CN106544331B - New directed evolution technology SNDS and obtained heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme - Google Patents
New directed evolution technology SNDS and obtained heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme Download PDFInfo
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Abstract
The invention discloses a novel directed evolution technology SNDS and a heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme obtained by the same. The invention uses complementary single-chain DNA of two parental enzyme genes as starting sequence to carry out enzyme digestion respectively, products after digestion are directly mixed without recovery to carry out primer-free PCR, construct mutant library and screen beneficial mutants. The method of the invention realizes the mutation of two parent genes with lower sequence similarity, and effectively expands the application of the DNA shuffling method; the small fragment recovery step is simplified; the recombination preference of the traditional DNA shuffling is obviously reduced, better gene diversity and recombination types are generated, and beneficial mutation can be screened from few recombinants. The method of the invention is adopted to carry out mutation rearrangement on OPHC2 and MPH genes of methyl parathion hydrolase with sequence similarity of only 50.9 percent, and high-efficiency heat-resistant methyl parathion hydrolysis heterozygote enzyme is obtained by screening.
Description
Technical Field
The invention relates to a novel protein directed evolution technology, in particular to a directed evolution technology SNDS, and also relates to a heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme obtained by applying the technology, belonging to the field of protein directed evolution.
Background
Since Stemmer firstly proposed DNA shuffling technology on Nature in 1994 and improved the minimum inhibitory concentration of cefotaxime by 32,000 times compared with original strain, DNA shuffling has been a research hotspot in protein evolution technology, such as modified by structural-based recombination (restricted to selected design points) and less efficient than random mutation (producing a large number of null mutations), it mimics certain aspects of natural recombination and can rapidly detect more regions on the target DNA in one experiment, thus making it more effective on protein evolution and more efficient, original DNA shuffling technology is easily obtained, making it widely used, although these approaches are similar to DNA shuffling and similar to DNA shuffling, such as DNA shuffling, and the most of DNA shuffling, such as DNA shuffling, homology, strain.
Organophosphorus pesticides, which are produced in large quantities at home and abroad and used as insecticides in large areas, can destroy the activity of acetylcholinesterase, thereby causing a series of neurotoxic symptoms and even death. Therefore, with the improvement of the quality of life and the enhancement of environmental awareness of people, the pollution of the organophosphorus pesticide to the environment and the damage of the ecological balance are more and more concerned, and how to effectively remove the organophosphorus pesticide residue and reduce the toxicity of the organophosphorus pesticide residue becomes a hot problem which is generally concerned and struggled by researchers in various countries in the world.
Organophosphorus degrading enzyme (EC3.1.8.1), an enzyme capable of hydrolyzing phosphoester bond, on one hand, it can break the phosphoester bond of Organophosphorus pesticides to detoxify it, on the other hand, since the different Organophosphorus pesticides are mostly different in substituent groups, one Organophosphorus degrading enzyme can often hydrolyze multiple Organophosphorus pesticides, wherein, Methyl Parathion Hydrolase (MPH), the most suitable substrate of which is Methyl parathion, the results of enzyme molecular structure analysis and evolutionary tree analysis show that it belongs to β lactamase family (PFAM accession No. PF00753).
The methyl parathion hydrolase is mainly found and separated in China and mainly divided into two categories, one category is the Methyl Parathion Hydrolase (MPH) derived from strains such as Pseudomonas sp.WBC-3, Plesiomonas sp.M6, Ochrobactrum sp.M231 and the like, and the similarity of the amino acid sequences of the methyl parathion hydrolase is very high and is 98.2% at the lowest. The common characteristics of the compounds on the aspect of enzymology properties are high efficiency of catalyzing methyl parathion, favorable catalysis under alkaline conditions and poor thermal stability. In another class, OPHC2 isolated from Pseudomonas pseudoalcaligenes has the highest similarity of only 50.9% to MPH amino acid sequence, and the enzymes have good thermal stability but relatively poor ability to catalyze methyl parathion.
In recent years, the present inventors' laboratories have been devoted to research work on organophosphorus degrading enzymes to isolate some bacteria highly effective in degrading organophosphorus pesticides from pesticide-contaminated soil, and to isolate two genes encoding methylparathion hydrolase: OPHC2(GenBank Accession No. CAE53631) derived from Pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes) and MPH-Och (GenBank Accession No. ACC63894) derived from Ochrobactrum sp. Wherein the similarity of MPH-Och and reported MPH derived from Pseudomonas sp.WBC-3 is very high, reaching 98.2 percent, and the enzymological properties are basically the same. Comparison of MPH-Och with OPHC2 revealed that the amino acid sequence similarity of these two enzyme proteins was 47.7%, and that the properties of the enzyme proteins were significantly different. OPHC2 has good heat resistance, the optimum temperature for degrading methyl parathion is 65 ℃, and the activity is nearly 40% after the temperature is kept at 70 ℃ for 30 minutes. The optimal reaction temperature of the MPH-Och enzyme reaction is only 20 ℃, the relative enzyme activity is rapidly reduced to below 10 percent when the temperature is kept at 70 ℃ for less than 2 minutes, the enzyme activity is basically lost after the temperature is kept for 5 minutes, and the thermal stability of the MPH-Och enzyme reaction is much lower than that of OPHC 2. However, MPH-Och has a catalytic efficiency for methyl parathion at room temperature significantly superior to OPHC2, and its specific activity is 23.23U/mg, which is about 5.8 times that of OPHC 2. Therefore, the method combines the advantages of the two enzymes to develop novel efficient heat-resistant methyl parathion hydrolase, and has great significance for further reducing the production cost of the enzymes and promoting the industrialization of the enzymes.
