CN107151265B - Polyhydroxyalkanoate PHA granule binding protein PhaP mutant and preparation method and application thereof - Google Patents

Polyhydroxyalkanoate PHA granule binding protein PhaP mutant and preparation method and application thereof Download PDF

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CN107151265B
CN107151265B CN201610124594.6A CN201610124594A CN107151265B CN 107151265 B CN107151265 B CN 107151265B CN 201610124594 A CN201610124594 A CN 201610124594A CN 107151265 B CN107151265 B CN 107151265B
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phap
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CN107151265A (en
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陈国强
赵泓宇
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Beijing micro structure factory Biotechnology Co.,Ltd.
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Tsinghua University
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
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    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides

Abstract

The invention discloses a polyhydroxyalkanoate PHA granule binding protein PhaP mutant and a preparation method and application thereof. The protein provided by the invention is A) or B): A) a protein which is obtained by performing amino acid modification on at least one amino acid residue in the amino acid sequence of the polyhydroxyalkanoate particle binding protein (PhaP) and has the same function as the PhaP; B) a protein which is derived from A) and has the same function by adding a tag sequence to the end of the amino acid sequence of the protein shown in A). Experiments prove that the invention provides a plurality of PHA granule binding protein PhaP mutants, which have stronger emulsibility, higher heat resistance and stronger capacity of reducing the interfacial surface tension of a solution compared with wild type protein.

Description

Polyhydroxyalkanoate PHA granule binding protein PhaP mutant and preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a polyhydroxyalkanoate PHA granule binding protein PhaP mutant and a preparation method and application thereof.
Background
Biosurfactants are a large class of biosynthetic amphipathic molecules (Mullgan et al, Environ Pollut,133(2005), 183-98; Khire et al, Adv Exp Med Biol,672(2010),146-57) including low molecular weight glycolipids, lipopeptides, phospholipids, neutral lipids, and the like, and high molecular weight lipid-containing polymers such as lipopolysaccharides, lipoproteins, polysaccharide-protein-fatty acid complexes, and the like (Syldatk et al, Z Naturforsch C, 40(1985), 61-7; Ogawa et al, Biosci Biotechnol biochem,64(2000), 2466-8; Morita et al, J Biosci Bioeng,104(2007), 78-81; Wicke et al, JNAT Prod, 63 (621), 621-6). These molecular structures are essentially composed of two parts, respectively an oleophobic and hydrophilic polar group, such as monosaccharides, polysaccharides, amino acids and phosphate groups, and a non-polar group composed of a hydrophobic and oleophilic hydrocarbon chain, such as saturated or unsaturated fatty alcohols and fatty acids. Due to the amphiphilic molecular structure which is both oleophylic and hydrophilic, the biosurfactant can obviously reduce the interfacial tension or be adsorbed on the interface to form compact directional arrangement to change the hydrophilic/oleophylic performance of the interface, so that the oil/water two phases can be well dispersed. Most representative of these are glycolipids (Mata-Sandoval et al, Microbiol Res,155(2001), 249-56; Guilmanov et al, Biotechnol Bioeng,77(2002), 489-94;).
Polyhydroxyalkanoates (PHA) is a storage polymer of intracellular carbon and energy sources synthesized by many microorganisms under non-equilibrium growth conditions (Anderson et al, Microbial Rev.,54(1990) 450-.
PHA particle surface-binding protein PhaP (hereinafter referred to as PhaP) is widely present in various PHA synthesis bacteria, although not essential for PHA synthesis, but directly affects the size of PHA-containing bulk particles and the rate of accumulation of PHA synthesis (Pieper-F ü rst et al, J.Bacteriol.,176 (1994)) 4328. PHA particles are observed to increase when its own phaP gene is overexpressed in Ralstonia eutropha, whereas R.eutropha lacking mutations in the phaP gene greatly reduces the number of synthesized PHA particles (Wieczorek et al, J.Bacteriol.,177(1995) 2425. F2435.) expression of the phaP of Rosoccus rubber in E.coli, increases the number of synthesized PHA particles, reduces the size thereof (Wieczorek et al, J.Bacteriol.,177(1995) 2425. P) 24235. PhaP is considered to have a hydrophobic region which is involved in the formation of PHA particles in PHA-particle surface-binding protein Phap and PHA-particle-surface-binding protein (Piepp-H) 1037, which is considered to have both hydrophilic and hydrophobic regions (Biophyte-protein-alpha-2517, which are reported to be involved in the formation of PHA-particle-protein (Biophyte et al, Biophyte-11, AHP), and PHA-protein, Inc. (accounts for the formation of PHA-protein).
Disclosure of Invention
An object of the present invention is to provide PhaP mutants of polyhydroxyalkanoate PHA granule binding protein.
