CN107151265B - Polyhydroxyalkanoate PHA particle-binding protein PhaP mutant and its preparation method and application - Google Patents

Abstract

The invention discloses a polyhydroxyalkanoate PHA particle binding protein PhaP mutant and a preparation method and application thereof. The protein provided by the present invention is the following A) or B): A) amino acid modification is performed 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 PhaP; A) A protein derived from A) with a tag sequence added to the end of the amino acid sequence of the protein shown and having the same function. The experiments of the present invention prove that the present invention provides a variety of PhaP mutants of polyhydroxyalkanoate PHA ester particle-binding protein, the mutants have stronger emulsifying properties, higher heat resistance, and higher heat resistance than the wild-type protein. Strong ability to reduce the interfacial surface tension of solutions.

Description

Polyhydroxyalkanoate PHA particle-binding protein PhaP mutant and preparation method thereof with application

technical field

The invention relates to the field of biotechnology, in particular to a polyhydroxyalkanoate PHA particle-binding protein PhaP mutant and a preparation method and application thereof.

Background technique

Biosurfactants are a large class of biosynthetic amphiphilic molecules (Mulligan 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, etc. and high molecular weight lipid-containing polymers, such as lipopolysaccharides, lipoproteins, polysaccharide-protein-fatty acid complexes, etc. (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, J Nat Prod, 63 (2000), 621- 6). These molecular structures are basically composed of two parts, namely oleophobic and hydrophilic polar groups, such as monosaccharides, polysaccharides, amino acids and phosphate groups, and non-polar groups composed of hydrophobic and lipophilic hydrocarbon chains. Groups, such as saturated or unsaturated fatty alcohols and fatty acids. It is precisely because of this amphiphilic molecular structure that is both lipophilic and hydrophilic that biosurfactants can significantly reduce the interfacial tension, or adsorb on the interface to form a tight alignment to change the hydrophilic/lipophilic properties of the interface. The oil/water phase is well dispersed. The most representative of them are glycolipids (Mata-Sandoval et al., Microbiol Res, 155 (2001), 249-56; Guilmanov et al., Biotechnol Bioeng, 77 (2002), 489-94;).

Polyhydroxyalkanoic acids (PHA) are intracellular carbon and energy storage polymers synthesized by many microorganisms under non-equilibrium growth conditions (Anderson et al., Microbial Rev., 54 (1990) 450- 472; Lee et al., Biotech. Bioeng., 49 (1996) 1-14). Under natural conditions, PHA exists in the form of insoluble fat particles in bacteria. PHA molecules form a hydrophobic core, and the particle surface is wrapped with a special monolayer membrane structure composed of particle-binding protein (PhaP), regulatory protein (PhaR), PHA synthase (PhaC), PHA-degrading enzyme (PhaZ) and monolayer phospholipids Membrane composition (Steinbüchel et al., Can. J. Microbiol. 41 (1995) 94-105; York et al., J. Bacteriol. 183 (2001) 2394-2397).

PHA particle surface binding protein PhaP (hereinafter referred to as PhaP) is widely present in various PHA synthesizing bacteria. Although PhaP is not necessary for PHA synthesis, it directly affects the size of PHA inclusion body particles and the rate of PHA synthesis and accumulation (Pieper- Fürst et al., J. Bacteriol., 176 (1994) 4328-4337). In Ralstonia eutropha, overexpression of its own phaP gene observed an increase in the number of PHA particles; while in R. eutropha with a deletion mutation of the phaP gene, the number of synthesized PHA particles was greatly reduced (Wieczorek et al., J. Bacteriol., 177 (1995) 2425-2435 ). Expression of phaP of Rodococcus rubber in E. coli increases the number and size of PHA particles (Wieczorek et al., J. Bacteriol., 177 (1995) 2425-2435). PhaP is considered to be an amphiphilic protein with a hydrophobic region and a hydrophilic region, the hydrophobic region is in contact with PHA particles, and the hydrophilic region faces the cytoplasm. This structure is conducive to its participation in the formation of PHA particles (Pieper-Fürst et al., J. . Bacterio 1., 177 (1995) 2513-2523). Meanwhile, PhaP in Aeromonas hydrophila species was reported to function as a biosurfactant (Wei et al., Applied Microbiology and Biotechnology., 91 (2011) 1037-1047).

