CN105695430B - Improved phospholipase and preparation method and application thereof - Google Patents

Abstract

The present application describes improved phospholipases which are fusion proteins comprising a phospholipase moiety and a hydrophobic domain portion of a protein. Methods of making and uses of the improved phospholipases, as well as related nucleic acids, vectors, cells, and the like, are also described.

Description

Improved phospholipase and preparation method and application thereof

Technical Field

The present application relates generally to phospholipases and industrial applications thereof. In particular, the application relates to modification of phospholipase by molecular biology, and the modified phospholipase can be applied to degumming process of oil and fat refining.

Background

The phospholipid in the vegetable oil brings certain technical problems for oil refining. Degumming is an important step in vegetable oil refining, and refers to a process of removing various phospholipid substances dissolved in oil and fat. In general, degumming regimes can be divided into three types, including chemical degumming (e.g., aqueous degumming, acid degumming, etc.), physical degumming (e.g., membrane filtration degumming), and biological degumming (e.g., enzymatic methods). With the development of biotechnology, degumming of vegetable oils has been increasingly carried out enzymatically. Enzymatic degumming utilizes phospholipase to hydrolyze fatty acid chains of phospholipids to produce hydrated lysophospholipids, which are then removed by a hydration process. Compared with the traditional degumming method, the enzymatic degumming reaction condition is mild, the consumption of chemical substances can be greatly saved, almost no wastewater is generated, and the method has potential advantages in the aspects of environmental protection, economy, quality and the like [1 ].

The efficiency of phospholipase degumming is affected by a number of factors. For example, since the phospholipase reaction occurs at the oil-water interface, the oil-water interface area between the enzyme and the substrate is increased, and the phospholipase can efficiently function. At present, the common practice in the art is to use strong shear stirring and the like to keep the phospholipase on the oil-water interface during the reaction.

Therefore, there remains a need in the art for new methods for increasing phospholipase activity and reaction efficiency.

Summary of The Invention

In a first aspect, the present application provides a fusion protein comprising a phospholipase moiety and a hydrophobic domain portion of a protein.

In some embodiments, the phospholipase is phospholipase C.

In some embodiments, the phospholipase is C L OP L C8(GenBank: D49969.1).

In some embodiments, the sequence of the phospholipase moiety is as set forth in SEQ ID NO. 1, or is a homologue, variant or fragment of SEQ ID NO. 1 that retains phospholipase activity, preferably a conservative variant.

In some embodiments, the protein is an oil body protein.

In some embodiments, the oil body protein is selected from the group consisting of oil body proteins from soybean, peanut, sesame, mustard, canola, sunflower, corn, and cotton.

In some embodiments, the oil body protein is soy oil body protein.

In some embodiments, the hydrophobic domain portion of the oil body protein is a hydrophobic domain portion comprising a proline knot, preferably the hydrophobic domain portion has a sequence as shown in SEQ ID No. 5, or a homologue, variant or fragment of SEQ ID No. 5, preferably a conservative variant, that retains hydrophobicity.

In some embodiments, the homologue, variant or fragment of SEQ ID NO. 5 is a homologue, variant or fragment of SEQ ID NO. 5 retaining the proline knot, said variant preferably being a conservative variant.

In some embodiments, the hydrophobic domain portion of the protein is a transmembrane domain portion of the protein.

In a second aspect, the present application provides a nucleic acid molecule capable of encoding a fusion protein according to the first aspect.

In some embodiments, the nucleic acid molecule comprises the polynucleotides set forth in SEQ ID NO. 2 and SEQ ID NO. 6.

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 a hydrophobic domain of a protein.

In some embodiments, the expression vector comprises the polynucleotides set forth in SEQ ID NO. 2 and SEQ ID NO. 6.

In a fourth aspect, the present application provides a host 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 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 host cell according to the fourth aspect, for the preparation of a phospholipase.

In a sixth aspect, the present application provides a method of increasing phospholipase activity comprising fusing a phospholipase to a hydrophobic domain of a protein.

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, the method comprises the step of subjecting the fusion protein or the fusion phospholipase to an emulsification treatment prior to the contacting.

In a ninth aspect, the present application provides degummed fats and oils prepared according to the method of the eighth aspect.

