Protein with lipopolysaccharide degrading enzyme activity and gene and application thereof
By developing the protein ALP3 and its recombinant fusion protein, which have lipopolysaccharide degrading enzyme activity, the problem of intestinal endotoxin degradation in aquatic animals has been solved, achieving efficient degradation of lipopolysaccharides, significantly improving enzyme activity, and promoting animal health.
Patent Information
- Application Number
- CN202511151737.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-18
- Publication Date
- 2025-11-21
AI Technical Summary
Research on intestinal endotoxin-degrading enzymes in aquatic animals is still lacking in current technology, making it difficult to effectively degrade lipopolysaccharides released by Gram-negative bacteria, leading to enterogenic endotoxemia and affecting animal health and growth performance.
ALP3, a protein with lipopolysaccharide degrading enzyme activity, and its recombinant fusion protein were developed. By attaching a tag protein to the amino terminus or carboxyl terminus, efficient purification and detection were achieved. The enzyme protein was obtained through biological expression and prepared by combining a recombinant vector and a yeast expression system.
It achieves efficient degradation of lipopolysaccharides and significantly improves enzyme activity. The enzyme activity of ALP3 is 75 times higher than that of existing enzymes, reaching 370,000 U/mg, effectively reducing the risk of endotoxin and promoting animal health.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of genetic engineering, and particularly relates to a protein with lipopolysaccharide-degrading enzyme activity, a gene thereof and application. BACKGROUND
[0002] Lipopolysaccharide (LPS) is the main component of gram-negative bacteria, which is released when the bacteria die and rupture (such as antibiotic treatment) or proliferate, and can enter the animal body through the gill, stomach and intestinal mucosa, etc. The intestine is the most vulnerable organ. Intestinal barrier damage under stress conditions, mucosal ischemia atrophy, damage, shedding and increased mucosal permeability can all cause endotoxin translocation, leading to intestinal endotoxemia. Endotoxin can cause decreased feed intake, slow growth, reduced digestibility, and high mortality in severe cases.
[0003] Currently, there are three research directions: biodegradation, enzymatic degradation and chemical adsorption (bile acid). LPS is composed of O-antigen, core polysaccharide and lipid A. Lipid A is generally considered to be the "biologically active center" of LPS, which participates in the induction of local or systemic inflammatory response. For dephosphorylating enzymes acting on lipid A, the specific activity of existing alkaline phosphatase is more than 5000 U / mg. The research on aquatic endotoxin-degrading enzymes is still in the blank. SUMMARY
[0004] The main problem to be solved by the present application is how to obtain an enzyme protein with high lipopolysaccharide-degrading enzyme activity.
[0005] In order to solve the above problems, the present application provides the following protein, which can be any of the following proteins: 1) the amino acid sequence is SEQ ID No: 9 or SEQ ID No: 5 in the sequence listing; 2) a protein obtained by substitution, deletion and / or addition of one or more amino acid residues of the protein in 1) and having the same function; 3) a fusion protein obtained by connecting a protein tag to the N terminus or / and C terminus of 1) or 2).
[0006] The above protein is a lipopolysaccharide-degrading enzyme, named ALP3 or recombinant fusion protein ALP3.
[0007] In order to facilitate the purification or detection of the protein in 1), a tag protein can be connected to the amino terminal end or carboxyl terminal end of the protein consisting of the amino acid sequence shown in SEQ ID No: 5 in the sequence listing.
[0008] The above protein can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically.
[0009] The tag proteins include, but are not limited to, a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.
[0010] The nucleotide sequence encoding the protein ALP3 of the present application can be easily mutated by a person of ordinary skill in the art using known methods, such as a method of directed evolution or a method of point mutation. Those nucleotides which are artificially modified to have 75% or more identity with the nucleotide sequence of the protein ALP3 isolated from the present application are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application, as long as they encode the protein ALP3 and have the function of the protein ALP3.
[0011] The present application also provides a nucleic acid molecule encoding the above-mentioned protein, which can be a DNA molecule as described below: 1) a DNA molecule having the nucleotide sequence of SEQ ID No: 6 in the sequence listing; 2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in 1) and encoding the above-mentioned protein; 3) a DNA molecule hybridizing to the nucleotide sequence defined in 1) or 2) under stringent conditions and encoding the protein of claim 1.
[0012] The 75% or more identity can be 80%, 85%, 90%, or 95% or more.
[0013] Herein, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, in Advanced BLAST 2.1, and then the value of the identity (%) can be obtained.
[0014] Herein, the over 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
[0015] Herein, the over 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
[0016] The present application also provides a biological material containing the nucleic acid molecule described above, which can be any one of the following: B1) an expression cassette containing the nucleic acid molecule described above; B2) a recombinant vector containing the nucleic acid molecule described above, or a recombinant vector containing the expression cassette of B1); B3) a recombinant microorganism containing the nucleic acid molecule described above, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2).
