A method for preparing D-pantoic acid by enzymatic conversion
The catalyzed conversion of DL-panolactone into D-panolactone by genetically engineered bacterial mutants or their crude enzyme liquid, and solved the problems of high cost of preparing D-panolactone and low product purity in the prior art, and achieved an efficient and low-cost preparation process.
Patent Information
- Application Number
- CN202411995523.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-04
AI Technical Summary
The existing preparation methods of D-panol acids have problems such as high cost, low product purity and long production cycle, which are difficult to meet the market's demand for high efficiency and low cost.
DL-panolactone was transformed by enzymatic method to produce D-panolactone by using genetically engineered bacteria mutants or their crude enzyme solution, and L-panolactone was obtained at the same time, and the reaction conditions were controlled at pH 6.5-8.0 and 25-37°C.
It realizes efficient, simple and low-cost D-panol acid preparation, improves enzyme catalytic activity and splitting efficiency, shortens production cycle and reduces costs.
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Figure CN119753041A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing D-pantoic acid through enzymatic conversion. Background Art
[0002] D-pantoic acid is the precursor of D-pantoic acid lactone. The aqueous solution containing D-pantoic acid is adjusted to pH 1 to 4 with acid and the temperature is controlled at 20 to 80°C to obtain D-pantoic acid lactone aqueous solution, which is then extracted with an organic solvent, concentrated, distilled, and crystallized to obtain D-pantoic acid lactone.
[0003] D-Pantolactone is a white hygroscopic crystal that is easily soluble in water. Its molecular formula is C6H 10 03, chemical name is α-hydroxy-β,β-dimethyl-γ-butyrolactone, relative molecular weight is 130.03. D-pantothenic acid lactone is an important chiral intermediate for the synthesis of D-calcium pantothenate and other pantothenic acid products. D-calcium pantothenate, also known as vitamin B5, is an essential vitamin for human life activities. It can be used as a feed additive and food additive. Its market size is huge and it is an important chiral pharmaceutical intermediate.
[0004] At present, there are generally three methods for preparing D-pantoic acid or D-pantoic acid lactone: (1) Chemical method 2,2-dimethyl-3-hydroxypropanal is chemically synthesized from formaldehyde and isobutyraldehyde, and then reacted with sodium cyanide or hydrocyanic acid to generate 2,2-dimethyl-3-cyanopropanediol, which is then hydrolyzed and esterified to obtain DL-pantoic acid lactone. Then, a chiral resolution agent such as D-camphorsulfonic acid, ephedrine, quinine, etc. is used to form a complex salt with DL-pantoic acid lactone. The D-type and L-type complex salts are separated by chemical treatment to obtain D-type pantoic acid lactone, and L-pantoic acid lactone and the resolution agent are recovered. This method is expensive due to the use of chiral resolution agents, and the chemical resolution process is cumbersome, which is not suitable for industrial production.
[0005] (2) Microbial fermentation method Compared with chemical methods, the microbial fermentation method uses renewable resources as raw materials and obtains D-pantothenate calcium in one step through fermentation of microorganisms that can synthesize pantothenic acid by themselves. In 2024, the team of Academician Zheng Yuguo of Zhejiang University of Technology systematically transformed Escherichia coli through metabolic engineering, significantly increasing the production of D-pantothenic acid, and achieving a D-pantothenic acid production of 86.03g / L in a 5-liter bioreactor. This result confirms the feasibility and economic potential of microbial fermentation to produce D-pantothenic acid.
[0006] It is reported that some domestic companies have achieved the production of D-calcium pantothenate by microbial fermentation. However, the product separation cost of this method is relatively high, and the product purity is lower than that of the chemical method. The production cost and product purity cannot be taken into account at the same time, and the product can only be used for purposes such as feed additives.
