Immobilization of an l-threonine aldolase mutant and its use in the synthesis of l-threo-para-methylthiophenylserine

Abstract

The application provides immobilization of L-threonine aldolase mutant and application thereof in synthesis of L-threo-p-methylsulfonyl phenylserine. Specifically, the application provides L-threonine aldolase immobilized enzyme using grafted polyethyleneimine silica nanoparticles (SNPs-PEI) as immobilization material. The application also provides a method for synthesizing L-threo-p-methylsulfonyl phenylserine from glycine and p-methylsulfonyl benzaldehyde using L-threonine aldolase immobilized enzyme as catalyst. The product obtained by the synthesis method has high optical purity and high reaction conversion rate, and provides a good foundation for industrial application of L-threonine aldolase mutant.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. Specifically, this invention relates to the immobilization of an L-threonine aldolase mutant and its application in the synthesis of L-threo-p-methylsulfonylphenylserine. Background Technology

[0002] L-threo-p-methylsulfonylbenzylserine is an important pharmaceutical intermediate, a key intermediate in the synthesis of the antibiotic florfenicol, and has great application value in the pharmaceutical field.

[0003] Currently, the main methods for industrial production of L-threo-p-methylsulfonylbenzylserine both domestically and internationally are divided into chemical methods and biocatalytic methods. The most common strategy in the chemical method is to use glycine and p-methylsulfonylbenzaldehyde as raw materials, employing copper sulfate as a catalyst, and utilizing the complexation of metal ions to condense them into a Schiff base. Then, a suitable reducing agent (such as hydrogen and palladium on carbon catalyst) is used to reduce the Schiff base to L-threo-p-methylsulfonylbenzylserine. However, the chemical method suffers from drawbacks such as low theoretical yield, complex processes, and the generation of large amounts of copper-containing wastewater and waste salts that pollute the environment. Biocatalysis, on the other hand, offers advantages such as mild conversion conditions, environmental friendliness, and strong stereoselectivity. The most significant characteristic of L-threonine aldolase is that, in the presence of the cofactor pyridoxal phosphate (PLP), it can catalyze the aldol condensation reaction between 4-methylsulfonylbenzaldehyde and glycine, directly forming an asymmetric C-C bond, thereby yielding L-threo-p-methylsulfonylbenzylserine.

[0004] Existing technology CN116254252A discloses a method for the asymmetric synthesis of L-threo-p-methylsulfonylphenylserine using a mutant N18S / Q39R / Y319L(SRL) obtained through genetic engineering, starting with wild-type aldolase from *Neptunomonas marine*. The substrate conversion rate is 80.1%, and the de value of L-threo-p-methylsulfonylphenylserine is >99%. This is the reaction with the highest conversion rate and the highest de value of the product reported to date.

[0005] In the above reaction, the feed ratio of glycine to 4-p-methylsulfonylbenzaldehyde is 10:1. Excess glycine increases production costs and is also detrimental to product purification; moreover, since the catalyst is a free enzyme, it cannot be reused.

[0006] In view of the aforementioned shortcomings of the prior art, there is a need in the art to optimize the reaction system and reduce the glycine feed ratio while ensuring the optical purity of the product. Simultaneously, suitable materials should be selected to immobilize L-threonine aldolase, enabling its reuse. Summary of the Invention

[0007] One of the objectives of this invention is to provide an immobilized L-threonine aldolase mutant.

[0008] Another object of the present invention is to provide a method for synthesizing L-threo-p-methylsulfonylbenzylserine using an immobilized L-threonine aldolase mutant.

[0009] In a first aspect of the present invention, a method for preparing an immobilized material is provided, wherein the immobilized material is silica nanoparticles grafted with polyethyleneimine (SNPs-PEI), the method comprising the steps of:

[0010] (S1) Provide silica nanoparticles, activate the silica nanoparticles to expose their surface hydroxyl groups, thereby obtaining hydroxylated silica nanoparticles (SNPs-OH);

[0011] (S2) The hydroxylated nano silica particles (SNPs-OH) are reacted with glycidyl ether trimethoxysilane to obtain epoxidized nano silica particles (SNPs-OH).

[0012] (S3) The epoxidized nano silica particles are reacted with polyethyleneimine to obtain polyethyleneimine-grafted silica nanoparticles (SNPs-PEI).

[0013] In another preferred embodiment, the silica nanoparticles are activated with hydrochloric acid in step (S1).

[0014] In another preferred embodiment, in step (S2), the hydroxylated nano silica particles react with glycidyl ether trimethoxysilane in a mass:volume ratio of 1:2-2:1 (preferably 1:1).