SUMMARY OF THE INVENTION

An object of the present invention is to provide a polyhydroxyalkanoate PHA particle-binding protein PhaP mutant.

The protein provided by the present invention is the following A) or B):

A) A protein that carries out amino acid modification to at least one amino acid residue in the amino acid sequence of the polyhydroxyalkanoate particle-binding protein PhaP, and has the same function as PhaP;

B) A protein derived from A) by adding a tag sequence to the end of the amino acid sequence of the protein shown in A) and having the same function.

The above-mentioned same function as PhaP may be an emulsifying function or other functions.

The amino acid sequence of the polyhydroxyalkanoate particle-binding protein PhaP is sequence 1, and the nucleotide sequence of the encoding gene is sequence 2.

In the above protein, the amino acid modification is amino acid substitution.

In the above protein, the amino acid is replaced with a hydrophilic amino acid with a hydrophobic amino acid.

In the above protein, the replacement of the hydrophilic amino acid with a hydrophobic amino acid is to replace any one, two or three of the hydrophilic amino acids at the following positions in the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with a hydrophobic amino acid: 23rd, 24th, 30th, 38th, 45th, 52nd, 72nd and 82nd.

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 proteins,

The protein shown in A is the protein obtained by any one of the following 1)-9) methods:

1) replacing the 23rd tyrosine of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with phenylalanine or alanine;

2) replacing asparagine at position 24 of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine or alanine;

3) replacing asparagine at position 30 of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine or alanine;

4) replacing glutamine at position 38 of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine, isoleucine or alanine;

5) replacing the 45th tyrosine of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with phenylalanine or alanine;

6) replacing glutamine at position 52 of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine, isoleucine or alanine;

7) replacing glutamine at position 72 of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine, isoleucine or alanine;

8) Substituting leucine, isoleucine or alanine for glutamine at position 82 of the polyhydroxyalkanoate particle binding protein PhaP amino acid sequence;

9) Any 2 or any 3 combinations of 1)-8).

Among the above proteins, the methods shown in 9) are as follows 9)-1), 9)-2), 9)-3), 9)-4):

9)-1): replacing the 38-position glutamine of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine, and replacing the 52-position glutamine of the PhaP amino acid sequence with leucine;

9)-2): replacing glutamine at position 38 in the amino acid sequence of polyhydroxyalkanoate particle binding protein PhaP with leucine, and replacing glutamine at position 72 in the amino acid sequence of PhaP with leucine;

9)-3): replacing the 38-position glutamine of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine, and replacing the 82-position glutamine of the PhaP amino acid sequence with leucine;

and The glutamine at position 82 of the PhaP amino acid sequence was replaced with leucine.

DNA molecules encoding the above proteins are also within the scope of the present invention.

The application of above-mentioned protein or above-mentioned DNA molecule as emulsifier is also the scope of protection of the present invention;

Or the application of above-mentioned protein or above-mentioned DNA molecule in the preparation of emulsifier is also the scope of protection of the present invention;

Or the application of above-mentioned protein or above-mentioned DNA molecule in emulsified oil is also the scope of protection of the present invention;

Or the application of above-mentioned protein or above-mentioned DNA molecule in the preparation of oil-water emulsion is also the scope of protection of the present invention;

The application of the above-mentioned protein or the above-mentioned DNA molecule in the preparation of surfactant is also within the protection scope of the present invention.

Another object of the present invention is to provide a product.

The product provided by the present invention includes the above-mentioned protein.

The above products are emulsifiers or surfactants.

The experiments of the present invention prove that the present invention provides a variety of PhaP mutants of polyhydroxyalkanoate PHA ester particle-binding protein through a large number of experiments and structural biology analysis, and the mutants are obtained through construction, expression and purification, and the mutants are relatively Compared with the wild-type protein, it has stronger emulsifying properties, higher heat resistance, and stronger ability to reduce the interfacial surface tension of the solution. High-performance mutants can be used as biosurfactants in medical, cosmetic, consumer goods, nanomaterials, polymer materials, oil exploration and other fields. Compared with wild-type proteins, they have the advantages of strong performance and low cost.

