CN116355872B - Galactose oxidase mutant GAO-5F/AR, plasmid, recombinant bacterium and application thereof - Google Patents
Galactose oxidase mutant GAO-5F/AR, plasmid, recombinant bacterium and application thereof Download PDFInfo
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- CN116355872B CN116355872B CN202310158953.XA CN202310158953A CN116355872B CN 116355872 B CN116355872 B CN 116355872B CN 202310158953 A CN202310158953 A CN 202310158953A CN 116355872 B CN116355872 B CN 116355872B
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- 108010015133 Galactose oxidase Proteins 0.000 title claims abstract description 71
- 239000013612 plasmid Substances 0.000 title claims abstract description 13
- 241000894006 Bacteria Species 0.000 title abstract description 5
- 102000004190 Enzymes Human genes 0.000 claims abstract description 27
- 108090000790 Enzymes Proteins 0.000 claims abstract description 27
- 150000001413 amino acids Chemical class 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 238000002741 site-directed mutagenesis Methods 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 4
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims description 3
- 241000223218 Fusarium Species 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 27
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 230000003197 catalytic effect Effects 0.000 description 17
- 238000000034 method Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 108010089807 chitosanase Proteins 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- -1 aromatic alcohols Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 108010037176 copper oxidase Proteins 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000007986 glycine-NaOH buffer Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000003138 primary alcohols Chemical class 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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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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- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03009—Galactose oxidase (1.1.3.9)
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Abstract
The application relates to a galactose oxidase mutant GAO-5F/AR, a plasmid, a recombinant bacterium and application thereof, belonging to the technical field of genetic engineering, wherein the amino acid sequence of the galactose oxidase mutant is shown as SEQ ID NO. 1, compared with wild galactose oxidase GAO-5F, site-directed mutagenesis is carried out on 403 and 484 amino acids, the nucleotide sequence is shown as SEQ ID NO.2, the application also provides a recombinant plasmid and a recombinant strain containing the nucleotide shown as SEQ ID NO.2, and the enzyme activity of the mutant is 6 times of that of the wild type.
Description
Technical Field
The application belongs to the technical field of genetic engineering, and particularly relates to a galactose oxidase mutant GAO-5F/AR, a plasmid, a recombinant bacterium and application thereof.
Background
Galactose oxidase (e.c. 1.1.3.9) is a monomeric free radical copper oxidase with copper ions as cofactor. This extracellular monomeric enzyme is secreted by some filamentous fungi, mainly fusarium. Galactose oxidase catalyzes the two-electron oxidation and oxidizes various primary alcohols and aldehydes, especially galactose with a C6 hydroxyl group, to produce the corresponding aldehyde, while reducing water to hydrogen peroxide in a catalytic reaction. Galactose oxidase has a strict stereoselectivity and a broad substrate specificity and can oxidize monosaccharides, polysaccharides, aliphatic and aromatic alcohols, polyols and many other galactose unit-containing compounds. These features enable galactose oxidase to be used in a variety of applications, such as biosensors, chemical synthesis, biomedical and molecular cross-linking.
The good catalytic activity, thermal stability and catalytic efficiency of the enzyme molecule are the basis for the industrial production and commercial application of the enzyme molecule. At present, the enzyme with higher catalytic activity is obtained, and one method is to excavate novel enzyme molecules, but the separation and screening process has large workload and low efficiency. The protein engineering is based on the guidance of structural bioinformatics, and can modify enzyme molecules to further reasonably improve the catalytic activity of the enzyme molecules. For subsequent industrial production, the catalytic activity of galactose oxidase needs to be further improved. By combining three-dimensional structure and sequence analysis, the activity, thermal stability or catalytic efficiency of the enzyme can be reasonably improved through protein engineering. Therefore, it is necessary to dig out and identify key amino acid sites affecting the catalytic activity of galactose oxidase, and on the basis of this, to perform directed modification on galactose oxidase, thereby obtaining galactose oxidase mutants with high catalytic activity.
