Sphingolipid ceramide N-deacylase mutant and application thereof in preparation of ganglioside

Abstract

The invention discloses a sphingolipid ceramide N-deacylase mutant. The mutant is formed by amino acid mutation in sphingolipid ceramide N-deacylase as shown in SEQ ID NO.1, and the amino acid mutation is selected from one or more of mutation of the 232 site Tyr, mutation of the 254 site Asn, mutation of the 416 site Phe and mutation of the 592 site Leu. The invention also discloses application of the mutant in preparation of ganglioside derivative molecules by efficient enzymatic synthesis. The stability of the sphingolipid ceramide N-deacylase SCDase mutant is improved, and the pyrolysis chain temperature Tm of the mutant is improved by 5.79 DEG C compared with that of a wild type; the sphingolipid ceramide N-deacylase SCDase mutant provided by the invention enhances the enzyme preparation efficiency of ganglioside, especially for short-chain fatty acids (carbohydrate chain length lt; and 4) the synthetic activity is improved by more than two times, and the result broadens the practical application value of the SCDase enzyme.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to sphingolipid ceramide N-deacylase mutants and their application in the preparation of gangliosides. Background Technology

[0002] Sphingolipids, as important components of biological membranes, play crucial roles in various physiological processes such as cell recognition, signal transduction, and immune regulation. Sphingolipid ceramide N-deacylase (SCDase) is a bifunctional enzyme that catalyzes the cleavage of the amide bond between the sphingoamino alcohol and fatty acid chain in sphingolipid (GSL) molecules, leading to reversible synthesis reactions. Figure 1 ).

[0003] Wild-type SCDase enzymes derived from the marine bacterium *Shewanella alga G8* exhibit a predominantly hydrolytic activity between pH 6.0 and 6.5, and a predominantly synthetic activity between pH 7.0 and 7.5. This characteristic makes this enzymatic method valuable for the hydrolysis of sphingolipids or the synthesis of sphingolipid analogs. However, the relatively insufficient thermostability of *Shewanella alga G8* SCDase enzymes affects their long-term storage stability to some extent, and their activity is relatively low when catalyzing short-chain fatty acid substrates in specific sphingolipids, limiting their application potential in related fields. Therefore, improving the thermostability and catalytic activity of SCDase for specific substrates through protein engineering, and developing SCDase mutants with superior catalytic performance, is of great value. Summary of the Invention

[0004] The purpose of this invention is to provide a mutant sphingolipid ceramide N-deacylase (SCDase) with improved thermal stability and catalytic activity. Through analysis of the wild-type SCDase crystal structure, point mutations were designed using computational methods, and extensive screening was conducted to obtain an enzyme mutant with improved thermal stability and catalytic activity. The parental SCDase protein sequence was mutated at amino acid substitution positions. The amino acids of the enzyme mutant are represented as "original residue / position / substitute residue". The optimal mutant sphingolipid ceramide N-deacylase (SCDase) is the Y232L / N254T / F416A / L592I four-point mutant, which exhibits improved thermal stability and catalytic activity against short-chain fatty acid substrates.

[0005] The "sphingolipid ceramide N-deacylase SCDase enzyme mutant with improved thermal stability and catalytic activity" described in the application refers to the mutant enzyme SCDase that has enhanced thermal denaturation resistance, the thermal denaturation temperature Tm of the mutant enzyme relative to the wild-type enzyme is improved, and the mutant enzyme SCDase relative to the wild-type enzyme improves the catalytic activity on short-chain fatty acid ganglioside substrates.

[0006] To achieve the above-mentioned purpose, the application provides the following technical scheme: a sphingolipid ceramide N-deacylase SCDase enzyme mutant with improved thermal stability and catalytic activity, the mutant SCDase is constructed by analyzing the high-resolution crystal structure of the wild-type SCDase enzyme, and then based on the substrate-enzyme protein structure molecular docking, the point mutation of the corresponding site is designed and constructed with the aid of calculation, and the mutant is obtained through stability and activity screening.

[0007] A SCDase mutant with improved thermal stability and catalytic activity, the mutant is formed by amino acid mutation in the sphingolipid ceramide N-deacylase SCDase shown in SEQ ID NO. 1, and the amino acid mutation is selected from one or more mutations of tyrosine at position 232, asparagine at position 254, phenylalanine at position 416, and leucine at position 592.