Description of drawings

Fig. 1 is an electrophoresis image of the recombinant plasmid pGEX-6P-1-PhaP.

Figure 2 is an SDS-PAGE chart of the polyhydroxyalkanoate PHA particle-binding protein PhaP.

Figure 3 is a graph of the purification of PhaP by gel filtration chromatography.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Preparation of PHA particle-binding protein PhaP and its mutants

1. Preparation of PHA particle-binding protein PhaP

The amphiphilic protein used in the present invention is the polyhydroxyalkanoate particle binding protein phaP gene from Aeromonas hydrophila, which is overexpressed in prokaryotic or eukaryotic and affinity purified with GST tag.

The amino acid sequence of the polyhydroxyalkanoate particle-binding protein PhaP from Aeromonas hydrophila is the sequence 1 in the sequence listing, and the nucleotide sequence of the encoding gene phaP is the sequence 2 in the sequence listing. The specific operations are as follows:

1. Construction of recombinant plasmid pGEX-6P-1-PhaP

The recombinant plasmid pGEX-6P-1-PhaP is a vector obtained by replacing the gene phaP shown in sequence 2 in the sequence table with the fragment between the BamHI and XhoI restriction sites of the pGEX-6P-1 (VT1258, Youbao Bio) vector. , the gene phaP is co-expressed with the GST tag on the vector and the fusion protein PhaP is co-expressed.

2. Expression and purification of protein PhaP

The recombinant plasmid pGEX-6P-1-PhaP was introduced into Escherichia coli BL21 (DE3) competent cells (purchased from Quanshijin Biotechnology Co., Ltd.), and a resistant plate (containing 100 μg/ml of ampicillin) was smeared to screen positive colonies. The positive colonies were identified by bacterial liquid PCR, and the primers were 5'-CGGGATCCATGATGAATATG-3'; 5'-CACTCGAGGGCCTT GCCCGTG-3'. A fragment of 350 bp in size that was basically consistent with the expected theoretical value was positive, and the positive bacteria were named BL21/pGEX-6P-1-PhaP.

Pick a single colony of BL21/pGEX-6P-1-PhaP, add it to 100ml of LB liquid medium (containing 100μg/ml of ampicillin), shake and culture at 37°C until the OD 600 is 0.5-0.8, and add IPTG with a final concentration of 0.5mM. , 16 ℃ overnight induction and expression of fusion protein, to obtain BL21/pGEX-6P-1-PhaP bacterial liquid after induction. At the same time, the bacterial solution without IPTG was set as blank control.

The above BL21/pGEX-6P-1-PhaP bacterial solution was centrifuged at 8000rpm for 12min, the supernatant was discarded, the precipitate was collected and resuspended in binding buffer solution (0.5M NaCl, 50mM Tris-HCl, pH=8.0), ice bath, ultrasonication The cells were disrupted; centrifuged at 13,000 rpm for 60 minutes, and the supernatant was the clarified crude cell extract.

The crude cell extract was loaded onto a GST affinity chromatography medium (DP201-01, full gold) self-packed purification column, the column volume was 4mL, and 200mL binding buffer solution (0.5M NaCl, 50mM Tris-HCl, pH= 8.0) Wash off impurity proteins, and finally add 2 column volumes of eluent (0.5M NaCl, 50mM Tris-HCl pH=8.0) to the affinity chromatography column, and add 500uL 3C PreScission protease enzyme (Department of Biology, Tsinghua University). Prepared by the Center for Structural Biology) digested overnight at 4 degrees, and GST was excised to obtain the purified product by affinity chromatography.

