CN117965487A - Engineering enzyme composition, preparation method thereof, coding gene and application - Google Patents
Engineering enzyme composition, preparation method thereof, coding gene and application Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 123
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 123
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 46
- 239000000203 mixture Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 229920002527 Glycogen Polymers 0.000 claims abstract description 33
- 229940096919 glycogen Drugs 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 230000003197 catalytic effect Effects 0.000 claims abstract description 22
- 102000003925 1,4-alpha-Glucan Branching Enzyme Human genes 0.000 claims abstract description 19
- 108090000344 1,4-alpha-Glucan Branching Enzyme Proteins 0.000 claims abstract description 19
- 238000002823 phage display Methods 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 13
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 12
- 108020000005 Sucrose phosphorylase Proteins 0.000 claims abstract description 11
- 238000000338 in vitro Methods 0.000 claims abstract description 11
- 108050004944 Alpha-glucan phosphorylases Proteins 0.000 claims abstract description 10
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 9
- 238000010367 cloning Methods 0.000 claims abstract description 4
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- 238000003776 cleavage reaction Methods 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 230000007017 scission Effects 0.000 claims description 5
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 claims description 4
- 229920002472 Starch Polymers 0.000 claims description 4
- 239000007795 chemical reaction product Substances 0.000 claims description 4
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- 238000004064 recycling Methods 0.000 claims description 4
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- 235000019698 starch Nutrition 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- 108091081021 Sense strand Proteins 0.000 claims description 3
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- 108010006785 Taq Polymerase Proteins 0.000 claims description 3
- 238000012512 characterization method Methods 0.000 claims description 3
- 230000000295 complement effect Effects 0.000 claims description 3
- 238000006555 catalytic reaction Methods 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 3
- 102000004169 proteins and genes Human genes 0.000 description 9
- 229920000310 Alpha glucan Polymers 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 5
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- 238000001727 in vivo Methods 0.000 description 3
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102100023321 Ceruloplasmin Human genes 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 2
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
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- 229920001503 Glucan Polymers 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
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- 108091034117 Oligonucleotide Proteins 0.000 description 1
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- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- BNABBHGYYMZMOA-AHIHXIOASA-N alpha-maltoheptaose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O[C@@H]4[C@H](O[C@H](O[C@@H]5[C@H](O[C@H](O[C@@H]6[C@H](O[C@H](O)[C@H](O)[C@H]6O)CO)[C@H](O)[C@H]5O)CO)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O BNABBHGYYMZMOA-AHIHXIOASA-N 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
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- 239000002537 cosmetic Substances 0.000 description 1
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- 108020001507 fusion proteins Proteins 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
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- 239000002539 nanocarrier Substances 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 150000008135 α-glycosides Chemical group 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01007—Sucrose phosphorylase (2.4.1.7)
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Abstract
The invention relates to the field of enzyme engineering, in particular to an engineering enzyme composition, a preparation method, a coding gene and application thereof. An engineered enzyme composition comprising three enzymes: sucrose phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.1 and a coding gene; alpha glucan phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.2 and a coding gene; and glycogen branching enzyme, which consists of an amino acid sequence shown in SEQ ID NO.3 and a coding gene. The preparation method of the engineering enzyme composition comprises the following steps: cloning the gene of the enzyme into a phage display vector and characterizing the catalytic properties of the phage enzyme; constructing a mutant library of phage display enzyme by using a random mutation method; before starting selection, characterizing the mutant library; the evolved enzyme clone with the best catalytic properties was selected. The invention improves the reaction process of enzyme catalysis and the separation process of enzyme and substrate, so that the in-vitro synthesis efficiency of glycogen analogues is greatly improved, thereby reducing the cost.
Description
Technical Field
The invention relates to the field of enzyme engineering, in particular to an engineering enzyme composition, a preparation method, a coding gene and application thereof.