[0015] In another preferred embodiment, in step (S2), the hydroxylated nano-silica particles react with glycidyl ether trimethoxysilane at 60-80°C, preferably 70°C.

[0016] In another preferred embodiment, in step (S3), the epoxidized nano-silica particles react with polyethyleneimine in a mass ratio of 1:2.5-2.5:1 (preferably 1:1.25-1.25:1).

[0017] In another preferred embodiment, the epoxidized nano-silica particles in step (S3) react with polyethyleneimine at 20-30°C, preferably 25°C.

[0018] In a second aspect of the invention, an immobilization material for immobilizing L-threonine aldolase is provided, said material being silica nanoparticles grafted with polyethyleneimine, and said material being prepared by the method described in the first aspect of the invention.

[0019] In a third aspect of the present invention, a method for preparing immobilized L-threonine aldolase is provided, comprising the following steps:

[0020] Immobilized L-threonine aldolase is obtained by contacting the immobilized material as described in the second aspect of the present invention with L-threonine aldolase.

[0021] In another preferred embodiment, the mass ratio of the immobilized material to L-threonine aldolase is 5:1-50:1, more preferably 10:1-20:1.

[0022] In another preferred embodiment, the concentration of the L-threonine aldolase is 0.1-5 mg / mL, more preferably 0.5-0.6 mg / mL.

[0023] In another preferred embodiment, the immobilized material is contacted with L-threonine aldolase in HEPES buffer.

[0024] In another preferred embodiment, the immobilized material is contacted with L-threonine aldolase at 20-30°C, preferably 25-27°C.

[0025] In another preferred embodiment, the immobilized material is contacted with L-threonine aldolase in a shaker at 200 rpm.

[0026] In another preferred embodiment, the immobilized material is contacted with L-threonine aldolase for 1-10 hours, preferably 4-6 hours.

[0027] In a fourth aspect of the invention, an immobilized L-threonine aldolase is provided, the immobilized L-threonine aldolase comprising silica nanoparticles grafted with polyethyleneimine and L-threonine aldolase immobilized thereon.

[0028] In another preferred embodiment, the immobilized L-threonine aldolase is prepared by the method described in the third aspect of the present invention.

[0029] In a fifth aspect of the invention, the use of immobilized L-threonine aldolase as described in the fourth aspect of the invention is provided in the preparation of L-threo-p-methylsulfonylbenzeneserine.

[0030] In a sixth aspect of the invention, a method for preparing L-threo-p-methylsulfonylbenzylserine is provided, the method comprising the steps of: reacting 4-p-methylsulfonylbenzaldehyde and glycine as substrates in the presence of an immobilized L-threonine aldolase as described in the fourth aspect of the invention, thereby synthesizing L-threo-p-methylsulfonylbenzylserine.

[0031] In another preferred embodiment, the reaction system of the reaction comprises:

[0032] 4-p-methylsulfonylbenzaldehyde and glycine were used as substrates.

[0033] Pyridoxal 5-phosphate (PLP), as a coenzyme, and

[0034] Immobilized L-threonine aldolase as a catalyst.

[0035] In another preferred embodiment, the concentration of immobilized L-threonine aldolase in the reaction system is 1-3 g / L, preferably 1.5-1.85 g / L.

[0036] In another preferred embodiment, the molar ratio of 4-p-methylsulfonylbenzaldehyde to glycine is 1:1 to 1:10, more preferably 1:5.

[0037] In another preferred embodiment, the concentration of 4-p-methylsulfonylbenzaldehyde in the reaction system is 50-200 mmol / L, preferably 100 mmol / L.

[0038] In another preferred embodiment, the concentration of glycine in the reaction system is 300-1000 mmol / L, more preferably 500 mmol / L.

[0039] In another preferred embodiment, the reaction is carried out in a hydrophilic organic solvent, wherein the amount of organic solvent added is ≤20% of the total reaction volume.

[0040] In another preferred embodiment, the organic solvent is dimethyl sulfoxide (DMSO), N,N dimethylamide (DMF), or acetonitrile.

[0041] In another preferred embodiment, the organic solvent is acetonitrile.

[0042] In another preferred embodiment, the concentration of the acetonitrile is 10%-20%, more preferably 15%.

[0043] In another preferred embodiment, the reaction system further includes additives for regulating enzyme activity.

[0044] In another preferred embodiment, the additive used to regulate enzyme activity is an additive that either enhances or inhibits enzyme activity.