The purified product of affinity chromatography was centrifuged at 4000 rpm with an ultrafiltration concentration tube with a filter membrane pore size of 10kD at 4°C. Since the protein was larger than the filter membrane pore size, it was trapped in the upper layer, and the buffer and salt were smaller than the filter membrane. The pore size of the membrane is thrown to the lower layer in the process of centrifugation, so as to achieve the effect of concentration. Concentrate to a final volume of about 1ml. Collect the concentrated product and perform gel column filtration chromatography. The pore size of the gel column filtration chromatography is Superdex 200 10 /300 column, the column volume was 25 mL, the elution time was 60 min, the flow rate of the eluent (0.5 M NaCl, 50 mM Tris-HCl pH=8.0) was 0.5 mL/min, and 6 mL of the eluent was collected (Figure 3).

The collected 6 mL of eluate was concentrated with an ultrafiltration concentration tube with a pore size of 10 kD, and the concentrated solution was collected to obtain protein PhaP.

The protein PhaP is subjected to SDS-PAGE, and the electrophoresis results are shown in Figure 2. It can be seen that the size of the obtained protein PhaP is about 14 kDa, which is consistent with the molecular weight reported in the literature.

The empty vector pGEX-6P-1 was introduced into Escherichia coli BL21 to obtain BL21/pGEX-6P-1. The above-mentioned method was used to express and purify the protein, but the target protein with a size of 14k Da was not obtained.

2. Preparation of PhaP mutants

A PhaP mutant is a protein obtained by replacing at least one amino acid in the amino acid sequence of the PhaP protein with amino acids. If one amino acid is replaced, a PhaP single point mutant is obtained, and if two amino acids are replaced, a PhaP double point mutant is obtained. If three amino acids are replaced, a PhaP double point mutant is obtained. The substitution yielded the PhaP three-point mutant.

PhaP single point mutants are 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, PhaP/Q82A.

PhaP double point mutants are specifically PhaP/Q38L-Q52L, PhaP/Q38L-Q72L, PhaP/Q38L-Q82L, PhaP/Q52L-Q72L;

The PhaP three-point mutant is specifically PhaP/Q38L-Q52L-Q72L.

The amino acid sequence of the mutant PhaP/Y23F is that the 23rd tyrosine (Tyr) of sequence 1 is mutated to phenylalanine (Phe), and the nucleotide sequence of the gene encoding the mutant PhaP/Y23F is the 67th position of sequence 2. TAC at position -69 is mutated to TTC.

The amino acid sequence of the mutant PhaP/Y23A is to mutate the tyrosine (Tyr) at the 23rd position of the sequence 1 to alanine (Ala), and the nucleotide sequence of the gene encoding the mutant PhaP/Y23A is the 67th position of the sequence 2. - TAC at position 69 is mutated to GCC.

The amino acid sequence of the mutant PhaP/N24L is to mutate the asparagine (Asn) at the 24th position of the sequence 1 to leucine (Leu), and the nucleotide sequence of the gene encoding the mutant PhaP/N24L is the 70th position of the sequence 2. -AAC at position 72 is mutated to CTG.

The amino acid sequence of the mutant PhaP/N24A is that the asparagine (Asn) at the 24th position of the sequence 1 is mutated to alanine (Ala), and the nucleotide sequence of the gene encoding the mutant PhaP/N24A is the 70th position of the sequence 2. -AAC at position 72 is mutated to GCC.

The amino acid sequence of the mutant PhaP/N30L is to mutate the asparagine (Asn) at the 30th position of the sequence 1 to leucine (Leu), and the nucleotide sequence of the gene encoding the mutant PhaP/N30L is the 88th position of the sequence 2. -AAC at position 90 is mutated to CTG.

The amino acid sequence of the mutant PhaP/N30A is to mutate the asparagine (Asn) at the 30th position of the sequence 1 to alanine (Ala), and the nucleotide sequence of the gene encoding the mutant PhaP/N30A is the 88th position of the sequence 2. - AAC at position 90 is mutated to GCC.

The amino acid sequence of the mutant PhaP/Q38L is to mutate the glutamine (Gln) at the 38th position of the sequence 1 to leucine (Leu), and the nucleotide sequence of the gene encoding the mutant PhaP/Q38L is the 112th position of the sequence 2. -CAG at position 114 is mutated to CTG.