[0008] SEQ ID NO. 1: MTTQAVDSLAQQCFIIQSPTNGQYLHRFHQGGTVDDGLSYRFDNIS QAEASAFYFKPSRRGHFMMTDADGRFFASHLPAEVSAGRYPGEFAEWRVDAETAPSGEFSYRFHAVGLNLGLRHNYSGGGLYFFDLLNPGNNTSEASFKLVASDACSAFPEVEVNASGDFSALKGDASLPVRGLVDAHTHITSYEFMGGKMMHGKPFHRWGVTQALNDSAVIHGPNGSLDLIGNLYSFNDANFRYDTRGWPDFPWWPNHEQMTHSGYYYKWIERAWLGGLRLMVTHLVENEVLCNAQKTINPASWVNPNDCNTMNSIQLQINRLKQMQEYIDVQSGGPGKGFFRLVSSPQEAREVIADGKLAVLMGIEASELFNCGIKDDCNRRQIEEQLQQVYAKGVRILFPTHKFDNQLGGSVVEDGFINIGEVLATGHFFETQACDADTQGRPFKSGFPILGEIPVLKDILNAVGLNPQYDENMLHCNKHGLSEKGVYLVNRMIDMGMLIELDHMSAQTATSVMDIVEQRQYGGVITSHSWMTDGTQGRLHPNTLRLAKVGGFMAPYNSNANHLGGSIDRYLQLIADTPFLPGVGLGTDMSGLGAQAGPRDDAATNPLHYPFVSEFGIQFERQVSGNRVFDFNQDGMAHYGMLADHLQDVREQLGGSTYEALMNSAEAYLQMWERAEAHLEHHHHHHHHHH.

[0009] Preferably, the amino acid sequence of the four-point mutant Y232L / N254T / F416A / L592I is shown as SEQ ID No. 2.

[0010] SEQ ID No. 2: MTTQAVDSLAQQCFIIQSPTNGQYLHRFHQGGTVDDGLSYRFDNIS QAEASAFYFKPSRRGHFMMTDADGRFFASHLPAEVSAGRYPGEFAEWRVDAETAPSGEFSYRFHAVGLNLGLRHNYSGGGLYFFDLLNPGNNTSEASFKLVASDACSAFPEVEVNASGDFSALKGDASLPVRGLVDAHTHITSYEFMGGKMMHGKPFHRWGVTQALNDSAVIHGPNGSLDLIGNLLSFNDANFRYDTRGWPDFPWWPTHEQMTHSGYYYKWIERAWLGGLRLMVTHLVENEVLCNAQKTINPASWVNPNDCNTMNSIQLQINRLKQMQEYIDVQSGGPGKGFFRLVSSPQEAREVIADGKLAVLMGIEASELFNCGIKDDCNRRQIEEQLQQVYAKGVRILFPTHKFDNQLGGSVVEDGAINIGEVLATGHFFETQACDADTQGRPFKSGFPILGEIPVLKDILNAVGLNPQYDENMLHCNKHGLSEKGVYLVNRMIDMGMLIELDHMSAQTATSVMDIVEQRQYGGVITSHSWMTDGTQGRLHPNTLRLAKVGGFMAPYNSNANHLGGSIDRYLQLIADTPFLPGVGLGTDMSGIGAQAGPRDDAATNPLHYPFVSEFGIQFERQVSGNRVFDFNQDGMAHYGMLADHLQDVREQLGGSTYEALMNSAEAYLQMWERAEAHLEHHHHHHHHHH.

[0011] Preferably, the sphingolipid ceramide N-deacylase variant further comprises a derivative protein having homology of > 80% to the amino acid sequence shown in SEQ ID No. 2 and having sphingolipid ceramide N-deacylase activity.

[0012] Preferably, the sphingolipid ceramide N-deacylase mutant expression vector is a prokaryotic or eukaryotic expression vector capable of recombinantly expressing the SCDase enzyme.

[0013] Preferably, the microorganism host cell transformed with the vector is an E. coli or a Bacillus subtilis or a yeast expression system.

[0014] The preparation method of the sphingolipid ceramide N-deacylase SCDase mutant is disclosed, which comprises introducing the coding nucleotide sequence of the sphingolipid ceramide N-deacylase SCDase mutant into a biological cell to express the coding gene of the sphingolipid ceramide N-deacylase SCDase mutant, so as to obtain the sphingolipid ceramide N-deacylase SCDase mutant recombinant expression cell or protein.

[0015] Technical effects and advantages of the present application:

[0016] 1. The stability of the sphingolipid ceramide N-deacylase SCDase mutant of the present application is improved, and the thermal dissociation temperature Tm is increased by 5.79℃ compared with the wild type.

