Proline dehydrogenase, gene, mutant and application of proline dehydrogenase

Abstract

The invention provides proline dehydrogenase, a gene, a mutant and application of the proline dehydrogenase. The enzyme is derived from Pseudomonas xanthophysigenes and is subjected to recombinant expression in escherichia coli, a target gene is subjected to codon optimization when a plasmid is constructed, and the optimized DNA (Deoxyribose Nucleic Acid) sequence is as shown in SEQ ID NO. 2. The proline dehydrogenase is subjected to mutation modification. The enzyme and the mutant thereof can be used for efficiently catalyzing degradation of L-proline so as to complete biological resolution of DL-proline, high-purity D-proline is obtained, and the enzyme and the mutant thereof have great economic value in the field of green production of D-proline.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a proline dehydrogenase, a gene, a mutant and their applications. Background Art

[0002] D-proline is a five-membered cyclic imino acid, which is relatively rare in nature but has various important functions. It can be used as a chiral intermediate for synthesizing certain chiral drugs. For example, D-proline can be used as a key chiral intermediate for eletriptan, and can also be used as a key precursor for synthesizing various alkaloids. In addition, D-proline can also participate in catalyzing multiple asymmetric reactions as a chiral catalyst.

[0003] Currently, the production methods of D-proline mainly include chemical production methods and biological production methods. Among them, the chemical production methods mainly obtain D-proline through a series of chemical reactions. For example, in the chemical asymmetric synthesis method (CN 107827802A), pyrrolidin-2-methanol is used as a raw material, and D-prolinol is obtained by reduction under the catalysis of a chiral metal, and then D-proline is obtained by oxidation. However, this scheme uses expensive catalysts and the reaction ee value is not high, so the final industrial value is low; while the chemical asymmetric transformation method for synthesizing D-proline uses chemical reagents to achieve the purpose of resolving DL-proline, with high yield and efficiency but high production cost, which will cause greater pollution to the environment and does not meet the requirements of green production. The biological production method is mainly biological enantioselective degradation method. Currently, there is a patent study (CN111424060A) that uses Lysinibacillus xylosus to degrade L-proline in DL-proline to obtain D-proline, and produces a by-product 1-pyrroline-5-carboxylic acid to improve the industrial value. However, the efficiency of this method is low, the fermentation and conversion time are both long, the total substrate concentration is low, only 40 g / L, and it is necessary to batch-feed 10 g / L DL-proline each time for reaction, and the operation is relatively complex. Considering the total fermentation and conversion time, it takes more than 168 h to obtain 20 g / L D-proline. Therefore, this method still cannot meet the requirements of industrial production. Summary of the Invention

[0004] In order to solve the problem of low production efficiency of D-proline caused by insufficient catalytic activity of proline dehydrogenase in the prior art, the present invention provides a proline dehydrogenase, a gene, a mutant and their applications. The main purpose of the present invention is to increase the substrate concentration of the conversion reaction, simplify the production process, shorten the reaction time to improve the production efficiency, and make the overall reaction more suitable for industrial production.

[0005] In order to achieve the above invention purpose, the present invention is realized through the following technical solutions.

[0006] A proline dehydrogenase, derived from Pseudomonas xantholysinigenes, has an amino acid sequence as shown in SEQ ID NO.1. The target gene is codon-optimized, and the optimized nucleotide sequence is as shown in SEQ ID NO.2.

[0007] A mutant of proline dehydrogenase, the amino acid sequence of the mutant is an amino acid sequence obtained by mutating one or more of the tyrosine at position 549, tyrosine at position 434, tyrosine at position 568, and alanine at position 368 in the amino acid sequence shown in SEQ ID NO.1. The mutation includes at least one of the following mutation sites: Y434Q, Y434M, Y568I, Y549L, A368M, A368K, Y549L + Y434M, A368M + Y434M; where the meanings of amino acid abbreviations are as follows: A - alanine, L - leucine, I - isoleucine, Y - tyrosine, K - lysine, Q - glutamine, M - methionine.

