CN105734029B - Phospholipase antibacterial peptide - Google Patents
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- CN105734029B CN105734029B CN201410767157.7A CN201410767157A CN105734029B CN 105734029 B CN105734029 B CN 105734029B CN 201410767157 A CN201410767157 A CN 201410767157A CN 105734029 B CN105734029 B CN 105734029B
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Abstract
The invention provides a fusion protein comprising a phospholipase moiety and an antimicrobial peptide moiety. The fusion protein has phospholipid hydrolysis activity and antibacterial activity, and can be used for reducing the bacterial contamination risk of the oil horn in phospholipase degumming.
Description
Technical Field
The invention relates to a fusion protein, in particular to a phospholipase antibacterial peptide.
Background
With the development of biotechnology, vegetable oil degumming process has been increasingly carried out by using phospholipase, which hydrolyzes fatty acid chains of phospholipids to produce hydrated lysophospholipids, and removes the hydrolyzed lysophospholipids by a hydration process to obtain a byproduct called oil foot[1]。
The oil foot contains 20-25% of phospholipid (wherein choline phospholipid, ethanolamine phospholipid and inositol phospholipid account for 1/3), 25-30% of neutral oil, 45-50% of water, 0.5-1.0% of sterol and 5-8% of other components, such as cake powder, protein, saccharide, wax, pigment and organic and inorganic impurities. So that it can be used in the fields of producing fatty acid, recovering neutral oil, preparing compound fertilizer, preparing feed and extracting vitamin E, etc. However, the literature reports that the oil residue is easy to ferment when heated (above 20 ℃), seriously pollutes the environment and also greatly reduces the nutritional value of the oil residue in the feed[2]。
Antimicrobial peptides (AMPs) are short peptide substances with a certain bactericidal effect generated by a natural immune system in a host defense system, are widely distributed in the natural world, and have broad-spectrum antibacterial property. Many antibacterial peptides not only have bactericidal action on gram-negative bacteria and gram-positive bacteria, but also have a certain inhibiting action on some fungi, protists, even tumor cells and viruses.
The antibacterial action mechanism of the antibacterial peptide is closely related to the secondary structure of the antibacterial peptide. The antibacterial peptide is a short peptide with highly conserved sequence consisting of 20-60 amino acids, and the N end of the variant substituted by the conserved amino acid is rich in hydrophilic basic amino acids, such as lysine and arginine, and has positive charge; the C terminal is rich in hydrophobic amino acid, is usually amidated, is neutral or weakly acidic, and has electric neutrality. The secondary structure of antimicrobial peptides is most commonly the alpha helix, and in addition, the beta-sheet and Loop structures (Loop) make the spatial structure of the peptide chain more compact. First, the electropositive regions of the antimicrobial peptides bind and disrupt the bacterial cell membrane through electrostatic interactions. Secondly, the water-lipid amphipathy of the antibacterial peptide enables the antibacterial peptide to have the characteristics similar to phospholipid molecules, and can help the antibacterial peptide to melt small holes in a human membrane structure, so that the membrane structure is damaged, and the main mechanism for the antibacterial peptide to play a biological role is provided.
[1]Clausen K.Enzymatic oil-degumming by a novel microbialphospholipase.European journal of lipid science and technology.2001；103:333-40.
[2] The research progress of the comprehensive utilization of soybean oil foot in China, oil engineering and technology 201306:62-64
Disclosure of Invention
The inventors of the present invention found that by introducing a polypeptide fragment having antibacterial activity (e.g., an antibacterial peptide) into phospholipase by fusion protein technology (fusion protein technology), a fusion enzyme having high phospholipid hydrolysis activity and antibacterial (e.g., against escherichia coli, bacillus subtilis) activity is obtained, which can be used to reduce the risk of contamination of oil horns in phospholipase degumming.
Accordingly, it is a first object of the present invention to provide a fusion protein comprising a phospholipase moiety and an antimicrobial peptide moiety.
In a specific embodiment of the invention, the phospholipase is phospholipase C.
In a specific embodiment of the invention, the sequence of the phospholipase moiety is shown in SEQ ID NO 3.
In a particular embodiment of the invention is a homologue, variant or fragment of SEQ ID NO. 3 which retains phospholipase activity. In a preferred embodiment of the invention, the variant is a variant with conservative amino acid substitutions.
