLQMLCEIGAHYPGFETDIHGVHRDGATGKYMVPVLKQGADPGDKPSDTVKKAPGKKPSAAKKS*

[0096] Further, in the above technical solution, the primer is shown in Table 1, the composition of the PCR reaction system is shown in Table 2, and the PCR reaction condition is shown in Table 3.

[0097] Table 1: Primers used in PCR mutation

[0098] Primers Sequence (5'-3') LR-F GGGTCGGGATCCGAATATGAAAAAAGCAACCCTGTGGGC LR-R AGTGCGGCCGCAAGCTTTTATTTAACGACCTTCGGATTG LDC-F GGGTCGGGATCCGAATATGAACATCATCGCATTATGAA LDC-R GTGCGGCCGCAAGCTTTTAGGACTTTTTAGCCGCAGAC

[0099] Table 2: PCR reaction system

[0100]

[0101]

[0102] Table 3: PCR reaction condition

[0103]

[0104] Compared with the prior art, the present application has the following technical advantages:

[0105] A、The racemization and decarboxylation steps of the present application both use enzyme method, and two enzymes, racemase and decarboxylase, are used to prepare D-lysine hydrochloride, which is more green and environmentally friendly, and avoids the deficiency that the prior art uses chemical method for racemization and enzyme method for decarboxylation.

[0106] B、The substrate concentration of the reaction and the yield calculated according to the theoretical value of D-lysine hydrochloride are both superior to the method in the prior art, and the production efficiency is obviously improved. DETAILED DESCRIPTION

[0107] Figure 1 is the HPLC purity spectrum of D-lysine hydrochloride;

[0108] Figure 2 is the chiral purity (ee value) detection spectrum of D-lysine hydrochloride. DETAILED DESCRIPTION

[0109] The engineering bacteria EcLR and EcLDC are activated and cultured by using LB culture medium:

[0110] LB medium configuration method: 5 g / L of yeast powder, 10 g / L of proteose peptone, 10 g / L of sodium chloride, after configuration, sterilize by 121℃ high pressure steam for 20 min, and then add 2% (w / v) agar to make LB solid medium for strain plate culture and single colony isolation and purification. Before use, add kanamycin to a final concentration of 50 μg / mL. Activation and culture method: the glycerol tube containing the engineering bacteria was streaked on the LB solid medium plate and incubated at 37℃ overnight. Then, a single colony was picked from the plate and inoculated into 5 mL of LB resistant liquid medium, and incubated at 37℃, 220 rpm overnight. The bacterial cells were collected by centrifugation, and the plasmid was extracted according to the operation manual of the plasmid extraction kit.

[0111] Preparation of racemase and decarboxylase crude enzyme solution

[0112] The racemase and decarboxylase engineering bacteria were inoculated into 5 mL of LB liquid medium (kanamycin concentration 50 μg / mL) respectively, and incubated at 37℃ for 12 h to obtain seed liquid. Then, 1 mL of seed liquid was transferred into 500 mL of LB liquid medium with the same resistance, and incubated at 37℃ with shaking until the OD 600 When the OD reached 0.8, IPTG inducer was added to a final concentration of 0.3 mM. The culture was induced at 28℃ for 20 h. After the culture was completed, the bacterial cell culture solution was centrifuged at 8000 rpm for 20 min, and the supernatant was discarded. The bacterial cells were resuspended in PBS buffer with pH=7.0, and configured into 25% (w / v) bacterial suspension for low-temperature ultrasonic disruption. The supernatant was collected as crude enzyme solution.

[0113] Enzyme activity determination of racemase and decarboxylase

[0114] 2.1 Enzyme activity determination of racemase

[0115] A 50 g / L L-lysine hydrochloride solution was prepared using PBS buffer, 0.1 g / L pyridoxal phosphate (PLP) was added, and ammonia was added to adjust the pH to 7.5. 1 mL of racemase crude enzyme solution was added, and the chiral amino acid was analyzed by high performance liquid chromatography after constant temperature shaking at 40℃ for 20 min. One unit of racemase activity was defined as 1 micromole of amino acid enantiomer enzyme produced from its corresponding enantiomer per minute under the determination conditions.

[0116] 2.2 Enzyme activity determination of decarboxylase

[0117] The PBS buffer was used to prepare a 50 g / L L-lysine hydrochloride solution, 0.1 g / L pyridoxal phosphate (PLP) was added, and ammonia was added to adjust the pH to 6.0. 1 mL of crude decarboxylase enzyme solution was added, and after 30 min of constant temperature oscillation reaction at 35°C, the sample was taken for HPLC detection. The 1,5-pentanediamine content generated in the reaction was calculated by detecting the peak area and the standard curve of 1,5-pentanediamine, and thus the enzyme activity was calculated. The enzyme activity U of the decarboxylase was defined as: 1 micromole of 1,5-pentanediamine generated per minute using L-lysine hydrochloride as the substrate, and the enzyme amount was defined as one enzyme activity unit, i.e. 1 U.

[0118] Preparation of D-lysine hydrochloride by enzyme catalysis cascade reaction in a small test

[0119] Example 1

[0120] In a 1 L reaction system, 50 g of L-lysine hydrochloride was added, ammonia was added to adjust the pH to 7.2, 50 mL of crude racemase enzyme solution, 0.1 g of PLP was added, the reaction kettle was stirred at a speed of 200 rpm, and the reaction was carried out at 37°C for 8 h. The optical rotation value was 0. Then the temperature was raised to 80°C to inactivate the racemase, and then cooled to room temperature. The pH value was adjusted to 6.5 with hydrochloric acid, 0.1 g of PLP was added, and then 50 mL of crude decarboxylase enzyme solution was slowly added, and the decarboxylation reaction was started at a temperature of 30°C. The pH was adjusted to 6.5 during the reaction, and after 20 h, the bacterial debris was removed by centrifugation at 8000 rpm, 0.5% activated carbon was added, and decolorization was carried out at 60°C for 1 h. The decolorized liquid was added to an aqueous hydrochloric acid solution to adjust the pH to 5.5, and then rotary evaporation was carried out under reduced pressure until all the solids were precipitated. Methanol was added and stirred for 1 h, and then the product was washed once more with methanol after filtration. After drying, 19.1 g of D-lysine hydrochloride was obtained. The sample was sent for HPLC detection, and the product purity was >99%, the enantiomer content ee was 99.7%, and the yield of D-lysine hydrochloride was 76.4% of the theoretical value.