The amino acid sequence of the mutant PhaP/Q38I is that the glutamine (Gln) at the 38th position of sequence 1 is mutated to isoleucine (IIe), and the nucleotide sequence of the gene encoding the mutant PhaP/Q38I is the 112th position of sequence 2. CAG at position -114 was mutated to ATC.

The amino acid sequence of the mutant PhaP/Q38A is to mutate the glutamine (Gln) at the 38th position of the sequence 1 to alanine (Ala), and the nucleotide sequence of the gene encoding the mutant PhaP/Q38A is the 112th position of the sequence 2. -CAG at position 114 is mutated to GCC.

The amino acid sequence of the mutant PhaP/Y45F is that the tyrosine (Tyr) at position 45 of sequence 1 is mutated to phenylalanine (Phe), and the nucleotide sequence of the gene encoding the mutant PhaP/Y45F is the 133rd position of sequence 2. TAC at position -135 is mutated to TTC.

The amino acid sequence of the mutant PhaP/Y45A is to mutate the tyrosine (Tyr) at the 45th position of the sequence 1 to alanine (Ala), and the nucleotide sequence of the gene encoding the mutant PhaP/Y45A is the 133rd position of the sequence 2. -TAC at position 135 is mutated to GCC.

The amino acid sequence of the mutant PhaP/Q52L is that the glutamine (Gln) at position 52 of sequence 1 is mutated to leucine (Leu), and the nucleotide sequence of the gene encoding the mutant PhaP/Q52L is the 154th position of sequence 2. -CAG at position 156 is mutated to CTG.

The amino acid sequence of the mutant PhaP/Q52I is that the glutamine (Gln) at position 52 of sequence 1 is mutated to isoleucine (IIe), and the nucleotide sequence of the gene encoding the mutant PhaP/Q52I is the 154th position of sequence 2. CAG at position -156 was mutated to ATC.

The amino acid sequence of the mutant PhaP/Q52A is that the glutamine (Gln) at position 52 of sequence 1 is mutated to alanine (Ala), and the nucleotide sequence of the gene encoding the mutant PhaP/Q52A is the 154th position of sequence 2. -CAG at position 156 is mutated to GCC.

The amino acid sequence of the mutant PhaP/Q72L is that the glutamine (Gln) at position 72 of sequence 1 is mutated to leucine (Leu), and the nucleotide sequence of the gene encoding the mutant PhaP/Q72L is the 214th position of sequence 2. -CAG at position 216 is mutated to CTG.

The amino acid sequence of the mutant PhaP/Q72I is that the glutamine (Gln) at position 72 of sequence 1 is mutated to isoleucine (IIe), and the nucleotide sequence of the gene encoding the mutant PhaP/Q72I is the 214th position of sequence 2. CAG at position -216 was mutated to ATC.

The amino acid sequence of the mutant PhaP/Q72A is that the glutamine (Gln) at position 72 of sequence 1 is mutated to alanine (Ala), and the nucleotide sequence of the gene encoding the mutant PhaP/Q72A is the 214th position of sequence 2. -CAG mutation at position 216 to GCC.

The amino acid sequence of the mutant PhaP/Q82L is to mutate the glutamine (Gln) at the 82nd position of the sequence 1 to leucine (Leu), and the nucleotide sequence of the gene encoding the mutant PhaP/Q82L is the 244th position of the sequence 2. -CAG at position 246 is mutated to CTG.

The amino acid sequence of the mutant PhaP/Q82I is that the glutamine (Gln) at position 82 of sequence 1 is mutated to isoleucine (IIe), and the nucleotide sequence of the gene encoding the mutant PhaP/Q82I is the 244th position of sequence 2. CAG at position -246 was mutated to ATC.

The amino acid sequence of the mutant PhaP/Q82A is to mutate the glutamine (Gln) at the 82nd position of the sequence 1 to alanine (Ala), and the nucleotide sequence of the gene encoding the mutant PhaP/Q82A is the 244th position of the sequence 2. -CAG at position 246 is mutated to GCC.

The amino acid sequence of the mutant PhaP/Q38L-Q52L is to mutate glutamine (Gln) at position 38 of sequence 1 to leucine (Leu), and mutate glutamine (Gln) at position 52 of sequence 1 to leucine Amino acid (Leu); the nucleotide sequence of the gene encoding the mutant PhaP/Q38L-Q52L is to mutate CAG at positions 112-114 of sequence 2 into CTG, and mutate CAG at positions 154-156 into CTG.