The protein provided by the invention is A) or B):
A) a protein which is obtained by performing amino acid modification on at least one amino acid residue in the amino acid sequence of the polyhydroxyalkanoate particle binding protein (PhaP) and has the same function as the PhaP;
B) a protein which is derived from A) and has the same function by adding a tag sequence to the end of the amino acid sequence of the protein shown in A).
The same function as PhaP may be an emulsifying function or other functions.
The amino acid sequence of the polyhydroxyalkanoate particle binding protein PhaP is shown as sequence 1, and the nucleotide sequence of the coding gene is shown as sequence 2.
In the above protein, the amino acid modification is an amino acid substitution.
In the above protein, the amino acid substitution is a hydrophilic amino acid and a hydrophobic amino acid.
In the protein, the replacement of the hydrophilic amino acid with the hydrophobic amino acid is to replace any one or any 2 or any 3 of the hydrophilic amino acids at the following sites in the amino acid sequence of the polyhydroxyalkanoate particle binding protein PhaP with the hydrophobic amino acid: 23, 24, 30, 38, 45, 52, 72 and 82.
In the above protein, the hydrophobic amino acid is glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan or methionine;
the hydrophilic amino acid is serine, threonine, cysteine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine or histidine.
Among the above-mentioned proteins, the proteins,
the protein A is obtained by any one of the following methods 1) to 9):
1) replacing tyrosine at position 23 of amino acid sequence PhaP of polyhydroxyalkanoate particle binding protein with phenylalanine or alanine;
2) replacing asparagine at the 24 th position of the amino acid sequence of the polyhydroxyalkanoate particle binding protein (PhaP) with leucine or alanine;
3) replacing asparagine at the 30 th position of the amino acid sequence of the polyhydroxyalkanoate particle binding protein (PhaP) with leucine or alanine;
4) replacing glutamine at position 38 of an amino acid sequence of polyhydroxyalkanoate particle binding protein (PhaP) with leucine, isoleucine or alanine;
5) replacing tyrosine at the 45 th position of the amino acid sequence of the PHP with phenylalanine or alanine;
6) replacing glutamine at position 52 of an amino acid sequence of polyhydroxyalkanoate particle binding protein (PhaP) with leucine, isoleucine or alanine;
7) replacing glutamine at position 72 of an amino acid sequence of polyhydroxyalkanoate particle binding protein (PhaP) with leucine, isoleucine or alanine;
8) replacing glutamine at position 82 of an amino acid sequence of polyhydroxyalkanoate particle binding protein (PhaP) with leucine, isoleucine or alanine;
9)1) -8) or any combination of 2 or 3.
In the above protein, the method indicated by 9) is the following 9) -1), 9) -2), 9) -3), 9) -4):
9) -1): replacing glutamine at position 38 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, and replacing glutamine at position 52 of the PhaP amino acid sequence with leucine;
9) -2): replacing glutamine at position 38 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, and replacing glutamine at position 72 of the PhaP amino acid sequence with leucine;
9) -3): replacing glutamine at position 38 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, and replacing glutamine at position 82 of the PhaP amino acid sequence with leucine;
9) -4): replacing glutamine at position 38 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, replacing glutamine at position 52 of the PhaP amino acid sequence with leucine, and replacing glutamine at position 82 of the PhaP amino acid sequence with leucine.
DNA molecules encoding the above proteins are also within the scope of the present invention.
The application of the protein or the DNA molecule as an emulsifier is also within the protection scope of the invention;
or the application of the protein or the DNA molecule in the preparation of the emulsifier is also within the protection scope of the invention;
or the application of the protein or the DNA molecule in emulsified oil is also the protection scope of the invention;
or the use of the above-mentioned protein or the above-mentioned DNA molecule in the preparation of an oil-water emulsion is also within the scope of the present invention;
or the use of the above protein or the above DNA molecule in the preparation of a surfactant is also within the scope of the present invention.
It is another object of the invention to provide a product.
The invention provides a product comprising the protein.
The above product is emulsifier or surfactant.
Experiments prove that the invention provides a plurality of PHA particle binding protein PhaP mutants through a large number of experiments and structural biology analysis, the mutants are obtained through construction, expression and purification, and compared with wild type protein, the mutants have stronger emulsibility, higher heat resistance and stronger capacity of reducing the interfacial surface tension of a solution. The high-performance mutant can be used as a biosurfactant to be applied to the fields of medical treatment, cosmetics, mass consumption products, nano materials, high polymer materials, petroleum exploitation and the like, and has the advantages of strong performance and low cost compared with wild protein.
Drawings
FIG. 1 is an electrophoretogram of recombinant plasmid pGEX-6P-1-PhaP.