Disclosure of Invention
The first technical problem to be solved by the present invention is to provide a novel method for directed evolution of proteins, which comprises: a Single-stranded Nucleotide DNA direct recombination technique (SNDS, Single-stranded Nucleotide DNA directlyShuffling);
the second technical problem to be solved by the invention is to apply the SNDS technology to carry out mutation rearrangement on the methyl parathion hydrolase OPHC2 and MPH so as to obtain the high-efficiency heat-resistant methyl parathion hydrolysis hybrid enzyme.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention firstly discloses a directed evolution method (SNDS) of protein, which comprises the following steps:
(1) taking two complementary single-chain parent DNA sequences for coding the protein as starting sequences, and performing enzyme digestion by DNase I respectively;
(2) directly mixing products subjected to enzyme digestion in the step (1) without recovery to perform primer-free PCR;
(3) carrying out primer PCR by taking a product of primer-free PCR as a template, and recovering a PCR product;
(4) inserting the PCR product recovered in the step (3) into an expression vector, transforming host bacteria, and constructing a mutant library;
(5) and screening the beneficial mutants from the mutant library to obtain the mutant.
Wherein, the similarity of the two single-chain parental DNA sequences is more than or equal to 50.9 percent; preferably, the protein is methyl parathion hydrolase.
The two single-chain parental DNA sequences can be obtained by adopting various methods such as an asymmetric PCR amplification method or an artificial synthesis method. For example, the relevant single-stranded DNA is directly amplified from a plasmid containing the parent enzyme gene by asymmetric PCR. The primer-free PCR is to rearrange the randomized fragment digested by DNase I by PCR without adding primers; the primer PCR is to utilize primers on two sides of a gene to carry out PCR amplification to obtain a rearrangement product of the full-length gene, and the selected primer is selected according to the type of hybrid enzyme to be obtained.
The invention uses DNase I to digest the single-stranded DNA of two parent enzyme genes respectively, then directly mixes and recombines the products after DNase I digestion without recovery, thereby leading the partially digested large fragment to be capable of serving as the function similar to 'scaffold' in RACHITT (Coco, W.M.et al.DNA shuffling method for generating high efficiency combined genes and expressed enzymes. Nature biotechnology19,354-359, Doi: Doi 10.1038/86744(2001)) in the mixed recombination, thereby achieving the mutation of the two parent genes with the sequence similarity of only 50.9 percent and the heterozygosity of the new gene after SNDS reorganization is 100 percent.
Previously, it was widely recognized by those skilled in the art that recovery of the product after DNase I digestion in the DNA shuffling technique helps to reduce wild-type incorporation into the mutant pool and to improve recombination. From the experimental results of the invention, it is more effective to directly carry out mixed recombination on the product after DNase I digestion. The new gene shuffled by applying the SNDS of the invention has a heterozygosity of 100% no matter the parent with high similarity or the parent with low similarity, while the traditional DNAsuffling requires that the similarity of the parent gene is at least more than 80%, and the heterozygosity of the generated new gene is generally about 1%. The SNDS technique of the present invention expands the application of DNA shuffling methods, making them more versatile and generating more efficient mutation types.
The SNDS technology of the invention obviously reduces the recombination preference existing in the traditional DNA shuffling, thereby generating better gene diversity. Moreover, the process of the invention produces a better type of recombination: for highly similar parents, multiple crossover recombinant types will be generated, thereby generating more desirable mutant types; whereas for low similarity parents, single crossover recombinant forms will be generated, thus avoiding too low activity rates in the mutant pool. In conclusion, the SNDS technology of the invention is more effective and can screen few recombinants for beneficial mutations.
The invention further discloses a method for obtaining heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme by directed evolution, which comprises the following steps:
(1) taking complementary single-stranded DNA of a methyl parathion hydrolase OPHC2 gene and a methyl parathion hydrolase MPHM gene as starting sequences, and performing enzyme digestion by DNase I respectively;
(2) directly mixing products subjected to enzyme digestion in the step (1) without recovery, and then carrying out primer-free PCR;
(3) carrying out primer PCR by taking a product of primer-free PCR as a template, and recovering a PCR product;
(4) inserting the PCR product recovered in the step (3) into an expression vector, transforming host bacteria, and constructing a mutant library;
(5) and screening beneficial mutant enzyme from the mutant library to obtain the mutant enzyme.
Wherein the GenBank accession number of the methyl parathion hydrolase OPHC2 is as follows: CAE 53631; the GenBank accession number of the methyl parathion hydrolase MPHM is as follows: ACC 63894. GenBank of the nucleotide sequence of the methyl parathion hydrolase OPHC2 gene: AJ 605330; GenBank of the nucleotide sequence of the methyl parathion hydrolase MPHM gene: KX 982227.
The system for carrying out enzyme digestion by using DNase I in the step (1) comprises: the total digestion rate is 50 mu L, wherein, 10-15 ng/mu L of single-stranded DNA is 42.5 mu L, 10 XDnase I buffer is 5 mu L, and 40U/mL of Dnase I is 2.5 mu L; the digestion conditions include: the DNase I is inactivated by enzyme digestion at 25 ℃ for 30min and treatment at 75 ℃ for 10 min.
The system of the primer-free PCR in the step (2) comprises: the total volume is 50. mu.L, wherein each of the digested products is 5. mu.L, 5 XSastpfu buffer 10. mu.L, 2.5mM dNTPs 4. mu.L, Fast pfu 1.5. mu.L, and the balance dd H2O; the primer-free PCR procedure included: 5min at 94 ℃; 30s at 94 ℃, 30s at 45 ℃, 1min at 72 ℃ and 30s for 45 cycles.
