Beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H and application thereof
By modifying the β1,3-galactosyltransferase Pmβ3GalT, a mutant enzyme Pmβ3GalT-S207H was obtained, which solved the problems of insufficient resources and poor stability of existing enzymes, and realized the efficient and low-cost biosynthesis of LNT.
Patent Information
- Application Number
- CN202610058828.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-16
- Publication Date
- 2026-04-14
AI Technical Summary
Limited existing β1,3-galactosyltransferase resources, unclear catalytic transglycosylation mechanism, low solubility and poor stability of heterologous expression have hindered the rapid development of lactose-N-tetrasaccharide (LNT) biosynthesis.
By modifying the β1,3-galactosyltransferase Pmβ3GalT, a mutant enzyme Pmβ3GalT-S207H was obtained, which improved its enzyme activity, thermostability and alkali resistance. Recombinant engineered bacteria were then constructed for the synthesis of LNT.
The mutant enzyme Pmβ3GalT-S207H exhibits a 157.21% increase in enzyme activity, significantly improved thermal stability and alkali resistance, supporting the efficient and low-cost industrial production of LNT.
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Abstract
Description
Technical Field
[0001] This invention relates to a β1,3-galactosyltransferase Pmβ3GalT-S207H and its applications, belonging to the field of functional enzyme technology. Background Technology
[0002] Lactose-N-tetrasaccharide (lacto- N LNT (leukotriene tetraose) is one of the core components of human milk oligosaccharides (HMOs), which have functions such as regulating intestinal microbiota balance, enhancing immunity, and promoting infant brain development. It has been approved by the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) as a food additive for infant formula. Currently, LNT production methods mainly include chemical synthesis and biosynthesis. Compared to the more complex chemical synthesis method, biosynthesis is lower in cost and has a simpler, more stable, and sustainable process.
[0003] β1,3-galactosyltransferase (β3GalT) can transfer galactose to lactoyl-N-trisaccharide II (Lacto-Gal) via a β1,3-glycosidic bond using UDP-galactose (UDP-Gal) as a donor. N LNTs are synthesized by synthesizing β-triose II (LNT II). Numerous studies have demonstrated that β3GalT is a key rate-limiting enzyme in the biosynthesis of LNTs. However, β-1,3-GalT suffers from limitations such as limited existing resources, an unclear catalytic transglycosylation mechanism, low solubility due to heterologous expression, and poor stability. These factors hinder the rapid development of LNT biosynthesis methods. Therefore, it is essential to modify β-1,3-GalT to obtain β3GalT with high transglycosylation activity.
[0004] In recent years, the widespread application of protein engineering technology in the field of enzyme preparations has laid a solid foundation for the development of novel, highly active, and highly stable β1,3-galactosyltransferases. Modifying β1,3-galactosyltransferases is expected to improve LNT production efficiency, further reduce production costs, and promote the industrialization of its biosynthesis process, possessing significant theoretical and practical value. Summary of the Invention
[0005] In view of the above-mentioned prior art, the present invention provides a β1,3-galactosyltransferase Pmβ3GalT-S207H and its application, belonging to the field of functional enzyme technology.
[0006] This invention is achieved through the following technical solution: A β1,3-galactosyltransferase Pmβ3GalT-S207H, the amino acid sequence of which is shown in SEQ ID NO.3.
[0007] The nucleotide sequence of the gene encoding the β1,3-galactosyltransferase Pmβ3GalT-S207H is shown in SEQ ID NO.4.
[0008] Application of the β1,3-galactosyltransferase Pmβ3GalT-S207H in the preparation of lactose-N-tetrasaccharide.
[0009] A recombinant expression vector containing the encoding gene for the aforementioned β1,3-galactosyltransferase Pmβ3GalT-S207H.
[0010] A recombinant engineered bacterium whose genome contains the gene encoding β1,3-galactosyltransferase Pmβ3GalT-S207H and can express β1,3-galactosyltransferase Pmβ3GalT-S207H.
