Method for synthesizing gal-g2-cnp by enzyme and application thereof

Abstract

The application discloses a method for synthesizing Gal-G2-CNP by using an enzyme and application of the method, wherein the Gal-G2-CNP is obtained by reacting G2-CNP, a sugar nucleotide donor UDP-Gal, under the action of an LgtE enzyme and an enzyme activator, at pH 5-9, at 25-42 DEG C, for 1-4 hours; compared with a complex chemical synthesis method, the enzyme synthesis method provided by the application improves conversion rate, and greatly simplifies a synthesis path and efficiency; and the application in preparation of an alpha-amylase detection kit has remarkable significance for promoting application and popularization of the alpha-amylase detection kit as an in-vitro diagnostic reagent.

Description

Technical Field

[0001] This invention relates to the field of amylase detection reagent synthesis technology, and in particular to a method for the enzymatic synthesis of Gal-G2-CNP and its application. Background Technology

[0002] Serum and urine α-amylase (AMY) assays have been used as diagnostic markers for acute pancreatitis for over half a century. A novel substrate for AMY assay, Gal-G2-α-CNP (2-chloro-4-nitrobenzene-α-galactose-maltodextrin), employs a chromophore release method. This involves attaching a chromophore—chloro-p-nitrophenol (CNP)—to a maltodextrin. When AMY hydrolyzes the substrate, the chromophore is released, and the rate of chromophore formation can be measured to calculate enzyme activity. This method is unaffected by endogenous glucose and does not require multiple enzymes, resulting in lower costs. Compared to G3-CNP (2-chloro-4-nitrobenzene-maltotriose) and other substrates, the use of galactose instead of glucose at the non-reducing end avoids interference from endogenous glucosidase. The application of this substrate as an in vitro diagnostic reagent has significant advantages and market potential.

[0003] Gal-G2-CNP, as a substrate for α-amylase hydrolysis, is widely used as a clinical in vitro diagnostic reagent for pancreatitis. However, its preparation in China is still in a critical stage, relying heavily on imports. The synthesis of Gal-G2-α-CNP generally employs chemical synthesis methods, but the optimized synthetic route is complex and has a low conversion rate, significantly limiting its application and promotion as an in vitro diagnostic reagent.

[0004] LgtE enzyme is a β-1,4-galactosyltransferase derived from Neisseriagonorrhoeae strain F62. Literature reports that LgtE enzyme can mediate the transfer of galactose to various LOS structures of the Neisseri series of low-fat sugars (LOSs) using UDP-Gal as an acceptor, demonstrating its catalytic activity utilizing long-chain liposuction substrates. β-1,4-galactosyltransferases are known to exist in organisms from various sources. Utilizing this enzyme to synthesize Gal-G2-CNP from the maltose derivative G2-CNP could achieve efficient synthesis of Gal-G2-CNP, which is of significant value and importance for the domestic production of the substrate reagent Gal-G2-CNP. Currently, no reports have been found regarding this enzymatic synthetic route. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the enzymatic synthesis of Gal-G2-CNP and its application, which solves the defects of existing technologies such as complex chemical synthesis routes and low conversion efficiency, so as to promote its application and popularization as an in vitro diagnostic reagent.

[0006] Therefore, the solution of the present invention is as follows:

[0007] A method for the enzymatic synthesis of Gal-G2-CNP involves reacting G2-CNP, glyconucleotide donor UDP-Gal, and LgtE enzyme and enzyme activator at pH 5–9 and 25–42°C for 1–4 h to obtain Gal-G2-CNP.

[0008] Furthermore, the amino acid sequence of the LgtE enzyme is shown in SEQ ID NO:2.

[0009] Furthermore, the nucleotide sequence encoding the LgtE enzyme is shown in SEQ ID NO:1.

[0010] Preferably, the preparation process of the LgtE enzyme includes the following steps:

[0011] S1. Based on codon optimization of the LgtE gene of the strain, the cloned LgtE gene was ligated into an expression plasmid to obtain a recombinant plasmid;

[0012] S2. Recombinant plasmids were transformed into competent E. coli cells, and engineered E. coli strains were obtained by screening.

[0013] S3. LgtE enzyme protein was obtained by fermentation and induced expression using engineered bacteria.

[0014] Furthermore, the reaction process is carried out at a pH of 7-8 and a temperature of 37°C.

[0015] Furthermore, the amount of the LgtE enzyme used is 1-2 g / L.