[0045] In another preferred embodiment, the additive used to regulate enzyme activity is Mn. 2+ K + Ba 2+ Zn 2+ Mg 2+ NH4 + Ca 2+ Fe 2+ Fe 3+ Cu 2+ Mn 2+ , or Ni 2+ .

[0046] In another preferred embodiment, the additive for regulating enzyme activity is selected from the group consisting of: Ba 2+ Ca 2+ K + Mn 2+ Ni 2 + , or combinations thereof.

[0047] In another preferred embodiment, the additive used to regulate enzyme activity is K. + .

[0048] In another preferred embodiment, the concentration of pyridoxal 5-phosphate (PLP) in the method is 10 μM-100 μM, preferably 20 μM-40 μM.

[0049] In another preferred embodiment, the reaction is carried out at pH 5.0-9.0, more preferably pH 7.5-9.0, and even more preferably pH 8.5.

[0050] In another preferred embodiment, the reaction temperature conditions in the method are: 25℃-42℃, preferably 40℃.

[0051] In another preferred embodiment, the amount of immobilized enzyme in the reaction system is 1.85 g / L, the amount of 4-p-methylsulfonylbenzaldehyde is 100 mmol / L, the amount of glycine is 500 mmol / L, and the amount of 5'-pyridoxal phosphate is 20 μmol / L.

[0052] In another preferred embodiment, the reaction temperature is 37°C; the reaction solvent is HEPES buffer solution with 15% acetonitrile added; the reaction pH is 9.0; and 1 mM K+ is added to the reaction system. + The reaction time was 140 min.

[0053] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0054] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0055] Figure 1 The image shows the SDS-PAGE electrophoresis results after purification of threonine aldolase SRL.

[0056] Figure 2 The Fourier transform infrared spectra of the nanoparticles before and after immobilization are shown.

[0057] Figure 3 The figure shows the optimal organic solvents required for the synthesis of L-threo-p-methylsulfonylbenzylserine.

[0058] Figure 4 The figure shows the optimal temperature required for the synthesis of L-threo-p-methylsulfonylbenzylserine.

[0059] Figure 5 The diagram shows the optimal pH values ​​required for the synthesis of L-threo-p-methylsulfonylbenzylserine.

[0060] Figure 6 The figure shows the optimal PLP concentration required for the synthesis of L-threo-p-methylsulfonylbenzylserine.

[0061] Figure 7 The diagram shows the optimal metal ion required for the synthesis of L-threo-p-methylsulfonylbenzylserine.

[0062] Figure 8 The graph shows the effect of the initial concentration of added protein on the immobilization yield.

[0063] Figure 9 The figure shows the effect of the initial concentration of added protein on the activity of the immobilized enzyme.

[0064] Figure 10 The graph shows a comparison of the thermal stability of immobilized and free enzymes.

[0065] Figure 11 The figure shows a comparison of the acid-base stability of immobilized and free enzymes.

[0066] Figure 12 The graph shows the results of the recycling of immobilized enzymes. Detailed Implementation

[0067] Through extensive and in-depth research, the inventors have, for the first time, developed an immobilization method for an L-threonine aldolase mutant and its application in the synthesis of L-threo-p-methylsulfonylphenylserine. Through screening, this invention discovered that using polyethyleneimine-grafted silica nanoparticles (SNPs-PEI) as the immobilization material to prepare an immobilized L-threonine aldolase exhibits high enzyme activity recovery, high acid-base stability, and high thermal stability. Its application in the synthesis of L-threo-p-methylsulfonylphenylserine yields a product with high optical purity and high reaction conversion rate. Based on this, this invention was completed.

[0068] the term

[0069] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0070] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0071] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0072] threonine aldolase

[0073] Threonine aldolases are a type I folding enzyme dependent on pyridoxal 5-phosphate, which can further catalyze the condensation of aldehydes and glycine to produce β-hydroxy-α amino acid derivatives with two chiral centers.

[0074] The threonine aldolase L-Tanem, derived from Neptunomonas marine, has a broad substrate spectrum, capable of catalyzing a range of aldehydes and α-amino acid substrates to generate corresponding β-hydroxy-α-amino acid compounds. Mutations at specific sites on the wild-type threonine aldolase yielded mutants with significantly enhanced activity. The threonine aldolase of this invention is preferably a variant of L-TAnem with the N18S / Q39R / Y319L mutation, specifically based on the sequence shown in SEQ ID NO:2 (wild-type), where N at position 18 is mutated to S, Q at position 39 is mutated to R, and Y at position 319 is mutated to L.