The primer of the primer PCR in the step (3) comprises: a primer pair 1 consisting of the nucleotide sequences shown in SEQ ID No.7 and SEQ ID No.6, or a primer pair 2 consisting of the nucleotide sequences shown in SEQ ID No.5 and SEQ ID No. 8; the primer PCR program comprises: 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃, 1min at 72 ℃ for 10s, and 20 cycles.
The expression vector in the step (4) comprises a pET30 vector; the host bacteria include Escherichia coli.
The invention applies SNDS technology to reorganize two genes of methyl parathion hydrolase OPHC2 and MPH with sequence similarity of only 50.9 percent, and randomly selects 50 positive strains from the constructed escherichia coli mutant library for sequence determination, so that the result genes are all subjected to heterozygous mutation, and the mutation rate is 100 percent. Through the activity determination of methyl parathion hydrolase on 500 clones, 33% of strains are screened out to have enzyme activity, and mutant enzymes with improved thermal stability and enzyme activity compared with MPH are further screened out from the strains.
The traditional DNA shuffling is utilized to carry out reorganization on two genes of methyl parathion hydrolase OPHC2 and MPH, and analysis and sequencing results show that a correct target band cannot be formed when the traditional DNA shuffling is used for mutant construction, and large fragments are deleted.
The invention further discloses the heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme obtained by the method and a coding gene thereof.
The amino acid sequence of the heat-resistant high-efficiency methyl parathion hydrolysis hybrid enzyme B-C1 obtained by the method is shown in SEQ ID No.2, and the nucleotide sequence of the coding gene is shown in SEQ ID No. 1.
The amino acid sequence of the heat-resistant high-efficiency methyl parathion hydrolysis hybrid enzyme B-A5 obtained by the method is shown in SEQ ID No.4, and the nucleotide sequence of the coding gene is shown in SEQ ID No. 3.
The invention further discloses a recombinant expression vector containing the gene and a recombinant host cell containing the recombinant expression vector. The coding gene of the heat-resistant high-efficiency methyl parathion hydrolytic heterozygote is inserted between proper restriction enzyme cutting sites of an expression vector, so that the nucleotide sequence of the heat-resistant high-efficiency methyl parathion hydrolytic heterozygote is operably connected with an expression regulation sequence. The recombinant host cells of the present invention may be prokaryotic or eukaryotic, including but not limited to E.coli cells.
The invention also discloses a method for preparing the heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme, which comprises the following steps: (1) transforming host cells by using a recombinant expression vector containing the coding gene of the heat-resistant high-efficiency methyl parathion hydrolytic heterozygote enzyme to obtain a recombinant strain; (2) culturing the recombinant strain, and inducing the expression of heat-resistant high-efficiency methyl parathion hydrolytic heterozygote enzyme; (3) recovering and purifying the expressed heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme to obtain the compound.
In the process of PCR with primers, two pairs of primers are selected for amplification respectively, one pair is used for screening to obtain primers (shown by SEQ ID No.7 and SEQ ID No. 6) of mph in the first half of the heterozygous gene and the ophc2 in the second half of the heterozygous gene, and the other pair is used for screening to obtain primers (shown by SEQ ID No.5 and SEQ ID No. 8) of ophc2 in the first half of the heterozygous gene and the mph in the second half of the heterozygous gene. The sequence analysis result shows that the 5 'end of the B-C1 mutant gene contains 789 nucleotides of the 5' end of the MPH gene, and the nucleotide sequence is the nucleic acid sequence of OPHC2, namely the N end of the B-C1 mutant enzyme is 263 amino acids of the N end of the MPH protein, and the C end is 33 amino acids of the C end of the OPHC2 protein. The 5 'end of the B-A5 mutant gene contains 66 nucleotides at the 5' end of the OPHC2 gene, and the nucleic acid sequence is MPH thereafter, namely the N end of the B-A5 mutant enzyme contains 22 amino acids at the end of OPHC2N, and the C end is 274 amino acids at the C end of the MPH protein.
The enzyme property analysis shows that the activity of the B-C1 and B-A5 hybrid enzyme is improved compared with that of MPH and OPHC2 of the parents, particularly the relative enzyme activity of B-A5 is 248.77U/mg which is 10.68 times that of MPH. The result of the thermal stability determination shows that after the temperature is kept at 70 ℃ for 10 minutes, the enzyme activity is determined at 37 ℃, and the MPH enzyme activity is completely lost; by taking the parent OPHC2 as a control, the residual enzyme activity of the hybrid enzyme B-C1 can reach 70 percent, and the residual enzyme activity of the hybrid enzyme B-A5 is still 2.95 times of that of OPHC 2. Determination of protein T Using circular dichroism SpectroscopymThe value of hybrid enzyme B-A5 was 58 ℃ which was 2 ℃ higher than that of MPH (56 ℃).
The heat-resistant efficient methyl parathion hydrolytic heterozygote enzyme or the coding gene thereof has wide application prospect in hydrolyzing organophosphorus pesticides, particularly methyl parathion.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
(1) compared with the traditional DNA shuffling, the SNDS technology of the invention has simpler experimental steps: the conventional DNAsuffling technology needs to recover fragments after DNA digestion, and the method of the invention simplifies the step.
(2) The SNDS technology of the invention realizes the mutation of two genes of OPHC2 and MPH with the sequence similarity of only 50.9 percent, while the similarity of the traditional DNA shuffling at least needs more than 80 percent. The SNDS technology of the invention expands the application of the DNA shuffling method and makes the DNA shuffling method more universal.