Brief description of the drawings

FIG. 1 shows a representation of the data obtained by Protscale software (A)http://web.expasy.org/protscale/) Graph of analysis of the hydrophobic domain of soybean oil body protein.

FIG. 2 shows analysis of the expression levels of phospholipase (C L O) and phospholipase-oil body protein hydrophobic domain fusion protein (C L O-O) by gel electrophoresis and staining, the leftmost lane is a molecular weight marker, lane 1 shows protein expression of a strain sample before addition of an inducer, lane 2 shows the expression level of phospholipase (C L O) after addition of an inducer, and lane 3 shows the expression level of phospholipase-oil body protein hydrophobic domain fusion protein (C L O-O) after addition of an inducer.

FIG. 3 shows the quantitative analysis of phospholipase (C L O) and phospholipase-oil body protein hydrophobic domain fusion protein (C L O-O). Lane No. 1-5 shows the electrophoretic staining pattern of BSA solutions at concentrations of 50, 100, 200, 500 and 1000. mu.g/m L in sequence, Lane No. 6 shows the expression level of phospholipase (C L O), Lane No. 7 shows the expression level of phospholipase-oil body protein hydrophobic domain fusion protein (C L O-O), and the rightmost lane is the molecular weight marker.

FIG. 4 shows a comparison of the phosphate group hydrolysis activities of phospholipase C L O and fusion protein C L O-O.

FIG. 5 shows a comparison of phospholipid hydrolysis activities before and after emulsification for phospholipase C L O, fusion protein C L O-O, and control P L C.

FIG. 6 shows a standard curve in the phospholipid hydrolysis viability assay.

Brief description of the sequences

1, amino acid sequence of phospholipase C L OP L C8;

2, phospholipase C L OP L C8;

3, SEQ ID NO: the full-length amino acid sequence of soybean oil body protein (FJ 864730.1);

4, SEQ ID NO: encoding nucleic acid sequence of soybean oil body protein (FJ864730.1)

5, SEQ ID NO: an amino acid sequence of the hydrophobic domain of soybean oil body protein;

6 of SEQ ID NO: nucleic acid sequence encoding the hydrophobic domain of soybean oil body protein:

7, SEQ ID NO: a positive amplification primer of a hydrophobic structure domain of soybean oil body protein;

8, SEQ ID NO: a reverse amplification primer of the hydrophobic domain of the soybean oil body protein;

9, phospholipase C L OP L C8 as a forward amplification primer;

10 phospholipase C L OP L C8 reverse amplification primer.

Detailed description of the invention

Definition of

Unless otherwise indicated, each term in the present application has the same meaning as commonly understood by one of ordinary skill in the art.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and generally refer to a polymer of a plurality of amino acids joined by peptide bonds.

The terms "hydrophobic domain", "hydrophobic fragment", "hydrophobic region" or similar terms as used herein are to be understood in the same sense and refer to the region of the hydrophobic structure that constitutes a protein.

The international common single or three letter abbreviations for amino acids are used herein.

The terms "nucleic acid" and "polynucleotide" as used herein are used interchangeably and include, but are not limited to, DNA, RNA, and the like. Nucleotides may be naturally occurring or synthetic analogs.

The term "cell" as used herein may be a eukaryotic cell or a prokaryotic cell, such as, but not limited to, a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell or a plant cell.

Detailed description of the embodiments

The present application is based, at least in part, on engineering phospholipases with Fusion Protein Technology (Fusion Protein Technology) to improve their performance.

In general, the phospholipase degumming process involves the problem of oil-water interface based on phospholipid microcapsule structure [2, 3], i.e., phospholipid fatty acid chains and triacylglycerols form a hydrophobic core, phospholipid hydrophilic heads are exposed to form an oil-in-water microcapsule structure, and phospholipase reaction occurs at the oil-water interface. Therefore, in practical applications, emulsification (strong shear stirring) is often used to increase the oil-water interface area between the phospholipase and the substrate, so that the phospholipase can function efficiently. However, since phospholipases are often hydrophilic, continuous shear emulsification is theoretically required to retain them at the oil-water interface.