Disclosure of Invention
Aiming at the defects of the prior art, the application provides a galactose oxidase mutant GAO-5F/AR, a plasmid, a recombinant bacterium and application thereof, and the improvement of the catalytic activity of galactose oxidase is realized by carrying out molecular transformation on galactose oxidase, so that a foundation is laid for industrial application of galactose oxidase.
The GAO-5F is galactose oxidase constructed in escherichia coli in the early stage of the laboratory, has excellent heat stability, can be suitable for industrial production process, and has better application prospect. In order to improve the catalytic activity of the galactose oxidase, the application selects galactose oxidase with higher catalytic activity as a template based on an expression platform of the galactose oxidase in escherichia coli, selects a potential site influencing the catalytic activity of the galactose oxidase based on multi-sequence comparison, and carries out molecular transformation on galactose oxidase GAO-5F through site-directed mutagenesis to obtain a galactose oxidase mutant with improved catalytic activity.
The application is realized by the following technical scheme:
a galactose oxidase mutant GAO-5F/AR has an amino acid sequence shown in SEQ ID NO. 1, and site-directed mutagenesis is performed on amino acids 403 and 484 compared with wild-type galactose oxidase GAO-5F (GenBank ID: XM_ 031208735.1).
Further, the wild-type galactose oxidase GAO-5F is derived from Fusarium odoratissimum.
The application also provides a gene for encoding the galactose oxidase mutant GAO-5F/AR, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
The application also provides a recombinant plasmid which carries the nucleotide shown in SEQ ID NO.2, and the expression vector is preferably pET28a.
The application also provides a recombinant engineering strain obtained by transforming the recombinant plasmid, and the expression host is preferably E.coli BL21 (DE 3).
The application also provides application of the galactose oxidase mutant GAO-5F/AR enzyme.
Compared with the prior art, the application has the beneficial effects that:
aiming at the problem of low catalytic activity of the existing galactose oxidase, the application adopts a multi-sequence comparison method based on galactose oxidase GAO-5F, selects a plurality of potential amino acid sites which can influence the catalytic activity of the galactose oxidase, reforms the galactose oxidase by site-directed mutagenesis, and obtains double mutation GAO-5F/AR for mutating aspartic acid at position 403 and glutamine at position 484 into alanine and arginine respectively for the first time, and the enzyme activity of the obtained mutant is obviously improved by 6 times compared with that of a wild type mutant. The mutant with the discovery and the improved catalytic activity lays a foundation for development, transformation and application of galactose oxidase.
Drawings
Fig. 1: influence of temperature and pH on the activity of galactose oxidase mutant GAO-5F/AR enzyme;
fig. 2: influence of temperature and pH on the stability of galactose oxidase mutant GAO-5F/AR enzyme.
Detailed Description
The process according to the application is further illustrated by way of example in the accompanying drawings. The experimental conditions used in the examples may be selected according to the prior art. For experimental methods in which specific conditions are not noted in the examples, it is generally possible to run the method under conventional conditions or under conditions recommended by the manufacturer.
The application will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present application and are not intended to limit the scope of the present application.
EXAMPLE 1 construction of galactose oxidase mutation site
PCR amplification was performed using the plasmids of the wild-type galactose oxidase GAO-5F as a template and the primer pairs of 403F, 403R, 484F and 4844R in Table 1, respectively, to construct galactose oxidase mutants GAO-5F/403A and GAO-5F/484R, in which GAO-5F/403A mutated aspartic acid at position 403 to alanine and GAO-5F/484R mutated glutamine at position 484 to arginine, as compared with the wild-type chitosanase GAO-5F.