[0011] Furthermore, the host of the recombinant engineered bacteria is Escherichia coli.
[0012] Application of the recombinant engineered bacteria in the preparation of β1,3-galactosyltransferase Pmβ3GalT-S207H.
[0013] This invention involves the mutation of the 1,3-galactosyltransferase Pmβ3GalT to obtain the mutant enzyme—β1,3-galactosyltransferase Pmβ3GalT-S207H. Compared with the wild-type enzyme, this mutant enzyme exhibits significantly increased enzyme activity (157.21%), as well as marked improvements in thermostability and alkali tolerance. The β1,3-galactosyltransferase Pmβ3GalT-S207H of this invention provides theoretical and practical support for the application of β1,3-galactosyltransferases in LNT synthesis, and has significant theoretical and practical value for the industrial production of LNTs.
[0014] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description
[0015] Figure 1 : Schematic diagram of SDS-PAGE electrophoresis detection results, where M represents the standard protein marker, WT crude enzyme represents the crude enzyme solution of Pmβ3GalT, crude enzyme flow-through solution represents the flow-through solution during the purification of crude Pmβ3GalT, and WT pure enzyme represents the pure enzyme solution of Pmβ3GalT.
[0016] Figure 2: Schematic diagram of temperature stability test results for β1,3-galactosyltransferase Pmβ3GalT and mutant enzyme, where WT represents β1,3-galactosyltransferase Pmβ3GalT and S207H represents β1,3-galactosyltransferase Pmβ3GalT-S207H.
[0017] Figure 3 Schematic diagram of pH stability assay results for β1,3-galactosyltransferase Pmβ3GalT.
[0018] Figure 4 : Schematic diagram of the relative enzyme activities of wild-type enzyme and 8 mutant enzymes. The control is the wild-type enzyme, namely β1,3-galactosyltransferase Pmβ3GalT.
[0019] Figure 5 Schematic diagram of pH stability assay results for β1,3-galactosyltransferase Pmβ3GalT-S207H. Detailed Implementation
[0020] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0021] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0022] Example 1: Expression and purification of β1,3-galactosyltransferase Pmβ3GalT This invention is derived from Proteus mirabilis ( Proteus mirabilis A β1,3-galactosyltransferase, Pmβ3GalT (GenBank: WP_196547312.1), was discovered in the database. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. This invention involves heterologous expression of this enzyme and investigation of its enzymatic activity to determine its suitability for industrial production of lactose-1-thiotransferases (LNTs).
[0023] The amino acid sequence of β1,3-galactosyltransferase Pmβ3GalT is shown in SEQ ID NO.1, as follows: MNNLPLVSIIIPINKDNPYLLEAINSVLNQTYKNIEVIVIANRCNKLLLDKLSQLDNIKIIITEIAYISFSLNLGIHQAKGKYIARMDSDDISLPSRIDTQVSYLEKNTDIAIVGSNIELIDENSKLLNK RITYPTLDKDIKKALIYKCCLAHPTVIFRKDVIESIGGYLYGPISEDYELWLRASLVHNIKFGNINEVLLNYRIHSSQTTSNKNRVKVFTNDITIKFRYLLQTKKISLFGGILYSTLVFIKNILIKAKK.
[0024] The nucleotide sequence of the gene encoding β1,3-galactosyltransferase Pmβ3GalT is shown in SEQ ID NO.2, as shown below (direction 5'-3'): .
[0025] The gene for synthesizing Pmβ3GalT (nucleotide sequence shown in SEQ ID NO.2) was synthesized. This gene fragment was amplified and ligated into the empty pMAL-c5E plasmid to construct the pMAL-c5E-Pmβ3GalT expression vector. The constructed recombinant plasmid was then transformed into *E. coli* BL21(DE3) using conventional methods to construct the recombinant engineered bacterium.
