Beta-1, 4-galactosyl transferase mutant and application thereof

Abstract

The invention discloses a beta-1, 4-galactosyl transferase mutant and application thereof, and relates to the technical field of gene engineering and enzyme engineering, and the beta-1, 4-galactosyl transferase mutant at least has one of I13W, L130R and F157W mutations on the basis of beta-1, 4-galactosyl transferase AaGT25; the amino acid sequence of the AaGT25 is as shown in SEQ ID NO. 1. The invention also discloses an application of the mutant in catalyzing a glycosylation reaction. By combining bioinformatics, molecular docking and site-directed mutagenesis technologies, the catalytic performance of the beta-1, 4-galactosyl transferase is efficiently improved, and the beta-1, 4-galactosyl transferase has great industrial application value.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and enzyme engineering, and in particular to a beta-1,4-galactosyltransferase mutant and application thereof. Background Art

[0002] β-1,4-Galactosyltransferases are key enzymes that catalyze the transfer of a galactosyl group from UDP-galactose (UDP-Gal) to an N-acetylglucosamine (GlcNAc) acceptor molecule, forming a β-1,4-glycosidic bond. This reaction is widely involved in the biosynthesis of glycoproteins, glycolipids, and polysaccharides, and has important applications in medicine (e.g., antibody drug glycosylation modification), food (e.g., oligosaccharide synthesis), and biomaterials. However, naturally derived β-1,4-Galactosyltransferases generally suffer from low catalytic efficiency and poor stability, limiting their industrial application. Summary of the Invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a β-1,4-galactosyltransferase mutant and its application.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: providing a β-1,4-galactosyltransferase mutant, which has at least one of the I13W, L130R and F157W mutations based on the β-1,4-galactosyltransferase AaGT25; the amino acid sequence of the β-1,4-galactosyltransferase AaGT25 is shown in SEQ ID NO.1.

[0005] Furthermore, the mutant I13W has an amino acid sequence as shown in SEQ ID NO.2.

[0006] Furthermore, the mutant L130R has an amino acid sequence as shown in SEQ ID NO.3.

[0007] Furthermore, the mutant F157W has an amino acid sequence as shown in SEQ ID NO.4.

[0008] The present invention also provides the use of the above-mentioned β-1,4-galactosyltransferase mutant in catalyzing glycosylation reactions.

[0009] The present invention also provides a gene encoding the above-mentioned β-1,4-galactosyltransferase mutant.

[0010] The present invention also provides primers for amplifying the above-mentioned gene.

[0011] Furthermore, the primer pair for amplifying the gene encoding the mutant I13W is shown in SEQ ID NO.5 and SEQ ID NO.6.

[0012] Furthermore, the primer pair for amplifying the gene encoding the mutant L130R is shown in SEQ ID NO. 7 and SEQ ID NO. 8.

[0013] Furthermore, the primer pair for amplifying the gene encoding mutant F157W is shown in SEQ ID NO.9 and SEQ ID NO.10.

[0014] The present invention also provides a recombinant expression vector, characterized in that it comprises the above-mentioned gene.

[0015] The present invention also provides a recombinant cell, characterized in that it comprises the above-mentioned recombinant expression vector.

[0016] The present invention also provides a method for preparing a beta-1,4-galactosyltransferase mutant, which comprises transferring the above gene into Escherichia coli for expression, and then separating and purifying the gene to obtain the beta-1,4-galactosyltransferase mutant.

[0017] The present invention has the following beneficial effects: The present invention combines bioinformatics, molecular docking, and computer simulation techniques to analyze and obtain the key active sites of AaGT25, and uses site-directed mutagenesis technology to prepare β-1,4-galactosyltransferase mutants through hospital expression. The enzyme activities of the mutants I13W, L130R, and F157W of the present invention are significantly improved compared to the wild type, approximately 1.7, 1.2, and 1.85 times that of the wild type, respectively. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is the result of gel electrophoresis verification of β-1,4-galactosyltransferase AaGT25; Figure 2 The figure shows the comparison of the enzyme activities of β-1,4-galactosyltransferase and mutants. DETAILED DESCRIPTION

[0018] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0019] Example 1 A β-1,4-galactosyltransferase mutant has an I13W mutation based on β-1,4-galactosyltransferase AaGT25 (SEQ ID NO. 1), and its amino acid sequence is shown in SEQ ID NO. 2.

[0020] Example 2 A β-1,4-galactosyltransferase mutant has an L130R mutation based on β-1,4-galactosyltransferase AaGT25 (SEQ ID NO. 1), and its amino acid sequence is shown in SEQ ID NO. 3.

[0021] Example 3 A β-1,4-galactosyltransferase mutant has an F157W mutation based on β-1,4-galactosyltransferase AaGT25 (SEQ ID NO. 1), and its amino acid sequence is shown in SEQ ID NO. 4.

[0022] Experimental Example 1 Mutant Construction The three-dimensional structure of β-1,4-galactosyltransferase (AaGT25) was modeled using the SWISS-MODEL tool. The key active sites were predicted through molecular docking and computer simulation techniques. Alanine scanning and virtual saturation mutagenesis were performed on sites within 3 Å of substrate binding using Discovery Studio. Mutation sites were selected based on sequence conservation analysis, and the activity of the mutants was verified. The key active sites of AaGT25 were preliminarily analyzed through changes in the interaction between the mutants and the substrate, and the mutants I13W, L130R, and F157W with enhanced activity were obtained.