The inventors of the present application have found that by combining the hydrophobic domain of the protein with a phospholipase, the resulting fusion protein can remain at the oil-water interface during the reaction, avoiding the continuous shear emulsification process typically necessary for degumming with phospholipases.

In addition, the inventors of the present application have also found that particularly, the hydrophobic domain of oleosin has an embedded structure (e.g., proline knot) similar to the microcapsule structure of the phospholipase degumming reaction (i.e., when the phospholipid hydrolyzes the phospholipid, the phospholipid forms an internal hydrophobic, external hydrophilic microcapsule structure similar to the internal hydrophobic, external hydrophilic structure of oil body), so that the inventors of the present application imagine that the expected results of modifying phospholipase with the hydrophobic domain of oleosin may be better, and further experiments prove that the results of modifying phospholipase with the hydrophobic domain of oleosin are significantly better.

The fused phospholipase provided by the application can have one or more of the following advantages:

1. compared with the original phospholipase, the phosphoric ester bond hydrolysis capacity of the fused phospholipase is obviously improved;

2. when the substrate is mixed phospholipid, the phospholipid hydrolysis capacity is obviously improved by means of a simple emulsification process;

3. continuous shearing emulsification is not needed in the degumming process, so that the production cost is saved.

In a first aspect, the present application provides a fusion protein comprising a phospholipase moiety and a hydrophobic domain portion of a protein.

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.

Many proteins have hydrophobic domains and are useful in this application. Typical examples are transmembrane proteins. Since both cell membranes or organelle membranes (e.g., mitochondrial membranes) contain lipid components, the transmembrane domains of transmembrane proteins tend to be hydrophobic and are therefore useful in this application.

Phospholipase enzymes are well known to those skilled in the art and function to hydrolyze glycerophospholipids the phospholipase enzymes are generally classified as phospholipase enzymes of type a1, a2, B, C, D, depending on the position of the respective ester bond acting inside the phospholipid molecule, the phospholipase enzymes of the present application can be natural phospholipase enzymes, synthetic or modified phospholipase enzymes, or one or a mixture of at least two of the above mentioned phospholipase enzymes, in one embodiment of the present invention, the phospholipase enzyme used is phospholipase C (P L C), isoenzymes of various phospholipase C enzymes are also found in the art, such as P L C- β, P L C- γ, P L C-, P L C-, etc. as an example, the phospholipase of the present application can be phospholipase C L OP L C8(GenBank: D49969.1), hereinafter also abbreviated as "C L O", the amino acid sequence and the encoding nucleic acid sequence of which are shown in SEQ ID NOs: 1 and 2, respectively.

In some embodiments, the phospholipase moiety is a homologue, variant, or fragment of SEQ ID No. 1 that retains phospholipase activity.

The term "homologue" as used herein is meant to be well known in the art and refers to a substance, such as a homologous gene, a homologous protein, etc., that is produced by chemotaxis or evolution of a common progenitor molecule, and that exhibits similarity in sequence or structure.

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 other of these aliphatic amino acids with any 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, substitution of asparagine (N) with glutamine (Q), and vice versa, substitution of arginine (R) with lysine (K), and vice versa, substitution of any other of these aromatic amino acids with phenylalanine (F), tyrosine (Y), and tryptophan (W), and substitution of cysteine (C) with methionine (M), and vice versa.

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 protein is an oil body protein. The oil body protein is a protein from the oil body (oil body) as the organelle in plant seed cells [4,5 ]. Oil body proteins are widely found in a variety of oil-producing crops, including, but not limited to, soybean, peanut, sesame, mustard, canola, sunflower, corn, cotton, and the like. In some embodiments, the oil body protein is soy oil body protein. The amino acid sequence and the coding nucleic acid sequence of the soybean oil body protein are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4.

The hydrophobic region of oil body proteins is a stalk-like structure of about 11nm length, extending into the hydrophobic acyl portion of phospholipids and the triacylglycerol matrix inside the oil body [6] according to Tzen and Huang (1992) analysis of the hydrophobic region of oil body proteins, it is presumed that the hydrophobic region is a trans-parallel β folded structure topped with a "proline knot" consisting of 3 proline (P) and 1 serine (S). in oil body proteins derived from various plants, the hydrophobic region is highly conserved, especially a "proline knot". in some embodiments, the hydrophobic domain of soybean oil body protein can be used, the amino acid sequence and encoding nucleic acid sequence of which are shown in SEQ ID NO:5 and 6, respectively.