(3) The heterozygosity of the new gene after the SNDS is shuffled is 100% no matter the parent with high similarity or the parent with low similarity, while the heterozygosity of the traditional DNA shuffling is about 1%.
(4) The heat-resistant high-efficiency methyl parathion hydrolytic hybrid enzyme obtained by applying the SNDS technology has higher relative enzyme activity and higher heat stability than that of a parent, and is more suitable for industrial application.
Definitions of terms to which the invention relates
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell. probes 8:91-98 (1994)).
The term "recombinant host cell" or "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.
The term "expression" means the transcription and/or translation of an endogenous gene or transgene in a cell.
Drawings
FIG. 1 is an electropherogram of single-stranded mph and ophc2 DNA; wherein, A is a single chain obtained by PCR amplification; b is the single-stranded DNA obtained by recovery;
FIG. 2 is an electrophoretogram of a hybrid gene.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
1. Experimental Material
1.1 strains and plasmids
Plasmid pET30a (+) -ophc2, pET30a (+) -mph, strain E.coli BL21, E.coli Top10 were preserved in our laboratory, and vector pGM-T was purchased from Tiangen Biochemical technology (Beijing) Ltd.
1.2 reagents
The plasmid extraction and gel recovery kit is purchased from Axygen, Taq polymers from Tiangen Biochemical technology (Beijing) Ltd4Ligase, restriction endonucleaseEnzymes were purchased from NEB, recombinase, Fast Pfu from holo-gold biotechnology, ltd, peptone (Tryptone) and Yeast extract (Yeast extract) from Oxford, uk, YNB from kyoto kodada biotechnology, ltd, and other chemical reagents were all in-house analytical purity.
1.3 test apparatus
Constant temperature shaking table (Taicang scientific and technological equipment factory), PCR instrument (U.S. Bio-Rad company), constant temperature box (Tester instruments Co., Tianjin), gel imaging analysis system (Shanghai Peishen science Co., Ltd.), connection instrument (Kayoudi Biotechnology Co., Ltd.), constant temperature metal bath (gold and silver apricot biotechnology Co., Ltd.), vortex oscillator (Yangbei instruments manufacturing Co., Ltd., Haiman city), vacuum freeze dryer (Germany Christ company), an autoclave (SANYO, Japan), a desktop high-speed refrigerated centrifuge (Eppendorf, Germany), a vertical high-speed refrigerated centrifuge (Sigma, USA), an electric heating constant temperature water bath (Hei Heng instruments, Ltd.), an electric shock conversion instrument (Bio-Rad, USA), an enzyme-labeling instrument (Thermo, Finland), a protein electrophoresis instrument (Hebeijing hexakis instruments), and a Leichi MM400 grinding instrument (Retsch, Germany).
Example 1 obtaining of thermostable and highly efficient methyl parathion hydrolytic heterozygote enzyme by applying directed evolution technology SNDS
1. Experimental methods
1.1 acquisition of mph and ophc2 Single Strand
Carrying out asymmetric PCR amplification by taking plasmid pET30a (+) -ophc2 stored in a laboratory as a template and using ophc2-up-BamHI (ggatcgcCGCACCGGCACACACACACACAACAGAAG) and ophc2-down-XhoI (ctcgagtCAGC GGTCGCTACGGATCGG) primers to obtain a single-chain ophc 2; meanwhile, taking pET30a (+) -mph plasmid as a template, mph-up-BamHI (ggatcCGCTCCACAAGTTAAGAACTTC) and mph-down-XhoI (ctcgagtACTTTGGGTTAACGACGG) as primers, carrying out asymmetric PCR amplification to obtain single-chain mph.
The PCR system was as follows:
the single-stranded PCR procedure was as follows:
amplification conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 1min at 72 ℃ for 10s, 31 cycles. And recovering the amplification product by a recovery kit.
1.2 Single Strand digestion step
The recovered mph and ophc2 single-stranded DNA are digested with Dnase I, which is then inactivated by digestion at 25 ℃ for 30min and treatment at 75 ℃ for 10 min.
The digestion system was as follows:
1.3 obtaining of mph and ophc2 hybrid genes
After the digestion, mph and ophc2 fragments were mixed and subjected to primer-free PCR as follows:
the procedure is as follows:
amplification conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 45 ℃, 1min at 72 ℃ and 30s for 45 cycles.
1.4 PCR with primers
And (3) carrying out primer-containing PCR by taking a primer-free PCR product as a template, and selecting the selected primer according to the type of the hybrid enzyme to be obtained.
Two pairs of primers are selected for amplification respectively, one pair is used for screening and obtaining primers (mph-up-BamHI: ggatccGCTGCTCCACAAGTTAGAACTTC and ophc 2-down-XhoI: ctcgagTCAGC GGTCGCTACGGATCGG) of mph in the first half of a heterozygous gene and ophc2 in the second half; the other pair is primers for screening the first half of the hybrid gene for oph 2 and the second half of the mph gene (oph 2-up-BamHI: ggatccGCCGCACCGGCACAACAGAAG and mph-down-XhoI: ctcgagTTACTTTGGGTTAACGACGG).
The procedure is as follows: amplification conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃, 1min at 72 ℃ for 10s, and 20 cycles. And (4) carrying out agarose gel electrophoresis on the PCR product, and recovering for later use.
1.5 construction of E.coli mutant pools
Carrying out BamHI/XhoI double enzyme digestion on the recovered PCR product and pET30 vector respectively, recovering the fragment after enzyme digestion, and adding T4The ligase is used for ligation, and the ligation system is as follows: mutant Gene 6. mu.L, pET 302. mu.L, 10 XT4DNA LigaseBuffer 1μL，T4DNA Ligase 1. mu.L was ligated overnight at 16 ℃.
