CN114774385B - Trypsin-like enzyme and pepsin resistance improved zearalenone hydrolase - Google Patents
Trypsin-like enzyme and pepsin resistance improved zearalenone hydrolase Download PDFInfo
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- CN114774385B CN114774385B CN202210242251.5A CN202210242251A CN114774385B CN 114774385 B CN114774385 B CN 114774385B CN 202210242251 A CN202210242251 A CN 202210242251A CN 114774385 B CN114774385 B CN 114774385B
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- zearalenone
- zhd101
- trypsin
- hydrolase
- pepsin
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a zearalenone hydrolase with improved trypsin resistance and pepsin resistance and application thereof. The zearalenone hydrolase with improved trypsin resistance and pepsin resistance is produced by producing a plurality of amino acid substitutions in the zearalenone hydrolase with the amino acid sequence of SEQ ID NO.1 and derived from the Paenispora faricola, and the amino acid substitutions are 115 and 254 substitutions. The invention screens a strain of zearalenone hydrolase mutant by mutating the zearalenone hydrolase, experiments show that the obtained mutant has no influence on the hydrolysis function of the zearalenone, the resistance half-life period of trypsin is prolonged by 32% compared with that of wild zearalenone hydrolase, the resistance half-life period of pepsin is prolonged by 43% compared with that of wild zearalenone hydrolase.
Description
Technical Field
The invention relates to zearalenone hydrolase, in particular to zearalenone hydrolase with improved resistance to trypsin and pepsin.
Background
Zearalenone hydrolase (ZHD 101) is also known as zearalenone degrading enzyme. The zearalenone degrading enzyme ZHD gene is a gene encoding pantolactone hydrolase in the Paenibacillus roseus, and the protein ZHD101 encoded by the gene can specifically bind and degrade the zearalenone. The reaction degradation mechanism is ZHD that the ester bond of ZEN is broken to become dihydroxyphenyl derivative with an open side chain, and then CO is lost 2 A non-toxic alkyl resorcinol product is obtained, which is non-toxic. Therefore, it is often used as a feed additive to improve feed utilization.
ZHD 101A 101 is divided into hydrolase folding center domain and cap structure. The junction of the two structures forms a larger groove, this groove is confirmed by the substrate complex structure as a substrate binding site. The benzene ring portion of the substrate ZEN is mainly fixed by hydrogen bonds, and the lactone ring portion is mainly bonded to the active center through hydrophobic forces. As a result of structural analysis, the catalytic triplets of ZHD101 were S102, H242 and E126, and the structure S102 of the collection substrate complex was mutated to a 102. S102 attacks carbonyl C in the lactone ring of the substrate as a nucleophile, allowing the substrate to be hydrolyzed.
Heretofore, studies on zearalenone hydrolases and zearalenone degrading enzymes have focused mainly on finding and screening strains producing zearalenone hydrolases of different characteristics from different sources or obtaining strains producing zearalenone hydrolases of different characteristics by a gene recombination method, and the preparation and application of zearalenone hydrolases, and the like. Searching Chinese patent library, about 70 patents related to zearalenone degrading enzyme are found, and the patent related to the discovery or screening of zearalenone degrading enzyme with different characteristics from different microorganism sources comprises a plurality of aspects: among the patents related to the improvement of the application range of zearalenone degrading enzyme PH are: CN202010272789.1, CN201711180443.3, etc.; patents on improvement of the enzymatic activity of zearalenone degrading enzyme are: CN201910825318.6, CN201910823208.6, CN201911024030.5, cn20111008679. X, etc.; patents for improving stability of zearalenone degrading enzyme are: CN202011381340.5, CN201810010538.9, CN201710516347.5, etc.; patents on increasing the expression level of zearalenone degrading enzyme protein are: CN201910823034.3, cn20191082888. X, CN201910321167.0, cn201610156145.X, etc.; patents for obtaining a strain or mutant