In a specific embodiment of the invention, the antimicrobial peptide is derived from a mammal.
In one embodiment of the present invention, the antimicrobial peptide SMAP29 is preferred.
In a specific embodiment of the invention, the sequence of the sequence part of the antibacterial peptide is shown as SEQ ID NO. 1.
In a particular embodiment of the invention, to a homologue, variant or fragment of SEQ ID NO. 1 which retains phospholipase activity, in a particular embodiment of the invention, the variant is preferably a conservative amino acid substitution variant.
It is a second object of the present invention to provide a nucleic acid molecule capable of encoding the fusion protein of the present invention.
In a specific embodiment of the invention, the nucleic acid molecule comprises the polynucleotides shown in SEQ ID NO. 2 and SEQ ID NO. 4.
It is a third object of the present invention to provide an expression vector comprising the nucleic acid molecule of the present invention.
In a specific embodiment of the invention, the expression vector comprises the polynucleotides shown in SEQ ID NO. 2 and SEQ ID NO. 4.
It is a fourth object of the invention to provide a host cell comprising a fusion protein, a nucleic acid molecule, or an expression vector of the invention.
The fifth object of the present invention is to provide a method for increasing phospholipase activity, comprising fusing phospholipase with an antibacterial peptide.
The sixth object of the present invention is to provide a method for degumming oils and fats, comprising contacting the fusion protein of the present invention or a disrupted solution of a host cell with oils and fats.
Brief description of the drawings
FIG. 1 shows the results of gel electrophoresis detection of the fusion protein, wherein lane 1 shows the results of detection of cells containing empty plasmids, lane 2 shows the results of detection of cells containing the target gene CL, and lane 3 shows the results of detection of cells containing the target gene CL-S.
FIG. 2 shows the electrophoresis results of the CL and CL-S protein solutions, wherein lanes 1 to 5 show the electrophoresis results of BSA solutions at concentrations of 50, 100, 200, 500 and 1000. mu.g/mL, lane 6 shows the electrophoresis result of the CL protein solution, and lane 7 shows the electrophoresis result of the CL-S protein solution.
FIG. 3 shows the results of comparison of the hydrolysis activities of the fusion protein CL-S and phospholipase CL.
FIG. 4 shows the structure of the fusion protein CL-S antibacterial activity assay.
FIG. 5 is a standard curve prepared using phospholipase C with different enzyme activities.
Detailed Description
It is understood that the sum of the percentages by weight of the components contained in the composition according to the invention is equal to 100%.
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
In the present invention, the percentage (%) or parts refers to the weight percentage or parts by weight with respect to the composition, unless otherwise specified.
In the present invention, the respective components referred to or the preferred components thereof may be combined with each other to form a novel embodiment, if not specifically stated.
In the present invention, all embodiments and preferred embodiments mentioned herein may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, the sum of the contents of the components in the composition is 100% if not indicated to the contrary.
In the present invention, the sum of the parts of the components in the composition may be 100 parts by weight, if not indicated to the contrary.
In the present invention, unless otherwise stated, the numerical range "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "0 to 5" indicates that all real numbers between "0 to 5" have been listed herein, and "0 to 5" is only a shorthand representation of the combination of these numbers.
In the present invention, unless otherwise indicated, the integer numerical range "a-b" represents a shorthand representation of any combination of integers between a and b, where a and b are both integers. For example, an integer numerical range of "1-N" means 1, 2 … … N, where N is an integer.
In the present invention, unless otherwise specified, "combinations thereof" mean multicomponent mixtures of the elements described, for example two, three, four and up to the maximum possible.
The term "a" or "an" as used herein means "at least one" if not otherwise specified.
All percentages (including weight percentages) stated herein are based on the total weight of the composition, unless otherwise specified.
The "ranges" disclosed herein are in the form of lower and upper limits. There may be one or more lower limits, and one or more upper limits, respectively. The given range is defined by the selection of a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this manner are inclusive and combinable, i.e., any lower limit can be combined with any upper limit to form a range. For example, ranges of 60-120 and 80-110 are listed for particular parameters, with the understanding that ranges of 60-110 and 80-120 are also contemplated. Furthermore, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5.
Herein, unless otherwise specified, the proportions or weights of the components are referred to as dry weights.
In this context, each reaction is carried out at normal temperature and pressure unless otherwise specified.