[0075] The wild-type L-TAnem nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0076] SEQ ID NO:1:

[0077]

[0078] SEQ ID NO:2:

[0079] MASNDSCIEDTVSFTSDNIAAAAPEIVQAMAQACQGNAQPYGGDALTQNVEAQLKAIFECDLQLFLVPTGSAANAISLAALTPPWGAILCHQESHINNDECGAPEFFTAGAKLIAVAGTHGKLDPQALTQAARNKRGDVHSVEPTTVSITQATEVGSIYALDELNEIGQICRNEGLKL HMDGARFANALSALGCTPAEMTWKAGVDVLSFGATKNGSLCAEAIILFDKSYAQEIAFRRKRGGHLLSKMRFLSAQMHAYLADDLWLTNARHANLMAARLAAGLSALSRVSLIAPTESNIIFCRMPTKMIAALQQQGFQFYHDRWGDGIVRLVTSFATTQAQVDTFIAAAAQLNQNTD

[0080] Immobilized L-threonine aldolase

[0081] As used in this invention, the terms "immobilized L-threonine aldolase" or "immobilized enzyme" have the same meaning and both refer to the immobilized L-threonine aldolase of the fourth aspect of this invention.

[0082] The immobilized L-threonine aldolase of this invention utilizes polyethyleneimine (PEI), a polycationic polymer containing numerous amino (-NH2) groups. These groups can be protonated and become positively charged in aqueous solution, while L-threonine aldolase is negatively charged at pH 8.0. Therefore, the enzyme molecules adsorb onto the positively charged PEI-grafted silica surface through electrostatic interactions. This electrostatic adsorption is a rapid and mild binding method that does not affect enzyme activity. Simultaneously, the immobilization process is mild, preserving the enzyme's native structure or activity and avoiding the influence of strong acids, strong bases, or organic solvents. The use of nano-silica promotes rapid and uniform distribution of enzyme molecules on the carrier surface, increasing the surface area of ​​contact between the enzyme molecules and the substrate, thereby improving the activity, loading capacity, and stability of the immobilized enzyme.

[0083] This invention provides a method for preparing immobilized L-threonine aldolase, comprising the following steps:

[0084] (1) Obtain L-threonine aldolase solution;

[0085] (2) Prepare immobilized carriers and graft polyethyleneimine-containing silica nanoparticles;

[0086] (3) The immobilization vector SNPs-PEI was mixed with L-threonine aldolase solution to obtain immobilized L-threonine aldolase.

[0087] Preferably, the L-threonine aldolase solution in step (1) is obtained by breaking down engineered bacteria expressing L-threonine aldolase.

[0088] Preferably, in step (2), the silica nanoparticles are first activated with HCl to expose the hydroxyl groups on their surface to obtain hydroxylated silica nanoparticles (SNPs-OH), then glycidyl ether trimethoxysilane is added and refluxed to obtain epoxidized silica nanoparticles (SNPs-EPO), and finally polyethyleneimine is grafted to obtain SNPs-PEI.

[0089] Preferably, during the fixation in step (3), the mixing ratio of silica nanoparticles and L-threonine aldolase solution is: 50 mg of SNPs-PEI is mixed with 5 mL of L-threonine aldolase solution with a concentration of 0.5 mg / mL.

[0090] More preferably, SNPs-PEI and free L-threonine aldolase solution are mixed at a ratio of 50 mg nanoparticles to 5 mL of 0.5 mg / mL L-threonine aldolase solution, and then added to an appropriate amount of HEPES buffer and immobilized in a shaker at 25 °C and 200 rpm for 4 h.

[0091] application

[0092] The L-threo-aldolase immobilized enzyme of the present invention can catalyze a condensation reaction to generate L-threo-p-methylsulfonylbenzaldehyde using glycine and p-methylsulfonylbenzaldehyde as substrates and pyridoxal phosphate as a coenzyme.

[0093] The present invention also provides a composition containing an effective amount of the immobilized enzyme of the present invention, and a food- or industrially acceptable carrier or excipient. Such carriers include (but are not limited to): water, buffer solutions, glucose, glycerol, ethanol, and combinations thereof.

[0094] The composition may also contain substances that regulate the activity of the threonine aldolase of the present invention. Any substance that enhances enzyme activity is acceptable.

[0095] After obtaining the threonine aldolase of the present invention, those skilled in the art can conveniently use the enzyme to perform in vitro enzymatic synthesis, especially for the conversion of substrates 4-p-methylsulfonylbenzaldehyde and glycine.