producing zearalenone hydrolase by a gene recombination method are as follows: CN201911024030.5, CN201911023315.7, CN201711180443.3, CN202110157570.1, CN201810010538.9, CN201710322734.5, and the like; patents on the preparation method of zearalenone hydrolase are as follows: CN201611249978.7, CN201510943066.9, CN201910308008.7, CN201611250033.7, etc.; the method for constructing the genetic engineering bacteria for producing zearalenone toxin degrading enzyme comprises the following steps: CN202110124614.0, cn202110124609.X, cn201610156145.X, CN201911018173.5, CN202011381340.5, CN201911023315.7, etc.; patents concerning the discovery or screening of strains of different microbial origin that produce zearalenone degrading enzymes are: CN202011325114.5, CN201910556331.6, CN202010991141.X, cn201911186458.X, CN201910556331.6, CN201410147140.1, CN201410047870.4, etc.; patents concerning the use of zearalenone degrading enzymes are: CN201911327678.X, CN201911038267.9, CN201910693874.2, CN201910298924.7, CN201610372613.7, CN201480055221.7, CN200910241454.7, CN201310356776.2, CN201310083469.1, CN 2015115441. X, CN201910938276.7, CN201910228804.X, and the like. The main researches related to the zearalenone degrading enzyme in the Chinese national potential theory library comprise the steps of excavating gene resources of the zearalenone degrading enzyme with high-efficiency degradation rate, researching enzymatic properties, catalyzing reaction mechanisms, cloning and expressing enzyme genes, directionally modifying enzymes, and the like, and the application of the zearalenone degrading enzyme in the feed industry and the like. Studies and patents on zearalenone hydrolases with increased trypsin and pepsin resistance have not been reported to date.
Disclosure of Invention
The primary object of the present invention is to provide a zearalenone hydrolase with improved resistance to trypsin and pepsin.
The invention is to carry out site-directed mutagenesis on a gene of zearalenone hydrolase (called ZDH101 gene). The gene sequence of zearalenone hydrolase obtained from Saprolegnia rosea (Clonostachys rosea) has GENBANK accession number KR363960.1. The amino acid sequence of the mature protein of this enzyme is ALI16790.1 (SEQ ID NO. 1).
According to the invention, a zearalenone hydrolase mutant is obtained by carrying out mutation screening on the zearalenone hydrolase, and experiments show that the obtained ZHD mutant has no influence on the hydrolysis function of zearalenone, has a resistance half-life of trypsin prolonged by 32% compared with wild ZHD101, has a resistance half-life of pepsin prolonged by 43% compared with wild ZHD101, and is named ZHD101 K254Q/Y115H 。
The zearalenone hydrolase with improved trypsin resistance and pepsin resistance is produced by producing a plurality of amino acid substitutions in the zearalenone hydrolase with the amino acid sequence of SEQ ID NO.1 and derived from the Paenispora faricola (Clonostachys rosea), and the amino acid substitutions are 115 and 254 substitutions.
According to a further feature of the site-directed mutagenesis modified zearalenone hydrolase according to the present invention, the amino acid substitution at position 115 is a substitution of tyrosine with histidine; the amino acid substitution at position 254 is substitution of lysine with glutamine. The amino acid sequence of the site-directed mutagenesis modified zearalenone hydrolase is SEQ ID NO.2.
The zearalenone hydrolase mutant (ZHD 101) K254Q/Y115H ) After digestion with simulated artificial pancreatic juice (pH 6.8, trypsin concentration 1mg/mL at 40 ℃) for 100 minutes, mutant ZHD101 was obtained K254Q/Y115H Half-life was about 166min, whereas wild-type ZHD101 wt Half-life was about 125min, showing mutant ZHD101 K254Q/Y115H Resistance to trypsin is higher than wild type ZHD101 wt And improves the quality of the product. The zearalenone hydrolase mutant is digested by simulated artificial gastric fluid (pepsin with pH of 1.2 and concentration of 0.012mg/mL at 40deg.C) for 80min, and the mutant ZHD is obtained K254Q/Y115H Half-life was about 66min, whereas wild-type ZHD101 wt Half-life was about 46min. Thus showing mutant ZHD101 K254Q/Y115H Compared with wild type ZHD101 wt The resistance to pepsin is improved.