[0017] Further, the recombinant vector of B2) is a recombinant vector obtained by inserting the nucleotide sequence of SEQ ID No: 6 between the secretion signal peptide sp23 sequence and the tag sequence of the vector pGAP_SP23Q6.
[0018] In a specific embodiment, the recombinant vector is pGAP_SP23Q6_ALP3. The structure of the recombinant vector pGAP_SP23Q6_ALP3 is described as follows: a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No: 6 between the 5'-AAGATCTTGTCTGCTTTGTTGTTGTTGTTCACTTTGGCTTTCGCTCAACAACAACAACAACAAATG-3' and 5'-CATCATCATCATCATCATCATCATCAT-3' fragments of the starting vector pGAP_SP23Q6, while keeping other sequences of the starting vector pGAP_SP23Q6 unchanged.
[0019] B3) the recombinant bacteria is a recombinant Pichia pastoris obtained by introducing the recombinant vector into a yeast; and the yeast is Pichia pastoris GS115.
[0020] The present application also provides a method for preparing the protein described above, which comprises introducing a DNA molecule capable of expressing the protein described above into a microorganism of interest, obtaining a recombinant microorganism, culturing the recombinant microorganism, collecting the supernatant of the fermentation broth, and obtaining the protein.
[0021] Further, the culturing the recombinant microorganism comprises the following step: inoculating the recombinant microorganism into a fermentation medium to culture, thereby obtaining the protein.
[0022] The present application also provides the use of the protein as described above in any of the following: 1) the use in preparing a lipopolysaccharide-degrading enzyme; 2) the use in preparing a product containing lipopolysaccharide-degrading enzyme activity; 3) the use in degrading lipopolysaccharide.
[0023] The present application also provides the use of the nucleic acid molecule as described above in any of the following: 1) the use in preparing a lipopolysaccharide-degrading enzyme; 2) the use in preparing a product containing lipopolysaccharide-degrading enzyme activity; 3) the use in degrading lipopolysaccharide.
[0024] The present application also provides the use of the biological material as described above in any of the following: 1) the use in preparing a lipopolysaccharide-degrading enzyme; 2) the use in preparing a product containing lipopolysaccharide-degrading enzyme activity; 3) the use in degrading lipopolysaccharide.
[0025] The present application also provides the use of the method as described above in any of the following: 1) the use in preparing a lipopolysaccharide-degrading enzyme; 2) the use in preparing a product containing lipopolysaccharide-degrading enzyme activity; 3) the use in degrading lipopolysaccharide.
[0026] The present application provides a protein having lipopolysaccharide-degrading enzyme activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, and a method for preparing a lipopolysaccharide-degrading enzyme, which lays a foundation for low-cost production of a novel lipopolysaccharide-degrading enzyme with high yield, and has important application potential in the biological degradation of endotoxins in the field of aquatic products. BRIEF DESCRIPTION OF DRAWINGS
[0027] Figure 1 Figure 1 is a western blotting diagram of proteins ALP1-ALP4. Lane 1, recombinant bacteria crude enzyme solution of protein ALP1; Lane 2, recombinant bacteria crude enzyme solution of protein ALP2; Lane 3, recombinant bacteria crude enzyme solution of protein ALP3; Lane 4, recombinant bacteria crude enzyme solution of protein ALP4.
[0028] Figure 2 Figure 2 is an enzyme activity diagram of proteins ALP1-ALP4 degrading lipopolysaccharide. DETAILED DESCRIPTION
[0029] The present application is further described in detail to which specific embodiments refer, the examples given are only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.
[0030] The experimental methods in the following examples are all routine methods, unless otherwise specified, according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.
[0031] Unless otherwise specified, the quantitative tests in the following examples are all set up with three repeated experiments, and the results are taken as the average value.
[0032] The pGAP_SP23Q6 in the following examples has been described in: Yao YY, Zhang QS, Liu SB, Yang HW, Chen XY, Yang YL, Gao CC, Ran C, Teame T, Zhang Z, Zhou ZG. Pichia pastoris composition expressed aerolysin mutant of Aeromonas veronii as an oral vaccine evaluated in zebrafish (Danio rerio). Mar Life Sci Technol. 2024 Aug 20;6(3):475-487. The biological material can be obtained from the applicant, which is only used for repeating the experiments of the present application, and cannot be used for other purposes.
[0033] The E. coli BL21(DE3) competent cells and GS115 competent cells in the following examples are commercially available products of Beijing Coolab Technology Co., Ltd.
[0034] The data in the following examples are processed by SPSS11.5 statistical software, the experimental results are expressed as mean ± standard deviation, and One-way ANOVA test is used, P<0.05 (*), P<0.01 (**), P<0.001 (***), P<0.0001 (****).