The ligation product was transformed into E.coli expression strain BL21 by heat shock. And (3) carrying out shake liquid culture on the monoclonal strains growing in the transformed plate at 37 ℃ and 200rpm for 10-12h, carrying out plasmid extraction and enzyme digestion identification on the transformed strains, determining positive strains, randomly selecting the positive strains, carrying out sequence determination, and analyzing mutation rate.
1.6 Methylphosphine Activity assay screening of beneficial mutant enzymes
1.6.1 Primary screening
Adding kana resistant 300 microliter LB culture medium into a sterilized 96 deep-well plate, inoculating bacteria on the plate into the well, 300rpm/min, 10h, 37 ℃, then adding 100 microliter LB culture medium of 4mM IPTG, 750rpm/min, 4h, 37 ℃, taking out 50 microliter per well for methyl parathion hydrolase activity determination, and primarily screening out the strain with enzyme activity.
The method for measuring the activity of the methyl parathion hydrolase comprises the following steps: adding 100 μ L enzyme solution into a system containing 5 μ L10 mg/mL methyl parathion and 900 μ L50 mmol/L Tris-Cl (pH8.0) buffer solution, maintaining at 37 deg.C for 10min, adding 1mL 10% trichloroacetic acid to stop reaction, and adding 1mL 10% Na2CO3And (3) developing the solution, measuring the light absorption value at 410nm, and calculating the content of the hydrolysate on the nitrophenol and the activity of the enzyme. One unit of enzyme activity (U) is defined as: the amount of enzyme required to release 1. mu. mol of p-nitrophenol per minute at 37 ℃. The clones were screened for enzymatically active strains.
1.6.2 rescreening mutant enzymes with increased thermostability and enzyme Activity
In order to screen heat-resistant and efficient mutant enzymes, the strains with activity in the primary screening are inoculated into a 96-hole deep-hole plate again, the strains are cultured for 10 hours at 300rpm/min overnight, then 100 mu L of LB culture medium with 4mM IPTG is added, the strains are cultured for 4 hours at 750rpm, 50 mu L of the strains are taken out of each hole and are placed at 70 ℃ for treatment for 5 minutes, 10 minutes and 15 minutes, and then the activity of methyl parathion hydrolase is measured at 37 ℃. Screening the mutant enzyme with improved thermal stability and enzyme activity compared with MPH.
1.7 mutant enzyme purification
The mutant enzyme-containing strain was transferred to LB medium containing 50. mu.g/mL kanamycin, cultured with shaking at 37 ℃ and 200r/min, the culture broth was transferred to 50mL of liquid medium containing 50. mu.g/mL ampicillin in an amount of 1%, cultured at 37 ℃ and 200r/min to OD600After 0.6, IPTG was added to a final concentration of 0.4mmol/L and induced at 16 ℃ for 20h with shaking at 200 r/min. Centrifuging the induced bacteria liquid at 4 ℃ and 8000r/min for 10min, and then removing the supernatant to collect bacteria. The cells were resuspended in 20mmol/L Tris-HCl (pH8.0) and sonicated on ice. The crushed crude enzyme solution was centrifuged at 12000r/min at 4 ℃ for 20min, and the supernatant was carefully removed and purified by affinity chromatography.
1.8 determination of enzymatic Properties
The purified enzyme protein was analyzed for its enzymatic properties, including enzyme activity (method 1.6.1) and thermal stability (enzyme activity was measured at 37 ℃ after 10 minutes at 70 ℃). At the same time, protein T was determined by circular dichroismmValue (temperature at which protein is denatured to half).
1.9 sequencing and analysis of the mutant enzymes
The plasmid with the mutant gene was extracted and subjected to sequence analysis.
2. Results of the experiment
2.1 acquisition of mph and ophc2 Single Strand
Obtaining single-chain ophc2 and single-chain mph through asymmetric PCR amplification, recovering the amplification product through a recovery kit, and showing the amplification result and the recovery result in figure 1.
2.2 obtaining of hybrid Gene
And (3) carrying out primer-containing PCR by using a primer-free PCR product as a template. The PCR product was subjected to agarose gel electrophoresis to reveal a desired band of about 900bp (FIG. 2), and recovered for use.
2.3 construction of E.coli mutant libraries
Connecting the recovered PCR product with pET30 vector, transforming Escherichia coli expression strain BL21, growing about 500 monoclonals in the transformed plate, and determining positive strain through enzyme digestion identification. Randomly selecting 50 positive strains for sequence determination, and analyzing and sequencing to obtain result genes with heterozygous mutation and mutation rate of 100%.
2.4 preliminary screening of beneficial mutant enzymes
After screening 500 clones, 33% of the strains had enzyme activity as measured by methyl parathion hydrolase activity.
2.5 rescreening of mutant enzymes with increased thermostability and enzyme Activity
The invention screens the mutant enzymes B-C1 and B-A5 with improved thermal stability and enzyme activity compared with MPH from the initially screened active strains.
2.6 determination of the enzymatic Properties of the mutant enzymes
The mutant enzymes B-C1 and B-A5 were purified, and the purified enzyme proteins were analyzed for enzymatic properties (Table 1).
Compared with the parent MPH and OPHC2, the relative enzyme activity of the screened B-C1 and B-A5 hybrid enzyme is improved, particularly the relative enzyme activity of B-A5 is 248.77U/mg, which is 10.68 times of that of MPH.