[0026] Transformants were inoculated into 5 mL of LB medium and activated overnight at 37°C and 220 r / min. Then, 1% of the transformants were inoculated into 50 mL of ZYP-5052 fermentation medium and induced to ferment in shake flasks at 20°C and 220 r / min for 40 h. After fermentation, the cells were collected by centrifugation, resuspended in an appropriate amount of buffer, and sonicated at low temperature. After freezing and centrifugation for 10 min, the supernatant was collected as the crude enzyme solution. The crude enzyme solution was loaded onto amylose resin, and impurities were washed with purification buffer and fusion protein was eluted with elution buffer. The eluent was collected and concentrated by ultrafiltration using a 50 kDa ultrafiltration tube to obtain pure enzyme solution.
[0027] The crude and purified enzyme solutions of β1,3-galactosyltransferase Pmβ3GalT were detected by SDS-PAGE electrophoresis, and the results are as follows: Figure 1 As shown in the figure, the purified protein band was single, with a molecular weight of approximately 75 kDa, consistent with the theoretical value. This indicates that the purified protein is a Pmβ3GalT protein fused with the fusion-aiding tag MBP. The absence of obvious contaminating proteins near the target band suggests high purity of the purified protein. It can be further used for studies on proteomic properties.
[0028] Example 2 Enzymatic characterization of β1,3-galactosyltransferase Pmβ3GalT The reaction system for determining the transglycosylation activity of β1,3-galactosyltransferase Pmβ3GalT was as follows: 1.5 mM LNT II, 1.5 mM UDP-Gal, 10 μL crude enzyme solution (pure enzyme concentration: 20–80 μg / mL), and 20 mM MgCl2 solution were mixed thoroughly in a 50 mM Tris-HCl (pH 7.5) buffer solution, and the volume was brought to 100 μL with distilled water. The mixture was incubated in a 30°C water bath for 2 h, and the reaction was terminated by boiling for 10 min.
[0029] The enzymatic kinetic parameters of β1,3-galactosyltransferase Pmβ3GalT were determined using different concentrations of donor and acceptor solutions (0.1 mM–10 mM) as substrates. With a fixed UDP-Gal concentration of 1.5 mM, LNT II concentrations of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, and 3.0 mM were set; with a fixed LNT II concentration of 1.5 mM, UDP-Gal concentrations of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, and 4.0 mM were set. The parameters were calculated using nonlinear regression analysis. K m and k cat The values are shown in Table 1.
[0030]
[0031] The purified enzyme solution of β1,3-galactosyltransferase Pmβ3GalT was incubated at different temperatures (25–60 °C) for 2 h, and its residual enzyme activity under standard reaction conditions was measured to analyze its thermostability. The initial enzyme activity before incubation was defined as 100%, and the residual relative activities after incubation at different temperatures were calculated. The results are as follows: Figure 2 As shown in the figure, the results indicate that as the temperature increases, the residual enzyme activity decreases until it is completely inactivated.
[0032] The purified enzyme solution of β1,3-galactosyltransferase Pmβ3GalT was incubated for 2 h in different pH buffers (CBS buffer at pH 4.0–5.0; MES buffer at pH 5.0–6.5; HEPES buffer at pH 6.5–7.5; and Tris-HCl buffer at pH 7.0–9.5). The residual enzyme activity under standard reaction conditions was then measured to analyze its pH stability. The initial enzyme activity before incubation was defined as 100%. The residual relative activities after incubation at different pH values were calculated, and the results are shown below. Figure 3 As shown in the figure. The results indicate that the acid and alkali tolerance of β1,3-galactosyltransferase Pmβ3GalT is generally moderate.
[0033] Example 3 Semi-rational design of β1,3-galactosyltransferase Pmβ3GalT The study in Example 2 showed that the β1,3-galactosyltransferase Pmβ3GalT has only moderate transglycosylation activity, which cannot meet the requirements for efficient and low-cost LNT synthesis. Therefore, this invention utilizes semi-rational design to modify it, aiming to obtain a mutant enzyme with superior function, further improve its transglycosylation activity, and reduce the synthesis cost of LNT.