In some embodiments, the sequence of the hydrophobic domain portion of the oil body protein is as shown in SEQ ID NO. 5, or is a homolog, variant or fragment of SEQ ID NO. 5 that retains hydrophobicity. In some embodiments, the homolog, variant, or fragment of SEQ ID No. 5 is a homolog, variant, or fragment of SEQ ID No. 5 that retains the proline knot.

In some embodiments, the phospholipase moiety and the hydrophobic domain moiety are directly linked by a covalent bond.

In some embodiments, the fusion protein consists essentially of a phospholipase moiety and a hydrophobic domain portion of a protein. In some embodiments, the phospholipase moiety and the hydrophobic domain 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 hydrophobic domain portion of the protein, and the optional linker peptide, the fusion protein may also include other moieties, including, but not limited to, signal peptide sequences, starting Met, etc., as well as ancillary moieties for identification, isolation, and purification of the fusion protein, e.g., histidine tags, fluorescent tags, etc.

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. 6.

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 a hydrophobic domain of a protein. In some embodiments, the expression vector comprises the polynucleotides set forth in SEQ ID NO. 2 and SEQ ID NO. 6. 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 to a hydrophobic domain of a protein. 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, the method comprises the step of subjecting the fusion protein or the fusion phospholipase to an emulsification treatment prior to the contacting.

In a ninth aspect, the present application provides degummed fats and oils prepared according to the method of the eighth aspect.

It is to be understood that the embodiments of the technical features described in the first aspect are also applicable to the second to ninth aspects.

It should also be understood that the above detailed description is only for the purpose of making the present application more clear to a person skilled in the art, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will be apparent to those skilled in the art.

Examples

The following examples are provided to further illustrate the present application and are not intended to be limiting in any way.

Example 1 construction of phospholipase-oil body protein hydrophobic fragment fusion protein (C L O-O) expression vector

By means ofhttp://web.expasy.org/protscale/Protscale analysis tool of the website, on soy oil body protein (FJ864730.1, SEQ ID)ID NO:3) was analyzed (see FIG. 1). From the results of FIG. 1, the sequence of the hydrophobic region (hereinafter, abbreviated as "O") of soybean oil body protein was obtained as follows and synthesized by Shanghai Bioengineering Co., Ltd:

ATIGITLLLLSGLTLTGTVIGLIIATPLLVIFSPILVPAAFVLFLVASGFLFSGGCGVAAIAALS(SEQID NO:5)

according to the coding sequence of the hydrophobic region and the enzyme cutting sites (BamHI and XhoI) in the used expression vector pCold, primers for amplifying the hydrophobic region of the soybean oil body protein are designed, and the primer sequences are as follows:

5'CGCCTCGAGGCCACTATTGGCATCACACTC 3'(SEQ ID NO:7)

5'GGCGGATCCAGACAAAGCAGCAATGG 3'(SEQ ID NO:8)

the phospholipase selected in this experiment was phospholipase C L OP L C8(GenBank: D49969.1), hereinafter also abbreviated as "phospholipase C L O", and the sequence thereof is shown in SEQ ID NO: 1.

Based on the coding sequence of phospholipase C L O (SEQ ID NO:2) and the restriction sites (NdeI and XhoI) in the expression vector pCold used, primers for amplifying phospholipase C L O were designed, and the sequences of the primers were as follows:

5’GGAATTCCATATGAAAAGAAA GATTTGTAAG GC 3’(SEQ ID NO:9)