[0008] An application of proline dehydrogenase, the application includes the following steps:

[0009] (1) Construction of recombinant Escherichia coli: The DNA fragment shown in SEQ ID NO.2 is cloned into the expression vector pET28a(+) using NheI and HindIII restriction enzyme sites to obtain the pET28a(+)-PxPutA recombinant plasmid, which is transformed into Escherichia coli BL21(DE3) competent cells to obtain highly expressed recombinant Escherichia coli;

[0010] (2) Fermentation culture: Pick a single colony from step (1), inoculate it into a 5 mL LB test tube (resistant to kanamycin sulfate) and culture it overnight, transfer it to a TB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 2%, culture it at 37 °C for 2 h, add the inducer IPTG to a final concentration of 0.2 mmol / L, and continue to culture it at 18 °C for 24 hours to end the fermentation. The cells in the fermentation broth are directly added to the substrate for reaction without separation;

[0011] (3) Degradation of L-proline in DL-proline: The bacterial liquid obtained in step (2) by fermentation is used to degrade L-proline by whole-cell catalysis to obtain high-purity D-proline. The initial concentration of DL-proline is 30 - 50 g / L, the addition amount of the recombinant Escherichia coli wet cell bacterial liquid is 20 - 30 g / L, the reaction temperature range is 20 °C to 40 °C, and the degradation time is 24 - 36 h.

[0012] An application of a proline dehydrogenase mutant, including the following steps:

[0013] (1) Construction of recombinant Escherichia coli: Site-directed mutagenesis PCR was carried out using the DNA fragment shown in SEQ ID NO.2 as a template to construct a recombinant plasmid containing the coding gene with the mutation site. The mutant plasmid was transferred into the competent cells of Escherichia coli BL21(DE3) to obtain highly expressed recombinant Escherichia coli;

[0014] (2) Fermentation culture: Pick a single colony from step (1) and inoculate it into a 5 mL LB test tube (resistant to kanamycin sulfate) for overnight culture. Transfer it to TB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 2%, culture at 37 °C for 2 h, add the inducer IPTG to a final concentration of 0.2 mmol / L, and continue to culture at 18 °C for 24 hours to end the fermentation. The cells in the fermentation broth were directly added to the substrate for reaction without separation;

[0015] (3) Degradation of L-proline in DL-proline: The bacterial liquid obtained from the fermentation in step (2) was used to degrade L-proline by whole-cell catalysis to obtain high-purity D-proline. The initial concentration of DL-proline was 30 - 50 g / L, the addition amount of the wet cell bacterial liquid of recombinant Escherichia coli was 20 - 30 g / L, the reaction temperature range was 20 °C to 40 °C, and the degradation time was 24 - 36 h.

[0016] The beneficial effects of the present invention are as follows:

[0017] (1) The proline dehydrogenase provided by the present invention has a homology of 77.4% with the amino acid sequence of PutA derived from Pseudomonas pseudoalcaligenes. The PxPutA provided by the present invention is a new proline dehydrogenase.

[0018] (2) The present invention provides a proline dehydrogenase derived from Pseudomonas xantholysinigenes. By constructing a highly expressed recombinant engineering bacterium, it is found that this enzyme can efficiently catalyze the dehydrogenation degradation of L-proline, thereby producing high-purity D-proline.

[0019] (3) The present invention provides an optimized mutant of the proline dehydrogenase derived from Pseudomonas xantholysinigenes. By constructing a highly expressed recombinant engineering bacterium, it is found that this enzyme has higher catalytic activity than the wild type, thus greatly improving the reaction efficiency. Further applying this enzyme to the preparation of D-proline, it is found to have advantages such as rapid reaction and good stability, overcoming the defect of long time-consuming in the biological resolution method of D-proline, and having important application value for the industrial preparation of D-proline. Specific embodiments

[0020] The following examples facilitate a better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. In the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged. For the chromatograms of different reactions under the same condition parameters, there will be a certain error range for the retention time of the target peak. Generally, a difference within 0.1 min can be regarded as an error and can be considered the same target substance.

[0021] The present invention will be described in more detail below with reference to specific examples.

[0022] In the pre-column chiral derivatization-high performance liquid chromatography detection method of L-glutamic acid, D-proline, L-proline, and L-1-pyrroline-5-carboxylic acid in the examples, the pre-column chiral derivatization-high performance liquid chromatography conditions are as follows: After centrifuging the fermentation broth, the supernatant is diluted and then 4 g / L triethylamine / acetonitrile solution and 2 g / L 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) / acetonitrile solution are added. Heat in a metal bath at 30 °C for 30 min. Centrifuge, and use HPLC to separate the derivatives of D-proline, L-proline, and L-glutamic acid and L-1-pyrroline-5-carboxylic acid in the supernatant. Calculate the concentration according to the internal standard method. Chromatographic column: C18 column; Mobile phase: 0.1% trifluoroacetic acid aqueous solution / methanol (70:30, v / v), gradient elution, and the changes in the mobile phase concentration are shown in Table 1; Flow rate: 1.0 mL / min; Detection wavelength: 254 nm; Column temperature: 30 °C.