TABLE 1 primer sequences
EXAMPLE 2 Activity detection of galactose oxidase mutant
The two mutants constructed in example 1 above were subjected to enzyme activity detection and compared with the wild-type galactose oxidase GAO-5F. The activity of galactose oxidase is measured by a peroxidase coupling method, and the specific operation is as follows: the reaction mixture containing ABTS (4.41 mg), horseradish peroxidase (45U), D-galactose (90 mM), a proper amount of galactose oxidase and potassium dihydrogen phosphate buffer (100 mM, pH 7.0) was reacted at 40℃for 3min, and OD was measured 420 The amount of change in absorbance. One unit (U) of galactose oxidase activity is defined as oxidizing 2. Mu. Mol ABTS or 1. Mu. Mol O per minute under the above conditions 2 (galactose) enzyme amount required. The catalytic activity of GAO-5F was 62.84U/mg, the relative enzyme activity was 100%, and the relative enzyme activities of the galactose oxidase mutants GAO-5F/403A and GAO-5F/484R were 149.40% and 144.39%, respectively.
EXAMPLE 3 construction, inducible expression and purification of galactose oxidase mutant GAO-5F/AR
The two sites are subjected to combined mutation, plasmids of GAO-5F/403A are used as templates, 484F and 4844R are used as primer pairs for PCR amplification, products are digested by DpnI and are transformed into escherichia coli BL21 (DE 3), and a chitosanase mutant GAO-5F/AR is constructed, and compared with wild-type chitosanase GAO-5F, the GAO-5F/AR mutates aspartic acid at position 403 and glutamine at position 484 into alanine and arginine respectively.
The recombinant strain containing galactose oxidase mutant GAO-5F/AR was inoculated into LB medium, cultured at 37℃for about 3 hours, and then further cultured with IPTG at a final concentration of 0.1mM at 25℃for 18 hours. Centrifugally collecting thalli, carrying out ultrasonic crushing, purifying protein by using a Ni-NTA affinity column, eluting target protein by using NPI-200, and then carrying out ultrafiltration desalination and concentration at 4 ℃ to obtain a purified galactose oxidase mutant GAO-5F/AR.
Amino acid sequence of galactose oxidase mutant GAO-5F/AR:
VAISQPAAKAETPEGSLQFLSLRASAPIGTAINRDKWRVTCDSQHEGDECSKAI
DGDRDTFWHTAWAAGATNDPKPPHTITIDMGSSQNVNGLSVLPRQDGSDHG
WIGRHNVFLSTDGKNWGDAVATGTWFADNTEKYSNFETRPARYVRLVAVTE
ANDQPWTSIAEINVFKAASYTSPQPGLGRWGPTLDFPIVPVAAAVEPTSGKVL
VWSSYRNDAFGGSPGGVTLTSTWDPSTGVISQRTVTVTKHDMFCPGISMDGN
GQVVVTGGNDAQKTSLYDSSSDSWIPGPDMKVARGYQSSATLSNGRVFTIGG
SWSGGIFEKNGEVYDPSSKTWTSLPKALVKPMLTADQQGLYRSDNHGWLFG
WKKGSVFQAGPSTAMNWYYTTGNGDVKSAGKRQSSRGTAPDAMCGNAVM
YDAVKGKILTFGGSPSYQDSDATTNAHIITISEPGSTPKTVFASNGLYYPRTFHT