Herein, unless otherwise specified, the individual reaction steps may or may not be performed sequentially. For example, other steps may be included between the various reaction steps, and the order may be reversed between the reaction steps. Preferably, the reaction processes herein are carried out sequentially.
The present application is based, at least in part, on engineering phospholipases with Fusion Protein Technology (Fusion Protein Technology) to improve their performance.
The inventor introduces the polypeptide fragment with antibacterial activity into phospholipase by fusion protein technology (fusion protein technology), obtains the fusion enzyme with high phospholipid hydrolysis activity and anti-Escherichia coli and Bacillus subtilis, and is expected to be used in the frontier field of reducing the contamination risk of oil horn in phospholipase degumming. The fused phospholipase provided by the application can have one or more of the following advantages:
introducing antibacterial activity into the field of phospholipase degumming for reducing the risk of contamination of the degummed product;
the phospholipid hydrolysis activity of the antimicrobial peptide-introduced phospholipase is maintained, and in preferred embodiments, the phospholipid hydrolysis activity of the antimicrobial peptide-introduced phospholipase is significantly increased.
In a first aspect, the present application provides a fusion protein comprising a phospholipase moiety and an antimicrobial peptide moiety.
Techniques for constructing fusion proteins are well known in the art. For example, the respective proteins or polypeptides to be fused can be introduced into the same expression vector for expression by enzymatic cleavage.
Phospholipases are a class of enzymes well known to those skilled in the art, which function to hydrolyze glycerophospholipids. Phospholipase enzymes are generally classified into phospholipase enzymes of A1, A2 and B, C, D types according to the position of each ester bond acting on the inside of phospholipid molecules. The phospholipase in the application can be natural phospholipase, synthesized or modified phospholipase, or one of the above phospholipases or a mixture of at least two thereof. In a particular embodiment of the invention, the phospholipase used is phospholipase c (plc). A variety of isoenzymes for phospholipase C have also been found in the art, such as PLC- β, PLC- γ, PLC-, and the like. As an example, the phospholipase of the present application can be a phospholipase CL-PLC, the amino acid sequence and the encoding nucleic acid sequence of which are shown in SEQ ID NO 3 and 4, respectively.
In some embodiments, the phospholipase moiety is a homolog, variant, or fragment of SEQ ID No. 3 that retains phospholipase activity.
Antimicrobial peptides (AMPs) are short peptide substances with a certain bactericidal effect generated by a natural immune system in a host defense system, are widely distributed in the natural world, and have broad-spectrum antibacterial property. Many antibacterial peptides not only have bactericidal action on gram-negative bacteria and gram-positive bacteria, but also have a certain inhibiting action on some fungi, protists, even tumor cells and viruses.
The antibacterial action mechanism of the antibacterial peptide is closely related to the secondary structure of the antibacterial peptide. The antibacterial peptide is a short peptide with highly conserved sequence consisting of 20-60 amino acids, and the N end is rich in hydrophilic basic amino acids such as lysine and arginine and has positive charges; the C terminal is rich in hydrophobic amino acid, is usually amidated, is neutral or weakly acidic, and has electric neutrality. The secondary structure of antimicrobial peptides is most commonly the alpha helix, and in addition, the beta-sheet and Loop structures (Loop) make the spatial structure of the peptide chain more compact. First, the electropositive regions of the antimicrobial peptides bind and disrupt the bacterial cell membrane through electrostatic interactions. Secondly, the water-lipid amphipathy of the antibacterial peptide enables the antibacterial peptide to have the characteristics similar to phospholipid molecules, and can help the antibacterial peptide to melt small holes in a human membrane structure, so that the membrane structure is damaged, and the main mechanism for the antibacterial peptide to play a biological role is provided.
In a specific embodiment of the invention, the antibacterial peptide used is an antibacterial peptide derived from mammals, such as from sheep bone marrow cells, bovine neutrophils, porcine small intestine. In a specific embodiment of the invention, the antimicrobial peptide used is antimicrobial peptide SMAP29, the amino acid sequence and the coding nucleic acid sequence of which are shown in SEQ ID NO 1 and 2, respectively.
In some embodiments, the antimicrobial peptide portion is a homolog, variant, or fragment of SEQ ID NO. 1 that retains antimicrobial peptide activity.