[0007] (3) Enzymatic splitting method Enzymatic resolution is a method of using enzymes in microorganisms or animal and plant tissues to perform selective asymmetric decomposition to separate the corresponding enantiomers from the racemate. D-pantoic acid lactone hydrolase can stereospecifically split DL-pantoic acid lactone to produce D-pantoic acid, and has the advantages of high stereoselectivity, mild reaction conditions, high catalytic efficiency, and environmental friendliness. In 2022, Zheng Pu's team at Jiangnan University heterologously expressed three D-pantoic acid lactone hydrolases from Fusarium in Pichia pastoris, and compared their enzyme activity and resolution efficiency. The final DL-pantoic acid lactone resolution rate reached 39.2%.
[0008] At present, the process of enzymatic splitting is relatively mature, but the amount of enzymes and enzyme activity produced by the existing fermentation process need to be further improved. With the continuous expansion of the market of D-panthenol and D-calcium pantothenate, the demand for D-pantoic acid lactone is increasing day by day. Exploring its efficient enzymatic splitting process has always been the goal pursued by researchers and producers. Therefore, it is imperative to develop an efficient, simple and low-cost process for preparing D-pantoic acid. . Summary of the invention
[0009] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing D-pantoic acid by enzymatic conversion.
[0010] In order to achieve the purpose of the present invention, the present invention will be implemented by adopting the following technical solutions.
[0011] A method for preparing D-pantoic acid by enzymatic conversion, characterized in that the method is to use 50 to 300 g / L of DL-pantoic acid lactone as a substrate, add a genetically engineered bacterial mutant having D-pantoic acid lactone hydrolase activity or a crude enzyme solution thereof, react at pH 6.5 to 8.0 and 25 to 37°C to generate D-pantoic acid and simultaneously obtain L-pantoic acid lactone.
[0012] As a preferred embodiment of the present invention, the genetically engineered bacterial mutant is a E. coli The invention relates to a genetically engineered bacterium with BL21 (DE3) as the host and plasmid pET-28a as the expression vector, carrying the D-pantoic acid lactone hydrolase mutant gene.
[0013] As a preferred embodiment of the present invention, the D-pantoic acid lactone hydrolase mutant gene can encode a D-pantoic acid lactone hydrolase mutant.
[0014] As a preferred embodiment of the present invention, the D-pantoic acid lactone hydrolase mutant is formed by mutation of one or more amino acid residue sites in the D-pantoic acid lactone hydrolase shown in the amino acid sequence SEQ ID NO.1, wherein the mutated amino acid residue sites are glutamic acid at position 80 mutated to isoleucine, asparagine at position 96 mutated to arginine, asparagine at position 197 mutated to valine, and aspartic acid at position 311 mutated to lysine.
[0015] As a preferred embodiment of the present invention, the crude enzyme solution is obtained by subjecting the whole cells of the genetically engineered bacterial mutant to ultrasonic or high-pressure homogenization.
[0016] As a preferred embodiment of the present invention, the substrate for the catalytic reaction is a pure DL-pantolactone or a crude DL-pantolactone with a content of more than 50%.
[0017] Application of a D-pantoic acid lactone hydrolase mutant or a genetically engineered bacterium expressing the D-pantoic acid lactone hydrolase mutant in the preparation of D-pantoic acid.
[0018] Compared with the prior art, the beneficial effect of the present invention is to provide an enzyme mutant and a new process method for preparing D-pantoic acid in an efficient, simple and low-cost manner.
[0019] (1) The present invention provides a mutant of D-pantoic acid lactone hydrolase. Specifically, the amino acid sequence of the D-pantoic acid lactone hydrolase mutant undergoes a specific mutation, thereby improving the enzyme catalytic activity and resolution efficiency.
[0020] (2) The present invention provides a new process method, using a mutant of D-pantoic acid lactone hydrolase to catalyze the substrate DL-pantoic acid lactone to generate D-pantoic acid, and simultaneously prepare L-pantoic acid lactone. The process method provided by the present invention has the advantages of being green, environmentally friendly, mild and efficient, greatly shortening the production cycle and reducing the production cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 A reaction scheme for the enzymatic conversion of D-pantoic acid. DETAILED DESCRIPTION
[0022] The technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0023] The experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the technical field, and can be specifically operated according to Molecular Cloning: A Laboratory Manual (Fourth Edition).