[0017] 2. The sphingolipid ceramide N-deacylase SCDase mutant of the present application enhances the preparation efficiency of gangliosidase method, especially the synthesis activity of short-chain fatty acids (sugar chain length <4) is improved by more than 2 times, which widens the practical application value of SCDase enzyme. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a schematic diagram of the SCDase catalyzed sphingolipid hydrolysis and synthesis reversible reaction of the present application;

[0019] Figure 2 It is a SCDase protein structure diagram analyzed by the present application, wherein the blue ball is the active center metal ion;

[0020] Figure 3 It is the interaction between sphingolipid and surrounding residues of SCDase protein based on molecular docking structure model analysis of the present application;

[0021] Figure 4 It is a SCDase mutant protein purification electrophoresis map, lane M represents Marker, lanes 1-5 are different mutant SCDase enzyme mutant expression and purification conditions;

[0022] Figure 5 It is the Tm value of the wild type SCDase and the optimal mutant obtained by the present application. DETAILED DESCRIPTION

[0023] The specific embodiments of the present application will be further described in combination with the drawings. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0024] The Tm value of the enzyme is the temperature at which half of the protein structure of the enzyme is unfolded, which can be determined by differential scanning calorimetry (DSC) and differential scanning fluorescence (DSF) to obtain the protein dissolution curve.

[0025] Primer synthesis: The primers used in the present application are synthesized by commercial companies.

[0026] The DpnI enzyme and other enzyme preparations used in the experiment were purchased from Shanghai Xinggenyuan Biotechnology Co., Ltd.; PrimeSTAR Max high-fidelity polymerase was purchased from Takara Company; DNA gel recovery kit and plasmid extraction kit were purchased from Axygen Company.

[0027] The E. coli BL21 (DE3), E. coli Origami B (DE3), E. coli DH5a strains and pET series plasmids involved in the following examples are from the laboratory (the above strains of Escherichia coli and plasmids can also be purchased)

[0028] The culture medium involved in the following examples is as follows:

[0029] LB liquid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, ampicillin 60 μg / L.

[0030] LB solid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L, ampicillin 60 μg / L.

[0031] Example 1:

[0032] Enzyme structure analysis: The complete expression framework of SCDase was constructed into the pET-51b expression vector, and the expression level of SCDase protein was explored in BL21 (DE3) and Origami B (DE3) expression strains, respectively. It was found that the expression level of SCDase in Origami B (DE3) strain was the highest, and the recombinant enzyme with C-terminal 10x His tag was purified. Finally, we obtained SCDase protein crystals under the optimized conditions of 45% v / v Polypropylene glycol P 400, 0.1 M BIS-TRIS (pH 6.5), and after diffraction at BL19U of Shanghai Synchrotron Radiation Facility, the diffraction data were collected. Since SCDase has no homologous structure, after collecting the diffraction data of the protein, the main chain model of SCDase protein structure was built using AlphaFold2, the crystal structure was analyzed by molecular substitution, and the resolution of 2.2 A was obtained by optimizing the model. SCDase protein crystal structure. The SCDase protein structure consists of two domains, a large and a small one, with the active site located in the large domain, the center of which is composed of 7 parallel β-sheets, surrounded by 8 α-helices, while the small domain folds into a β-barrel structure, as shown in Figure 2 .

[0033] Example 2

[0034] Molecular docking: Since in flexible docking, the backbone of the protein will swing to a certain extent, which is more in line with the actual situation of the enzyme catalytic site and the substrate binding state, the SCDase and the substrate are adopted by the flexible docking method. Because the molecular side chain of the substrate GM1 of SCDase is too large, the SCDase and two products lyso-GM1 and stearic acid are docked. First, the conformational sampling of the receptor protein and the ligand substrate is carried out using the centroid mode of Rosetta, the conformational set is used as the input, and the processing is carried out, and finally the docking simulation of the substrate and the protein is carried out. The catalytic site of SCDase is located in the large domain, and the catalytic pocket region can accommodate long sugar chains and fatty acid chains. The optimal docking results of SCDase and glycosphingolipid substrates are shown in Figure 3 , the fatty acid is surrounded by aromatic residues, the sphingosine chain of lysoglycosphingolipid is located near the active site and interacts with the nearby hydrophilic residues, and the sugar chain extends out of the catalytic pocket. These enzyme-substrate binding analyses provide a structural basis for subsequent mutant design.