[0016] Furthermore, the amount of G2-CNP used is 40-80 mM.

[0017] Preferably, the amount of G2-CNP used is 40mM or 80mM.

[0018] Furthermore, the enzyme activator is Mn 2+ The dosage is 2-12 mM.

[0019] This invention also provides the application of Gal-G2-CNP obtained by the above method in the preparation of α-amylase detection kits. Gal-G2-CNP uses galactose instead of glucose at the non-reducing end, avoiding interference from endogenous glucosidase. The application of this substrate as an in vitro diagnostic reagent has obvious advantages and market potential.

[0020] Compared with the prior art, the beneficial effects of the present invention include, but are not limited to:

[0021] 1. This invention is the first to apply LgtE enzyme to the synthesis of Gal-G2-CNP substrates. This enzymatic synthesis has the advantages of high efficiency and safety. Compared with the existing complex chemical synthesis methods, it improves the conversion rate and greatly simplifies the synthesis route and efficiency.

[0022] 2. The Gal-G2-CNP synthesized by the enzymatic method described in this invention can be mass-produced industrially and applied to the preparation of α-amylase detection kits, which is of great significance for promoting its application and popularization as an in vitro diagnostic reagent. Attached Figure Description

[0023] Figure 1 The results show the protein expression and purification of the LgtE enzyme described in this invention.

[0024] Figure 2 The results show the effect of pH on the reaction conversion rate as described in this invention.

[0025] Figure 3 Mn as described in this invention 2+ Results of the effect on reaction conversion rate.

[0026] Figure 4 The results show the effect of substrate G2-CNP concentration on conversion rate as described in this invention.

[0027] Figure 5 This is the LC-MS mass spectrum of the reaction product Gal-G2-CNP of this invention.

[0028] Figure 6 This is the HPLC chromatogram of the reaction product Gal-G2-CNP described in this invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention.

[0030] This invention utilizes the β-1,4-galactosyltransferase function of LgtE enzyme to successfully catalyze the synthesis of Gal-G2-CNP, which is of significant value and importance for the domestic production of the substrate reagent Gal-G2-CNP. This invention employs an enzymatic synthesis method, using the applicant's self-made maltose derivative G2-CNP as the substrate and UDP-Gal as the donor, utilizing LgtE enzyme catalysis to greatly simplify the synthetic pathway and improve conversion efficiency, enabling industrial-scale mass production.

[0031] Our preliminary screening revealed that, apart from the LgtE enzyme used in this invention, which specifically acts on maltose and can efficiently catalyze the formation of Gal-G2-CNP, other enzymes had low conversion rates or low specificity.

[0032] In an embodiment of the present invention, a β-1,4-galactosyltransferase LgtE gene from Neisseriagonorrhoeae strain F62 is provided, which encodes the LgtE protein, a glycosyltransferase. The gene sequence of the protein is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:2.

[0033] In an embodiment of the present invention, a method for the efficient enzymatic catalytic synthesis of Gal-G2-CNP is provided, comprising the following steps:

[0034] I. Construction of genetically engineered bacteria for the production of glycosyltransferase LgtE

[0035] In actual implementation, the following specific processes are included:

[0036] 1. Construct a genetically engineered bacterium that produces the glycosyltransferase LgtE;

[0037] 2. Production of glycosyltransferase LgtE using genetically engineered bacteria.

[0038] Step 1, constructing the genetically engineered bacteria for producing glycosyltransferase, can utilize any existing known expression vector and its corresponding expression host bacteria. The process follows conventional transformation procedures or the instructions for use of commercial host bacteria to construct the genetically engineered bacteria containing the glycosyltransferase. As a preferred embodiment, *Escherichia coli* is selected as the expression host. Specifically, the steps include:

[0039] 1) The codons of the β-1,4-galactosyltransferase LgtE gene sequence of Neisseriagonorrhoeae were optimized. The nucleotide sequence of the gene is shown in SEQ ID No:1.

[0040] 2) The glycosyltransferase gene obtained by cloning above was ligated into the Escherichia coli expression plasmid pET15b(+) to obtain the recombinant plasmid pET15b-LgtE.

[0041] 3) The recombinant plasmid pET15b-LgtE was transformed into E. coli BL21(DE3) competent cells.

[0042] 4) The engineered strain of Escherichia coli E.coli BL21(DE3) / pET15b-LgtE was obtained through screening.