[0024] The Chinese full names, three-letter abbreviations and single-letter abbreviations of the 20 natural amino acids in the present invention are shown in Table 1: Table 1 Chinese full names, three-letter abbreviations and single-letter abbreviations of 20 natural amino acids Amino Acid Chinese Name Three letter abbreviation Single letter abbreviation Arginine Arg R Histidine His H Lysine Lys K Aspartic acid Asp D Glutamate Glu E Serine Ser S Threonine Thr T Asparagine Asn N Glutamine Gln Q Cysteine Cys C Glycine Gly G Proline Pro P Alanine Ala A Isoleucine Ile I Valine Val V Leucine Leu L Methionine Met M Phenylalanine Phe F Tyrosine Tyr Y In this patent, the term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example, E80I means that the amino acid E at position 80 is mutated to amino acid I, and so on.
[0025] The technical scheme of the present invention is based on the wild-type D-pantoic acid lactone hydrolase shown in SEQ ID NO: 1 (the amino acid sequence of the corresponding encoding gene is shown in SEQ ID NO: 1), and adopts the molecular biology method of whole plasmid PCR amplification to mutate the gene of D-pantoic acid lactone hydrolase, thereby changing the amino acid sequence of the enzyme, realizing the change of the enzyme structure and function, and then obtaining the D-pantoic acid lactone hydrolase mutant through a directed screening method.
[0026] Specifically, the preparation method comprises the following steps: (1) constructing the gene of the corresponding mutation site of D-pantoic acid lactone hydrolase (D-Lac) into the pET-28a expression vector to obtain a recombinant plasmid carrying the target enzyme gene. (2) transferring the recombinant plasmid into the host bacterial cell, preferably Escherichia coli BL21 (DE3), to obtain the corresponding engineered strain. The engineered strain is inoculated into LB resistance medium for culture, and then amplified and cultured at a certain ratio. After a certain period of culture, an inducer isopropyl-β-D-galactoside (IPTG) or lactose is added to induce the culture for a certain period of time. The bacterial cells are collected by centrifugation and crushed under high pressure. (3) Determination of the enzyme activity of the D-pantoic acid lactone hydrolase mutant.
[0027] Example 1 Construction of D-pantolactone hydrolase (D-Lac) mutant library This embodiment relates to a mining and mutation process of a D-pantoic acid lactone hydrolase, comprising the following steps: (1) Select template sequence The amino acid sequences of D-pantolactone hydrolase reported in the existing literature were searched, and their species origin, catalytic activity data (substrate spectrum, activity, enzymatic properties, etc.), protein structure information, key amino acids in the protein structure, and catalytic mechanism were sorted out. Five candidate template enzymes were obtained, and an evolutionary tree was constructed using MEGA 6 to analyze the relationship between the sequences. Combined with their catalytic data, the final candidate template enzyme was selected. Fusarium fujikuroi The D-Lac amino acid sequence was used as the template sequence.
[0028] (2) Prediction of D-Lac mutation hotspots The present invention obtains the protein structure prediction database of AlphaFold 2 F The three-dimensional structure prediction file of D-Lac further determined the mutation site through analysis of substrate channel, binding pocket and molecular docking structure. F D-pantolactone hydrolase Deinococcus radiodurans (PDB: 3E5Z) and Xanthomonas campestris pv. Campestris The protein crystal structure of the protein was coupled and aligned with that of the peptide (PDB: 3DR2), and 10 key amino acid mutation sites were finally identified, including H79, E80, N96, N146, N197, Y215, Q219, N252, D311, and A349.