[0035] Example 1, Construction of ALP expression vector and obtaining of transformant 1. ALP gene synthesis Firstly, the coding sequences of ALP1-ALP4 proteins are synthesized, the coding sequence (CDS) of ALP1 is SEQ ID No: 2, the amino acid sequence of ALP1 protein is SEQ ID No: 1; the coding sequence (CDS) of ALP2 is SEQ ID No: 4, the amino acid sequence of ALP2 protein is SEQ ID No: 3; the coding sequence (CDS) of ALP3 is SEQ ID No: 6, the amino acid sequence of ALP3 protein is SEQ ID No: 5; the coding sequence (CDS) of ALP4 is SEQ ID No: 8, the amino acid sequence of ALP4 protein is SEQ ID No: 7.
[0036] 2. Construction of recombinant vector The coding gene of the protein in step 1 is inserted into the signal peptide sequence and 8×His-tag site of the pGAP_SP23Q6 vector by using the seamless cloning technology to construct the expression vectors pGAP_SP23Q6_ALP1, pGAP_SP23Q6_ALP2, pGAP_SP23Q6_ALP3 and pGAP_SP23Q6_ALP4.
[0037] The structure of the recombinant vector pGAP_SP23Q6_ALP3 is described as follows: the DNA fragment with the sequence of SEQ ID No: 6 is inserted between the 5'-AAGATCTTGTCTGCTTTGTTGTTGTTGTTCACTTTGGCTTTCGCTCAACAACAACAACAACAAATG-3' and 5'-CATCATCATCATCATCATCATCATCAT-3' fragments of the starting vector pGAP_SP23Q6, and the other sequences of the starting vector pGAP_SP23Q6 are kept unchanged to obtain the recombinant vector. The recombinant vector pGAP_SP23Q6_ALP3 can express the fusion protein ALP3 containing the protein tag with the amino acid sequence of SEQ ID No: 9.
[0038] The differences between the recombinant vectors pGAP_SP23Q6_ALP1, pGAP_SP23Q6_ALP2 and pGAP_SP23Q6_ALP4 and pGAP_SP23Q6_ALP3 are that the SEQ ID No: 6 in the recombinant vector pGAP_SP23Q6_ALP3 is replaced by SEQ ID No: 2, SEQ ID No: 4 and SEQ ID No: 8.
[0039] 3. Obtaining of recombinant transformant The expression vector was transformed into Pichia pastoris GS115, and the inserted sequence was verified by PCR, gel recovery, and sequencing. The sequencing primers were as follows: GAP-F: 5'-ACGCATGTCTTGAGATTATTGGA-3'; 3AOX-R: 5'-GGCAAATGGCATTCTGACATCCT-3'.
[0040] The transformants were successfully obtained and verified, and were named as GS115_pGAP_SP23Q6_ALP1, GS115_pGAP_SP23Q6_ALP2, GS115_pGAP_SP23Q6_ALP3, and GS115_pGAP_SP23Q6_ALP4.
[0041] Example 2, preparation of recombinant degradation enzymes ALP1-ALP4 The specific steps of the secretory expression of the degradation enzymes ALP1-ALP4 are as follows: 1. The transformant bacterial liquid was streaked on a YPD resistant plate (G418 0.5 mg / mL), and was cultured at 30°C and 200 rpm overnight to obtain a single colony.
[0042] 2. The single colony was inoculated in 5 mL of YPD resistant medium (G418 0.5 mg / mL), and was cultured at 30°C and 200 rpm until the bacterial liquid OD 600 =0.6-0.8.
[0043] 3. The transformant was inoculated in 20 mL of YPD liquid medium (prepared ingredients: 1% yeast extract, 2% peptone, and 2% glucose) at a 1% inoculation amount, and was cultured at 30°C and 200 rpm for 48 h.
[0044] 4. Centrifugation was performed at 4°C and 5000 rpm for 5 min, and the obtained supernatant was the crude enzyme liquid, which was stored at -20°C.
[0045] Subsequently, SDS-PAGE protein electrophoresis and western blot verification were performed.
[0046] The experimental results show that single protein bands appear at 48, 45, 55, and 16 kDa, which are close to the size of the predicted ALP1-ALP4 fusion His-Tag, indicating that the fusion proteins ALP1, ALP2, ALP3, and ALP4 are successfully expressed in GS115. Figure 1 ).
[0047] Example 3, determination of degradation activity of the recombinant degradation enzymes ALP1-ALP4 Experimental materials: Degradation enzymes ALP1-ALP4 crude enzyme solution; positive control quick CIP (5000 units / ml), purchased from Neb (Ipswich, MA, USA), item number M0525L; substrate LPS from Escherichia coli O55:B5 (MCE), purchased from MedChemexpress.cn (Monmouth Junction, NJ, USA), item number HY-D1056; ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit, purchased from Nanjing Kingsriver Biotechnology Co., Ltd.