Background
Glycogen is a natural nanoscale dendritic glucan (bound to small amounts of proteins or polypeptides of important importance) present in animal and plant cells. The minimum constituent unit of glycogen is alpha-D-glucose, and as shown in FIG. 1, the synthetic pathway in vivo is: 1) Self-glycosylation of glycogen proteins: under the autocatalysis of the glycogen protein, tyrosine residue 195 of the glycogen protein is valence-bound to glucose residue of uridine diphosphate glucose and persists until the length of the glucose chain approaches 10 glucose units, at which time glycosylated protein primers are formed. 2) Catalysis of Glycogen Synthase (GS): glycogen synthase transfers the glucose residues of uridine diphosphate glucose to the non-reducing end of glycosylated protein primers and allows them to be linked by alpha-1, 4-glycosidic bonds. 3) Catalysis of glycogen Branching Enzyme (BE): glycogen branching enzymes transfer one of the above-formed alpha-glycoside chains containing about 8-12 glucose residues to the other and allow them to be linked by an alpha-1, 6-glycosidic bond. 4) Under the combined action of GS and BE, stable glycogen beta particles with the particle size of about 20-50nm are finally formed.
Glycogen is an important part of the in vivo synthesis and catabolic cycle, and is a key substance for maintaining blood glucose and cell hydration. In vitro, glycogen spontaneously forms spherical nanostructures in water, which can be used as moisturizers, rheology modifiers and emulsifiers, and also as nanocarriers for functional substances. In addition, glycogen can also be used for activating signal molecules of cells, so that the tissue or organism can generate immunoregulation enhancement and even show anti-tumor activity. Therefore, glycogen has great potential in the fields of foods, cosmetics and medical and health.
Currently, researchers have prepared glycogen analogs (also referred to as glycogenic alpha-glucans or biological glycogen) in vitro by a variety of enzymatic tandem synthetic routes, such as the alpha-glucan phosphorylase-glycogen branching enzyme route, the sucrose phosphorylase-alpha-glucan phosphorylase-glycogen branching enzyme route, the amylosucrase-glycogen branching enzyme route, and the isoamylase-maltogenic glycosylase-glycogen branching enzyme route, among others. This is the case in food products. Development and application in the fields of medicine and the like are possible. However, in several in vitro synthetic routes, the procedures of product separation and purification are complicated due to the limitations of catalytic capacity and efficiency of enzymes, resulting in lower and expensive yields of glycogen analogues, which greatly limit the wide application thereof in various fields.
Disclosure of Invention
The first technical purpose of the invention is to provide an engineering enzyme composition which can improve the catalytic efficiency through directed evolution.
The second technical purpose of the invention is to provide a preparation method of an engineering enzyme composition capable of improving the catalytic efficiency through directed evolution.
A third technical object of the present invention is to provide a gene encoding the above-described engineered enzyme composition.
The fourth technical object of the present invention is to provide an application of an engineering enzyme composition.
The first technical purpose of the invention is realized by the following technical proposal:
an engineered enzyme composition comprising three enzymes:
Sucrose phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.1 and a coding gene;
Alpha glucan phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.2 and a coding gene;
and glycogen branching enzyme, which consists of an amino acid sequence shown in SEQ ID NO.3 and a coding gene.
The second technical purpose of the invention is realized by the following technical proposal:
The preparation method of the engineering enzyme composition comprises the following steps:
1) Cloning the gene of the enzyme into a phage display vector and characterizing the catalytic properties of the phage enzyme;
2) Constructing a mutant library of phage display enzyme by using a random mutation method;
3) Before starting selection, characterizing the mutant library;
4) The evolved enzyme clone with the best catalytic properties was selected.
Among the phage capsid proteins of the present invention, pIII and pVIII proteins are commonly used for displaying foreign proteins; methods for constructing the mutant library include error-prone polymerase chain reaction, DNA rearrangement, degenerate primer PCR, degenerate oligonucleotide cloning and the like; the phage-enzyme display library may be selected by catalytic elution, substrate labeling, TSA affinity, suicide, etc.
The invention selects the path of sucrose phosphorylase-alpha glucan phosphorylase-glycogen branching enzyme, and uses phage-enzyme display method to directionally evolve the sucrose phosphorylase, alpha glucan phosphorylase and glycogen branching enzyme used in the path to obtain super sucrose phosphorylase, super alpha glucan enzyme and super glycogen branching enzyme, and compared with the combination of three enzymes before evolution, the catalytic efficiency of super enzyme is greatly improved, and simultaneously, the reaction procedure of enzyme catalysis and the separation procedure of enzyme and substrate are improved, so that the in vitro synthesis efficiency of glycogen analogues is greatly improved, thereby reducing the cost.