Further, the present invention provides a DNA molecule encoding the zearalenone hydrolase of the invention having increased resistance to trypsin and pepsin.
The nucleotide sequence of the mutant DNA molecule is SEQ ID NO.3.
It is a further object of the present invention to provide a vector comprising a DNA molecule according to the present invention.
It is a further object of the present invention to provide a host cell comprising a DNA molecule according to the invention or comprising a vector according to the invention.
Both the vectors and host cells described above can be prepared by techniques well known in the art.
The invention also provides a production method of the zearalenone hydrolase with improved trypsin resistance and pepsin resistance, comprising the following steps: culturing the host cell of the invention under conditions suitable for expression of zearalenone hydrolase and isolating the zearalenone hydrolase from the culture medium.
When the DNA molecules of the invention are inserted into the vector in the proper orientation and correct reading frame, or transferred into the host cell, the DNA molecules can be expressed in any eukaryotic or prokaryotic expression system. A variety of host-vector systems can be used to express the protein coding sequence. Host-vector systems include, but are not limited to: bacteria transformed with phage, plasmid, or cosmid; microorganisms containing yeast vectors, such as yeasts; mammalian cell systems infected with viruses; insect cell systems infected with viruses; a plant cell system infected with bacteria. Preferred vectors of the invention include viral vectors, plasmids, cosmids, or oligonucleotides.
Preferred hosts of the invention are eukaryotic systems such as pichia pastoris; the preferred method of protein expression of the present invention is Pichia pastoris secretory expression.
In another aspect, the invention provides the use of the zearalenone hydrolase with improved trypsin resistance and pepsin resistance, in particular the use of the zearalenone hydrolase with improved trypsin resistance and pepsin resistance in the preparation of a food or feed additive.
Drawings
FIG. 1 shows SDS-PAGE of proteins, wherein the black arrow indicates a Marker 25Kd band, and the black box indicates a target protein with a size of about 29 Kd. Lane 1 is a wild-type pichia pastoris GS115 control sample without the gene of interest; lane 2 is wild-type zearalenone hydrolase protein culture supernatant; lane 3 is mutant zearalene ketohydrolase protein culture supernatant.
FIG. 2 is a wild-type ZHD101 according to the invention wt And mutant ZHD101 K254Q/Y115H Results of protein gray scale scan data. The artificial pancreatic juice contains trypsin.
FIG. 3 is a wild-type ZHD101 according to the invention wt And mutant ZHD101 K254Q/Y115H Results of protein gray scale scan data. The artificial gastric juice contains pepsin.
FIG. 4 is a wild-type ZHD101 according to the invention wt And mutant ZHD101 K254Q/Y115H Is set in the temperature range of the temperature sensor.
FIG. 5 is a wild-type ZHD101 according to the invention wt And mutant ZHD101 K254Q/Y115H Is not limited, and is not limited.
FIG. 6 is a wild-type ZHD101 according to the invention wt And mutant ZHD101 K254Q/Y115H Is the most suitable pH of the sample.
FIG. 7 is a schematic illustration of the present inventionSaid wild type ZHD101 wt And mutant ZHD101 K254Q/Y115H pH stability of (C).
Detailed Description
The terms used herein, unless otherwise indicated, are intended to have meanings commonly understood by those skilled in the art. The following provides definitions of some specific terms used in the present invention.
“ZHD101 wt "indicates wild-type zearalenone hydrolase whose gene is in italics" ZHD101 wt "means.