[0121] Example 2:

[0122] To a 1L reaction system, 200g of L-lysine hydrochloride was added, and aqueous ammonia was added to adjust the pH to 7.5. Then, 50mL of crude racemase solution and 0.05g of PLP were added. The reaction was stirred at 200rpm and allowed to react at 37°C for 6h, with the optical rotation value being 0. The temperature was then raised to 80°C to inactivate the racemase, and the mixture was cooled to room temperature. The pH was adjusted to 6.5 with hydrochloric acid, 0.05g of PLP was added, and 50mL of crude decarboxylase solution was slowly added. The decarboxylation reaction was initiated at 30°C. During the reaction, the pH was adjusted to 6.5. After 15h, the mixture was centrifuged at 8000rpm to remove bacterial debris, and 0.5% activated carbon was added for decolorization at 60°C for 1h. The decolorized solution was then added with aqueous hydrochloric acid to adjust the pH to 5.5. Vacuum evaporation was performed until all solids precipitated. Methanol was added and stirred for 1h. After filtration, the product was washed once more with methanol, filtered, and dried to yield 81.8g of D-lysine hydrochloride. The sample was sent for HPLC detection, and the product purity was >99%, the enantiomer content ee was 99.9%, and the yield of D-lysine hydrochloride was 81.8% of the theoretical value.

[0123] Example 3:

[0124] 1 kg of L-lysine hydrochloride was added to a 5L reaction system, the pH value was adjusted to 7.5, and then 200 mL of crude racemase solution and 0.2 g of PLP were added. The stirring speed of the reactor was 200 rpm, and the reaction was carried out at 37 ° C for 4 hours. The optical rotation value was detected to be 0. The temperature was then raised to 80 ° C to inactivate the racemase and then cooled to room temperature. The pH value of the filtrate was adjusted to 6.5 with hydrochloric acid, 0.2 g of PLP was added, and then 100 mL of crude decarboxylase solution was slowly added, and the temperature was controlled at 30 ° C to start the decarboxylation reaction. During the reaction, the pH was adjusted to 6.5. After 12 hours, the bacterial debris was removed by centrifugation at 8000 rpm, and 0.5% activated carbon was added for decolorization at 60 ° C for 1 hour. The decolorized solution was added with hydrochloric acid aqueous solution to adjust the pH to 5.5, and vacuum rotary evaporation was carried out until all solids were precipitated. Methanol was added and stirred for 1 hour. After filtration, the product was washed with methanol again, and 432 g of D-lysine hydrochloride was obtained after filtration and drying. The sample was sent for HPLC detection. The product purity was >99% ( Figure 1 ), enantiomeric content ee 100% ( Figure 2 ), the yield of D-lysine hydrochloride was 86.4% of the theoretical value.

[0125] The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. For those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing D-lysine hydrochloride by enzyme-catalyzed cascade reaction, characterized in that: The method comprises the following steps: taking L-lysine hydrochloride as a raw material and treating it with racemase and deacidase respectively to obtain D-lysine hydrochloride.

2. The method for preparing D-lysine hydrochloride by enzyme-catalyzed cascade reaction according to claim 1, characterized in that: After L-lysine hydrochloride is freed by adding alkali, it is converted into DL-lysine using racemase, and then reacted in the presence of decarboxylase. After adding hydrochloric acid treatment, D-lysine hydrochloride is obtained.

3. The method for preparing D-lysine hydrochloride by enzyme-catalyzed cascade reaction according to claim 1 or 2, characterized in that: The amino acid sequence of the racemase is SEQ ID NO.

2.

4. The method for preparing D-lysine hydrochloride by enzyme-catalyzed cascade reaction according to claim 3, characterized in that: The method for constructing a racemase expression engineered bacterium comprises the following steps: codon-optimizing and synthesizing a racemase LR amino acid sequence to obtain a gene sequence; using primers LR-F / LR-R to PCR amplify the racemase gene and inserting it between the BamHI and EcoRI restriction sites on a pET-28a(+) expression vector to obtain a racemase expression vector pET28a-LR; and heat-shock-transforming the recombinant plasmid into competent E. coli BL 21(DE3) cells to obtain racemase expression engineered bacteria EcLR.

5. The method for preparing D-lysine hydrochloride by enzyme-catalyzed cascade reaction according to claim 1 or 2, characterized in that: The amino acid sequence of the decarboxylase is SEQ ID NO.

4.

6. The method for preparing D-lysine hydrochloride by enzyme-catalyzed cascade reaction according to claim 5, characterized in that: The method for constructing a decarboxylase-expressing engineered bacterium comprises the following steps: codon-optimizing and synthesizing a decarboxylase LDC amino acid sequence to obtain a gene sequence; using primers LDC-F / LDC-R to PCR amplify a racemase gene and inserting the gene between BamHI and EcoRI restriction sites on a pET-28a(+) expression vector to obtain a racemase expression vector pET28a-LDC; and heat-shock-transforming the recombinant plasmid into Escherichia coli BL21(DE3) competent cells to obtain a decarboxylase-expressing engineered bacterium EcLDC.

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