[0035] Example 3

[0036] Enzyme molecular design and mutant construction: Based on the analysis of the high-resolution structure of SCDase, the bioinformatics tools Pythia, EVcouplings, FRESC, EVOLVEpro, and Consurf were used to scan the entire protein structure to determine the combination of stable disulfide bonds, point mutation sites, ΔΔG, and protein conserved site analysis. Combined with kinetic simulation optimization of the mutant library, 42 potential single-point mutation sites to improve enzyme stability were selected; the gene of the parent sphingolipid ceramide N-deacylase SCDase wild type, sequence SEQ ID No. 3, was used as a template, and the full-plasmid PCR method was used to obtain the corresponding enzyme point mutant gene and expression vector.

[0037]

[0038] (Y232L) primer pair 1:

[0039] Upstream primer: Y232L-F: TCGGCAACCTGTTATCATTCAATGATGCGAAC;

[0040] Downstream primer: Y232L-R: ACAGGTTGCCGATCAGATCCAGGGAAC;

[0041] (N254T) primer pair 2:

[0042] Upstream primer: N254T-F: CGTGGTGGCCGACCCATGAACAGATGAC;

[0043] Downstream primer: N254T-R: TCGGCCACCACGGAAAATCCGGC;

[0044] (F416A) primer pair 2:

[0045] Upstream primer: F416A-F: GTGGAAGATGGCGCTATTAACATCGGTGAAG;

[0046] Downstream primer: F416A-R: GCCATCTTCCACCACCGAGCCAC;

[0047] (L592I) primer pair 2:

[0048] Upstream primer: L592I-F: GATATGAGTGGTATCGGTGCACAGGCAGGTCC;

[0049] Downstream primer: L592I-R: ACCACTCATATCCGTACCCAGGCCAACAC.

[0050] Using the above primers, the full plasmid PCR was carried out with the parent sphingolipid ceramide N-deacylase SCDase nucleotide sequence as a template to introduce corresponding double-point mutations at specific positions of the gene. The total volume of the reaction was 50 μl, and the components and amounts of the reaction system are shown in Table 1. After centrifugal mixing, the PCR amplification instrument was used for amplification. The full plasmid PCR site-directed mutation reaction program was set as follows: 98 ℃ pre-denaturation for 3 min, 30 cycles of amplification (98 ℃ denaturation for 15 s, 50-65 ℃ annealing for 15 s, 72 ℃ extension for 2 min); 72 ℃ extension for 5 min; 4 ℃ temporary storage of the PCR product. Among them, the annealing temperature depends on the Tm value of the upstream and downstream primers, and the extension time depends on the length of the full plasmid gene (generally 30 s / kb).

[0051] After gel purification of the PCR product, it was treated with DpnI enzyme at 37°C for 2 h and transformed into E. coli DH5α chemocompetent cells. The transformation product was plated on LB solid medium with the corresponding antibiotic resistance. After incubation at 37°C for 12 h, transformants were picked and inoculated into LB liquid medium. After shaking culture at 37°C for 10 h, the transformed cells were extracted and sent for commercial sequencing. The pET51-SCDase mutant plasmid was preserved after correct sequencing.

[0052] Table 1. Mutant PCR Reaction System

[0053]

[0054] Example 4:

[0055] Recombinant expression and purification of the enzyme: The correctly sequenced plasmid was transformed into E. coli BL21(DE3) and Origami B(DE3) competent cells. Recombinant SCDase mutant protein was purified using nickel affinity chromatography. Since SCDase protein readily forms polymers, molecular sieves were subsequently used to further assess protein folding quality and for further purification. First, the 200 mM imidazole elution buffer from the nickel column purification was concentrated and loaded onto a Superdex 200 column at a flow rate of 0.7 ml / min and a column pressure not exceeding 2.5 MPa. The target protein peak was collected. The purified SCDase protein was analyzed using 12% SDS-PAGE. (See attached table). Figure 4 Finally, the protein was concentrated to approximately 10 mg / mL using a 30 kDa ultrafiltration concentrator, aliquoted, and stored at -80°C.

[0056] Example 5:

[0057] Thermostability Study of SCDase Mutants: The thermal stability of the protein was comprehensively analyzed by determining the Tm values ​​of the protein using differential scanning calorimetry (DSC) and differential scanning fluorescence (DSF). The SCDase mutant protein was compared with SYPRO... TM By binding with Orange fluorescent dye and setting up the relevant measurement program on a real-time qPCR instrument, the temperature is gradually increased from 25 to 90℃, and mutant proteins with improved thermostability are screened by differential scanning fluorometry (DSF).