FIG. 2 is an SDS-PAGE pattern of PHA granule-binding protein PhaP.
FIG. 3 is a diagram of purification of PhaP by gel filtration chromatography.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of PHA granule-binding protein PhaP and mutants thereof
Preparation of PHA granule-binding protein PhaP
The invention uses the amphipathic protein which is the polyhydroxyalkanoate particle binding protein phaP gene from Aeromonas hydrophila, expresses the amphipathic protein in a procaryon or eucaryon in an excessive way, and carries out affinity purification by GST (glutathione S-transferase) tag.
The amino acid sequence of the polyhydroxyalkanoate particle binding protein PhaP from Aeromonas hydrophylla is shown as a sequence 1 in the sequence table, and the nucleotide sequence of the coding gene phaP is shown as a sequence 2 in the sequence table. The specific operation is as follows:
1. construction of recombinant plasmid pGEX-6P-1-PhaP
The recombinant plasmid pGEX-6P-1-PhaP is a vector obtained by replacing a gene phaP shown in a sequence 2 in a sequence table with a fragment between BamHI and XhoI enzyme cutting sites of a pGEX-6P-1(VT1258, Youbao organism) vector, and the gene phaP and a GST label on the vector are commonly expressed to jointly express the fusion protein PhaP.
2. Expression and purification of protein PhaP
The recombinant plasmid pGEX-6P-1-PhaP was introduced into competent cells of Escherichia coli BL21(DE3) (purchased from holo-gold Biotech Co., Ltd.), and a resistant plate (containing 100. mu.g/ml of ampicillin) was applied thereto to screen positive colonies. Carrying out PCR identification on the positive bacterial colony with 5'-CGGGATCCATGATGAATATG-3' primers; 5'-CACTCGAGGGCCTT GCCCGTG-3' are provided. Fragments of 350bp which were almost identical to the expected theoretical value were obtained as positive, and the positive strain was named BL 21/pGEX-6P-1-PhaP.
BL21/pGEX-6P-1-PhaP single colonies are picked up and respectively added into 100ml LB liquid medium (containing 100 ug/ml of ampicillin), shaking culture is carried out at 37 ℃ until OD 600 is 0.5-0.8, IPTG with the final concentration of 0.5mM is added, and the fusion protein is induced and expressed overnight at 16 ℃, so as to obtain BL21/pGEX-6P-1-PhaP bacterial liquid after induction. Meanwhile, a bacterial solution without IPTG is set as a blank control.
Centrifuging the BL21/pGEX-6P-1-PhaP bacterial solution at 8000rpm for 12min, discarding the supernatant, collecting the precipitate, suspending in binding buffer solution (0.5M NaCl, 50mM Tris-HCl, pH 8.0), performing ice bath, and ultrasonically crushing the cells; centrifuging at 13000rpm for 60 minutes, and obtaining supernatant as clarified crude cell extract.
Loading the crude cell extract with GST affinity chromatography medium (DP201-01, all-gold) in a column volume of 4mL, washing off the impure proteins with 200mL binding buffer solution (0.5M NaCl, 50mM Tris-HCl, pH 8.0), adding 2 column volumes of eluent (0.5M NaCl, 50mM Tris-HCl, pH 8.0) into the affinity chromatography column, adding 500uL 3C PreScission protease (prepared by Qinghua university student's center of structural biology) for 4-degree enzyme digestion overnight, and cutting off GST to obtain the purified product of affinity chromatography.
Centrifuging the affinity chromatography purified product at 4000rpm at 4 ℃ by using an ultrafiltration concentration tube with the filter membrane aperture size of 10kD, trapping the protein on the upper layer due to the protein being larger than the filter membrane aperture, and throwing the buffer solution and salt to the lower layer in the centrifuging process so as to achieve the concentration effect, concentrating to a final volume of about 1mL, collecting the concentrated product to perform gel column filtration chromatography, wherein the gel column filtration chromatography adopts a column with the aperture of Superdex 20010/300, the column volume is 25mL, the elution time is 60min, the flow rate of eluent (0.5M NaCl, 50mM Tris-HCl pH is 8.0) is 0.5mL/min, and 6mL of eluent is collected (figure 3).
And concentrating the collected 6mL of eluent by using an ultrafiltration concentration tube with the pore size of 10kD, and collecting a concentrated solution to obtain the protein PhaP.
Protein PhaP was subjected to SDS-PAGE, and the result of electrophoresis is shown in FIG. 2, and it can be seen that protein PhaP was obtained with a size of about 14kDa, which is consistent with the molecular weight reported in the literature.