[0096] As a preferred embodiment of the present invention, a method for forming L-threo-methanesulfonylphenylserine is also provided, the method comprising: treating a substrate to be transformed with the threonine aldolase described in the present invention, the substrate comprising 4-p-methylsulfonylbenzaldehyde and glycine.

[0097] The main advantages of this invention include:

[0098] 1) This invention screened immobilization vectors suitable for L-threonine aldolase mutants, providing immobilized enzymes with high enzyme activity recovery, high acid-base stability and high thermal stability, which is beneficial for the reuse of enzymes in industrial production.

[0099] 2) This invention optimizes the reaction system for the synthesis of L-threo-p-methylsulfonylbenzylserine. Compared with existing methods, the method of this invention has higher raw material utilization, higher product yield and chiral purity; compared with chemical catalysis, the process is simpler and less polluting.

[0100] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0101] Main instruments, materials and reagents: Silica nanoparticles were purchased from Jiangsu Xianfeng Nanotechnology; Polyethyleneimine was purchased from Ron Reagent; Glycine, 4-p-methylsulfonylbenzaldehyde, and pyridoxal 5-phosphate were all commercially available analytical grade.

[0102] Example 1: Expression and purification of threonine aldolase

[0103] A recombinant expression plasmid containing the L-threonine aldolase gene was heat-shock transformed into *E. coli* BL21(DE3) competent cells. The gene was derived from *Neptunomonas marine*, accession number WP_127693843.1, and mutated to N18S / Q39R / Y319L. Gene expression and protein purification were performed in *E. coli*. When the recombinant bacterial concentration reached OD600 of 0.6-0.9, IPTG was added to a final concentration of 0.5 mM, and the cells were induced overnight at 25°C and 220 rpm. The cells were collected by centrifugation and resuspended in 50 mM HEPES buffer (pH 8.0, 500 mM NaCl, 50 mM imidazole). 250 mL of cultured cells were resuspended in 40 mL of buffer and disrupted using a high-pressure cell disruptor (4–6 °C, 700 Pa). The cell disruption buffer was then centrifuged at 12,000 rpm for 45 min (4 °C). The supernatant was collected, and the protein was purified using a Ni-NTA column affinity chromatography. Impurities were eluted with 50 mM HEPES buffer (pH 8.0, 500 mM NaCl, 50 mM imidazole). Finally, the target protein was eluted with 50 mM HEPES buffer (pH 8.0, 500 mM NaCl, 250 mM imidazole). The eluted protein was concentrated and desalted to obtain the purified protein. The purified protein was stored in 50 mM HEPES buffer (pH 8.0) and analyzed by 12% SDS-PAGE. Protein concentration was determined using a Bradford protein assay kit (Shanghai Sangon Biotech). Results are as follows: Figure 1 As shown in the figure. The results indicate that a clear band was obtained at 41.9 kDa, suggesting that the target protein SRL has been purified.

[0104] Example 2: Construction of the immobilized vector SNPs-PEI

[0105] 10g of commercially available nano-silica particles (SNPs) were added to 200mL of 1mol / L HCl solution, sonicated for 30min, and magnetically stirred for 36h to obtain hydroxylated nano-silica particles (SNPs-OH). The nanoparticles were placed in a three-necked flask, and 100mL of anhydrous toluene was added. The mixture was then sonicated to form a homogeneous solution. 10mL of glycidyl ether trimethoxysilane was added dropwise, and the mixture was refluxed at 70℃ for 24h to obtain epoxidized nano-silica particles. 2g of the epoxidized nanoparticles were weighed and placed in a 50mL Erlenmeyer flask, and 4mL of HEPES buffer solution containing PEI was added. The reaction system was then incubated in a water bath at 25℃ and 170rpm for 24h to allow PEI to graft onto the surface of the nano-silica particles, resulting in polyethyleneimine-grafted silica nanoparticles (SNPs-PEI). Material characterization results are as follows: Figure 2As shown in the figure, the SNPs-PEI was successfully constructed.