[0058] Melting curves were obtained using differential scanning calorimetry (DSC). The results showed that the optimal 4-point combination mutant had a thermal stability (Tm) value 5.79℃ higher than that of the wild-type SCDase. Figure 3 .

[0059] Example 6:

[0060] Application of SCDase mutant in ganglioside enzyme method synthesis: the SCDase mutant of the application is applied to the ganglioside enzyme method derivatization synthesis reaction system, the efficiency and yield of ganglioside synthesis using wild type SCDase and mutant are compared, and the advantage of the mutant in ganglioside enzyme method synthesis is verified. The standard SCDase synthesis activity measurement system is 30 μL, 3 μL of 5 mM lyso-GM3(d18:1) and 3 μL of 5 mM fatty acid substrate with different chain lengths are first added to 19 μL of 25 mM pH 7.5 Tris-HCl buffer containing 0.1% Triton X-100 and 10% DMSO, then 5 μL of enzyme solution with a certain dilution factor is added, and the reaction is carried out at 37°C for 10 min, then the reaction is terminated by placing in a boiling water bath for 5 min, centrifuged at 12000 rpm for 20 min to remove inactivated protein, and the supernatant is analyzed by HPLC, and the chromatographic conditions are as follows:

[0061] Chromatographic column: Zorbax Eclipse Plus C18(4.6×100mm 3.5μm); mobile phase: acetonitrile / water(80 / 20,v / v), containing 0.03% triethylamine in the water phase, pH adjusted to 7.5 with phosphoric acid; flow rate: 1 mL / min; injection volume: 10 μL; detector: ultraviolet detector, detection wavelength 195 nm.

[0062] In the analysis of the obtained SCDase optimal mutant enzyme and wild type SCDase enzyme utilization of different carbon chain length fatty acid derivatization synthesis of ganglioside ability, the yield is increased by more than 2 times when using fatty acid with short chain carbon chain C8 or less, especially C chain length <4.

[0063] Table 2. Analysis of synthesis of ganglioside activity of mutant SCDase (Y232L / N254T / F416A / L592I) and wild type SCDase using fatty acids with different chain lengths

[0064]

[0065]

[0066] In conclusion, the application uses protein structure as guide to design and replace the amino acids of sphingosine ceramide N-deacylase SCDase wild type gene sequence with Tyr232Leu, Asn254Thr, Phe416Ala, Leu592Ile to obtain sphingosine ceramide N-deacylase SCDase (Y232L / N254T / F416A / L592I) four-site mutant, wherein the thermal stability Tm value of the obtained optimal mutant is increased by 5.79 ℃, and in the ganglioside derivatization synthesis reaction system, the relative catalytic activity is improved for different fatty acid synthesis activities, especially the activity for short-chain fatty acids (sugar chain length <4) is improved by more than 2 times, which improves the practical application value of SCDase enzyme.

[0067] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A mutant of a sphingolipid ceramide N-decase, characterized in that: The mutant is formed by amino acid mutations in the sphingolipid ceramide N-decase shown in SEQ ID NO. 1, and the amino acid mutations are selected from one or more of the following: mutation of Tyr at position 232, mutation of Asn at position 254, mutation of Phe at position 416, and mutation of Leu at position 592.

2. A mutant of a sphingolipid ceramide N-decase and use thereof according to claim 1, characterized in that: The amino acid sequence of the four-point mutant Y232L / N254T / F416A / L592I is shown in SEQ ID No.

2.

3. A mutant of a sphingolipid ceramide N-decase and use thereof according to claim 1, characterized in that: The sphingolipid ceramide N-decase mutant has a derived protein with a homology of ≥80% to the amino acid sequence shown in SEQ ID No. 2 and has sphingolipid ceramide N-decase activity.

4. The mutant of a sphingolipid ceramide N-decase and use thereof according to claim 1, wherein the mutant is a mutant of a sphingolipid ceramide N-decase having a mutation in the amino acid sequence of SEQ ID NO:

1. The expression vector of the sphingolipid ceramide N-decase mutant is a prokaryotic or eukaryotic expression vector for expressing the sphingolipid ceramide N-decase mutant.

5. A mutant of a sphingolipid ceramide N-decase and use thereof according to claim 4, characterized in that: The expression vector is for transforming a microbial host cell into an E. coli or yeast expression system.

6. Use of a mutant sphingolipid ceramide N-decase according to any one of claims 1 to 5, characterized in that: The sphingolipid ceramide N-decase mutant is used in the high-efficiency enzymatic synthesis of ganglioside-derived molecules.

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