The empty vector pGEX-6P-1 was introduced into E.coli BL21 to obtain BL 21/pGEX-6P-1. The protein is expressed and purified by the method, and the target protein with the size of 14k Da is not obtained.
Second, preparation of PhaP mutant
The PhaP mutant is a protein obtained by substituting at least one amino acid in the amino acid sequence of the PhaP protein with an amino acid, and if one amino acid is substituted, a single-point PhaP mutant is obtained, if 2 amino acids are substituted, a double-point PhaP mutant is obtained, and if 3 amino acids are substituted, a three-point PhaP mutant is obtained.
PhaP single-point mutants are specifically PhaP/Y23F, PhaP/Y23A, PhaP/N24L, PhaP/N24A, PhaP/N30L, PhaP/N30A, PhaP/Q38L, PhaP/Q38I, PhaP/Q38A, PhaP/Y45F, PhaP/Y45A, PhaP/Q52L, PhaP/Q52I, PhaP/Q52A, PhaP/Q72L, PhaP/Q72I, PhaP/Q72A, PhaP/Q82L, PhaP/Q82I and PhaP/Q82A.
The PhaP double-point mutant is specifically PhaP/Q38L-Q52L, PhaP/Q38L-Q72L, PhaP/Q38L-Q82L and PhaP/Q52L-Q72L;
the PhaP triple-point mutant is particularly PhaP/Q38L-Q52L-Q72L.
The amino acid sequence of the mutant PhaP/Y23F is that tyrosine (Tyr) at the 23 rd position of the sequence 1 is mutated into phenylalanine (Phe), and the nucleotide sequence of the coding gene of the mutant PhaP/Y23F is that TAC at the 67 th position to the 69 th position of the sequence 2 is mutated into TTC.
The amino acid sequence of the mutant PhaP/Y23A is that the tyrosine (Tyr) at the 23 rd position of the sequence 1 is mutated into alanine (Ala), and the nucleotide sequence of the mutant PhaP/Y23A coding gene is that the TAC at the 67 th position to the 69 th position of the sequence 2 is mutated into GCC.
The amino acid sequence of the mutant PhaP/N24L is characterized in that asparagine (Asn) at the 24 th position of the sequence 1 is mutated into leucine (Leu), and the nucleotide sequence of the encoding gene of the mutant PhaP/N24L is characterized in that AAC at the 70 th position to the 72 th position of the sequence 2 is mutated into CTG.
The amino acid sequence of the mutant PhaP/N24A is characterized in that asparagine (Asn) at the 24 th position of the sequence 1 is mutated into alanine (Ala), and the nucleotide sequence of the encoding gene of the mutant PhaP/N24A is characterized in that AAC at the 70 th position to the 72 th position of the sequence 2 is mutated into GCC.
The amino acid sequence of the mutant PhaP/N30L is characterized in that asparagine (Asn) at the 30 th position of the sequence 1 is mutated into leucine (Leu), and the nucleotide sequence of the coding gene of the mutant PhaP/N30L is characterized in that AAC at the 88 th position to the 90 th position of the sequence 2 is mutated into CTG.
The amino acid sequence of the mutant PhaP/N30A is characterized in that asparagine (Asn) at the 30 th position of the sequence 1 is mutated into alanine (Ala), and the nucleotide sequence of the encoding gene of the mutant PhaP/N30A is characterized in that AAC at the 88 th position to the 90 th position of the sequence 2 is mutated into GCC.
The amino acid sequence of the mutant PhaP/Q38L is characterized in that glutamine (Gln) at the 38 th position of the sequence 1 is mutated into leucine (Leu), and the nucleotide sequence of the encoding gene of the mutant PhaP/Q38L is characterized in that CAG at the 112 th position to the 114 th position of the sequence 2 is mutated into CTG.
The amino acid sequence of the mutant PhaP/Q38I is characterized in that glutamine (Gln) at the 38 th position of the sequence 1 is mutated into isoleucine (IIe), and the nucleotide sequence of the encoding gene of the mutant PhaP/Q38I is characterized in that CAG at the 112 th position to the 114 th position of the sequence 2 is mutated into ATC.
The amino acid sequence of the mutant PhaP/Q38A is characterized in that glutamine (Gln) at the 38 th position of the sequence 1 is mutated into alanine (Ala), and the nucleotide sequence of the mutant PhaP/Q38A encoding gene is characterized in that CAG at the 112 th position to the 114 th position of the sequence 2 is mutated into GCC.
The amino acid sequence of the mutant PhaP/Y45F is that tyrosine (Tyr) at the 45 th position of the sequence 1 is mutated into phenylalanine (Phe), and the nucleotide sequence of the coding gene of the mutant PhaP/Y45F is that TAC at the 133 th position to the 135 th position of the sequence 2 is mutated into TTC.