5’CGC CTCGAG TTTTATATTTATAAGTTGAATTTCC3’(SEQ ID NO:10)

the hydrophobic region of oil body protein and phospholipase C L O were PCR amplified with Primerstar Hi-Fi polymerase (Takara Corp.) for denaturation: 95 ℃ for 30s, annealing: 55 ℃ for 30s, extension: 72 ℃ for 90s, 30 cycles, the hydrophobic region amplification product of oil body protein was ligated with expression vector pColdTF (from Takara) using T4 ligase (from Fermentas.) the ligated vector was transformed into E.coli DH 2 competent cells (from Shanghai Producer) and cultured overnight at 37 ℃ in solid L B medium plate (1.5% yeast powder 5%, yeast powder 10%, NaCl 5%) containing 50. mu.g/m L ampicillin, followed by transformation at 37 ℃ with 200rpm in 5m L L B medium (5% yeast powder, 10% peptone, 5% NaCl) and extraction with Qiagen plasmid extraction plasmid, sent to Shanghai sequencing kit, the phospholipase I was inserted into the amplified product of XhoI, phospholipase C L O, and the amplified product was expressed as a recombinant plasmid of recombinant plasmid (NdeI, phospholipase C6778, XhoO-fusion plasmid).

In addition, according to a similar method, phospholipase C L O was separately ligated into the expression vector pCold to obtain an expression vector for phospholipase (C L O).

Example 2 expression of fusion protein C L O-O and phospholipase C L O

Respectively transfecting Escherichia coli B L21 competent cells (purchased from Shanghai biological engineering Co., Ltd.) with a C L O-O expression vector and a C L O expression vector by a heat shock method, selecting a single colony, inoculating the single colony into a L B culture medium, carrying out shaking culture at 37 ℃ and 200rpm until the OD600 value is 0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM as an inducer, carrying out shaking culture at 150rpm at 16 ℃ overnight, and carrying out centrifugation collection on thalli 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) at 5S intervals for 20min, and then centrifuged at 12000rpm for 20min to obtain the lysate supernatant.

The results of the cleavage of the sample were shown in FIG. 2 by polyacrylamide gel electrophoresis (SDS-PAGE). As seen in FIG. 2, no significant expression of the target protein was observed before IPTG induction (lane 1), while the large expression of the target proteins C L O-O (lane 3) and C L O (lane 2) was observed in the 110 to 130kD region after IPTG induction.

Example 3 quantitative determination of fusion protein C L O-O and phospholipase C L O

Bovine Serum Albumin (BSA) at a concentration of 1mg/m L was used as a stock solution of the standard substance, diluted to different concentrations of 50, 100, 200 and 500. mu.g/m L, and subjected to co-electrophoresis with the C L O-O and C L O samples obtained in example 2, and the results are shown in FIG. 3.

The bands were analyzed using the 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), the C L O and C L O — O sample concentrations were calculated to be 1.25mg/m L and 1.46mg/m L, respectively.

Example 4 detection of phosphate group hydrolysis Activity of fusion protein C L O-O and phospholipase C L O (pNPPC method)

Drawing a standard curve:

0.01391g of p-nitrophenol was weighed and dissolved in 50m L of sterile water to prepare 2 mmol/L working solution and substrate solutions 1-7 for establishing a standard curve were prepared according to the following formulation.

Substrate buffer formulation: 0.1M boric acid-sodium borate buffer (pH 7.6), 20mM pNPPC, 1% Triton-X-100, 5mM CaCl₂。

After mixing the solutions, they were incubated at 37 ℃ for 15 minutes, and then 500. mu. L0.5.5N NaOH was added to determine absorbance at 410nm, and a standard curve of pNP amount versus absorbance was plotted.

The reaction process is as follows:

the reaction buffer solution 600. mu. L was added to 25. mu. L of an enzyme solution to be assayed (containing 0.5, 1 and 2pmol of C L O-O or C L O, respectively, adjusted according to the quantitation result in example 3), reacted at 37 ℃ for 15min, and 500. mu. L0.5.5N NaOH was added to terminate the reaction, and the absorbance was measured at 410 nm.

And (4) processing a result:

the amount of pNP from the reaction was calculated from the standard curve and the comparison is shown in FIG. 4. according to the results of FIG. 4, the hydrolytic activity of the phosphate group of C L O-O was significantly higher than that of C L O at 0.5, 1 and 2pmol (P < 0.01).

Thus, this example demonstrates that the phosphate group hydrolysis activity of the fusion protein C L O-O is superior to that of the unfused phospholipase C L O.