SVVLPDGNVFITGGQRRGIPFADSTPQLTPELYVPNDDTFYKQQPNSIVRVYHS
ISLLLPDGRVFNGGGGLCGDCDTNHFDAQIYTPNNLYDSNGKLARRPKITKVS
AKSVKVGGKITITADTSIKQASLIRYGTSTHTVNTDQRRIPLSLRRTGTGNSYS
FQVPSDSGIALPGYWMLFVMNSAGVPSVASTLLVTQ
nucleotide sequence of galactose oxidase mutant GAO-5F/AR:
gtggctatcagccagccggcggctaaagctgaaaccccggaaggctctctgcagttcctgtctctgcgtgctagcgcacc
gatcggcaccgctatcaaccgtgataaatggcgtgtgacctgtgactctcagcacgaaggcgacgaatgctctaaagcga
tcgacggcgaccgtgacaccttctggcacaccgcatgggctgcgggcgctaccaacgacccgaaaccgccgcacacg
atcaccatcgacatgggttcctctcagaacgtgaacggtctgtctgttctgccgcgtcaggacggttctgaccacggttgga
ttggtcgtcacaacgtttttctgtctaccgacggcaaaaactggggcgacgcggttgcgaccggcacctggttcgcagaca
acaccgaaaaatactctaacttcgaaacccgtccggcgcgttacgttcgtctggttgcggttaccgaagcgaacgaccagc
cgtggacctctatcgcggaaatcaacgttttcaaagctgcttcctacacctctccgcagccgggtctgggtcgttggggtcc
gaccctggacttcccgatcgttccggttgcagcggccgttgaaccgacctccggtaaagtgctggtttggtcctcttaccgt
aacgacgctttcggtggttcgccgggtggtgttaccctgacctccacctgggacccgtccaccggtgttatctctcagcgta
ccgttaccgttactaaacacgacatgttctgccctggtatctctatggacggcaacggtcaggttgttgttaccggtggtaac
gacgcgcagaaaacctctctgtacgactcctcttctgattcttggattccgggtccggacatgaaagtggcgcgtggctacc
agtctagcgctaccctgtctaacggtcgtgttttcaccatcggtggttcttggtctggtggtatcttcgagaaaaacggtgagg
tttatgacccgtcctctaaaacctggacctctctgccgaaagcgctggttaaaccgatgctgaccgctgaccagcagggtct
gtaccgttctgataaccacggttggctgttcggttggaaaaaaggttctgttttccaggctggtccgtctaccgctatgaactg
gtactacaccaccggtaacggcgatgttaaatctgcgggtaaacgtcagtctagccgtggtaccgccccggatgcaatgt
gcggtaacgcggttatgtacgatgcggttaaaggtaaaatcctgaccttcggtggttccccgtcttaccaggactctgatgc
gaccaccaacgcgcacatcatcaccatctccgaaccgggttctaccccgaaaaccgttttcgcgtctaacggtctgtactac
ccgcgtaccttccacactagcgttgttctgccggacggtaacgtgttcatcaccggtggccagcggcgtggtatcccgttc
gcggactctaccccgcagctgaccccggaactgtacgttccgaacgacgataccttctacaaacagcagccgaactctatt
gttcgtgtttaccactctatctccctgctgctgccggatggccgtgttttcaacggtggcggcggcctgtgcggtgactgcga
caccaaccacttcgacgcacagatctacaccccgaacaacctgtacgactctaacggtaaactggctcgtcgtccgaaaat
caccaaagtgtctgctaaatctgtgaaagttggtggtaaaatcactatcaccgcagacaccagcatcaaacaggcatctctg
atccgttacggtacctccacccacaccgttaacaccgaccagcgtcgtatcccgctgtctctgcgtcgtaccggtaccggta
actcttacagcttccaggttccgtctgactctggtatcgctctgccgggttactggatgctgttcgttatgaactctgcgggtgt
tccgtctgttgcgtctaccctgctggttacccag
example 4 detection of enzymatic Properties of galactose oxidase mutant
(1) Activity analysis of galactose oxidase mutant
The relative enzyme activity 451.1U/mg of the galactose oxidase mutant GAO-5F/AR was measured as 6.01 times that of the wild-type galactose oxidase GAO-5F by the above-mentioned galactose oxidase activity detection method of example 2.