The amino acid sequence of the mutant PhaP/Y45A is that tyrosine (Tyr) at the 45 th position of the sequence 1 is mutated into alanine (Ala), and the nucleotide sequence of the mutant PhaP/Y45A coding gene is that TAC at the 133 th position to the 135 th position of the sequence 2 is mutated into GCC.
The amino acid sequence of the mutant PhaP/Q52L is characterized in that glutamine (Gln) at the 52 th position of the sequence 1 is mutated into leucine (Leu), and the nucleotide sequence of the encoding gene of the mutant PhaP/Q52L is characterized in that CAG at the 154 th position to the 156 th position of the sequence 2 is mutated into CTG.
The amino acid sequence of the mutant PhaP/Q52I is characterized in that glutamine (Gln) at the 52 th position of the sequence 1 is mutated into isoleucine (IIe), and the nucleotide sequence of the encoding gene of the mutant PhaP/Q52I is characterized in that CAG at the 154 th position to the 156 th position of the sequence 2 is mutated into ATC.
The amino acid sequence of the mutant PhaP/Q52A is characterized in that glutamine (Gln) at the 52 th position of the sequence 1 is mutated into alanine (Ala), and the nucleotide sequence of the mutant PhaP/Q52A encoding gene is characterized in that CAG at the 154 th position to the 156 th position of the sequence 2 is mutated into GCC.
The amino acid sequence of the mutant PhaP/Q72L is characterized in that glutamine (Gln) at the 72 th position of the sequence 1 is mutated into leucine (Leu), and the nucleotide sequence of the encoding gene of the mutant PhaP/Q72L is characterized in that CAG at the 214 th position to the 216 th position of the sequence 2 is mutated into CTG.
The amino acid sequence of the mutant PhaP/Q72I is characterized in that glutamine (Gln) at the 72 th position of the sequence 1 is mutated into isoleucine (IIe), and the nucleotide sequence of the encoding gene of the mutant PhaP/Q72I is characterized in that CAG at the 214 th position to the 216 th position of the sequence 2 is mutated into ATC.
The amino acid sequence of the mutant PhaP/Q72A is characterized in that glutamine (Gln) at the 72 th position of the sequence 1 is mutated into alanine (Ala), and the nucleotide sequence of the mutant PhaP/Q72A coding gene is characterized in that CAG at the 214 th position to the 216 th position of the sequence 2 is mutated into GCC.
The amino acid sequence of the mutant PhaP/Q82L is characterized in that glutamine (Gln) at the 82 th position of the sequence 1 is mutated into leucine (Leu), and the nucleotide sequence of the encoding gene of the mutant PhaP/Q82L is characterized in that CAG at the 244 th position to the 246 th position of the sequence 2 is mutated into CTG.
The amino acid sequence of the mutant PhaP/Q82I is characterized in that glutamine (Gln) at the 82 th position of the sequence 1 is mutated into isoleucine (IIe), and the nucleotide sequence of the mutant PhaP/Q82I coding gene is characterized in that CAG at the 244 th position to the 246 th position of the sequence 2 is mutated into ATC.
The amino acid sequence of the mutant PhaP/Q82A is characterized in that glutamine (Gln) at the 82 th position of the sequence 1 is mutated into alanine (Ala), and the nucleotide sequence of the mutant PhaP/Q82A coding gene is characterized in that CAG at the 244 th position to the 246 th position of the sequence 2 is mutated into GCC.
The amino acid sequence of the mutant PhaP/Q38L-Q52L is that glutamine (Gln) at the 38 th position of the sequence 1 is mutated into leucine (Leu), and glutamine (Gln) at the 52 th position of the sequence 1 is mutated into leucine (Leu); the nucleotide sequence of the encoding gene of the mutant PhaP/Q38L-Q52L is characterized in that CAG at 112 th to 114 th positions of the sequence 2 is mutated into CTG, and CAG at 154 th to 156 th positions is mutated into CTG.
The amino acid sequence of the mutant PhaP/Q38L-Q72L is that glutamine (Gln) at the 38 th position of the sequence 1 is mutated into leucine (Leu), and glutamine (Gln) at the 72 th position of the sequence 1 is mutated into leucine (Leu); the nucleotide sequence of the encoding gene of the mutant PhaP/Q38L-Q72L is characterized in that CAG at 112 th to 114 th positions of the sequence 2 is mutated into CTG, and CAG at 214 th to 216 th positions is mutated into CTG.