[0034] The specific process of semi-rational design involves screening for potential mutation sites using three different methods, as follows: (1) Multiple sequence alignment of β3GalT with high transglycosylation activity for synthesizing LNT was performed using the multiple sequence alignment software ClustalX 2.1 and the website ESPript 3.0 to screen for potential amino acid sites that could improve transglycosylation activity and perform site-directed mutagenesis.
[0035] (2) The structure of Pmβ3GalT was predicted and analyzed based on AlphaFold2 and Swiss-Model, and the results were uploaded to the HotSpot Wizard 3.1 website to predict key amino acid mutation sites.
[0036] (3) The open protein model of Pmβ3GalT was molecularly docked with the small molecule ligand LNT II using Autodock Vina, and the docking results were visualized using Pymol software. Amino acids within 5 Å of the ligand molecule were searched.
[0037] Several key active sites were identified using the above screening methods. Site-directed and saturation mutagenesis will be conducted on these key active sites, and the mutant enzymes to be investigated are as follows: Sequence alignment mutant enzymes: I13V, N17D, S89A, T100K, C150N, R159K, E179D, T209A, K213S.
[0038] HotSpot predicts the following mutant enzymes: P18E, P18Y, A66K, I68A, I118S, L120D, I132T, Y134A, Y134K, C150T, A152H, T209M, N214M.
[0039] I11A, K15A, I132A, L151A, S207A, T209A, Y218A.
[0040] Saturation mutants at the S89 site: 19 types.
[0041] Saturation mutants at the S207 site: 19 types.
[0042] Example 4: Expression and extraction of various mutant enzymes For each mutant enzyme to be investigated in Example 3, specific amplification primers were designed. Using the gene of the wild-type enzyme synthesized in Example 1 as a template, the gene of each mutant enzyme was amplified. Recombinant expression vectors were constructed according to the method in Example 1, transformed into recombinant engineered bacteria, cultured, and crude enzyme solutions of each mutant enzyme were collected for the following studies.
[0043] Example 5: Determination of transglycosylation activity of mutant enzyme Preliminary screening revealed that most mutant enzymes exhibited significantly reduced transglycosylation activity compared to wild-type enzymes. Eight mutant enzymes with high crude enzyme activity (S207A, S207E, S207G, S207H, S207K, S207M, S207Q, and S207R) were selected for purification to obtain pure enzyme solutions. Enzyme activity was quantitatively determined using high-performance liquid chromatography (HPLC). The relative enzyme activity of each mutant enzyme was calculated with the wild-type enzyme activity as 100%. The results are as follows: Figure 4 As shown in the figure, S207H exhibits the highest transglycosylation activity, significantly higher than the wild-type enzyme and the other seven mutant enzymes, with an enzyme activity reaching 257.21% of that of the wild-type enzyme.
[0044] The mutant enzyme S207H is named β1,3-galactosyltransferase Pmβ3GalT-S207H, and its amino acid sequence is shown in SEQ ID NO.3. Compared with the amino acid sequence shown in SEQ ID NO.1, the amino acid at position 207 is mutated from tryptophan (Ser) to histidine (His); the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4. The nucleotide sequences of the overlapping primers S207H-F and S207H-R used to amplify this mutant enzyme are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.
[0045] The amino acid sequence of β1,3-galactosyltransferase Pmβ3GalT-S207H is shown in SEQ ID NO.3, as follows: MNNLPLVSIIIPINKDNPYLLEAINSVLNQTYKNIEVIVIANRCNKLLLDKLSQLDNIKIIITEIAYISFSLNLGIHQAKGKYIARMDSDDISLPSRIDTQVSYLEKNTDIAIVGSNIELIDENSKLLNK RITYPTLDKDIKKALIYKCCLAHPTVIFRKDVIESIGGYLYGPISEDYELWLRASLVHNIKFGNINEVLLNYRIHSHQTTSNKNRVKVFTNDITIKFRYLLQTKKISLFGGILYSTLVFIKNILIKAKK.