Phage-enzyme display and directed engineering of enzyme molecules: the phage display technology is to recombine the gene of target protein onto the gene of phage capsid protein by utilizing gene recombination method, and finally display the target protein on the surface of phage in the form of fusion protein. Although the most straightforward application of this technology is the screening of novel binders (ligands) to specific proteins, phage display technology can also be used for directed evolution of enzymes. The aim is to evolve the existing enzymes towards new or improved catalytic properties.
Preferably, the preparation method of the engineering enzyme composition specifically comprises the following steps:
(1) Directed evolution of engineered enzymes
1.1 Selecting a phage display vector of pCANTAB5E phagemid and a helper phage of M13KO7;
1.2 Amino acid sequences and coding genes of the three enzymes were determined as follows:
Sucrose phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.1 and a coding gene;
alpha glucan phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.2 and a coding gene; and glycogen branching enzyme, which consists of an amino acid sequence shown in SEQ ID NO.3 and a coding gene;
1.3 Designing a primer sequence for error-prone polymerase chain reaction;
1.4 Respectively amplifying DNA sequences of the three enzymes by adopting an error-prone polymerase chain reaction method;
1.5 Introducing the amplified target DNA into a vector phagemid (comprising cleavage of the target DNA and the vector phagemid, religation of the cleaved fragments);
1.6 Co-transforming the vector phagemid and the helper phage into E.coli;
1.7 Selecting a certain number of monoclonal clones for expansion culture respectively;
1.8 Characterization of the catalytic activity of each of the monoclonal phage display enzymes of the expansion culture, respectively;
1.9 Selecting an optimally evolved enzyme.
The invention focuses on the directional evolution of three common enzymes by using a phage display method to obtain three super enzymes with greatly improved catalytic activity, and then synthesizing glycogen analogues in vitro by using the three super enzymes. In addition, as the enzyme is displayed on the surface of the phage, the enzyme immobilization method can be understood, and the PEG can be used for separating the phage-enzyme precipitate after the subsequent catalytic reaction is finished, so that the product separation process is simplified, the enzyme can be reused, and the cost is reduced.
Preferably, the preparation method of the engineering enzyme composition specifically comprises the following steps:
(1) Directed evolution of engineered enzymes
1.1 Selecting a phage display vector of pCANTAB5E phagemid and a helper phage of M13KO7;1.2 Amino acid sequences and coding genes of the three enzymes were determined as follows:
Sucrose phosphorylase-)
Alpha glucan phosphorylase-)
Glycogen branching enzyme-)
(2) The DNA sequences of the three enzymes were amplified by error-prone polymerase chain reaction (error-prene PCR) methods, respectively.
Preferably, the DNA sequences of the three enzymes are amplified by error-prone polymerase chain reaction (error-prene PCR) methods, respectively, as follows:
① TaqDNA polymerase is adopted;
② Enzyme performs 4 times of DNA sequence amplification, and in the four times of amplification, the addition molar ratio of four bases is (ATGC) = (1:1:1:2), (1:1:2:1), (1:2:1:1) and (2:1:1:1);
③ Adding 5 mmol.L-1 MgCl 2、0.5mmol·L-1MnCl2 into the amplification reaction system;
④ The PCR procedure for each ATGC formulation was performed 2 times in succession.
More preferably, after the amplification reaction is completed, amplified fragments of three enzymes are collected separately, and amplified fragments of the same enzyme at different times are mixed together.
Preferably, the primer sequences are designed as follows:
Repeating the arrangement for three times according to four glycine and one serine (G4S) 3, adding enzyme cutting sites of Sfi I and Not I respectively at the 5' ends of the sense strand and the complementary strand, and adding a plurality of protecting bases, thereby obtaining two primer sequences:
E-Back：
5’-GTC CTC GCA ACT GCG GCC CAG CCG GC ATG GCC CAG GTG CAG CTG SWG SAG TCW GG-3’
F-for：
5’-GAG TCA TTC TCG ACT TGC GGC CGC TTT GAT CTC CAS CTT GGT CC-3’
Where r=a/G, s=g/C, w=a/T, m=a/C, k=g/T.