The amino acid sequence of mutant PhaP/Q38L-Q72L is to mutate glutamine (Gln) at position 38 of sequence 1 to leucine (Leu), and mutate glutamine (Gln) at position 72 of sequence 1 to leucine Amino acid (Leu); the nucleotide sequence of the gene encoding the mutant PhaP/Q38L-Q72L is to mutate CAG at positions 112-114 of sequence 2 into CTG, and mutate CAG at positions 214-216 into CTG.

The amino acid sequence of mutant PhaP/Q38L-Q82L is to mutate glutamine (Gln) at position 38 of sequence 1 to leucine (Leu), and mutate glutamine (Gln) at position 82 of sequence 1 to leucine Amino acid (Leu); the nucleotide sequence of the gene encoding the mutant PhaP/Q38L-Q82L is to mutate CAG at positions 112-114 of sequence 2 to CTG, and mutate CAG at positions 244-246 into CTG.

The amino acid sequence of mutant PhaP/Q52L-Q72L is to mutate glutamine (Gln) at position 52 of sequence 1 to leucine (Leu), and mutate glutamine (Gln) at position 72 of sequence 1 to leucine Amino acid (Leu); the nucleotide sequence of the gene encoding the mutant PhaP/Q52L-Q72L is to mutate CAG at positions 154-156 of sequence 2 into CTG, and mutate CAG at positions 214-216 into CTG.

The amino acid sequence of the mutant PhaP/Q38L-Q52L-Q72L is to mutate glutamine (Gln) at position 38 of sequence 1 to leucine (Leu), and mutate glutamine (Gln) at position 52 of sequence 1 is leucine (Leu), and the glutamine (Gln) at position 72 of sequence 1 is mutated to leucine (Leu); the nucleotide sequence of the gene encoding the mutant PhaP/Q38L-Q52L-Q72L is the sequence 2 The CAG at positions 112-114 was mutated to CTG, and the CAG at positions 154-156 was mutated to CTG, and the CAG at positions 214-216 was mutated to CTG.

1. Preparation of recombinant vector expressing mutant protein

The recombinant vectors expressing different mutant proteins are the fragments between the BamHI and XhoI restriction sites of the pGEX-6P-1 vector ((VT1258, Youbao Bio)) by replacing the encoding genes of different mutant proteins, and the obtained vectors have different mutations. The bulk protein was co-expressed with the GST tag on the vector to express different mutant proteins.

The preparation methods of recombinant vectors expressing different mutant proteins are as follows:

Using pGEX-6P-1-PhaP as a template, PCR was performed with 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/Q38A, pGEX-6P-1-PhaP/Y45F, pGEX-6P-1-PhaP/Y45A, pGEX-6P-1 -PhaP/Q52L, pGEX-6P-1-PhaP/Q52I, pGEX-6P-1-PhaP/Q52A, 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-Q72L.

2. Expression and purification of mutants

The method is the same as that in the above 1, except that the recombinant plasmid pGEX-6P-1-PhaP is replaced with the recombinant vector expressing different mutant proteins prepared in the above 1 to obtain mutant PhaP/Y23F, mutant PhaP/Y23A, mutant 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 PhaP/Q72A, Mutant PhaP/Q82L, Mutant PhaP/Q82I , Mutant PhaP/Q82A, Mutant PhaP/Q38L-Q52L, Mutant PhaP/Q38L-Q72L, Mutant PhaP/Q38L-Q82L, Mutant PhaP/Q52L-Q72L, Mutant PhaP/Q38L-Q52L-Q72L.

Table 1 is the mutant primers

Table 1 shows the primers for single-point mutation. The method for multiple-point mutation is the same as that for single-point mutation, that is, using mutation primers to introduce other mutation sites into the mutated vector.

Example 2. The ability of PhaP and its mutants to emulsify soybean oil

The protein PhaP prepared in Example 1 and different mutant proteins were prepared into aqueous solutions of the same concentration (50 μg/ml); the oil phase was soybean oil (Arowana brand).