[0023] Example 1

[0024] Construction, cultivation, and induction expression of a genetically engineered bacterium of Escherichia coli with high expression of proline dehydrogenase PxPutA

[0025] (1) Obtain the protein sequence of proline dehydrogenase from Pseudomonas xantholysinigenes in the NCBI database as shown in SEQ ID NO.1. After codon optimization, the optimized nucleotide sequence is SEQ ID NO.2, which is synthesized by Beijing Tsingke Biotechnology Co., Ltd. and cloned between the NheI and HindIII restriction enzyme sites of the pET28a(+) vector, thus obtaining the pET28a(+)-PxPutA recombinant plasmid.

[0026] (2) Transform the obtained recombinant plasmid into Escherichia coli BL21(DE3) competent cells.

[0027] (3) Pick a single colony and inoculate it into a 5 mL LB test tube (kanamycin sulfate resistant) for overnight culture. Then transfer it to TB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 2%, culture at 37 °C for 2 h, add the inducer IPTG to a final concentration of 0.2 mmol / L, and continue to culture at 18 °C for 24 h to end the fermentation. Collect the bacterial liquid for standby.

[0028] Example 2

[0029] Wild-type proline dehydrogenase PxPutA is used for the preparation of D-proline

[0030] In a 10 ml system, add 50 g / L of DL-proline to the fermentation broth containing 30 g / L of wet cells (the bacterial liquid prepared in Example 1), shake and oscillate (220 rpm) at 30 °C for 24 h, then take samples. After detection by HPLC, since the essence of the reaction is the specific dehydrogenation of L-proline, the final reaction result is expressed by the ee value of D-proline. The reaction results are shown in Table 1.

[0031] Example 3

[0032] Construction of proline dehydrogenase PxPutA mutant plasmid

[0033] 1) According to the amino acid sequence SEQ ID NO.1 of wild-type PxPutA, perform protein homology modeling analysis on the alphafold3 website, and then perform molecular docking of the simulated structure with the substrate (AutoDock 4.2.6 software) to analyze its substrate binding pocket. It is found that the positions of Y434, E286, K326, A368, E369, R428, A433, Y434, L510, and Y549 are relatively close to the active center, and it is speculated that these sites may affect the catalytic activity of PxPutA on the substrate.

[0034] 2) Use the wild-type plasmid pET28a(+)-PxPutA as a template for whole-plasmid single-point mutation PCR to construct recombinant plasmids containing the coding genes of PxPutA-Y434 site, A368 site, Y549, and Y568 sites mutated into Y434Q, Y434M, Y568I, Y549L, A368M, A368K, Y549L+Y434M, and A368M+Y434M (Y-tyrosine, A-alanine, Q-glutamine, M-methionine, I-isoleucine, L-leucine, K-lysine) respectively. After correct sequencing, extract the plasmids and store them.

[0035] Example 4

[0036] Construction, culture, and induction expression of proline dehydrogenase PxPutA mutant highly expressed Escherichia coli genetic engineering bacteria

[0037] The mutant plasmids obtained in Example 3 were separately transformed into competent cells of Escherichia coli BL21(DE3), spread on plates and grown overnight. Single colonies were picked and inoculated into 5 mL LB tubes (resistant to kanamycin sulfate) and cultured overnight. Then, they were transferred to TB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 2%, cultured at 37°C for 2 h, and then IPTG was added as an inducer to a final concentration of 0.2 mmol / L. The culture was continued at 18°C for 24 h, and the fermentation was terminated. The bacterial liquid was collected for standby. (The composition of LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride; the composition of TB medium: 12 g / L tryptone, 24 g / L yeast extract, 4 g / L glycerol, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4)

[0038] Example 5

[0039] The proline dehydrogenase PxPutA mutant is used for the preparation of D-proline

[0040] In a 10 ml system, 50 g / L of DL-proline was added to the fermentation broth containing 30 g / L of wet cells (the bacterial liquid prepared in Example 4). After shaking and incubating at 30°C (220 rpm) for 24 h, samples were taken. Since the essence of the reaction is the specific dehydrogenation of L-proline in DL-proline, the final reaction result is expressed by the ee value of D-proline. The ee values of D-proline produced by wild-type proline dehydrogenase PxPutA (WT) and the proline dehydrogenase PxPutA mutant are shown in Table 1 below.