The third technical purpose of the invention is realized by the following technical proposal:
genes encoding the above-described engineered enzyme compositions,
The nucleotide sequence of the gene comprises:
1) The nucleotide sequence of sucrose phosphorylase shown in SEQ ID NO. 1;
2) The nucleotide sequence of alpha glucan phosphorylase shown in SEQ ID NO. 2;
3) The nucleotide sequence of glycogen branching enzyme shown in SEQ ID NO. 3.
The fourth technical object of the present invention is achieved by the following technical scheme:
Use of an engineered enzyme composition for the in vitro synthesis of a glycogen analogue.
Preferably, the synthesis reaction temperature is 50-60 ℃, the pH of the system is 7-8, sucrose is used as a substrate, gelatinized starch is used as a primer, the reaction time is 14-18 hours, after the reaction is finished, 15-25% PEG is used for precipitating phage-enzyme, the enzyme can be separated out for convenient subsequent recycling, and the rest reaction products are dialyzed and dried to obtain glycogen analogues.
More preferably, the synthesis reaction temperature is 55 ℃, the pH of the system is 7.2, sucrose is used as a substrate, gelatinized starch is used as a primer, the reaction time is 16 hours, after the reaction is finished, 20 percent of PEG is used for precipitating phage-enzyme, the enzyme can be separated out for convenient subsequent recycling, and the rest reaction products are dried after dialysis, so that the glycogen analogue is obtained.
In summary, the invention has the following beneficial effects:
1. Compared with the original enzyme (commercial enzyme), the engineered enzyme composition (super enzyme) subjected to directional modification has the advantages that Km is greatly reduced, namely, the affinity of the super enzyme and a substrate is far higher than that of the original enzyme and the substrate, and the catalytic conversion efficiency is greatly improved;
2. the invention improves the reaction process of enzyme catalysis and the separation process of enzyme and substrate, so that the in-vitro synthesis efficiency of glycogen analogues is greatly improved, thereby reducing the cost.
Drawings
FIG. 1 is an in vivo synthetic route pattern for glycogen;
FIG. 2 is an electron micrograph of in vitro synthesis of glycogen analogues;
FIG. 3 is an H-NMR spectrum of the enzyme synthesis product of the present invention.
Detailed Description
The preparation method of the engineering enzyme composition specifically comprises the following steps:
1) Directed evolution of engineered enzymes
1.1 Selecting a phage display vector of pCANTAB5E phagemid and a helper phage of M13KO7;
1.2 Amino acid sequences and coding genes of the three enzymes were determined as follows:
Sucrose phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.1 and a coding gene;
Alpha glucan phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.2 and a coding gene;
and glycogen branching enzyme, which consists of an amino acid sequence shown in SEQ ID NO.3 and a coding gene.
The primer sequences were designed as follows:
Repeating the arrangement for three times according to four glycine and one serine (G4S) 3, adding enzyme cutting sites of Sfi I and Not I respectively at the 5' ends of the sense strand and the complementary strand, and adding a plurality of protecting bases, thereby obtaining two primer sequences:
G-Back：
5’-GTC CTC GCA ACT GCG GCC CAG CCG GC ATG GCC CAG GTG CAG CTG SWG SAG TCW GG-3’
H-for：
5’-GAG TCA TTC TCG ACT TGC GGC CGC TTT GAT CTC CAS CTT GGT CC-3’
Where r=a/G, s=g/C, w=a/T, m=a/C, k=g/T.
The DNA sequences of the three enzymes are amplified by adopting an error-prone polymerase chain reaction (error-prene PCR) method, and the method is concretely as follows:
① TaqDNA polymerase is adopted;
② Enzyme performs 4 times of DNA sequence amplification, and in the four times of amplification, the addition molar ratio of four bases is (ATGC) = (1:1:1:2), (1:1:2:1), (1:2:1:1) and (2:1:1:1);
③ Adding 5 mmol.L-1 MgCl2 and 0.5 mmol.L-1 MnCl2 into an amplification reaction system;
④ The PCR procedure for each ATGC formulation was performed 2 times in succession.
After the amplification reaction is completed, amplified fragments of the three enzymes are respectively collected, and amplified fragments of the same enzyme with different times are mixed together.