“ZHD101 K254Q/Y115H "means mutant zearalenone hydrolase whose gene is in italics" ZHD101 K254Q /Y115H "means.
Example 1: synthesis of zearalenone hydrolase gene
The invention adopts Clonostachys rosea-source wild zearalenone hydrolase gene (GenBank registration number is KR 363960.1) which is synthesized by Shanghai JieRui gene company (other commercial companies with complete gene synthesis can also be completed).
Example 2: ligation of zearalenone hydrolase Gene (ZHD 101) with cloning vector Taox+PgHT+BBPB
1. The pGH plasmid containing ZHD 101-purpose gene synthesized by the whole gene and the cloning vector Taox+PgHT+BBPB (self-constructed by the laboratory in which the inventors were located) were digested with restriction enzymes EcoRI and SpeI/XbaI at 37℃for 30min, respectively, under the following conditions of Table 1:
table 1:
2. separating two target fragments of the enzyme-digested product after electrophoresis by 1% agarose gel, and using T 4 DNA ligase ligation, ligation system as follows table 2:
table 2:
| ZHD101 enzyme cleavage products | 7.0μL |
| Cloning vector cleavage products | 1.0μL |
| T 4 DNA ligase | 1.0μL |
| T 4 DNA ligase buffer | 1.0μL |
| ddH 2 O | 1.0μL |
| Total volume of | 10.0μL |
The ligation was performed at 16℃for 16h with DNA ligase, the DH 5. Alpha. Competent cells transformed with the ligation product were amplified, plasmids were extracted with a plasmid extraction kit, and the electrophoresis results after double digestion with EcoRI and PstI showed two bands of 3.8kb and 6.3kb, indicating successful ligation, and the maize gibberellin hydrolase gene was determined by DNA sequencing.
The zearalenone hydrolase gene (ZHD) is successfully connected with the cloning vector Taox+PgHT+BBPB through the two steps, so that the cloning vector M+Taox+PgHT+PB is obtained.
Example 3: gene fragment Paox+Pgap+SS1 is connected with cloning vector M+Taox+PgHT+PB
1. The gene fragment Paox- +Pgap+SS1 is prepared by calling out a cloning vector Paox+SS1+PB stored by a research institute where the inventor is located (and a university microbiological institute of south China), double digestion with EcoRI and SpeI endonucleases, purification and recovery; inserting gene segment Paox+Pgap+SS1 into cloning vector M+Taox+PgHT+PB to realize extracellular expression of ZHD101 protein and facilitate later purification of protein.
2. Cloning vector M+Taox+PgHT+PB was obtained from example 2, and the ligation of the gene fragment Paox+Pgap+SS1 to cloning vector M+Taox+PgHT+PB was performed in the same manner as in example 2.
The gene fragment Paox+Pgap+SS1 is successfully inserted into the cloning vector M+Taox+PgHT+PB through the two steps, and the expression cassette is successfully constructed.
Example 4: determination of mutant mutation sites
Constructing a random mutation library of a zearalenone hydrolase gene (ZHD 101), screening a ZHD protein mutant library, determining the enzymatic properties, trypsin resistance and pepsin resistance of the mutant zearalenone hydrolase, and screening to obtain the mutant, wherein the gene is ZHD101 K254Q/Y115H The mutant is mutated at amino acid 115 and 254. The inventors determined that site-directed mutagenesis was performed on positions 115 and 254, and mutant ZHD101 after mutagenesis K254Q/Y115H Genes are synthesized by Shanghai Jieli Gene, but can also be synthesized by other commercial companies with complete gene synthesis.