[0041] Table 1 Summary table of ee values of D-proline produced by wild-type proline dehydrogenase PxPutA and the mutant

[0042]

[0043]

[0044] Example 6

[0045] The proline dehydrogenase PxPutA mutant Y434M is used for the preparation of D-proline (changing the concentration of DL-proline). In a 10 ml system, 30 g / L of DL-proline was added to the fermentation broth containing 30 g / L of Y434M wet cells (the bacterial liquid prepared in Example 4). After shaking and incubating at 30°C (220 rpm) for 24 h, samples were taken and detected by HPLC. The dehydrogenation of L-proline was complete, the ee value of D-proline was 100%, and the concentration of D-proline was 10.5 g / L.

[0046] Example 7

[0047] The proline dehydrogenase PxPutA mutant Y434M is used for preparing D-proline (changing the conversion time)

[0048] In a 10 ml system, 50 g / L of DL-proline was added to the fermentation broth containing 30 g / L of wet cells of Y434M, and the mixture was shaken (220 rpm) at 30 °C for 36 h for conversion. L-proline was completely dehydrogenated, the ee value of D-proline was 100%, and the concentration of D-proline was 17.5 g / L.

Claims

1. A proline dehydrogenase, characterized in that, Derived from Pseudomonas xantholysinigenes, the amino acid sequence is as shown in SEQ ID NO.

1. The target gene was codon-optimized, and the optimized nucleotide sequence is as shown in SEQ ID NO.

2.

2. A mutant of the proline dehydrogenase as described in claim 1, characterized in that, The amino acid sequence of the mutant is an amino acid sequence obtained by mutating one or more of the tyrosine at position 549, tyrosine at position 434, tyrosine at position 568, and alanine at position 368 in the amino acid sequence shown in SEQ ID NO.

1. The mutations include at least one of the following mutation sites: Y434Q, Y434M, Y568I, Y549L, A368M, A368K, Y549L + Y434M, A368M + Y434M; where the meanings of the amino acid abbreviations are as follows: A - alanine, L - leucine, I - isoleucine, Y - tyrosine, K - lysine, Q - glutamine, M - methionine.

3. Use of a proline dehydrogenase as described in claim 1, characterized in that, The application includes the following steps: (1) Construction of recombinant Escherichia coli: The DNA fragment shown in SEQ ID NO.2 was cloned into the expression vector pET28a(+) using the NheI and HindIII restriction sites to obtain the pET28a(+)-PxPutA recombinant plasmid, which was then transformed into Escherichia coli BL21(DE3) competent cells to obtain highly expressed recombinant Escherichia coli. (2) Fermentation culture: Pick a single colony from step (1) and inoculate it into a 5 mL LB test tube (resistant to kanamycin sulfate) for overnight culture. Transfer it to a TB medium containing 50 μg / mL of kanamycin sulfate at an inoculation amount of 2%, culture at 37°C for 2 h, add the inducer IPTG to a final concentration of 0.2 mmol / L, and continue to culture at 18°C for 24 hours to end the fermentation. The cells in the fermentation broth are directly added to the substrate for reaction without separation. (3) Degradation of L-proline in DL-proline: The bacterial liquid obtained in step (2) by fermentation is used to degrade L-proline by whole-cell catalysis to obtain high-purity D-proline. The initial concentration of DL-proline is 30 - 50 g / L, the addition amount of the wet cell bacterial liquid of recombinant Escherichia coli is 20 - 30 g / L, the reaction temperature range is 20°C to 40°C, and the degradation time is 24 - 36 h.

4. Use of the proline dehydrogenase mutant according to claim 2, characterized in that, It includes the following steps: (1) Construction of recombinant Escherichia coli: Site-directed mutagenesis PCR was performed using the DNA fragment shown in SEQ ID NO.2 as a template to construct a recombinant plasmid containing the coding gene with mutation sites. The mutant plasmid was transferred into the competent cells of Escherichia coli BL21(DE3) to obtain highly expressed recombinant Escherichia coli. (2) Fermentation culture: Pick a single colony from step (1) and inoculate it into a 5 mL LB test tube (resistant to kanamycin sulfate) for overnight culture. Transfer it to a TB medium containing 50 μg / mL of kanamycin sulfate at an inoculation amount of 2%, culture at 37°C for 2 h, add the inducer IPTG to a final concentration of 0.2 mmol / L, and continue to culture at 18°C for 24 hours to end the fermentation. The cells in the fermentation broth are directly added to the substrate for reaction without separation. (3) Degradation of L-proline in DL-proline: The bacterial liquid obtained in step (2) by fermentation is used to degrade L-proline through whole-cell catalysis to obtain high-purity D-proline. The initial concentration of DL-proline is 30 - 50 g / L, the addition amount of the wet cell bacterial liquid of recombinant Escherichia coli is 20 - 30 g / L, the reaction temperature range is 20°C to 40°C, and the degradation time is 24 - 36 h.

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