[0048] Determination of degradation activity of recombinant enzymes ALP1-4: 1. Take 100 μL of ALP1-4 crude enzyme solution (sample group), positive control (10, 20, 30 μL of quick CIP, add 20, 40, 60 μL of CIP buffer, respectively, and add endotoxin-free water to 100 μL), and negative control (endotoxin-free water) into endotoxin-free test tubes, respectively, add 2 μL of 1 mg / mL LPS, and react at 37°C for 1 h; 2. Determine the residual amount of endotoxin according to the operation manual of ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit.
[0049] Calculation of enzyme activity (enzyme activity unit U / mg contained in each milligram of enzyme):
[0050] Among them, S: endotoxin concentration of sample group, EU / mL.
[0051] N: endotoxin concentration of negative control, EU / mL.
[0052] P: endotoxin concentration of positive control, EU / mL.
[0053] V: volume of reaction system, mL.
[0054] 50: difference in enzyme activity units between each group of positive control, U.
[0055] C: protein concentration of ALP in sample group, mg / mL.
[0056] d: dilution multiple of sample to be tested.
[0057] The experimental results show that the enzyme activity of lipopolysaccharide degrading enzymes ALP1-ALP4 is significantly higher than that of the positive control group quickCIP (P<0.0001), and the enzyme activity of ALP3 is the highest, with a specific activity of more than 370,000 U / mg, which is significantly higher than that of other ALP (P<0.0001), and is 75 times higher than that of quick CIP Figure 2 .
[0058] The above has been described in detail. For those skilled in the art, the present application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the purpose and scope of the present application and without unnecessary experiments. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application.
Claims
1. A protein, which is any one of the following: 1) a protein having an amino acid sequence of SEQ ID No: 9 or SEQ ID No: 5 in the sequence listing; 2) a protein obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the protein of 1) and having the same function; 3) a fusion protein obtained by linking a protein tag to the N terminus or / and C terminus of the protein of 1) or 2).
2. A nucleic acid molecule encoding the protein of claim 1, characterized in that, The nucleic acid molecule is any one of the following DNA molecules: 1) a DNA molecule having a nucleotide sequence of SEQ ID No: 6 in the sequence listing; 2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in 1) and encoding the protein of claim 1; 3) a DNA molecule hybridizing to the nucleotide sequence defined in 1) or 2) under stringent conditions and encoding the protein of claim 1.
3. Biomaterials containing the nucleic acid molecule according to claim 2, characterized in that, The biological material is any one of the following: B1) an expression cassette containing the nucleic acid molecule of claim 2; B2) a recombinant vector containing the nucleic acid molecule of claim 2, or a recombinant vector containing the expression cassette of B1); B3) a recombinant microorganism containing the nucleic acid molecule of claim 2, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2).
4. The biomaterial of claim 3, wherein, B2) the recombinant vector is a recombinant vector obtained by inserting the nucleotide sequence of SEQ ID No: 6 into the multiple cloning site of the vector pGAP_SP23Q6; B3) the recombinant microorganism is a recombinant Pichia pastoris obtained by introducing the recombinant vector into a yeast; the yeast is Pichia pastoris GS115.
5. A method of producing the protein of claim 1, characterized by, The method is introducing into a microorganism a DNA molecule capable of expressing the protein of claim 1 to obtain a recombinant microorganism, culturing the recombinant microorganism, collecting the supernatant of the fermentation broth to obtain the protein.
6. The production method according to claim 5, wherein The culturing of the recombinant microorganism comprises the following steps: inoculating the recombinant microorganism into a fermentation medium to culture to obtain the protein.
7. Use of the protein of claim 1 in any one of the following: 1) for preparing a lipopolysaccharide-degrading enzyme; 2) for preparing a product containing lipopolysaccharide-degrading enzyme activity; 3) for degrading lipopolysaccharide.
8. Use of the nucleic acid molecule of claim 2 in any one of the following: 1) for preparing a lipopolysaccharide-degrading enzyme; 2) for preparing a product containing lipopolysaccharide-degrading enzyme activity; 3) for degrading lipopolysaccharide.
9. Use of the biological material of claim 3 or 4 in any one of the following: 1) for preparing a lipopolysaccharide-degrading enzyme; 2) for preparing a product containing lipopolysaccharide-degrading enzyme activity; 3) for degrading lipopolysaccharide.
10. Use of the method of claim 5 or 6 in any one of the following: 1) for preparing a lipopolysaccharide-degrading enzyme; 2) for preparing a product containing lipopolysaccharide-degrading enzyme activity; 3) for degrading lipopolysaccharide.