The result of thermal stability measurement shows that after the temperature is kept at 70 ℃ for 10 minutes, the enzyme activity is measured at 37 ℃, the MPH enzyme activity is completely lost, the enzyme activity of the hybrid enzyme B-C1 can reach 70 percent by taking the other parent OPHC2 as a control, and the residual enzyme activity of the hybrid enzyme B-A5 is still 2.95 times of that of OPHC 2. At the same time, protein T was determined by circular dichroismmThe value (temperature at which the protein is denatured to half) is 58 ℃ for B-A5, which is an increase of 2 ℃ over MPH (56 ℃).
TABLE 1 analysis of enzymatic Properties of the hybrid enzymes
2.7 sequencing and analysis of the mutant enzymes
As a result of sequence analysis, the 5 'end of the B-C1 mutant gene contains 789 nucleotides of the 5' end of the MPH gene, and the nucleotide sequence is the nucleic acid sequence of OPHC2, namely the N end of the B-C1 mutant enzyme is 263 amino acids of the N end of the MPH protein, and the C end is 33 amino acids of the C end of the OPHC2 protein. The nucleotide sequence of the B-C1 mutant gene is shown as SEQ ID No.1, and the amino acid sequence thereof is shown as SEQ ID No. 2.
The 5 'end of the B-A5 mutant gene contains 66 nucleotides at the 5' end of the OPHC2 gene, and the nucleic acid sequence is MPH thereafter, namely the N end of the B-A5 mutant enzyme contains 22 amino acids at the end of OPHC2N, and the C end is 274 amino acids at the C end of the MPH protein. The nucleotide sequence of the mutant gene is shown as SEQ ID No.3, and the amino acid sequence thereof is shown as SEQ ID No. 4.
Comparative example 1 (conventional DNA Shuffling experiment)
Using plasmid pET30a (+) -ophc2 stored in a laboratory as a template, and using ophc2-up-BamHI (ggatcgcCGCACCGGCACACACACACAACAGAAG) and ophc2-down-XhoI (ctcgagtCAGC GGTCGCTACGGATCGG) primers to amplify to obtain a full-length double-stranded ophc2 gene; meanwhile, using pET30a (+) -mph plasmid as a template, mph-up-BamHI (ggatcCGCTCCACAAGTTAAGAACTTC) and mph-down-XhoI (ctcgagtACTTTGGGTTAACGACGG) as primers, and amplifying to obtain the full-length double-stranded mph gene.
The amplification product is recovered by a recovery kit, and the final concentration of the recovered product is 30 ng/. mu.l. The recovered product was digested with Dnase I, 50 μ l system: 2U/mL DNaseI; about 800ng of recovered DNA (400 ng each for ophc2 and mph); the DNase I is inactivated by enzyme digestion at 25 ℃ for 30min and treatment at 75 ℃ for 10 min. And recovering the small fragment DNA of 50-100bp after enzyme digestion by a low-melting-point adhesive method. The fragments were recovered for primer-free PCR, and a 50. mu.l PCR reaction was as follows:
PCR procedure: 5min at 94 ℃; 94 ℃ for 30 sec; 30sec at 45 ℃; 1.5min at 72 ℃; 45 cycles, 72 ℃ for 10 min.
Then, using the PCR product without primers as a template, and selecting two pairs of primers for amplification respectively, wherein one pair is used for screening to obtain primers (mph-up-BamHI: ggatccGCTGCTCCACAAGTTAGAACTTC and ophc 2-down-XhoI: ctcgagTCAGC GGTCGCTACGGATCGG) of the mph gene in the first half of the heterozygous gene, and the other pair is used for screening to obtain primers (ophc 2-up-BamHI: ggatccGCCGCACCGGCACAACAGAAG and mph-down-XhoI: ctcgagTTACTTTGGGTTAACGACGG) of the oph gene in the first half of the heterozygous gene and the mph gene in the second half of the heterozygous gene.
The procedure was as follows: 94 ℃ for 5min, 1 cycle, 94 ℃ for 30sec, 50 ℃ for 30sec, 72 ℃ for 1min, 30 cycles, 72 ℃ for 10 min. The PCR product of about 900bp is recovered by a recovery kit.
Carrying out BamHI/XhoI double enzyme digestion on the recovered PCR product and pET30 vector respectively, recovering the fragment after enzyme digestion, and adding T4The ligase is used for ligation, and the ligation system is as follows: mutant Gene 6. mu.L, pET 302. mu.L, 10 XT4DNA Ligase Buffer 1μL，T4DNA Ligase 1. mu.L was ligated overnight at 16 ℃. The ligation product was transformed into E.coli expression strain BL21 by heat shock. And (3) carrying out shake liquid culture on the monoclonal strain growing in the transformed plate at 37 ℃ and 200rpm for 10-12h, and carrying out plasmid extraction and enzyme digestion identification on the transformed strain to determine a positive strain. Randomly selecting 20 positive strains for sequence determination, and finding out that a correct target band cannot be formed when the traditional DNA shuffling is used for mutant construction and a large fragment is deleted by analyzing a sequencing result.