[0029] (3) Site-directed mutagenesis The amino acids that play a key role in the enzyme catalytic efficiency were determined, and the degenerate codon NNK was used to design mutation primers. F D-Lac was used as a template pair derived from Fusarium fujikuroi The amino acid sequence of D-pantolactone hydrolase of the strain was subjected to site-directed mutagenesis. The sites of mutation for library construction were H79, E80, N96, N146, N197, Y215, Q219, N252, D311, and A349, and the beneficial mutation sites for improving enzyme activity were E80, N96, N197, and D311.
[0030] Example 2 Inducible expression of D-pantolactone hydrolase mutant The D-pantoic acid lactone hydrolase mutant constructed above is subjected to protein induction expression, and a crude enzyme solution is prepared. Specifically, the following steps are included: Activation strain: 10 μL pET28a- F The D-Lac mutant bacteria were inoculated into 5 mL of liquid LB medium containing 50 μg / mL kanamycin and activated in a shaker at 37°C and 220 r / min for 12 to 16 h.
[0031] Bacteria collection: Inoculate the activated bacterial solution into 50 mL LB liquid medium containing 50 μg / mL kanamycin at a 1% inoculum volume, and culture in a shaker at 37°C and 220 r / min for 3 h. 600 When the p-value was 0.6 to 0.8, the inducer IPTG was added at a final concentration of 0.2 mM, and the expression was induced for 12 to 14 h in a shaking incubator at 22°C and 180 r / min. The cultured bacterial solution was centrifuged at 6000 r / min for 5 min, the supernatant was discarded, and the bacterial precipitate was collected.
[0032] Preparation of crude enzyme solution: The collected bacteria were suspended in an appropriate amount of phosphate buffer (100 mM, pH 7.2), and crushed with an ultrasonic disruptor. Centrifuged again to obtain the intracellular supernatant and precipitate, and the supernatant was the crude enzyme solution of D-pantolactone hydrolase.
[0033] Example 3 Activity detection of D-pantolactone hydrolase mutants Enzyme activity determination method: Accurately weigh the wild-type and mutant crude enzyme solutions equivalent to 1 g of wet bacteria, take 40 mL of 25% (W / V) DL-pantolactone aqueous solution, control the temperature at 28 to 30°C for enzyme reaction, use 10% mass fraction ammonia water to control the pH at 6.8 to 7.2, react for 1 h, centrifuge at 10000 r / min for 10 min, take the supernatant to measure the optical rotation, and use HPLC to analyze the pantolactone content.
[0034] HPLC analysis conditions are: XDB-C18 HPLC column (250 mm × 4.6 mm, 5 μm) Mobile phase: 0.02 M potassium dihydrogen phosphate solution (adjust pH=3.0 with phosphoric acid): acetonitrile = 9:1 Flow rate: 1.0 mL / min, T = 30 °C, wavelength = 210 nm The relative enzyme activity results of the wild-type strain (WT) and different site combination mutants (M1-M6) are shown in Table 2.
[0035] Table 2 Relative enzyme activities of the wild-type strain (WT) and mutants with different site combinations (M1-M6) name Mutation site Relative enzyme activity WT - 100.0% M1 E80I 125.5% M2 N197V 113.2% M3 E80I-N197V 129.4% M4 E80I-N96R-N197V 134.2% M5 E80I-N197V-D311K 140.3% M6 E80I-N96R-N197V-D311K 168.7% From the results in Table 2, it can be seen that the enzyme activity of the mutant M6 (E80I-N96R-N197V-D311K) obtained in the present invention is significantly increased compared with the wild-type enzyme activity, and the enzyme activity is increased by 1.7 times.
[0036] Example 4 Preparation of D-pantoic acid by D-pantoic acid lactone hydrolase mutant 3 g of D-pantolactone hydrolase mutant M6 bacteria were added to a 1000 mL reaction system containing 50 g / L DL-pantolactone. The reaction temperature was 25°C and the pH was controlled at 6.5 with 10% ammonia water. The reaction was stirred for 3 hours and the resolution rate of DL-pantolactone was 49.1% according to HPLC analysis.