[0043] Step 2, which involves producing the glycosyltransferase LgtE using genetically engineered bacteria, includes the following steps:

[0044] a. Ferment Escherichia coli genetically engineered bacteria at 37℃ and induce at 20℃ for 9 hours;

[0045] b. Disrupt the induced E. coli genetically engineered bacteria and collect the supernatant;

[0046] c. Affinity chromatography using a Ni column was used to purify the target LgtE enzyme protein.

[0047] II. Efficient Synthesis of Gal-G2-CNP Using Enzymatic Methods

[0048] The specific process is as follows:

[0049] G2-CNP and glyconucleotide donor UDP-Gal, enzyme activator, and LgtE enzyme were added to the reaction vessel to carry out the reaction.

[0050] The reaction is carried out at pH 5–9 and temperature 25–42°C for 1–4 hours. Preferably, the conversion efficiency is highest at pH 7–8 and 37°C.

[0051] In one embodiment, the amount of LgtE enzyme used is 1-2 g / L.

[0052] In one embodiment, the amount of G2-CNP used is 40-80 mM; considering the conversion rate, 40 mM is preferred, and considering the production efficiency, 80 mM is preferred.

[0053] In one embodiment, the enzyme activator is Mn 2+ The dosage is 2-12 mM.

[0054] Under the conditions described above, the conversion rate can reach over 92% based on the amount of G2-CNP input; the purity of the product Gal-G2-CNP can be determined by HPLC detection and can reach over 99%.

[0055] In this scheme, the enzyme catalytic reaction formula is as follows:

[0056]

[0057] The above enzymatic synthesis method boasts advantages such as high catalytic efficiency, low cost, mild reaction conditions, high efficiency, and environmental safety. Compared to existing complex chemical synthesis methods (which yield less than 20% based on the first substrate), the conversion rate is significantly improved, reaching over 92%. Its application in the preparation of α-amylase detection kits is of great significance for promoting its use and widespread adoption as an in vitro diagnostic reagent.

[0058] Example

[0059] 1. Cloning, expression and purification of LgtE gene

[0060] After codon optimization of the β-1,4 galactosyltransferase LgtE gene sequence of Neisseria gonorrhoeae, the sequence SEQ ID No: 1 is as follows:

[0061] CATATGCAGAATCATGTGATTAGTCTGGCCAGTGCAGCAGAACGCCGCGCACATATTGCCGATACCTTTGGTAGTCGTGGCATTCCGTTTCAGTTTTTCGATGCACTGATGCCGAGTGAACGCCTGGAACAGGCAATGGCCGAACTGGTTCCGGGTCTGAGCGCCCATCCGTATCTGAGCGGCGTTGAAAAAGCATGCTTTATGAGCCATGCCGTTCTGTGGGAACAGGCCCTGGATGAAGGCCTGCCGTATATTGCCGTGTTTGAAGATGATGTGCTGCTGGGTGAAGGCGCCGAACAGTTTCTGGCCGAAGATACCTGGCTGGAAGAACGTTTTGATAAAGATAGTGCATTCATTGTGCGTCTGGAAACCATGTTTGCAAAAGTGATTGTGCGTCCGGATAAAGTGCTGAATTATGAAAATCGCAGCTTTCCGCTGCTGGAAAGCGAACATTGCGGTACCGCCGGCTATATTATTAGCCGTGAAGCAATGCGTTTCTTTCTGGATCGTTTTGCAGTTCTGCCGCCGGAACGCATTAAGGCCGTTGATCTGATGATGTTTACCTATTTCTTTGATAAGGAGGGCATGCCGGTGTATCAGGTGAGCCCGGCACTGTGCACCCAGGAACTGCATTATGCCAAATTTCTGAGCCAGAATAGTATGCTGGGCAGTGATCTGGAAAAAGATCGCGAACAGGGCCGCCGTCATCGCCGCTCACTGAAAGTGATGTTTGATCTGAAACGTGCACTGGGTAAATTTGGCCGTGAAAAGAAAAAACGTATGGAACGTCAGCGTCAGGCAGAACTGGAAAAAGTGTATGGTCGTCGTGTGATTCTGTTTAAATGAGGATCC

[0062] The protein sequence SEQ ID No:2 is:

[0063] MQNHVISLASAAERRAHIADTFGSRGIPFQFFDALMPSERLEQAMAELVPGLSAHPYLSGVEKACFMSHAVLWEQALDEGLPYIAVFEDDVLLGEGAEQFLAEDTWLEERFDKDSAFIVRLETMFAKVIVRPDKVLNYEN RSFPLLESEHCGTAGYIISREAMRFFLDRFAVLPPERIKAVDLMMFTYFFDKEGMPVYQVSPALCTQELHYAKFLSQNSMLGSDLEKDREQGRRHRRSLKVMFDLKRALGKFGREKKKRMERQRQAELEKVYGRRVILFK

[0064] DNA sequences were synthesized using a nucleotide synthesizer (BiolyticLabPerformance, Inc.) and inserted into the vector pet-15b(+), which was then transformed into *E. coli* BL21(DE3). DNA sequencing of positive clones confirmed that the target gene was correctly inserted into the vector, consistent with the design. This technique used *E. coli* Rosetta as the host strain, cultured in Luria-Bertani (LB) medium supplemented with ampicillin (50 μg / mL). When the OD of the cultured recombinant *E. coli* reached a certain level... 600 When the concentration is 0.6, add 0.2 mM IPTG and induce overnight at 20°C.

[0065] The bacterial culture was collected and centrifuged at 10,000 rpm for 15 min. The supernatant was discarded, and the weight of the bacterial sludge was recorded. A certain mass of bacterial sludge was taken and dissolved in 10 ml of buffer (20 mM Tris-HCl, H7.5 / 8.0 + 0.1 / 0.2% Triton) at a ratio of 1 g bacterial sludge to 10 ml buffer. Lysozyme (1:200) was added to the sample, mixed, and reacted on ice for 30 min. Then, the cells were sonicated and lysed at 4 °C using a Scientz JY92-IIN sonicator (working time: 3 seconds, interval: 4 seconds, 99 cycles, power not exceeding 200 W) and centrifuged (16,000 g, 4 °C, 30 min). This process was repeated at 16,000 g and 4 °C for 20 min. The supernatant and precipitate were collected and a sample was prepared for SDS-PAGE to examine protein expression. The supernatant was filtered through a 5 μm filter membrane, and the filtrate was purified by affinity purification using a nickel column after adding 10% 5 M NaCl.

[0066] The results showed that both soluble and inclusion body proteins of the LgtE enzyme were expressed. We purified the soluble protein by Ni column affinity chromatography, obtaining a highly pure LgtE protein with a size of approximately 35 kDa (e.g., ...). Figure 1 (As shown). Among them, Figure 1 Protein expression and purification of LgtE enzyme: 1. Cell lysis supernatant; 2. Cell lysis precipitate; 3. Protein permeation buffer; 4. Washing (20 mM imidazole); 5. Washing (50 mM imidazole); 6. Elution (200 mM imidazole).

[0067] 2. LgtE enzyme catalyzes the synthesis of Gal-G2-CNP from the substrate G2-CNP.

[0068] 2.1 Optimization of reaction conditions

[0069] 2.1.1 Reaction System and Materials

[0070] We selected a reaction temperature of 37℃, a pH value of 5-9, and Mn... 2+ The reaction was carried out with a concentration of 2-12 mM and a substrate concentration of 40-80 mM. The materials added are shown in Table 1.

[0071] Table 1: LgtE enzyme-catalyzed synthesis of Gal-G2-CNP reaction system

[0072]

[0073] 2.1.1 Effect of pH value on reaction conversion rate

[0074] The reaction was conducted using the materials listed in Table 1. We selected a reaction temperature of 37℃, a pH range of 5-9, and a reaction time of 1 hour for testing. Figure 2 The optimal pH for LgtE enzyme is found to be between 7 and 8.

[0075] 2.1.2Mn 2+ Effect of concentration on reaction conversion rate

[0076] The reaction was carried out according to the materials listed in Table 1. We selected a reaction temperature of 37℃, a pH value of 7, and Mn... 2+ The concentration was tested at 2–12 mM, and the reaction time was 1 hour. Figure 3 The optimal Mn can be obtained 2+ The concentration is 10 mM.

[0077] 2.1.3 Effect of G2-CNP concentration on reaction conversion rate

[0078] Following the reaction system listed in Table 1, we selected a reaction temperature of 37℃, a pH of 7, and a Mn content of... 2+The reaction system with a concentration of 10 mM was tested, and the reaction time was 1 hour. Different substrate concentrations ranging from 40 to 80 mM were selected for the reaction. Figure 4 It is known that 40 mM has a high conversion rate, but considering production efficiency, 80 mM M2-CNP can be selected as the optimal substrate concentration for the reaction.