The amino acid sequence of the mutant PhaP/Q38L-Q82L is characterized in that the 38 th glutamine (Gln) of the sequence 1 is mutated into leucine (Leu), and the 82 th glutamine (Gln) of the sequence 1 is mutated into leucine (Leu); the nucleotide sequence of the encoding gene of the mutant PhaP/Q38L-Q82L is characterized in that CAG at 112 th to 114 th positions of the sequence 2 is mutated into CTG, and CAG at 244 th to 246 th positions is mutated into CTG.
The amino acid sequence of the mutant PhaP/Q52L-Q72L is that glutamine (Gln) at the 52 th position of the sequence 1 is mutated into leucine (Leu), and glutamine (Gln) at the 72 th position of the sequence 1 is mutated into leucine (Leu); the nucleotide sequence of the encoding gene of the mutant PhaP/Q52L-Q72L is characterized in that CAG at 154 th to 156 th positions of the sequence 2 is mutated into CTG, and CAG at 214 th to 216 th positions is mutated into CTG.
The amino acid sequence of the mutant PhaP/Q38L-Q52L-Q72L is that glutamine (Gln) at the 38 th position of the sequence 1 is mutated into leucine (Leu), glutamine (Gln) at the 52 th position of the sequence 1 is mutated into leucine (Leu), and glutamine (Gln) at the 72 th position of the sequence 1 is mutated into leucine (Leu); the nucleotide sequence of the encoding gene of the mutant PhaP/Q38L-Q52L-Q72L is characterized in that the 112 th-114 th CAG of the sequence 2 is mutated into CTG, the 154 th-156 th CAG is mutated into CTG, and the 214 th-216 th CAG is mutated into CTG.
1. Preparation of recombinant vector for expression of mutant protein
The recombinant vector for expressing different mutant proteins is obtained by replacing BamHI and XhoI enzyme cutting sites of pGEX-6P-1 vector ((VT1258, Youbao organism)) with different mutant protein coding genes, and different mutant proteins and GST tags on the vector jointly express different mutant proteins.
The preparation method of the recombinant vector for expressing different mutant proteins comprises the following steps:
PCR was performed using pGEX-6P-1-PhaP as a template and the mutant primers shown in Table 1, respectively, to obtain recombinant vectors expressing different mutant proteins: pGEX-6P-1-PhaP/Y23F, pGEX-6P-1-PhaP/Y23A, pGEX-6P-1-PhaP/N24L, pGEX-6P-1-PhaP/N24A, pGEX-6P-1-PhaP/N30L, pGEX-6P-1-PhaP/N30A, pGEX-6P-1-PhaP/Q38L, pGEX-6P-1-PhaP/Q38I, pGEX-6P-1-PhaP/Q38, pGEX-6P-1-PhaP/Y45F, pGEX-6P-1-PhaP/Y45A, pGEX-6P-1-P/Q52L, pGEX-6P-1-P/Q52, pGEX-6P-1-PhaP/Q I, PhaP-6P-A P-P/Q A/A, pGEX-6P-1-PhaP/Q72L, pGEX-6P-1-PhaP/Q72I, pGEX-6P-1-PhaP/Q72A, pGEX-6P-1-PhaP/Q82L, pGEX-6P-1-PhaP/Q82I, pGEX-6P-1-PhaP/Q82A, pGEX-6P-1-PhaP/Q38L-Q52L, pGEX-6P-1-PhaP/Q38L-Q72L, pGEX-6P-1-PhaP/Q38L-Q82L, pGEX-6P-1-PhaP/Q52L-Q72L, pGEX-6P-1-PhaP/Q38L-Q52L-Q6772 72L.
2. Expression and purification of mutants
The same as the method in the first paragraph, except that mutant PhaP/Y23F, mutant PhaP/Y23A, mutant PhaP/N24L, mutant PhaP/N24A, mutant PhaP/N30L, mutant PhaP/N30A, mutant PhaP/Q38L, mutant PhaP/Q38I, mutant PhaP/Q38A, mutant PhaP/Y45F, mutant PhaP/Y45A, mutant PhaP/Q52L, mutant PhaP/Q52I, mutant PhaP/Q52A, mutant PhaP/Q72L, mutant PhaP/Q72I, mutant P/Q72A, mutant P/Q82L, mutant P/Q6382, mutant PhaP/Q68682, mutant PhaP/Q A, mutant PhaP/Q72 68638, mutant PhaP/Q8438-L, mutant PhaP/Q828652-L, mutant PhaP 82L, mutant PhaP/Q66 and mutant PhaP/Q8652L are obtained by replacing recombinant plasmid pGEX-PhaP with the recombinant vector prepared in the above 1 with the recombinant vector, Mutant PhaP/Q38L-Q82L, mutant PhaP/Q52L-Q72L and mutant PhaP/Q38L-Q52L-Q72L.