The term "homologue" as used herein is intended to mean a substance, such as a homologous gene, a homologous protein, etc., that is produced by chemotaxis or evolution of a common progenitor molecule and exhibits similarity in sequence or structure. For example, a homologue of a protein/nucleic acid may refer to a class of proteins/nucleic acids that are identical or similar in structure, sequence or function in different species. As a non-limiting example, the phospholipase CL-PLC in the present application is from CLOPLC8(GenBank: D49969.1), and its homologue can be from a phospholipase C of a type identical or similar in structure, sequence or function to CL-PLC of other microorganisms. By way of non-limiting example, the antimicrobial peptide SMAP29 of the present application is derived from sheep, and its homologue may be derived from another animal, such as a mammalian species, that is structurally, sequentially or functionally identical or similar to the antimicrobial peptide SMAP 29.
The term "variant" as used herein may refer to a natural variant or an artificial variant. As an example of a natural variant of a protein/nucleic acid, it may refer to a mutant produced during natural expression. As an example of an artificial variant of a protein/nucleic acid, it may refer to an artificial mutant obtained by artificial genetic engineering, including but not limited to amino acid substitutions, additions or deletions. A typical example of a genetic engineering approach is conservative amino acid substitutions. Certain amino acid substitutions, known as "conservative amino acid substitutions," can occur frequently in proteins without changing the conformation or function of the protein, a well-established rule in protein chemistry.
Conservative amino acid substitutions in this application include, but are not limited to, substitution of any one of these aliphatic amino acids with any one of glycine (G), alanine (a), isoleucine (I), valine (V), and leucine (L); substitution of threonine (T) with serine (S) and vice versa; substitution of glutamic acid (E) with aspartic acid (D), and vice versa; (ii) substitution of asparagine (N) with glutamine (Q), and vice versa; substitution of arginine (R) with lysine (K), and vice versa; substitution of any one of these aromatic amino acids with phenylalanine (F), tyrosine (Y) and tryptophan (W); and substitution of cysteine (C) with methionine (M) and vice versa. Other substitutions may also be considered conservative, depending on the particular amino acid environment and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often interchangeable, as are alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be exchanged for leucine and isoleucine, and sometimes for valine. Lysine (K) and arginine (R) are often interchanged at the following positions: the important characteristics of the amino acid residues are their charge and the different pKs of the two amino acid residues are not significant.
The term "fragment" as used herein generally refers to a "functional fragment", i.e., a fragment that retains the biological activity of the original sequence. By way of non-limiting example, functional fragments may refer to those fragments that retain a functional domain of a protein.
In some embodiments, the sequence of the antimicrobial peptide portion is as set forth in SEQ ID NO. 1, or is a homolog, variant, or fragment of SEQ ID NO. 1. In some embodiments, the variant of SEQ ID NO. 1 is a variant with conservative amino acid substitutions.
In some embodiments, the phospholipase moiety and the antimicrobial peptide moiety are directly linked by a covalent bond.
In some embodiments, the fusion protein consists essentially of a phospholipase moiety and an antimicrobial peptide moiety. In some embodiments, the phospholipase moiety and the antimicrobial peptide moiety are linked by a linker peptide. The selection and use of linker peptides is well known in the art.
It will be appreciated that in addition to the phospholipase moiety, the antimicrobial peptide moiety, and the optional linker peptide, the fusion protein may also include other moieties, including, but not limited to, a signal peptide sequence, an initial Met, and the like, as well as ancillary moieties for identification, isolation, and purification of the fusion protein, e.g., a histidine tag, a fluorescent tag, and the like.
In a second aspect, the present application provides a nucleic acid molecule capable of encoding a fusion protein according to the first aspect. The correspondence of the amino acid sequence of a protein to its encoding nucleic acid sequence is well known to those skilled in the art. The present application also contemplates encoding nucleic acid molecules that result from codon degeneracy as well as codon bias of different organisms. In some embodiments, the nucleic acid molecule comprises the polynucleotides set forth in SEQ ID NO. 2 and SEQ ID NO. 4.