[0079] 2.1.4 Optimal Conditions for Response

[0080] Based on the above single-factor verification experiment, the reaction system was selected as follows: pH 7-8, Mn 2+ When the concentration of G2-CNP was 10 mM and the concentration of G2-CNP was 40 mM, the conversion rate was calculated based on the substrate G2-CNP as the reaction time increased, and the results are shown in Table 2.

[0081] Table 2:

[0082] Conversion rate % 60.28 81.54 92.16 92.45 92.49

[0083] It is not difficult to conclude that the conversion rate increases with the extension of reaction time, but after 4 hours the conversion rate does not change much. Considering efficiency, the reaction time can be 3-4 hours, and the conversion rate can reach more than 92%.

[0084] 2.2 Reactions in the upper tank and post-processing and testing of the products

[0085] Take a 5L measuring cup that has been washed with deionized water and weigh the following solids according to Table 1 in the following order and specifications: UDP-Gal, G2-CNP, and MnCl2. After weighing, dissolve them in an appropriate amount of pure water (2.5L).

[0086] Upper chamber: Turn on the instrument and enter the SYS program. Remove the pH meter, rinse and wipe it clean with pure water, calibrate it, and insert it into the chamber. Pour the homogenized solution into a 5L reaction vessel (Bailun, BLB10-5M) through the injection port, set the rotation speed to 100 rpm, temperature to 37℃, and pH to 7-8 (±0.05); add LgtE enzyme (1 mg / mL, 1.5L) to initiate the Gal-G2-CNP synthesis reaction, with a final reaction volume of approximately 4L. Set the automatic alkali addition flow to add 1 MPa 9.0 Tris·HCl buffer, start the reaction, and perform TLC detection after 4 hours (developing solvent system: petroleum ether: ethyl acetate: methanol: water: acetic acid = 15:60:33:17:1). The results show that the reaction can effectively synthesize Gal-G2-CNP, i.e., the target product is generated, which was verified by mass spectrometry (LC-MS mass spectrometry as follows). Figure 5 As shown in the figure, the main peak contains the target product with a molecular weight of 682.334 (Gal-G2-CNPNa).

[0087] After removing the reaction solution, add 2L of ethanol, incubate at 4℃ for 2 hours to remove the enzyme, and then remove the ethanol by rotary evaporation. The ethanol-free sample is concentrated, filtered through a 0.45μm filter membrane, and purified by C18 column preparative HPLC (Jiangsu Zhirun Technology, Preparative HPLC 300). After removing acetonitrile from the purified sample, it is lyophilized to obtain approximately 100g of solid. The HPLC chromatogram after lyophilization is shown below. Figure 6 As shown, the peak elution time was 12.492 min, and the product purity was 99%.

[0088] This invention is not limited to the description in the specification and embodiments, and thus other advantages and modifications can be readily realized by those skilled in the art. Therefore, without departing from the spirit and scope of the general concept defined by the claims and their equivalents, this invention is not limited to the specific details, representative solutions and examples described herein.

Claims

1. A method for enzymatic synthesis of Gal-G2-CNP, characterized in that, Gal-G2-CNP was obtained by reacting G2-CNP, glyconucleotide donor UDP-Gal, and LgtE enzyme and enzyme activator at pH 7-8 and 25-42℃ for 2-4 hours. The amount of G2-CNP used is 40–80 mM, and the amount of LgtE enzyme used is 1–2 g / L; the enzyme activator is Mn. 2+ The dosage is 4–12 mM; the amino acid sequence of the LgtE enzyme is shown in SEQ ID NO:

2.

2. The method according to claim 1, characterized in that, The preparation process of the LgtE enzyme includes the following steps: S1. Based on strain LgtE Genetic codon optimization, obtained through cloning LgtE Genes are ligated into expression plasmids to obtain recombinant plasmids; S2. Recombinant plasmids were transformed into competent E. coli cells, and engineered E. coli strains were obtained by screening. S3. LgtE enzyme protein was obtained by fermentation and induced expression using engineered bacteria.

3. The method according to claim 1, characterized in that, The reaction temperature is 37°C.

4. The method according to claim 1, characterized in that, The dosage of G2-CNP is 40 mM.

5. The use of Gal-G2-CNP obtained by the method according to any one of claims 1-4 in the preparation of an α-amylase detection kit.

Images