[0037] Example 5 Preparation of D-pantoic acid by D-pantoic acid lactone hydrolase mutant 5 g of D-pantolactone hydrolase mutant M6 bacteria were added to a 1000 mL reaction system containing 100 g / L DL-pantolactone. The reaction temperature was 28°C and the pH was controlled at 6.8 with 10% ammonia water. The reaction was stirred for 5 hours and the resolution rate of DL-pantolactone was 48.9% according to HPLC analysis.
[0038] Example 6 Preparation of D-pantoic acid lactone hydrolase mutant 8 g of D-pantolactone hydrolase mutant M6 bacteria were added to a 1000 mL reaction system containing 150 g / L DL-pantolactone. The reaction temperature was 37°C and the pH was controlled at 8.0 with 10% ammonia water. The reaction was stirred for 10 h and the resolution rate of DL-pantolactone was 47.6% according to HPLC analysis.
[0039] Example 7 Preparation of D-pantoic acid by D-pantoic acid lactone hydrolase mutant 10 g of D-pantolactone hydrolase mutant M6 bacteria were added to a 1000 mL reaction system containing 200 g / L DL-pantolactone. The reaction temperature was 32°C and the pH was controlled at 7.5 with 10% ammonia water. The reaction was stirred for 15 h and the resolution rate of DL-pantolactone was 48.2% according to HPLC analysis.
[0040] Example 8 Preparation of D-pantoic acid lactone hydrolase mutant 15 g of D-pantolactone hydrolase mutant M6 bacteria were added to a 1000 mL reaction system containing 300 g / L DL-pantolactone. The reaction temperature was 30°C and the pH was controlled at 7.0 with 10% ammonia water. The reaction was stirred for 24 h and the resolution rate of DL-pantolactone was 48.1% according to HPLC analysis.
[0041] The technical solution of the present invention is described in detail above in conjunction with the embodiments / drawings, but the present invention is not limited to the above technical solution. For ordinary technicians in this technical field, after knowing the contents recorded in the present invention, they can make several equivalent changes and substitutions without departing from the principle of the present invention. These equivalent changes and substitutions should also be regarded as belonging to the protection scope of the present invention.
Claims
1. A method for preparing D-pantoic acid by enzymatic conversion, characterized in that: The method is to use 50 to 300 g / L of DL-pantoic acid lactone as a substrate, add a genetically engineered bacterial mutant with D-pantoic acid lactone hydrolase activity or a crude enzyme solution thereof, react at pH 6.5 to 8.0 and 25 to 37°C to generate D-pantoic acid and simultaneously obtain L-pantoic acid lactone.
2. The method for preparing D-pantoic acid by enzymatic conversion according to claim 1, characterized in that: The genetically engineered bacterial mutant is based on Escherichia coli E. coli The invention relates to a genetically engineered bacterium with BL21 (DE3) as the host and plasmid pET-28a as the expression vector, carrying the D-pantoic acid lactone hydrolase mutant gene.
3. The method for preparing D-pantoic acid by enzymatic conversion according to claim 2, characterized in that: The D-pantoic acid lactone hydrolase mutant gene can encode a D-pantoic acid lactone hydrolase mutant.
4. The method for preparing D-pantoic acid by enzymatic conversion according to claim 3, characterized in that: The D-pantoic acid lactone hydrolase mutant is formed by mutation of one or more amino acid residue sites in the D-pantoic acid lactone hydrolase shown in the amino acid sequence SEQ ID NO.1, wherein the mutated amino acid residue sites are glutamic acid at position 80 mutated to isoleucine, asparagine at position 96 mutated to arginine, asparagine at position 197 mutated to valine, and aspartic acid at position 311 mutated to lysine.
5. The method for preparing D-pantoic acid by enzymatic conversion according to claim 1, characterized in that: The crude enzyme solution is obtained by subjecting the whole cells of the genetically engineered bacterial mutant to ultrasonic or high-pressure homogenization.
6. Use of a D-pantoic acid lactone hydrolase mutant or a genetically engineered bacterium expressing a D-pantoic acid lactone hydrolase mutant in the preparation of D-pantoic acid.