Table 1 shows mutant primers
Figure BDA0000934828440000091
Figure BDA0000934828440000101
Table 1 shows the primers of single point mutation, and the method of multi-point mutation is the same as single point mutation, namely, other mutation sites are introduced into the mutated carrier by using the mutation primers.
Example 2 ability of PhaP and mutants thereof to emulsify Soybean oil
The protein PhaP prepared in example 1 and different mutant proteins were prepared in the same concentration of aqueous solution (50. mu.g/ml); the oil phase is soybean oil (Jinlongyu brand).
The specific operation is as follows:
1. preparation of an aqueous solution of a substance to be tested: the protein PhaP prepared in example 1 and the different mutant proteins were prepared as aqueous solutions (50. mu.g/ml) of the same concentrations, respectively.
2. Soybean oil is used as an oil phase, equal volumes (0.5 ml of the aqueous solution of the substance to be detected and 0.5ml of the oil phase) of the aqueous solution of the substance to be detected and the soybean oil are mixed at the temperature of 20 ℃ for emulsification treatment, the emulsification treatment adopts a vortex oscillator for oscillation treatment, and the vortex oscillator for oscillation treatment adopts a vortex oscillator (XH-C, jin Yi, medical instrument works of Jintan city) for oscillation treatment at the speed of 1300 revolutions per second for 120 seconds. And standing at constant temperature of 25 ℃ in a dark place after the emulsification treatment is finished, photographing for 2 days to observe and record the heights of an oil layer, an emulsion layer and a water layer. And calculating by formula (1) to obtain the relevant emulsification value. While ultrapure water was used as a control.
The formula for calculating the emulsification value is as follows:
emulsion value ═ emulsion layer/(oil layer + emulsion layer + water layer) × 100% (1)
Wherein, the total height of all layers refers to the total height of three layers of the oil layer, the emulsifying layer and the water layer. However, sometimes the aqueous or oil layer may disappear after emulsification, and the total height is the sum of the heights of the remaining two layers (layers). The larger the emulsification value, the stronger the emulsification ability.
The emulsification experiments were repeated three times, and the emulsification values are expressed as mean ± standard deviation.
As shown in Table 2, it can be seen that site-directed mutagenesis performed for 2 days by structural analysis all improved protein PhaP emulsibility to different degrees, wherein several mutations, such as mutants PhaP/Q38L, PhaP/Q52L, PhaP/Q72L, PhaP/Q82L, PhaP/Q38L-Q52L, PhaP/Q38L-Q72L, PhaP/Q38L-Q82L, PhaP/Q38L-Q52L-Q72L, are significantly improved.
In addition, the experiment increases the concentration of the aqueous solution of PhaP and its mutant protein to 150. mu.g/ml, and the emulsifying capacity of the mutant and the wild type are basically the same (Table 3), and the result shows that the protein after mutation has stronger emulsifying property at low concentration than the wild type, i.e. less dosage is needed to achieve the same emulsifying effect.
TABLE 2 comparison of emulsion layers of emulsified soybean oil with PhaP and its mutant protein in water (50. mu.g/ml)
Figure BDA0000934828440000111
Figure BDA0000934828440000121
TABLE 3 comparison of emulsion layers of emulsified soybean oil with PhaP and its mutant protein in water (150. mu.g/ml)
Figure BDA0000934828440000122
Figure BDA0000934828440000131
Thus, the PhaP mutant acts as an emulsifier.
Example 3 measurement of melting Point (Tm) for PhaP and its mutant proteins
The protein PhaP prepared in example 1 and different mutant proteins were prepared in the same concentration of aqueous solution (50 μ g/ml), and far-ultraviolet Circular Dichroism (CD) spectra were measured. The instrument used was a Jasco 715 spectrometer with a cuvette optical path of 1 mm. The resolution is 0.2nm, the bandwidth is 2nm, the scanning speed is 200nm/min, the response speed is 1 second, the photomultiplier voltage instrument is self-controlled, the detection wavelength range is 200nm-260nm, and the repetition times are 3 times. The intensity at 220nm of the CD spectrum is plotted against temperatureProtein secondary structure varies with temperature. The protein concentration was 0.1 mg/ml. Then, fitting the temperature-changing curve obtained by CD by using an S-shaped curve, and calculating the inflection point x of the curve by using a formula 20
y＝Ab+((At-Ab)/(1+exp((x0-x)/w))) (2)
x0The corresponding y value is (Ab + At)/2
Ab is the y value corresponding to the tangent line under the curve
At is the y value corresponding to the tangent on the curve
Will turn point x0The secondary structure of the corresponding temperature protein is obviously changed, and the temperature corresponding to the point is taken as the Tm value of the protein
After temperature swing tests, the following results (table 4) were obtained, and the Tm values of the mutated proteins were all improved, and in particular, the Tm values of several mutations, Q38L, Q38LQ52L, Q38LQ72L, Q38LQ82L, and Q38LQ52LQ72L, were improved by approximately 20 ℃.