SEQUENCE LISTING
<110> institute of biotechnology of Chinese academy of agricultural sciences
<120> a new directed evolution technology SNDS and obtained heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme
<130>BJ-2002-160849A
<160>8
<170>PatentIn version 3.5
<210>1
<211>891
<212>DNA
<213>artifical sequence
<400>1
gctgctccac aagttagaac ttctgctcca ggatactaca gaatgttgct gggtgacttc 60
gaaattactg ctttgtctga cggtaccgtt gctttgccag ttgacaagag attgaaccaa 120
ccagctccta agactcagtc tgctttggcc aagtacttcc agaaagctcc attggaaacc 180
tccgttaccg gttacttggt caacactggt tccaagttgg ttttggtcga cactggtgct 240
gccggtttgt tcggaccaac cttgggtaga cttgctgcca acttgaaggc cgctggttac 300
caaccagagc aagttgacga gatttacatc actcacatgc atcctgacca cgttggaggt 360
ttgatggttg gtgagcaatt ggctttccca aacgctgtcg ttagagctga ccaaaaggag 420
gccgatttct ggctttctca aaccaacttg gacaaggctc ctgacgattc taaaggtttc 480
ttcaagggtg ccatggcttc ccttaaccca tacgttaagg ccggtaagtt caagcctttc 540
tcgggtaaca ctgacttggt tcctggtatt aaagctttgg cctcccacgg acacaccgct 600
ggtcacacta cctacgttgt tgaatctcaa ggtcaaaagc ttgccttgtt gggtgacttg 660
atcttggtcg ctgctgttca attcgacgat ccatccgtta ctaaccaatt ggactctgac 720
tccaagtctg ctgccgttga gagaaagaaa gctttcgctg atgccgctaa gggaggttac 780
cttatcgctg gtgcgcacct gcccttcccc ggcctgggcc acgtacgcaa ggaagcccaa 840
ggctacgcct gggtacccgt cgagttcagc ccgatccgta gcgaccgctg a 891
<210>2
<211>296
<212>PRT
<213>artifical sequence
<400>2
Ala Ala Pro Gln Val Arg Thr Ser Ala Pro Gly Tyr Tyr Arg Met Leu
1 5 10 15
Leu Gly Asp Phe Glu Ile Thr Ala Leu Ser Asp Gly Thr Val Ala Leu
20 25 30
Pro Val Asp Lys Arg Leu Asn Gln Pro Ala Pro Lys Thr Gln Ser Ala
35 40 45
Leu Ala Lys Tyr Phe Gln Lys Ala Pro Leu Glu Thr Ser Val Thr Gly
50 55 60
Tyr Leu Val Asn Thr Gly Ser Lys Leu Val Leu Val Asp Thr Gly Ala
65 70 75 80
Ala Gly Leu Phe Gly Pro Thr Leu Gly Arg Leu Ala Ala Asn Leu Lys
85 90 95
Ala Ala Gly Tyr Gln Pro Glu Gln Val Asp Glu Ile Tyr Ile Thr His
100 105 110
Met His Pro Asp His Val Gly Gly Leu Met Val Gly Glu Gln Leu Ala
115 120 125
Phe Pro Asn Ala Val Val Arg Ala Asp Gln Lys Glu Ala Asp Phe Trp
130 135 140
Leu Ser Gln Thr Asn Leu Asp Lys Ala Pro Asp Asp Ser Lys Gly Phe
145 150 155 160
Phe Lys Gly Ala Met Ala Ser Leu Asn Pro Tyr Val Lys Ala Gly Lys
165 170 175
Phe Lys Pro Phe Ser Gly Asn Thr Asp Leu Val Pro Gly Ile Lys Ala
180 185 190
Leu Ala Ser His Gly His Thr Ala Gly His Thr Thr Tyr Val Val Glu
195 200 205
Ser Gln Gly Gln Lys Leu Ala Leu Leu Gly Asp Leu Ile Leu Val Ala
210 215 220
Ala Val Gln Phe Asp Asp Pro Ser Val Thr Asn Gln Leu Asp Ser Asp
225 230 235 240
Ser Lys Ser Ala Ala Val Glu Arg Lys Lys Ala Phe Ala Asp Ala Ala
245 250 255
Lys Gly Gly Tyr Leu Ile Ala Gly Ala His Leu Pro Phe Pro Gly Leu
260 265 270
Gly His Val Arg Lys Glu Ala Gln Gly Tyr Ala Trp Val Pro Val Glu
275 280 285
Phe Ser Pro Ile Arg Ser Asp Arg
290 295
<210>3
<211>891
<212>DNA
<213>artifical sequence
<400>3
gccgcaccgg cacaacagaa gacccaggta ccgggctact accgtatggc actcggtgac 60
ttcgaaatta ctgctttgtc tgacggtacc gttgctttgc cagttgacaa gagattgaac 120
caaccagctc ctaagactca gtctgctttg gccaagtact tccagaaagc tccattggaa 180
acctccgtta ccggttactt ggtcaacact ggttccaagt tggttttggt cgacactggt 240
gctgccggtt tgttcggacc aaccttgggt agacttgctg ccaacttgaa ggccgctggt 300
taccaaccag agcaagttga cgagatttac atcactcaca tgcatcctga ccacgttgga 360
ggtttgatgg ttggtgagca attggctttc ccaaacgctg tcgttagagc tgaccaaaag 420
gaggccgatt tctggctttc tcaaaccaac ttggacaagg ctcctgacga ttctaaaggt 480
ttcttcaagg gtgccatggc ttcccttaac ccatacgtta aggccggtaa gttcaagcct 540
ttctcgggta acactgactt ggttcctggt attaaagctt tggcctccca cggacacacc 600
gctggtcaca ctacctacgt tgttgaatct caaggtcaaa agcttgcctt gttgggtgac 660
ttgatcttgg tcgctgctgt tcaattcgac gatccatccg ttactagcca attggactct 720
gactccaagt ctgctgccgt tgagagaaag aaagctttcg ctgatgccgc taagggaggt 780
taccttatcg ctgctgccca cttgtccttc ccaggtattg gtcacatcag agctgaaggt 840
aagggatacc gtttcgttcc tgtcaactac tccgtcgtta acccaaagta a 891
<210>4
<211>296
<212>PRT
<213>artifical sequence
<400>4
Ala Ala Pro Ala Gln Gln Lys Thr Gln Val Pro Gly Tyr Tyr Arg Met