Example 5: wild type ZHD101 wt Gene and mutant ZHD101 K254Q/Y115H Secretory expression of gene integrated Pichia pastoris genome and recombinant protein
The purpose of this example is to screen mutants obtained in the previous example for secretory expression
The biobracks are linearized with restriction enzymes Xba I and Spe I to remove the biobracks sequence of the plasmid backbone, and then stable recombinants carrying the target gene can be obtained by homologous recombination double exchange using the integration sites Paox and Taox designed at both ends of the biobracks. The recipient strain of the experiment is Pichia pastoris GS115, after electric transformation, an MD plate is used for preliminary screening, then a monoclonal on the MD plate is selected and cultured in 2mL YPG liquid culture medium for 14-16 hours, and then pichia pastoris genome is extracted for PCR verification and positive clone recombinants are further screened.
And (3) obtaining positive clone recombinants with successful electrotransformation through MD plate screening and PCR verification.
Example 6: wild type ZHD101 wt Mutant ZHD101 K254Q/Y115H SDS-PAGE electrophoresis detection of recombinant proteins
(1) Preparing 10mL of 10% separating gel, uniformly mixing, filling the gel into a glass plate by using a micropipette until the gel is stopped at a position which is 2-3 cm away from the upper edge of a short glass plate, sealing the gel surface by using distilled water, slightly lifting one end of a gel preparation device, putting down the gel surface to be smooth, polymerizing for 40min until the distilled water is discarded, and sucking excessive water by using filter paper;
(2) Preparing 4mL of 5% concentrated gel, uniformly pouring the concentrated gel on the separating gel, inserting a comb with corresponding specification, avoiding generating bubbles, and polymerizing for 30min to be gelled and fixed;
(3) Filling an electrophoresis tank, filling electrophoresis liquid in the electrophoresis tank, preferably having a volume greater than half of the volume of the electrophoresis tank, transferring the prepared gel into the electrophoresis tank, and carefully pulling out the comb;
(4) Sequentially spotting, wherein the spotting amount is not too much, and 15 mu L of each hole is proper;
(5) Setting 90V running glue at the beginning of electrophoresis, changing the voltage of an indicator to 120V at a concentrated glue part to continue electrophoresis, and stopping electrophoresis when a target strip runs to a middle position (the target strip corresponds to a corresponding strip of a Maker and can be known in advance);
(6) Carefully peeling off gel, dyeing with Coomassie brilliant blue R-250 for 30min, and decolorizing with decolorizing solution until the background is light and the protein band is clear;
(7) The gel was imaged and the results were observed. SDS-PAGE of proteins shows the results of FIG. 1.
As shown in FIG. 1, the black arrow indicates a Marker 25Kd band, and the black box indicates a target protein with a size of about 29 Kd. Lane 1 is a pichia pastoris GS115 control sample without the gene of interest. The result shows that the target gene is successfully expressed in Pichia pastoris GS115, and the target protein can be amplified, cultured, separated and purified for the next experiment.
Example 7: detection of wild type ZHD101 by electrophoresis wt Mutant ZHD101 K254Q/Y115H Trypsin resistance and pepsin resistance assays of recombinant proteins
Wild ZHD and 101 wt And mutant ZHD101 K254Q/Y115H Digestion with artificial pancreatic juice (pH 6.8, trypsin concentration 1mg/mL at 40 ℃) (wild type ZHD 101) wt Protein and mutant ZHD101 K254Q/Y115H The addition amount of the protein is the same, and the content of the artificial pancreatic juice and the enzyme protein is 1: 50) were removed at 0, 10, 20, 30, 40, 60, 80, 100min, 20. Mu.l of protein running buffer was added to terminate digestion and immediately boiled for 5min, then SDS-PAGE electrophoresis was performed to check the digestion effect of trypsin, and the SDS-PAGE electrophoresis protein bands were subjected to gray-scale scanning to check the amount of residual protein, and wild type ZHD101 was calculated wt And mutant ZHD101 K254Q/Y115H Enzyme half-life of protein before and after trypsin treatment.