[0106] Example 3: Screening of the optimal immobilization carrier

[0107] Accurately weigh 0.2 g of epoxy resins Lx-1000EA, Lx-1000EP, Lx-EP120, Lx-1000ME, Lx-1000IDA, Lx-1000HA, SNPs, and SNPs-PEI, respectively, and add them to HEPES buffer (50 mmol / L, pH 8.0) containing an appropriate amount of purified protein. Incubate the immobilization system in a 25°C water bath shaker for 4 h. After incubation, wash three times with buffer to obtain the immobilized enzyme. The collected immobilized enzyme was then dissolved in HEPES buffer (20 mmol / L, pH 8.0) to form a homogenized solution. The immobilization yield (defined as the ratio of the amount of enzyme protein immobilized on the carrier to the initial total amount of enzyme protein added) and the immobilized enzyme activity were measured (the activity of the free enzyme was defined as 100%). All experimental data were measured three times and averaged. The results are shown in Table 1 below. The results indicate that the immobilization yield and immobilized enzyme activity were highest when using the SNPs-PEI carrier. Therefore, SNPs-PEI was subsequently selected as the immobilization carrier.

[0108] carrier type Immobilized enzyme activity (%) Immobilization yield (%) Enzyme load (mg / g) Lx-1000EA 58.5 65.3 32.7 Lx-1000EP 41.3 82.3 41.2 Lx-EP120 61.3 79.2 39.6 Lx-1000ME 45.6 72.8 36.4 Lx-1000IDA 38.5 78.3 39.2 Lx-1000HA 54.6 85.2 42.6 SNPs 88.8 73.2 36.6 SNPs-PEI 94.0 91.0 45.5

[0109] Example 4: Effect of different protein concentrations on immobilization yield

[0110] 50 mg of the SNPs-PEI nanocarrier was weighed and added to 5 mL of HEPES buffer (50 mmol / L, pH 8.0) containing 0.5 mg, 1 mg, 1.5 mg, and 2.5 mg of L-threonine aldolase, respectively. The mixture was incubated at 25°C for 4 h. After incubation, the unfixed free enzyme was washed away with buffer to obtain immobilized enzymes with different enzyme loadings. The protein content was determined by the Coomassie Brilliant Blue assay, and the immobilization yield was calculated (the yield of the enzyme immobilization process is defined as the ratio between the amount of enzyme protein immobilized on the carrier and the initial total amount of enzyme protein added). The detection results are as follows: Figure 8 As shown, the results indicate that the immobilization yield was highest when the initial enzyme concentration was 0.6 mg / mL.

[0111] Example 5: Effect of different reaction systems on the conversion rate and de value of L-threo-p-methylsulfonylphenylserine

[0112] 1. Determination of optimal pH, optimal temperature, optimal PLP concentration, optimal organic solvent type and concentration, and different metal ions for L-threonine aldolase (SRL).

[0113] The enzyme activity of SRL was investigated under different types and concentrations of organic solvents. The selected organic solvents were DMSO (10%-30%, in 5% gradients), DMF (10%-30%), and acetonitrile (10%-30%, in 5% gradients). The reaction system consisted of 20 μg / mL purified enzyme, 100 mM 4-p-methylsulfonylbenzaldehyde, 1 M glycine, and 50 μM PLP, with the final reaction volume adjusted to 1000 μL using 50 mM HEPES buffer. The yield and purity of the product L-threo-p-methylsulfonylbenzylserine were measured at 25 °C, and the relative enzyme activity under different organic solvent types and concentrations was calculated. Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of L-threo-p-methylsulfonylbenzylserine per minute under the above reaction conditions, which is defined as 1 activity unit (U). The results are as follows: Figure 3 As shown.

[0114] The results showed that SRL exhibited high activity under conditions of 10% DMSO, 10% acetonitrile, and 15% acetonitrile.

[0115] The enzyme activity of SRL was investigated at different temperatures (25℃, 30℃, 37℃, 40℃, and 42℃) in a 1000 μL volume. The reaction system consisted of 20 μg / mL purified enzyme, 100 mM 4-p-methylsulfonylbenzaldehyde, 1 M glycine, and 50 μM PLP, with the final reaction volume adjusted to 1000 μL using 50 mM HEPES buffer. After the reaction, the yield and purity of the product L-threo-p-methylsulfonylbenzylserine were measured, and the relative enzyme activity under different temperature conditions was calculated. The results are as follows: Figure 4 As shown in the figure. The results indicate that SRL exhibits high activity at 37℃ and 40℃.

[0116] The enzyme activity of SRL was investigated at different pH values ​​(5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0). The reaction system consisted of 20 μg / mL purified enzyme, 100 mM 4-p-methylsulfonylbenzaldehyde, 1 M glycine, and 50 μM PLP, with the final reaction volume adjusted to 1000 μL using 50 mM HEPES buffer. The yield and purity of the product L-threo-p-methylsulfonylbenzylserine were measured at 25 °C, and the relative enzyme activity under different pH conditions was calculated. The results are shown below. Figure 5 As shown in the figure. The results indicate that SRL exhibits high activity at pH 8.0, 8.5, and 9.0.