In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect. The present application also provides an expression vector comprising a nucleic acid encoding a phospholipase and a nucleic acid encoding an antimicrobial peptide. In some embodiments, the expression vector comprises the polynucleotides set forth in SEQ ID NO. 2 and SEQ ID NO. 4. In some embodiments, the expression vector is designed for expression in eukaryotic or prokaryotic cells. In some embodiments, the expression vector is designed for expression in a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell. In some embodiments, the expression vector is a plasmid. Suitable eukaryotic or prokaryotic vectors are well known to those skilled in the art, and a variety of maternal vectors are commercially available. Examples of carriers include, but are not limited to, the various carriers used in the examples of the present application.
In a fourth aspect, the application provides a cell comprising a fusion protein of the first aspect, or a nucleic acid molecule of the second aspect, or an expression vector of the third aspect.
In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell. In some embodiments, the cell is an escherichia coli (e. With respect to cells comprising a nucleic acid molecule of the present application, the nucleic acid molecule can be extrachromosomal (e.g., in a vector), or can be integrated into the chromosome of the host cell. Techniques for integrating nucleic acid molecules into the chromosome of a host cell and for introducing vectors into host cells by transformation or transfection are well known to those skilled in the art.
In a fifth aspect, the application provides the use of a nucleic acid molecule according to the second aspect, or an expression vector according to the third aspect, or a cell according to the fourth aspect, for the preparation of a phospholipase.
Techniques for producing a polypeptide or protein of interest using a nucleic acid molecule, expression vector, or genetically engineered host cell are well known to those skilled in the art.
In a sixth aspect, the present application provides a method of increasing phospholipase activity comprising fusing a phospholipase with an antimicrobial peptide. The phospholipase activity or property described herein includes, but is not limited to, the ability to hydrolyze phosphate ester bonds, the ability to hydrolyze mixed phospholipids, and the like.
In a seventh aspect, the present application provides a fused phospholipase obtainable by the method of the sixth aspect.
In an eighth aspect, the present application provides a method for degumming of oils and fats, comprising contacting the fusion protein of the first aspect or the fusion phospholipase of the seventh aspect with oils and fats. In some embodiments of the invention, a disruption solution of a host cell expressing the fusion protein or fusion phospholipase is contacted with the oil. In some embodiments, the method comprises the step of subjecting the fusion protein or the fusion phospholipase to an emulsification treatment prior to the contacting.
It is to be understood that the embodiments of the technical features described in the first aspect are also applicable to the second to eighth aspects.
Example 1 expression vector construction
The antibacterial peptide SMAP29 (synthesized by Biotechnology engineering Co., Ltd.) and phospholipase CL-PLC (synthesized by Biotechnology engineering Co., Ltd.) were introduced into an expression vector pET30a, wherein,
the amino acid sequence of the antibacterial peptide SMAP29 is shown as SEQ ID NO. 1;
the nucleotide sequence is shown as SEQ ID NO. 2.
The phospholipase CL-PLC amino acid sequence is shown in SEQ ID NO. 3;
the phospholipase CL-PLC nucleic acid sequence is shown in SEQ ID NO. 4.
The sequence of SMAP29 was digested with BamHI/XhoI, and antibacterial peptide SMAP29 was ligated into expression vector pET30a (available from Novagen) using T4 ligase (Fermentas) to construct a new vector, SMAP29-Pet30 a.
Phospholipase CL-PLC was PCR amplified with Primerstar high fidelity polymerase (Takara Inc.), and the amplification primers are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.
The PCR amplification procedure was as follows: 30cycles at 95 ℃ for 30s,55 ℃ for 30s and 72 ℃ for 1.5 min.
The PCR product of phospholipase CL-PLC and expression vector SMAP29-pET30a were digested with NdeI/BamHI, after tapping and purification, T4 ligase was ligated for 16 degrees for 1h, DH 5. alpha. competent cells (purchased from Biotechnology Ltd.) were transformed and cultured overnight at 37 ℃ in solid LB medium plates (1.5% agarose, yeast powder 5%, peptone 10%, NaCl 5%) containing 50. mu.g/mL antibiotic sodium, followed by shaking culture at 37 ℃ and 200rpm for 16h in 5mL LB medium (yeast powder 5%, peptone 10%, NaCl 5%).
Extracting plasmids by using the Qigen plasmid extraction kit, carrying out enzyme digestion verification, and sending the plasmids with correct enzyme digestion to the engineering bioengineering company Limited for sequencing. The plasmid with correct sequencing result is the expression vector of the phospholipase-antibacterial peptide.