The results are shown in FIG. 2. The zearalenone hydrolase mutant (ZHD 101) K254Q/Y115H ) After digestion with simulated artificial pancreatic juice (pH 6.8, trypsin concentration 1mg/mL at 40 ℃) for 100 minutes, half-life was measured at about 166min, whereas wild-type ZHD101 wt Half-life of about 125min, ZHD101 K254Q/Y115H The half-life of (2) is 32% longer than that of the wild type. Thus showing mutant ZHD101 K254Q/Y115H Resistance to trypsin is higher than wild type ZHD101 wt And improves the quality of the product.
Wild ZHD and 101 wt And mutant ZHD101 K254Q/Y115H Digestion with artificial gastric juice (pH 1.2, pepsin at concentration of 0.012mg/mL at 40deg.C) (wild-type ZHD 101) wt Protein and mutant ZHD101 K254Q/Y115H The same amount of protein added) were removed at 0, 10, 20, 30, 40, 50, 60, 80min, 20 μl was added and digestion was stopped by adding 7 μl of protein running buffer and immediately boiled for 5min, then SDS-PAGE electrophoresis was performed to detect the digestion effect of pepsin, and the SDS-PAGE electrophoresis protein bands were subjected to gray scanning to detect the amount of residual protein, and wild type ZHD101 was calculated wt And mutant ZHD101 K254Q/Y115H The half-life of the protein before and after pepsin treatment.
The results are shown in FIG. 3. The zearalenone hydrolase mutant (ZHD 101) K254Q/Y115H ) After 80 minutes of digestion with simulated artificial gastric fluid (pepsin at pH 1.2 at a concentration of 0.012mg/mL at 40 ℃), a half-life of about 66 minutes was measured, whereas wild-type ZHD101 wt Half-life of about 46min, ZHD101 K254Q/Y115H The half-life of (2) is extended by 43% compared with the wild type. Thus showing mutant ZHD101 K254Q/Y115H Resistance to pepsin is higher than wild type ZHD101 wt And improves the quality of the product. Example 8: wild type ZHD101 wt Mutant ZHD101 K254Q/Y115H Enzymatic Property analysis of recombinant proteins
1. Optimum temperature and temperature stability analysis
Wild type ZHD101 obtained after purification wt Mutant ZHD101 K254Q/Y115H After the recombinant protein was properly diluted, the enzyme activities were measured at a gradient temperature (10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃,60 ℃) in a reaction buffer having a pH of 7.4, and the relative enzyme activities at the other temperatures were calculated with the highest enzyme activity being 100%, with three parallel groups.
Wild type ZHD101 obtained after purification wt Mutant ZHD101 K254Q/Y115H After properly diluting the recombinant protein, the enzyme activity was measured after incubation at a gradient temperature (10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃,60 ℃) for 15 minutes, and the relative enzyme activities at other temperatures were calculated with the highest enzyme activity being 100%, three in parallel for each group.
The enzyme activity determination method comprises the following steps:
under certain reaction conditions, ZHD101 was able to react with ZEN as a hydrolysis substrate, and the change in peak area of ZEN was detected by HPLC to represent the reduction in substrate, and the enzyme activity of ZHD101 was further characterized. The reaction system is shown in Table 3:
table 3:
the reaction was stopped by adding 300. Mu.L of methanol at 30℃for 10 min. The product was filtered and detected by 20. Mu. LHPLC, absorbance detection wavelength at 233nm,60% acetonitrile eluting at 0.6 mL/min.
The amount of enzyme required to degrade 1 μg ZEN per minute under the assay conditions was defined as 1 unit of ZEN degrading enzyme activity. The calculation formula of the enzyme activity (U/mg) is as follows:
wherein X is the degradation amount of the substrate, and the unit is mug.
As shown in fig. 4, wild type ZHD101 wt And mutant ZHD101 K254Q/Y115H The optimum temperature of (2) is 40 ℃, and when the reaction temperature exceeds 50 ℃, the enzyme activity is rapidly reduced. As shown in fig. 5, wild type ZHD101 wt And mutant ZHD101 K254Q/Y115H After incubation at 50℃for 15min, the residual enzyme activity of each enzyme was reduced to below 30%.