1 5 10 15
Ala Leu Gly Asp Phe Glu Ile Thr Ala Leu Ser Asp Gly Thr Val Ala
20 25 30
Leu Pro Val Asp Lys Arg Leu Asn Gln Pro Ala Pro Lys Thr Gln Ser
35 40 45
Ala Leu Ala Lys Tyr Phe Gln Lys Ala Pro Leu Glu Thr Ser Val Thr
50 55 60
Gly Tyr Leu Val Asn Thr Gly Ser Lys Leu Val Leu Val Asp Thr Gly
65 70 75 80
Ala Ala Gly Leu Phe Gly Pro Thr Leu Gly Arg Leu Ala Ala Asn Leu
85 90 95
Lys Ala AlaGly Tyr Gln Pro Glu Gln Val Asp Glu Ile Tyr Ile Thr
100 105 110
His Met His Pro Asp His Val Gly Gly Leu Met Val Gly Glu Gln Leu
115 120 125
Ala Phe Pro Asn Ala Val Val Arg Ala Asp Gln Lys Glu Ala Asp Phe
130 135 140
Trp Leu Ser Gln Thr Asn Leu Asp Lys Ala Pro Asp Asp Ser Lys Gly
145 150 155 160
Phe Phe Lys Gly Ala Met Ala Ser Leu Asn Pro Tyr Val Lys Ala Gly
165 170 175
Lys Phe Lys Pro Phe Ser Gly Asn Thr Asp Leu Val Pro Gly Ile Lys
180 185 190
Ala Leu Ala Ser His Gly His Thr Ala Gly His Thr Thr Tyr Val Val
195 200 205
Glu Ser Gln Gly Gln Lys Leu Ala Leu Leu Gly Asp Leu Ile Leu Val
210 215 220
Ala Ala Val Gln Phe Asp Asp Pro Ser Val Thr Ser Gln Leu Asp Ser
225 230 235 240
Asp Ser Lys Ser Ala Ala Val Glu Arg Lys Lys Ala Phe Ala Asp Ala
245 250 255
Ala Lys Gly Gly TyrLeu Ile Ala Ala Ala His Leu Ser Phe Pro Gly
260 265 270
Ile Gly His Ile Arg Ala Glu Gly Lys Gly Tyr Arg Phe Val Pro Val
275 280 285
Asn Tyr Ser Val Val Asn Pro Lys
290 295
<210>5
<211>27
<212>DNA
<213>artifical sequence
<400>5
ggatccgccg caccggcaca acagaag 27
<210>6
<211>27
<212>DNA
<213>artifical sequence
<400>6
ctcgagtcag cggtcgctac ggatcgg 27
<210>7
<211>29
<212>DNA
<213>artifical sequence
<400>7
ggatccgctg ctccacaagt tagaacttc 29
<210>8
<211>26
<212>DNA
<213>artifical sequence
<400>8
ctcgagttac tttgggttaa cgacgg 26
Claims (3)
1. The heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme is characterized in that the amino acid sequence of the heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme is shown as SEQ ID No.2 or SEQ ID No. 4.
2. The gene of the heat-resistant high-efficiency methyl parathion hydrolysis heterozygote enzyme as claimed in claim 1, characterized in that the nucleotide sequence is shown as SEQ ID No.1 or SEQ ID No. 3.
3. The heat-resistant high-efficiency methyl parathion hydrolysis hybrid enzyme of claim 1 or the gene of claim 2 is applied to hydrolysis of methyl parathion pesticides.
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| CN103820404A (en) * | 2014-02-18 | 2014-05-28 | 南京农业大学 | Lipoxygenase mutant with simultaneously-improved enzymatic activity and thermal stability, and preparation method thereof |
| CN104263744A (en) * | 2014-09-11 | 2015-01-07 | 北京化工大学 | Artificially modified glucose oxidase gene and expression application thereof |
| CN104877977A (en) * | 2015-04-29 | 2015-09-02 | 江南大学 | Method for evolving enzyme based on synthetic single-stranded DNA (deoxyribonucleic acid) library |
| CN105255846A (en) * | 2015-10-19 | 2016-01-20 | 华东理工大学 | Engineering organophosphorus hydrolase, nucleic acid, mutant and application of engineering organophosphorus hydrolase and mutant |
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| CN103820404A (en) * | 2014-02-18 | 2014-05-28 | 南京农业大学 | Lipoxygenase mutant with simultaneously-improved enzymatic activity and thermal stability, and preparation method thereof |
| CN104263744A (en) * | 2014-09-11 | 2015-01-07 | 北京化工大学 | Artificially modified glucose oxidase gene and expression application thereof |
| CN104877977A (en) * | 2015-04-29 | 2015-09-02 | 江南大学 | Method for evolving enzyme based on synthetic single-stranded DNA (deoxyribonucleic acid) library |
| CN105255846A (en) * | 2015-10-19 | 2016-01-20 | 华东理工大学 | Engineering organophosphorus hydrolase, nucleic acid, mutant and application of engineering organophosphorus hydrolase and mutant |
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| Molecular Evolution of Adomet Synthetase by DNA Recombination with a Novel Separate-Mixing Method;Y. An et al.;《Molecular Biology》;20060630;第40卷(第3期);摘要，第486页左栏第1段至第491页右栏最后1段 * |
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