[0046] The nucleotide sequence of the gene encoding β1,3-galactosyltransferase Pmβ3GalT-S207H is shown in SEQ ID NO.4, as shown below (direction 5'-3'): atgaacaacctgccgctagtatcaattataatcccgatcaacaaagataacccgtatctattggaggcgatcaactcagttctgaatcaaacttacaaaaacattgaagtcatcgtgattgctaatcgttgtaataagctgttgctggacaagttgtcgcagctggacaacatcaagatcattatcaccgaaattgcgtatatttcttttagcctgaacctgggtattcaccaggcaaaaggcaaatacattgcgcgtatggattccgatgatatctccttgccgagccgtatcgacacccaggtttcctatcttgagaaaaacaccgatattgccattgtgggcagcaacatcgagctgatcgacgagaacagcaaactgctcaacaagcgcatcacgtacccgaccctggacaaggacattaagaaggctctcatctacaagtgctgcctggcgcatccaacggttatttttcgcaaagacgttatcgagagcattggtggctatctgtatggtccgattagtgaagactacgaactttggctgcgtgcatctctggttcataatatcaaattcggtaacatcaacgaagttctgctgaactaccgtatccacagccaccagaccaccagcaacaaaaaccgagtcaaagtgttcaccaatgatatcaccatcaagttccgttatctgttgcaaaccaaaaaaatttcgttgtttggtggcattctgtacagcaccctggtgttcatcaagaacatcctgattaaggcgaaaaaa。
[0047] The nucleotide sequence of the overlapping primer S207H-F for amplifying β1,3-galactosyltransferase Pmβ3GalT-S207H is shown in SEQ ID NO.5, as follows (direction 5'-3'): ccgtatccacagccaccagaccaccagcaacaaaaacc。
[0048] The nucleotide sequence of the overlapping primer S207H-R for amplifying β1,3-galactosyltransferase Pmβ3GalT-S207H is shown in SEQ ID NO.6, as shown below (direction 5'-3'): tggtggctgtggatacggtagttcagcagaacttcgttgatg.
[0049] Example 6: Temperature and pH stability of β1,3-galactosyltransferase Pmβ3GalT-S207H Following the method in Example 2, the temperature stability and pH stability of β1,3-galactosyltransferase Pmβ3GalT-S207H were investigated, and the results are as follows: Figure 2 , Figure 5 As shown in the figure. The results indicate that the β1,3-galactosyltransferase Pmβ3GalT-S207H retained more than 60% of its enzyme activity after incubation at 35–40 °C. Compared with the wild-type enzyme, the residual activity of the mutant enzyme at the optimal temperature was higher, indicating that the thermostability of the mutant enzyme was improved. The pH stability test results showed that the mutant enzyme had significantly improved tolerance to alkaline environments, and its residual activity was still more than 75% in the pH range of 7.5–8.5.
[0050] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.
Claims
1. A β1,3-galactosyltransferase Pmβ3GalT-S207H, characterized in that: The amino acid sequence is shown in SEQ ID NO.
3.
2. The gene encoding the β1,3-galactosyltransferase Pmβ3GalT-S207H according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.
4.
3. The application of the β1,3-galactosyltransferase Pmβ3GalT-S207H according to claim 1 in the preparation of lactose-N-tetrasaccharide.
4. A recombinant expression vector, characterized in that: It contains the gene encoding the β1,3-galactosyltransferase Pmβ3GalT-S207H as described in claim 2.
5. A recombinant engineered bacterium, characterized in that: The genome contains the encoding gene for the β1,3-galactosyltransferase Pmβ3GalT-S207H as described in claim 2, and is able to express the β1,3-galactosyltransferase Pmβ3GalT-S207H as described in claim 1.
6. The recombinant engineered bacteria according to claim 5, characterized in that: The host is Escherichia coli.
7. The use of the recombinant engineered bacteria according to claim 5 or 6 in the preparation of β1,3-galactosyltransferase Pmβ3GalT-S207H.