The results indicate that the PhaP mutant protein has higher thermostability.
TABLE 4 comparison of Tm values for the melting points of PhaP and its mutant proteins
Figure BDA0000934828440000132
Figure BDA0000934828440000141
Example 4 Effect of PhaP and its mutant proteins on interfacial surface tension of aqueous solutions
Due to hydrophobic effect, hydrophobic groups of surfactant molecules are close together to form a hydrophobic inner core, hydrophilic groups face outwards to be in contact with water, and self-assembly is carried out in an aqueous solution to form aggregates with various structures and sizes. The addition of a surfactant can reduce the interfacial surface tension of the aqueous solution.
The surface tension of the solution was measured using a JZ-200A interfacial tensiometer by the dyne loop method, and the surface tension of the solution was measured at the same concentration (Table 5) by preparing an aqueous solution (10mg/L) of the protein PhaP prepared in example 1 and different mutant proteins. The mutant protein has higher capacity of reducing the surface tension of the solution compared with the wild type, wherein the mutant PhaP/Q38L, mutant PhaP/Q72L, mutant PhaP/Q82L, mutant PhaP/Q38LQ52L, mutant PhaP/Q38LQ72L, mutant PhaP/Q38LQ82L and mutant PhaP/Q38LQ52LQ72L have more obvious effects. Under the same concentration (10mg/L), the mutant can reduce more surface tension compared with the wild type, and has better application prospect.
TABLE 5 surface tension values of PhaP and its mutant protein solutions (10mg/L)
Figure BDA0000934828440000151
Figure IDA0000934828520000011
Figure IDA0000934828520000021

Claims (4)

1. A protein obtained by any one of the following methods 1) to 9):
1) replacing tyrosine at position 23 of amino acid sequence PhaP of polyhydroxyalkanoate particle binding protein with phenylalanine or alanine;
2) replacing asparagine at the 24 th position of the amino acid sequence of the polyhydroxyalkanoate particle binding protein (PhaP) with leucine or alanine;
3) replacing asparagine at the 30 th position of the amino acid sequence of the polyhydroxyalkanoate particle binding protein (PhaP) with leucine or alanine;
4) replacing glutamine at position 38 of an amino acid sequence of polyhydroxyalkanoate particle binding protein (PhaP) with leucine, isoleucine or alanine;
5) replacing tyrosine at the 45 th position of the amino acid sequence of the PHP with phenylalanine or alanine;
6) replacing glutamine at position 52 of an amino acid sequence of polyhydroxyalkanoate particle binding protein (PhaP) with leucine, isoleucine or alanine;
7) replacing glutamine at position 72 of an amino acid sequence of polyhydroxyalkanoate particle binding protein (PhaP) with leucine, isoleucine or alanine;
8) replacing glutamine at position 82 of an amino acid sequence of polyhydroxyalkanoate particle binding protein (PhaP) with leucine, isoleucine or alanine;
9)1) -8) or any combination of 2 or 3;
the method shown in the 9) is as follows, 9) -1), 9) -2), 9) -3), 9) -4), and 9) -5):
9) -1): replacing glutamine at position 38 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, and replacing glutamine at position 52 of the PhaP amino acid sequence with leucine;
9) -2): replacing glutamine at position 38 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, and replacing glutamine at position 72 of the PhaP amino acid sequence with leucine;
9) -3): replacing glutamine at position 38 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, and replacing glutamine at position 82 of the PhaP amino acid sequence with leucine;
9) -4): replacing glutamine at position 52 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, and replacing glutamine at position 72 of the PhaP amino acid sequence with leucine;
9) -5): replacing glutamine at position 38 of a PhaP amino acid sequence of polyhydroxyalkanoate particle binding protein with leucine, replacing glutamine at position 52 of the PhaP amino acid sequence with leucine, and replacing glutamine at position 82 of the PhaP amino acid sequence with leucine.
2. A DNA molecule encoding the protein of claim 1.
3. Use of the protein of claim 1 or the DNA molecule of claim 2 as an emulsifier;
or the protein of claim 1 or the DNA molecule of claim 2 for use in the preparation of an emulsifier;
or the protein of claim 1 or the DNA molecule of claim 2, in emulsified oil;
or the protein of claim 1 or the DNA molecule of claim 2, in the preparation of an oil-in-water emulsion;
or the protein of claim 1 or the DNA molecule of claim 2 for use in the preparation of a surfactant.
4. A product comprising the protein of claim 1;
the product is an emulsifier or surfactant.
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