[0023] Experimental Example 2 Heterologous Expression and Purification (1) Expression and purification of β-1,4-galactosyltransferase (AaGT25) by Aggregatibacter actinomycetemcomitans The genomic DNA of AaGT25 was used as a template to amplify the coding gene of AaGT25 by PCR. After enzyme digestion, it was inserted into the pET-30a(+) vector and transformed into Escherichia coli BL21(DE3) competent cells. After culturing in a 37°C incubator, the plate with colonies was taken out and single colonies with good morphology were randomly selected and inoculated into a plate containing Kan + The cells were cultured in LB liquid medium containing 50 μg / mL of the antibiotic resistance factor at 37°C and 200 rpm for 12 h. After the bacteria grew to the logarithmic phase, samples were taken for colony PCR identification, and the PCR products were verified by sequencing and double enzyme digestion.

[0024] The verified bacterial solution was inoculated into 5 mL of Kan + The activated strain was cultured in LB liquid test tube medium containing antibiotics (50 μg / mL) at 37°C and 200 rpm overnight, and the next day it was transferred to 100 mL of Kan-containing liquid test tube medium at a 2% inoculum volume. + The culture was expanded in LB liquid medium containing antibiotics and cultured at 37°C and 200 rpm for 4-5 h until the OD600 When the value reached 0.6-0.8, IPTG was added to the bacterial solution with a final concentration of 0.5 mM, and the culture was induced at 15°C and 200 rpm for 16 h.

[0025] The induced expression bacterial solution was placed in a pre-cooled centrifuge tube and centrifuged at 4°C and 8000 rpm for 5 min. The supernatant was discarded, the bacterial pellet was collected, and the bacterial cells were suspended and washed with 10 mL of lysis buffer. The cells were disrupted for 15 min using an ultrasonic cell disruptor (ultrasound 2 s, interval 2 s) in an ice water bath. The cells were centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was collected as a crude enzyme solution and the protein was purified using nickel column affinity chromatography. The crude enzyme solution was passed through a 0.45 μm filter membrane and slowly loaded onto the column. The solution was incubated at 4°C for 1 h to allow the protein to fully bind to the nickel column. The impurities were then washed with 5 times the volume of washing buffer and the target protein was eluted with 10 times the volume of elution buffer. The eluate was collected and dialyzed to obtain purified β-1,4-galactosyltransferase (AaGT25). The gel electrophoresis verification results of the purified protein are shown in Figure 2. Figure 1 shown.

[0026] (2) Expression and purification of mutants Plasmids extracted from the E. coli cells verified as correct in step (1) were extracted using a plasmid miniprep kit. Whole-plasmid PCR was used to construct mutants I13W, L130R, and F157W, which were verified by sequencing. Expression was then induced and purified under the same conditions, and molecular weight was verified by SDS-PAGE.

[0027] Test Example 3 Enzymatic Property Determination The recombinant protein activity was measured using a Glycosyltransferase activity kit. Under the action of β-1,4-galactosyltransferase, the Gal molecule in UDP-Gal binds to the receptor N-GlcNAc. Simultaneously, the phosphate group in the uridine diphosphate (UDP) molecule is specifically hydrolyzed by the phosphatase. Finally, malachite green dye is added, and its characteristic color reaction with free phosphate ions is used for quantitative detection. One unit of enzyme activity is defined as the amount of 1 pmol of inorganic phosphate produced in one minute.

[0028] The specific steps are as follows: The concentrations of purified AaGT25 and mutants I13W, L130R, and F157W were measured using a BCA protein quantification kit and adjusted to the same concentration. As shown in Table 1, the donor (UDP-Gal) and acceptor substrate solution (N-GlcNAc) were mixed with the enzyme solution and incubated at 37°C for 15 min. Table 1 Enzyme reaction system

[0029] Add 30 μL of Malachite Green Reagent A to each well and mix gently. Then add 100 μL of distilled water and 30 μL of Malachite Green Reagent B. Incubate at room temperature for 20 minutes to stabilize the color development and measure the A620 value. Calculate the product amount and enzyme activity based on the phosphate standard curve. Compare the results as shown in the figure. Figure 2 shown.

[0030] Depend on Figure 2 It can be seen that compared with the wild-type β-1,4-galactosyltransferase (AaGT25), the enzyme activities of mutants I13W, L130R and F157W were increased to 1.7, 1.2 and 1.85 times, respectively.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A β-1,4-galactosyltransferase mutant, characterized in that The invention relates to a β-1,4-galactosyltransferase AaGT25, wherein the β-1,4-galactosyltransferase AaGT25 has at least one of the mutations I13W, L130R and F157W; the amino acid sequence of the β-1,4-galactosyltransferase AaGT25 is shown in SEQ ID NO.

1.

2. The β-1,4-galactosyltransferase mutant according to claim 1, characterized in that It is mutant I13W, and its amino acid sequence is shown in SEQ ID NO.

2.

3. The β-1,4-galactosyltransferase mutant according to claim 1, characterized in that It is the mutant L130R, and its amino acid sequence is shown in SEQ ID NO.

3.

4. The β-1,4-galactosyltransferase mutant according to claim 1, characterized in that It is mutant F157W, and its amino acid sequence is shown in SEQ ID NO.

4.

5. Use of the β-1,4-galactosyltransferase mutant according to any one of claims 1 to 4 in catalyzing a glycosylation reaction. A gene encoding the β-1,4-galactosyltransferase mutant according to any one of claims 1 to 4.

7. Primers for amplifying the gene according to claim 6.

8. A recombinant expression vector, characterized in that: Comprising the gene according to claim 6.

9. A method for preparing a β-1,4-galactosyltransferase mutant, characterized in that: The gene according to claim 6 is transferred into Escherichia coli for expression, and then separated and purified to obtain the β-1,4-galactosyltransferase mutant.

10. A recombinant cell, characterized in that Comprising the recombinant expression vector according to claim 8.

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