The specific operations are as follows:

1. Preparation of an aqueous solution of the substance to be tested: The protein PhaP prepared in Example 1 and different mutant proteins were prepared into aqueous solutions of the same concentration (50 μg/ml).

2. Using soybean oil as the oil phase, mix equal volumes (the aqueous solution of the substance to be tested and the oil phase are both 0.5 ml) of the aqueous solution of the substance to be tested and soybean oil for emulsification treatment, and the emulsification treatment adopts a vortex oscillator to vibrate , Vortex shaker was used for oscillating treatment using a vortex shaker (XH-C, Jinyi, Jintan Medical Instrument Factory) at a speed of 1300 rpm for 120 seconds. After the emulsification treatment was completed, let it stand at a constant temperature of 25°C in the dark, and stand for 2 days to take pictures and observe and record the heights of the oil layer, the emulsification layer and the water layer. And the relevant emulsification value is calculated by formula (1). At the same time, ultrapure water was used as a control.

The formula for calculating the emulsification value is as follows:

Emulsification value=Emulsion layer/(Oil layer+Emulsion layer+Water layer)×100% (1)

The total height of all layers refers to the total height of the oil layer, the emulsification layer and the water layer. But sometimes the water or oil layer may disappear after emulsification, and the total height is the sum of the heights of the remaining two layers. The larger the emulsification value, the stronger the emulsification ability.

The emulsification experiments were repeated three times, and the emulsification values were expressed as the mean ± standard deviation.

The results are shown in Table 2. It can be seen that after standing for 2 days, the site-directed mutations obtained by structural analysis all improved the emulsification of the protein PhaP to varying degrees. The mutants PhaP/Q38L, PhaP/Q52L, PhaP/Q72L, PhaP/Q82L , PhaP/Q38L-Q52L, PhaP/Q38L-Q72L, PhaP/Q38L-Q82L, PhaP/Q38L-Q52L-Q72L these mutations were significantly improved.

In addition, in this experiment, the concentration of the aqueous solution of PhaP and its mutant protein was increased to 150 μg/ml, and it was found that the emulsification ability of the mutant and the wild type was basically the same (Table 3). type, that is, less dosage is required to achieve the same emulsification effect.

Table 2 Comparison of emulsified layers of PhaP and its mutant protein aqueous solution (50 μg/ml) emulsified soybean oil

Table 3 Comparison of emulsified layers of PhaP and its mutant protein aqueous solution (150 μg/ml) emulsified soybean oil

Therefore, the PhaP mutants can act as emulsifiers.

Example 3. Melting point (Tm) measurement of PhaP and its mutant proteins

The protein PhaP prepared in Example 1 and different mutant proteins were prepared into aqueous solutions of the same concentration (50 μg/ml), and far-ultraviolet circular dichroism (CD) spectra were detected. The instrument used was a Jasco 715 spectrometer, and the optical path of the cuvette was 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 tube voltage instrument is automatically controlled, the detection wavelength range is 200nm-260nm, and the number of repetitions is 3 times. By plotting the intensity at 220 nm of the CD spectrum against temperature, it can be concluded that the secondary structure of the protein changes with temperature. The protein concentration was 0.1 mg/ml. Then use the S-shaped curve to fit the variable temperature curve obtained by CD, and use the formula 2 to calculate the curve inflection point x ₀

y=Ab+((At-Ab)/(1+exp((x ₀ -x)/w))) (2)

The y value corresponding to x ₀ is (Ab+At)/2

Ab is the y value corresponding to the tangent under the curve

At is the y value corresponding to the tangent on the curve

The temperature corresponding to the inflection point x ₀ has obvious changes in the secondary structure of the protein, and the temperature corresponding to this point is regarded as the protein Tm value

After the temperature change test, the following results were obtained (Table 4). The Tm values of the mutated proteins were increased, and the Tm values of the mutants Q38L, Q38LQ52L, Q38LQ72L, Q38LQ82L, Q38LQ52LQ72L were increased by nearly 20 °C.