Example 5 comparison of phospholipid hydrolysis Activity of fusion protein C L O-O and phospholipase C L O before and after emulsification treatment

We further verified the activity by the molybdenum-blue method (the substrates are mixed phospholipids: Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI) account for 30% each, and others account for 10%), and at the same time, we simulated emulsification treatment in practical applications and selected Disemann's phospholipase C (trade name: Purifine) as an enzyme control, hereinafter referred to as "P L C".

Reaction solution I was prepared according to the following table. Samples were taken before the emulsification treatment as emulsification treatment controls. The emulsification treatment adopts an emulsifying machine and emulsifies for 3min at 20000 rpm. The pre-emulsification sample and post-emulsification sample were reacted at 37 ℃ for 30 min.

Components of reaction solution I	Concentration of	Volume (m L)
			Mixed phospholipids	1％(w/v)	7.5
Tris-Cl buffer, pH 7.5	500mM	1.5
			CaCl2	100mM	0.75

C L O-O, C L O or P L C enzymeLiquid for treating urinary tract infection	200μg/mL	1.5
			H2O		3.75
In total		15

After the reaction, 200. mu. L reaction solution was taken and transferred to a new centrifuge tube, 200. mu. L chloroform was added, mixed well for 20s, centrifuged at 12000rpm for 1min, 80. mu. L of the centrifuged supernatant was taken, reaction solution II was prepared according to the following table, and reaction solution II was allowed to react at 37 ℃ for 30 min.

Components of reaction solution II	Concentration of	Volume (μ l)
			Centrifuging the supernatant		80
Tris-Cl buffer, pH9.0	1M	10
			MgCl2	500mM	4
Alkaline phosphatase of small intestine of Cattle (CIAP)	0.5U/μL	4
			H2O		102
Total of		200

The obtained reaction solution II was subjected to the following Table to prepare a reaction solution III. The reaction mixture III was reacted at 37 ℃ for 10 min.

Components of reaction solution III	Concentration of mother liquor	Volume (mu L)
			CIAP enzyme digestion reactant		200
H2O		740
			Ascorbic acid	10％(w/v)	20
Ammonium molybdate	2.5％(w/v)	40
			Total of		1000

And detecting the absorbance of the reacted reaction liquid III at the absorbance of 700nm, taking the inactivated enzyme liquid as a blank control, and converting the enzyme activity by using a standard curve, wherein the standard curve uses P L C with known activity as a standard substance, and the steps are repeated to obtain a standard curve chart shown in 6.

The data converted from the measured activity of the sample before emulsification being 100% are shown in FIG. 5. according to the results of FIG. 5, C L O-O has about 130% increase in activity after emulsification, about 10% increase in control P L C, and about 10% decrease in activity after emulsification for C L O.

This example demonstrates that the ability of the fusion protein C L O-O to hydrolyze mixed phospholipids is greatly improved after simple emulsification treatment, whereas phospholipase C L O and commercial phospholipase P L C do not have such excellent properties.

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Claims (9)

1. a fusion protein comprising a phospholipase moiety and a hydrophobic domain portion of an oil body protein, wherein the sequence of the phospholipase moiety is set forth in SEQ ID NO. 1 and the sequence of the hydrophobic domain portion of the oil body protein is set forth in SEQ ID NO. 5.

2. A nucleic acid molecule capable of encoding the fusion protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein the coding sequence for the phospholipase portion is set forth in SEQ ID No. 2 and the coding sequence for the hydrophobic domain portion of the oil body protein is set forth in SEQ ID No. 6.

4. An expression vector comprising the nucleic acid molecule of claim 2 or 3.

5. 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, wherein the host cell is a bacterial cell, a fungal cell, a mammalian cell, or an insect cell.

6. The host cell of claim 5, wherein the host cell is a yeast cell.

7. A method for improving phospholipase activity, comprising fusing phospholipase and hydrophobic domain of oil body protein, wherein the sequence of phospholipase is shown in SEQ ID NO. 1, and the sequence of hydrophobic domain of oil body protein is shown in SEQ ID NO. 5.

8. A method of degumming oil comprising contacting the fusion protein of claim 1 with oil.

9. The method of claim 8, further comprising the step of subjecting the fusion protein to an emulsification treatment prior to contacting.

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