In the following experiments, phospholipase CL-PLC is abbreviated as CL, a phospholipase CL-PLC expression vector is abbreviated as CL expression vector, a phospholipase antibacterial peptide fusion product is abbreviated as CL-S, and an expression vector of phospholipase-antibacterial peptide is abbreviated as CL-S expression vector.
Example 2
Respectively transfecting Escherichia coli BL21 competent cells (purchased from Biotechnology engineering Co., Ltd.) by a heat shock method of a CL-S expression vector and the CL expression vector, culturing overnight at 37 ℃ in a solid LB culture medium plate (1.5% agarose, 5% yeast powder, 10% peptone and 5% NaCl) containing 50 mu g/mL antibiotic kalin, selecting a single colony, inoculating the single colony in the LB culture medium at 37 ℃, carrying out shake culture at 200rpm, expanding culture until the OD600 value is 0.6-0.8, adding an isopropyl-beta-D-thiogalactopyranoside (IPTG) inducer with the final concentration of 1mM, and carrying out shake culture at 150rpm at 16 ℃ continuously. The cells were collected by centrifugation at 10000rpm for 10 min.
The cells were resuspended in Tris-Cl buffer (20mM, pH8.0) lysate, placed in ice, disrupted with an ultrasonicator 400W for 5S at intervals of 10S, disrupted in a co-ice bath for 20min at 12000rpm, and centrifuged for 20min to obtain the lysate supernatant.
The cleaved samples were examined by polyacrylamide gel electrophoresis (SDS-PAGE) and the control was an empty plasmid expression sample containing no target gene, and the results are shown in FIG. 1. As can be seen from FIG. 1, the expression of the target proteins CL-S (lane 3) and CL (lane 2) is seen in a large amount at around 50kD as compared with the empty plasmid expression sample (lane 1) containing no target gene.
Example 3 quantitative determination of fusion protein CL-S and phospholipase CL
As standard samples, Bovine Serum Albumin (BSA) solutions of 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 500. mu.g/mL and 1000. mu.g/mL were prepared, respectively.
The CL lysis supernatant and the CL-S lysis supernatant obtained in example 2 were subjected to polyacrylamide gel electrophoresis using Bovine Serum Albumin (BSA) at 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 500. mu.g/mL and 1000. mu.g/mL as a standard, and the results are shown in FIG. 2.
The bands were analyzed using AlphaImager HP software from the energy gel image processing system to obtain the standard curve equation y 0.3797 x-414.42 (R)20.9915) and the resulting CL and CL-S concentrations were calculated to be 0.47mg/mL and 0.40mg/mL, respectively.
Example 4 comparison of phospholipid hydrolysis Activity of fusion protein CL-S and phospholipase CL
According to the results of the test in example 3, the CL and CL-S concentrations were adjusted to be the same (0.40 mg/mL).
Reagents were prepared according to Table 1, and the samples were sequentially applied, and reacted at 37 ℃ for 30 minutes to obtain CL-S and CL reaction solutions, respectively.
TABLE 1
| No | Composition (I) | Concentration of mother liquor | Final concentration of reaction | Volume (μ l) | |
| 1 | |
1％(w/v) | 0.5％(w/v) | 100 | |
| 2 | Tris-Cl buffer, pH 7.5 | | 25mM | 20 | |
| 3 | CaCl2 | 100mM | 5mM | 10 | |
| 4 | |
20 | |||
| 5 | H2O | 50 | |||
| Total of | 200 |
mu.L of the reaction mixture was taken, 200. mu.L of chloroform was added thereto, and the mixture was mixed for 20 seconds and centrifuged at 12000rpm for 1 minute. mu.L of the centrifuged supernatant was taken out and put into a fresh centrifuge tube, and reagents (wherein CIAP is bovine small intestine alkaline phosphatase and purchased from New England Biolabs (NEB)) were added in the order and volume shown in Table 2 and mixed well, followed by reaction at 37 ℃ for 30 minutes.
TABLE 2
| No | Composition (I) | Concentration of mother liquor | Working fluid concentration | Volume (μ l) |
| 1 | |
80 | ||
| 2 | Tris-Cl buffer, pH9.0 | 1M | 50mM | 10 |
| 3 | MgCl2 | 500mM | 10mM | 4 |
| 4 | CIAP | 0.5U/μl | 10U/ |
4 |
| 5 | H2O | 102 | ||
| Total of | 200 |
In the above reaction system, other reagents were further added in the order and volume shown in Table 3, and the reaction was continued at 37 ℃ for 10 minutes.