This result indicates that the PhaP mutant protein has higher thermal stability.

Table 4 Comparison of melting point Tm values of PhaP and its mutant proteins

Example 4. Effects of PhaP and its mutant proteins on the interfacial surface tension of aqueous solutions

Due to the hydrophobic effect of surfactant molecules, the hydrophobic groups come together to form a hydrophobic core, and the hydrophilic groups contact with water outwards, and self-assembly occurs in the aqueous solution to form aggregates of various structures and sizes. Adding surfactants can reduce the interfacial surface tension of aqueous solutions.

The JZ-200A interfacial tensiometer used was used to measure the surface tension value of the solution by the Dyne ring method, and the protein PhaP prepared in Example 1 and different mutant proteins were configured into an aqueous solution of the same concentration (10 mg/L), and the measurement was performed at this concentration. Its surface tension values (Table 5). It was found that the ability of the mutant protein to reduce the surface tension of the solution was improved compared with the wild type, among which 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. At the same concentration (10 mg/L), the mutant can reduce the surface tension more than the wild type, and has a better application prospect.

Table 5 PhaP and its mutant protein solution (10mg/L) surface tension value

Claims (4)

1. A protein obtained by any one of the following methods 1) to 9): 1) Replace the 23rd tyrosine in the amino acid sequence of the polyhydroxyalkanoate particle binding protein PhaP with phenylalanine or alanine; 2) Replace the asparagine at position 24 of the amino acid sequence of the polyhydroxyalkanoate particle binding protein PhaP with leucine or alanine; 3) Replace asparagine at the 30th position of the amino acid sequence of the polyhydroxyalkanoate particle binding protein PhaP with leucine or alanine; 4) Replace glutamine at position 38 in the amino acid sequence of polyhydroxyalkanoate particle binding protein PhaP with leucine, isoleucine or alanine; 5) Replace the tyrosine at position 45 of the amino acid sequence of the polyhydroxyalkanoate particle binding protein PhaP with phenylalanine or alanine; 6) Replace glutamine at position 52 of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine, isoleucine or alanine; 7) Replace glutamine at position 72 of the polyhydroxyalkanoate particle binding protein PhaP amino acid sequence with leucine, isoleucine or alanine; 8) Replace glutamine at position 82 of the polyhydroxyalkanoate particle-binding protein PhaP amino acid sequence with leucine, isoleucine or alanine; 9) Any 2 or any 3 combinations of 1)-8); The methods shown in 9) are as follows 9)-1), 9)-2), 9)-3), 9)-4) and 9)-5): 9)-1): replace glutamine at position 38 of the amino acid sequence of polyhydroxyalkanoate particle-binding protein PhaP with leucine, and replace glutamine at position 52 in the amino acid sequence of PhaP with leucine; 9)-2): replace glutamine at position 38 of the amino acid sequence of polyhydroxyalkanoate particle-binding protein PhaP with leucine, and replace glutamine at position 72 of the PhaP amino acid sequence with leucine; 9)-3): replace glutamine at position 38 in the amino acid sequence of polyhydroxyalkanoate particle binding protein PhaP with leucine, and replace glutamine at position 82 in the amino acid sequence of PhaP with leucine; 9)-4): replace glutamine at position 52 in the amino acid sequence of polyhydroxyalkanoate particle binding protein PhaP with leucine, and replace glutamine at position 72 in the amino acid sequence of PhaP with leucine; and The glutamine at position 82 of the PhaP amino acid sequence was replaced with leucine. 2. A DNA molecule encoding the protein of claim 1. 3. the application of the described protein of claim 1 or the described DNA molecule of claim 2 as an emulsifying agent; Or the application of the described protein of claim 1 or the described DNA molecule of claim 2 in the preparation of emulsifier; Or the application of the described protein of claim 1 or the described DNA molecule of claim 2 in emulsified oil; Or the application of the described protein of claim 1 or the described DNA molecule of claim 2 in the preparation of oil-water emulsion; Or the application of the protein of claim 1 or the DNA molecule of claim 2 in the preparation of surfactants. 4. A product comprising the protein of claim 1; The product is an emulsifier or surfactant.

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