(2) Effect of temperature and pH on the enzyme Activity of galactose oxidase mutant GAO-5F/AR
The resulting galactose oxidase mutant GAO-5F/AR was diluted to an appropriate concentration with potassium dihydrogen phosphate buffer (100 mM, pH 7.0), and enzymatic reactions were carried out at 20-80℃respectively to investigate the effect of temperature on its enzyme activity. The resulting galactose oxidase mutant GAO-5F/AR was diluted with a buffer solution having a pH of 3.0 to 10.0 [ sodium citrate buffer solution (50 mM, pH 3.0 to 6.0), phosphate buffer solution (50 mM, pH6.0 to 8.0), tris-HCl buffer solution (50 mM, pH 8.0 to 9.0) and glycine-NaOH buffer solution (50 mM, pH 9.0 to 10.0) ] respectively, and then the reaction was carried out at 40℃to investigate the effect of pH on the enzyme activity thereof. The results are shown in FIG. 1, with the highest value of the enzyme activity being 100%, respectively. The results showed that the galactose oxidase mutant GAO-5F/AR had an optimum temperature of 60℃and an optimum pH of 7.0.
(3) Effect of temperature and pH on the stability of the galactose oxidase GAO-5F/AR enzyme
The resulting galactose oxidase mutant GAO-5F/AR was diluted to an appropriate concentration with potassium dihydrogen phosphate buffer (100 mM, pH 7.0), and the residual enzyme activities were tested after incubation at 30℃and 40℃and 50℃for different times, respectively, to investigate the effect of temperature on its stability. The resulting galactose oxidase mutant GAO-5F/AR was incubated in phosphate buffers (pH 6.0-8.0) at different pH's for different times, and then the residual enzyme activity was detected by reaction at 40℃to investigate the effect of pH on its stability. As a result, as shown in FIG. 2, 30% of the initial enzyme activity was retained when GAO-5F/AR was stored at 50℃for 24 hours. Exhibits maximum stability at pH 7.0, and retains more than 90% of its activity after 40h incubation.
The above embodiments are only for illustrating the technical concept and features of the present application, and should not be construed as limiting the scope of the present application. Various changes and modifications may be made by one skilled in the art in light of the present disclosure, and such equivalents are intended to be encompassed within the scope of the application as defined by the appended claims.
Claims (6)
1. The galactose oxidase mutant GAO-5F/AR is characterized in that the amino acid sequence of the galactose oxidase mutant is shown as SEQ ID NO. 1.
2. The galactose oxidase mutant GAO-5F/AR according to claim 1, wherein the galactose oxidase mutant GAO-5F/AR is derived from a wild type galactose oxidase GAO-5F having site-directed mutagenesis of amino acids 403 and 484, and wherein the wild type galactose oxidase GAO-5F is derived from fusarium odontissimum.
3. A gene encoding the galactose oxidase mutant GAO-5F/AR as set forth in claim 1, wherein the nucleotide sequence is as set forth in SEQ ID No. 2.
4. A recombinant plasmid is characterized in that the recombinant plasmid carries a nucleotide shown as SEQ ID NO.2, and an expression vector is pET28a.
5. A recombinant engineering strain, which contains the recombinant plasmid of claim 3, wherein the expression host is E.coli BL21 (DE 3).
6. Use of the galactose oxidase mutant GAO-5F/AR enzyme according to claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7115403B1 (en) * | 2000-05-16 | 2006-10-03 | The California Institute Of Technology | Directed evolution of galactose oxidase enzymes |
| CN112673097A (en) * | 2018-07-09 | 2021-04-16 | 科德克希思公司 | Engineered galactose oxidase variant enzymes |
| WO2022076263A1 (en) * | 2020-10-06 | 2022-04-14 | Codexis, Inc. | Engineered galactose oxidase variant enzymes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7115403B1 (en) * | 2000-05-16 | 2006-10-03 | The California Institute Of Technology | Directed evolution of galactose oxidase enzymes |
| CN112673097A (en) * | 2018-07-09 | 2021-04-16 | 科德克希思公司 | Engineered galactose oxidase variant enzymes |
| WO2022076263A1 (en) * | 2020-10-06 | 2022-04-14 | Codexis, Inc. | Engineered galactose oxidase variant enzymes |
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