TABLE 3
| No | Composition (I) | Concentration of mother liquor | Working fluid concentration | Volume (μ l) |
| 1 | CIAP enzyme digestion reactant | 200 | ||
| 2 | H2O | 740 | ||
| 3 | Ascorbic acid | 10％(w/v) | 0.2％(w/v) | 20 |
| 4 | Ammonium molybdate | 2.5％(w/v) | 0.1％(w/v) | 40 |
| Total of | 1000 |
And (3) taking the reaction liquid in the reaction system, detecting the absorbance at 700nm, carrying out parallel detection, and calculating the enzyme activity by using a standard curve. Wherein the blank control samples are respectively corresponding enzyme solutions inactivated by boiling with 100 deg.C water for 10min, and sodium phosphate (Na) with different concentrations is used in standard curve3PO4·12H2O) measuring the absorbance at 700nm and using phosphate radical (PO)4 3-) The concentration is plotted on the abscissa and the absorbance at 700nm is plotted on the ordinate, and the results are shown in FIG. 5. Since the higher the enzyme activity is, the PO is cut off by the phospholipase in the reaction solution4 3-The more, therefore, the more the enzyme activity of the sample can be obtained by substituting the absorbance of each reaction solution detected at 700nm into a standard curve equation to obtain PO4 3-And (4) expressing the concentration.
The results of CL-S and CL detection are shown in FIG. 3. According to the results of fig. 3, the phospholipid hydrolysis activity of the antimicrobial peptide-introduced phospholipase (CL-S) was significantly increased (. about.p < 0.01).
Example 5 test of antibacterial Properties of fusion protein CL-S
30. mu.L of Escherichia coli DH 5. alpha. (purchased from Biotechnology Ltd.) and Bacillus subtilis (purchased from Biotechnology Ltd.) were inoculated into 3mL of LB liquid medium (yeast powder 5%, peptone 10%, NaCl 5%), and cultured overnight at 37 ℃ with shaking at 200 rpm. After 400. mu.L of each bacterial solution was applied to LB solid medium plates containing no antibiotics, and dried, sample wells were punched out with a punch, 100. mu.L of CL-S (0.4mg/mL) sterilized by 0.22 μm filtration was added, and CL containing no antibacterial peptide and phospholipase A1 (PLA 1), phospholipase A2 (PLA 2) and phospholipase C (PLC) purchased from Dissman were used as controls, and the results are shown in FIG. 4.
According to the results in FIG. 4, CL-S has a significant inhibitory effect on both E.coli and B.subtilis, whereas the other control enzymes have no bacteriostatic activity.
It is to be understood that while the invention has been described in certain forms, it is not to be limited to the details shown and described in this specification. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention. Such variations are within the scope of the invention as claimed.
Claims (8)
1. A fusion protein comprising a phospholipase moiety and an antimicrobial peptide moiety, wherein the phospholipase is phospholipase C and the antimicrobial peptide is antimicrobial peptide SMAP 29; the sequence of the phospholipase part is shown as SEQ ID NO. 3, and the sequence of the antibacterial peptide part is shown as SEQ ID NO. 1.
2. A nucleic acid molecule capable of encoding the fusion protein of claim 1.
3. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule comprises the polynucleotide of SEQ ID No. 2 and SEQ ID No. 4.
4. An expression vector comprising the nucleic acid molecule of claim 2 or 3.
5. The expression vector of claim 4, wherein the expression vector comprises the polynucleotides of SEQ ID NO. 2 and SEQ ID NO. 4.
6. A host cell comprising the fusion protein of claim 1, or the nucleic acid molecule of claim 2 or 3, or the expression vector of claim 4 or 5.
7. A method of increasing a phospholipase activity comprising fusing a phospholipase with an antimicrobial peptide, wherein the phospholipase is phospholipase C and the antimicrobial peptide is antimicrobial peptide SMAP 29; the sequence of the phospholipase part is shown as SEQ ID NO. 3, and the sequence of the antibacterial peptide part is shown as SEQ ID NO. 1.
8. A method for degumming of oils and fats, comprising contacting the fusion protein of claim 1 or the disrupted solution of the host cell of claim 6 with oils and fats.
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