Vector DNApCANTAB E phagemid was prepared as follows:
① The digestion of vector DNA and PCR amplified fragments comprising:
Sfi I cleavage reaction:
two reaction systems were constructed with purified vector DNA and purified PCR amplification products, respectively:
| 10XM enzyme digestion buffer | 8.5μl |
| Sfi I(12U/μl) | 2μl |
| Adding sterilized double distilled water to | 50μl |
The reaction system is placed at 50 ℃ for enzyme digestion reaction for 4 hours.
After the cleavage reaction, DNA is recovered and Not I cleavage digestion reaction is performed:
the following two reaction systems are respectively constructed by using the PCR amplified fragment cut by Sfi I enzyme and the carrier DNA:
the reaction system was subjected to cleavage reaction at 37℃for 4 hours. After the completion, the cleavage product was recovered.
② The amplified fragment was inserted into the pcatab 5E vector as follows:
The PCR amplified fragment and pCANTAB5E vector after the double cleavage and purification described above were used to construct the following reaction system (amplified fragment: vector=3:1):
| PCR amplified fragment | 750ng |
| 10X ligation buffer | 2.5μl |
| PCANTAB5E vector | 250ng |
| T4DNA ligase (5-7U) | 1μl |
| Adding sterilized double distilled water to | 25μl |
The above system was connected at 16℃for 12 hours.
After co-transformation of E.coli TG1 with the vector phagemid prepared above and M13K07 helper phage, the culture product was diluted 100-fold and plated on 2 XYT-Amp-X-gal-IPTG solid medium plates.
Randomly selecting 40-50 monoclonal antibodies formed on the flat plate, performing amplification culture, and extracting and separating phage by using a catalytic elution method. The phage isolated at this point already displays the desired enzyme domain on its pIII protein.
The catalytic activity of each monoclonal phage-enzyme was characterized separately, i.e., km and Kcat were determined. The super with the largest Km and the smallest Kcat is selected for comparison with the primordial enzyme. The results obtained for each of the three enzymes were as follows:
Sucrose phosphorylase:
| Optimum pH | Optimum temperature | Catalytic efficiency | |
| Super enzyme | 7.2 | 53℃ | Km＝4.2mmol/L |
| Primordial enzyme | 7.6 | 55℃ | Km＝7.3mmol/L |
Alpha glucan phosphorylase:
| Optimum pH | Optimum temperature | Catalytic efficiency: | |
| Super enzyme | 7.8 | 82℃ | Km＝4.7mmol/L |
| Primordial enzyme | 8.0 | 80℃ | Km＝8.4mmol/L |
* And (3) injection: when maltoheptaose and phosphate are used as substrates.
Glycogen branching enzyme:
| Optimum pH | Optimum temperature | Catalytic efficiency | |
| Super enzyme | 7.2 | 70℃ | Km＝3.1mmol/L |
| Primordial enzyme | 7.4 | 75℃ | Km＝7.9mmol/L |
From the above results, it is clear that the Km of the directionally engineered super enzyme is greatly reduced compared with that of the original enzyme (commercially available enzyme), i.e., the affinity of the super enzyme to the substrate is far higher than that of the original enzyme to the substrate, and the catalytic conversion efficiency is greatly improved.
3) Three superases were used in combination for in vitro synthesis of glycogen analogues.
Combining the characterization data of the three super enzymes, determining that the reaction temperature is 55 ℃, the pH of the system is 7.2, sucrose is used as a substrate, gelatinized starch is used as a primer, the reaction time is 16 hours, and after the reaction is finished, precipitating phage-enzyme by using 20% PEG, so that the enzyme can be separated out for subsequent recycling, and the rest reaction products are dialyzed and dried to obtain glycogen analogues.
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.
Claims (10)
1. An engineered enzyme composition comprising three enzymes:
Sucrose phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.1 and a coding gene;
Alpha glucan phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.2 and a coding gene;
and glycogen branching enzyme, which consists of an amino acid sequence shown in SEQ ID NO.3 and a coding gene.
2. The preparation method of the engineering enzyme composition is characterized by comprising the following steps:
1) Cloning the gene of the enzyme into a phage display vector and characterizing the catalytic properties of the phage enzyme;
2) Constructing a mutant library of phage display enzyme by using a random mutation method;
3) Before starting selection, characterizing the mutant library;
4) The evolved enzyme clone with the best catalytic properties was selected.