2. Optimum pH and pH stability analysis
Wild type ZHD101 obtained after purification wt Mutant ZHD101 K254Q/Y115H After the recombinant protein was properly diluted, the relative enzyme activities at other pH values were calculated by measuring the enzyme activities according to the enzyme activity measurement method in "optimum temperature and temperature stability analysis" in reaction buffers having gradient pH values (4, 5, 6, 7, 7.5, 8, 9, 10, 11) at a temperature of 30℃and taking the highest enzyme activity as 100%, and three of them were arranged in parallel.
Wild type ZHD101 obtained after purification wt Mutant ZHD101 K254Q/Y115H After properly diluting the recombinant protein, placing the recombinant protein in a reaction buffer solution with gradient pH (4, 5, 6, 7.5, 9, 10 and 11) for incubation at 30 ℃ for 30min, measuring the enzyme activity according to an enzyme activity measuring method in 'optimal temperature and temperature stability analysis', and calculating the relative enzyme activities at other temperatures by taking the highest enzyme activity as 100%, wherein each group is provided with three parallel groups.
As shown in fig. 6, wild type ZHD101 wt And mutant ZHD101 K254Q/Y115H The reaction performed similarly under different pH conditions, the optimum pH was 9, the activity was inhibited in an acidic environment, and the enzyme activity increased with increasing pH, and the activity was active in an environment of pH5 to 11. As shown in the figure7, wild type ZHD101 wt And mutant ZHD101 K254Q/Y115H All showed good stability in the range of ph 7.5-10. As can be seen from a combination of the two experiments, mutant ZHD101 K254Q/Y115H Other enzymatic properties of (a) and wild-type ZHD101 wt The hydrolytic enzymes are substantially identical.
SEQUENCE LISTING
<110> and university of south China
<120> a zearalenone hydrolase with improved resistance to trypsin and pepsin
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 264
<212> PRT
<213> Clonostachys rosea
<400> 1
Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
1 5 10 15
Tyr Glu Gln Glu Gly Thr Gly Pro Asp Val Val Leu Val Pro Asp Gly
20 25 30
Leu Gly Glu Cys Gln Met Phe Asp Ser Ser Val Ser Gln Ile Ala Ala
35 40 45
Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
50 55 60
Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
65 70 75 80
Ala Ser Tyr Val Ile Ser Ile Leu Asp Ala Leu Asp Ile Lys His Ala
85 90 95
Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
100 105 110
Leu Gly Tyr Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
115 120 125
Thr Lys Leu Leu Asp His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
130 135 140
Glu Ile Ser Lys Ile Leu Ala Asn Val Met Leu Asn Asp Val Ser Gly
145 150 155 160
Gly Ser Glu Ala Trp Gln Ala Met Gly Asp Glu Val His Ala Arg Leu
165 170 175
His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
180 185 190
Pro Ser Ala Pro Val Lys Asp Leu Glu Ala Leu Arg Gly Lys Pro Leu
195 200 205
Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
210 215 220
Ile Val Thr Ala Thr Lys Ala Gly Val Asn Ile Gly Leu Leu Pro Gly
225 230 235 240
Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
245 250 255
Val Glu Thr Thr Gln Lys His Leu
260
<210> 2
<211> 264
<212> PRT
<213> Synthesis
<400> 2
Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
1 5 10 15
Tyr Glu Gln Glu Gly Thr Gly Pro Asp Val Val Leu Val Pro Asp Gly
20 25 30
Leu Gly Glu Cys Gln Met Phe Asp Ser Ser Val Ser Gln Ile Ala Ala
35 40 45
Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