[0117] The enzyme activity of SRL was investigated at different PLP concentrations (10 μM, 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM). The reaction system consisted of 20 μg / mL purified enzyme, 100 mM 4-p-methylsulfonylbenzaldehyde, 1 M glycine, and different concentrations of PLP. The final reaction volume was adjusted to 1000 μL using 50 mM HEPES buffer. The yield and purity of the product L-threo-p-methylsulfonylbenzylserine were measured at 25 °C, and the relative enzyme activity under different PLP concentrations was calculated. The results are as follows: Figure 6 As shown in the figure. The results indicate that SRL exhibits high activity at PLP concentrations of 20 μM, 30 μM, and 40 μM.

[0118] Different types of metal ions (Mn) 2+ K + Ba 2+ Ni 2+ Zn 2+ Cu 2+ Mg 2+ NH4 + Ca 2+ Fe 2+ Fe 3 + The enzyme activity of SRL was investigated using the following reaction system: 20 μg / mL purified enzyme, 100 mM 4-p-methylsulfonylbenzaldehyde, 50 μM PLP, 1 M glycine, and 1 mM of different metal ions. The final reaction volume was adjusted to 1000 μL using 50 mM HEPES buffer. The yield and purity of the product L-threo-p-methylsulfonylbenzylserine were measured at 25 °C, and the relative enzyme activity under different metal ion conditions was calculated. The results are as follows: Figure 7 As shown. The results indicate that SRL, with the addition of Ba metal ions... 2+ K + Ca 2+ It exhibits high activity.

[0119] 2. Orthogonal reaction reduces the amount of glycine substrate to be fed.

[0120] An orthogonal experiment was set up with five factors: Factor 1 is the type of organic solvent, with three levels of 10% DMSO, 10% acetonitrile, and 15% acetonitrile; Factor 2 is temperature, with two levels of 37℃ and 40℃; Factor 3 is pH, with three levels of 8.0, 8.5, and 9.0; Factor 4 is PLP concentration, with three levels of 20μM, 30μM, and 40μM; and Factor 5 is different types of metal ions, with three levels of 1mM Ba... 2+ 1mM K + 1mM Ca 2+An orthogonal system was established based on the above factors and levels. Reactions were carried out at feed ratios of 4-p-methylsulfonylbenzaldehyde and glycine of 1:1, 1:3, and 1:5. The yield and purity of the product L-threo-p-methylsulfonylbenzylserine were determined. Range analysis of the product purity was performed on the orthogonal results to determine the optimal levels of each factor. Under the feed ratio of 4-p-methylsulfonylbenzaldehyde and glycine of 1:5, the optimal reaction conditions were 15% acetonitrile, 20 μM PLP, 37℃, pH 9.0, and 1 mM K. + At this time, the conversion rate and product purity of the reaction are the highest.

[0121] Example 6: Determination of the activity of immobilized L-threonine aldolase

[0122] In a 1 mL reaction system (360 μg / mL immobilized enzyme, 100 mM 4-p-methylsulfonylbenzaldehyde, 20 μM PLP, 500 mM glycine, 1 mM K), + 15% (v / v) acetonitrile was added, and the final reaction volume was adjusted to 1000 μL using 50 mM HEPES buffer (pH 9.0). The reaction was carried out at 37 °C for 30 min. Then, 50 μL of the reaction solution was added to 950 μL of acetone to terminate the reaction. The supernatant was collected by centrifugation and analyzed by HPLC. Free enzyme activity under the same conditions was defined as 100%, and the measured immobilized enzyme activity was the relative activity. The detection results are as follows: Figure 9 As shown, the results indicate that the immobilized enzyme activity was highest when the initial protein concentration was 0.5 mg / mL, reaching 94% of the initial enzyme activity.

[0123] Thermal stability: Appropriate amounts of immobilized and free enzymes were incubated at 4, 25, 40, 50, and 60 °C for 4 hours, respectively. After cooling to room temperature, the remaining activity was measured to investigate the tolerance of the immobilized and free enzymes to different temperatures. Enzyme activity measured at 25 °C was defined as 100%, and enzyme activities measured at other incubation temperatures were relative activities. The test results are as follows: Figure 10 As shown, the results indicate that the thermostability of the immobilized enzyme is increased compared to that of the free enzyme.