3. The method for preparing an engineering enzyme composition according to claim 2, which is characterized by comprising the following steps:
(1) Directed evolution of engineered enzymes
1.1 Selecting a phage display vector of pCANTAB5E phagemid and a helper phage of M13KO7;
1.2 Amino acid sequences and coding genes of the three enzymes were determined as follows:
Sucrose phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.1 and a coding gene;
Alpha glucan phosphorylase, which consists of an amino acid sequence shown as SEQ ID NO.2 and a coding gene; and
Glycogen branching enzyme, which consists of an amino acid sequence shown in SEQ ID NO.3 and a coding gene;
1.3 Designing a primer sequence for error-prone polymerase chain reaction;
1.4 Respectively amplifying DNA sequences of the three enzymes by adopting an error-prone polymerase chain reaction method;
1.5 Introducing the amplified target DNA into a vector phagemid (comprising cleavage of the target DNA and the vector phagemid, religation of the cleaved fragments);
1.6 Co-transforming the vector phagemid and the helper phage into E.coli;
1.7 Selecting a certain number of monoclonal clones for expansion culture respectively;
1.8 Characterization of the catalytic activity of each of the monoclonal phage display enzymes of the expansion culture, respectively;
1.9 Selecting an optimally evolved enzyme.
4. A method of preparing an engineered enzyme composition in accordance with claim 3, wherein: the method for amplifying the DNA sequences of the three enzymes by adopting the error-prone polymerase chain reaction method comprises the following steps:
① TaqDNA polymerase is adopted;
② Enzyme performs 4 times of DNA sequence amplification, and in the four times of amplification, the addition molar ratio of four bases is (ATGC) = (1:1:1:2), (1:1:2:1), (1:2:1:1) and (2:1:1:1);
③ Adding 5 mmol.L-1 MgCl 2、0.5mmol·L-1MnCl2 into the amplification reaction system;
④ The PCR process of each ATGC ratio was performed 2 times in succession.
5. The method for preparing an engineered enzyme composition of claim 4, wherein: after the amplification reaction is completed, amplified fragments of the three enzymes are respectively collected, and amplified fragments of the same enzyme with different times are mixed together.
6. The method of preparing an engineered enzyme composition of claim 4, wherein the primer sequences are designed as follows:
Repeating the arrangement for three times according to four glycine and one serine (G4S) 3, adding enzyme cutting sites of Sfi I and Not I respectively at the 5' ends of the sense strand and the complementary strand, and adding a plurality of protecting bases, thereby obtaining two primer sequences:
E-Back：
5’-GTC CTC GCA ACT GCG GCC CAG CCG GC ATG GCC CAG GTG CAG CTG SWG SAG TCW GG-3’
F-for：
5’-GAG TCA TTC TCG ACT TGC GGC CGC TTT GAT CTC CAS CTT GGT CC-3’
Where r=a/G, s=g/C, w=a/T, m=a/C, k=g/T.
7. A gene encoding the engineered enzyme composition of claim 1 or 2 or an engineered enzyme composition prepared by the method of preparing the engineered enzyme composition of any one of claims 3-6.
8. The gene encoding the engineered enzyme composition of claim 7, wherein the nucleotide sequence comprises:
1) The nucleotide sequence of sucrose phosphorylase shown in SEQ ID NO. 1;
2) The nucleotide sequence of alpha glucan phosphorylase shown in SEQ ID NO. 2;
3) The nucleotide sequence of glycogen branching enzyme shown in SEQ ID NO. 3.
9. Use of an engineered enzyme composition for the in vitro synthesis of a glycogen analogue.
10. Use of an engineered enzyme composition as in claim 9, wherein:
The synthesis reaction temperature is 50-60 ℃, the pH of the system is 7-8, sucrose is used as a substrate, gelatinized starch is used as a primer, the reaction time is 14-18 hours, after the reaction is finished, 15-25% PEG is used for precipitating phage-enzyme, the enzyme can be separated out for convenient subsequent recycling, and the rest reaction products are dialyzed and dried to obtain glycogen analogues.
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