50 55 60
Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
65 70 75 80
Ala Ser Tyr Val Ile Ser Ile Leu Asp Ala Leu Asp Ile Lys His Ala
85 90 95
Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
100 105 110
Leu Gly His Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
115 120 125
Thr Lys Leu Leu Asp His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
130 135 140
Glu Ile Ser Lys Ile Leu Ala Asn Val Met Leu Asn Asp Val Ser Gly
145 150 155 160
Gly Ser Glu Ala Trp Gln Ala Met Gly Asp Glu Val His Ala Arg Leu
165 170 175
His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
180 185 190
Pro Ser Ala Pro Val Lys Asp Leu Glu Ala Leu Arg Gly Lys Pro Leu
195 200 205
Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
210 215 220
Ile Val Thr Ala Thr Lys Ala Gly Val Asn Ile Gly Leu Leu Pro Gly
225 230 235 240
Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Gln Tyr Val
245 250 255
Val Glu Thr Thr Gln Lys His Leu
260
<210> 3
<211> 792
<212> DNA
<213> Synthesis
<400> 3
atgcgcactc gcagcacaat ctcgaccccg aatggcatca cctggtacta tgagcaggag 60
ggtactggac ccgacgttgt cctcgtcccc gatggcctcg gagaatgcca gatgtttgac 120
agctccgtgt cgcaaattgc tgcccaaggc tttcgggtca ccacgtttga catgcccgga 180
atgtcccggt ctgcgaaggc accacccgag acctacactg aggtcacggc ccagaagctg 240
gcttcctatg tcatctccat cctggatgct cttgacatca agcacgctac tgtctggggc 300
tgcagctcag gagcttccac cgtcgtggcg ctgttgctcg gtcatcccga caggatacgc 360
aacgccatgt gccacgaact gccaacaaag ctactggacc acctttcaaa caccgctgtg 420
ctcgaagacg aggaaatctc aaagatcctg gccaatgtaa tgttgaacga cgtgtctgga 480
ggctcggagg cgtggcaagc catgggggac gaggtgcacg cgagactgca caagaactac 540
ccggtttggg ctcgaggata ccctcgcact attcctccct cagctccggt taaggatctg 600
gaggctctgc gtgggaagcc cctggactgg actgtcggcg ctgcgacacc aaccgagtct 660
ttctttgaca acattgttac cgctaccaag gctggtgtca acattgggtt gcttccaggg 720
atgcatttcc cttatgtttc ccacccggac gttttcgctc agtatgttgt ggaaactacg 780
cagaagcatc tt 792
Claims (7)
1. A zearalenone hydrolase with increased resistance to trypsin and pepsin, characterized in that: it is prepared from the Pachyrhizus rosea (SEQ ID NO. 1) as amino acid sequenceClonostachys rosea) An enzyme having increased resistance to trypsin and pepsin resulting from the production of a plurality of amino acid substitutions in zearalenone hydrolase, said amino acid substitutions being substitutions at positions 115 and 254; the amino acid substitution at position 115 is a substitution of histidine for tyrosine and the amino acid substitution at position 254 is a substitution of glutamine for lysine.
2. A DNA molecule characterized in that: encoding zearalenone hydrolase with increased resistance to trypsin and pepsin according to claim 1.
3. The DNA molecule of claim 2, wherein: the nucleotide sequence is SEQ ID NO.3.
4. A carrier, characterized in that: comprising the DNA molecule of claim 2 or 3.
5. A host cell, characterized in that: comprising the DNA molecule according to claim 2 or 3 or the vector according to claim 4.
6. A process for the production of zearalenone hydrolase having increased resistance to trypsin and pepsin according to claim 1, characterized in that the process comprises: culturing the host cell of claim 5 under conditions suitable for expression of zearalenone hydrolase and isolating said zearalenone hydrolase from the culture medium.
7. Use of zearalenone hydrolase with improved resistance to trypsin and pepsin as described in claim 1 for the preparation of a food or feed additive.
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