[0124] Acid-base stability: Appropriate amounts of immobilized and free enzymes were incubated at pH 6.0, 7.0, 7.5, 8.0, 8.5, and 9.0 for 4 hours, respectively. Residual activity was measured to investigate the acid and alkali tolerance of the immobilized and free enzymes. Enzyme activity measured at pH 8.0 was defined as 100%, and enzyme activities measured at other pH values ​​were defined as relative activities. The test results are as follows: Figure 11 As shown, the results indicate that the acid-base stability of the immobilized enzyme is increased compared to that of the free enzyme.

[0125] Recyclability: Immobilized enzymes with measured activity were collected by centrifugation, washed with HEPES buffer (50 mmol / L, pH 8.0) to remove residual substrate, and then the enzyme activity was measured again. This process was repeated for eight cycles to investigate the reusability of the immobilized enzyme. The enzyme activity measured in the first cycle was defined as 100%, and the enzyme activity measured in the remaining cycles was defined as relative activity. The test results are as follows: Figure 12 As shown, the results indicate that after 8 cycles, the immobilized enzyme still retains 89.4% of its initial activity.

[0126] Example 7: Scale-up of the reaction system for the synthesis of L-threo-p-methylsulfonylbenzylserine using immobilized enzymes.

[0127] The reaction conditions were as follows: 360 mg immobilized enzyme, 100 mM 4-p-methylsulfonylbenzaldehyde, 500 mM glycine, 20 μM PLP, and 15% (v / v) acetonitrile. The final reaction volume was brought to 1 L using HEPES (pH 9.0) buffer. The reaction was incubated at 25 °C with shaking at 250 rpm for 140 min. After the reaction, 50 μL of the reaction solution was added to 950 μL of acetone to terminate the reaction. The supernatant was collected by centrifugation and analyzed by HPLC. The results showed that the conversion rate of L-threo-p-methylsulfonylbenzylserine by L-threonine aldolase reached 74.8%, with a de value greater than 99%. Under these conditions, the conversion rate of L-threo-p-methylsulfonylbenzaldehyde and 1 M glycine by L-threonine aldolase reached 82.6%, with a de value greater than 99%.

[0128] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing an immobilized material, characterized in that, The immobilization material is silica nanoparticles grafted with polyethyleneimine (SNPs-PEI), and the method includes the following steps: (S1) Provide silica nanoparticles, activate the silica nanoparticles to expose their surface hydroxyl groups, thereby obtaining hydroxylated silica nanoparticles (SNPs-OH); (S2) The hydroxylated nano silica particles (SNPs-OH) are reacted with glycidyl ether trimethoxysilane to obtain epoxidized nano silica particles (SNPs-OH). (S3) The epoxidized nano silica particles are reacted with polyethyleneimine to obtain polyethyleneimine-grafted silica nanoparticles (SNPs-PEI).

2. An immobilization material for immobilizing L-threonine aldolase, characterized in that, The material is silica nanoparticles grafted with polyethyleneimine, and the material is prepared by the method as described in claim 1.

3. A method for preparing immobilized L-threonine aldolase, characterized in that, Includes the following steps: The immobilized material as described in claim 2 is contacted with L-threonine aldolase to obtain immobilized L-threonine aldolase.

4. An immobilized L-threonine aldolase, characterized in that, The immobilized L-threonine aldolase comprises silica nanoparticles grafted with polyethyleneimine and L-threonine aldolase immobilized thereon.

5. The immobilized L-threonine aldolase as described in claim 4, characterized in that, The immobilized L-threonine aldolase was prepared by the method described in claim 3.

6. The use of the immobilized L-threonine aldolase as described in claim 4 or 5 in the preparation of L-threo-p-methylsulfonylbenzeneserine.

7. A method for preparing L-threo-p-methylsulfonylphenylserine, characterized in that, The method includes the step of: reacting 4-p-methylsulfonylbenzaldehyde and glycine as substrates in the presence of the immobilized L-threo-p-methylsulfonylbenzylserine as described in claim 4 or 5, thereby synthesizing L-threo-p-methylsulfonylbenzylserine.

8. The method as described in claim 7, characterized in that, The reaction system of the reaction includes: 4-p-methylsulfonylbenzaldehyde and glycine were used as substrates. Pyridoxal 5-phosphate (PLP), as a coenzyme, and Immobilized L-threonine aldolase as a catalyst.

9. The method as described in claim 8, characterized in that, In the reaction system, the concentration of immobilized L-threonine aldolase is 1-3 g / L, preferably 1.5-1.85 g / L.

10. The method as described in claim 8, characterized in that, The molar ratio of 4-p-methylsulfonylbenzaldehyde to glycine is 1:1 to 1:10, preferably 1:5.

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