CN115125220B - Laccase EcLac and application thereof, laccase gene EcLac and application thereof - Google Patents
Laccase EcLac and application thereof, laccase gene EcLac and application thereof Download PDFInfo
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- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- 238000003259 recombinant expression Methods 0.000 claims description 70
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- 238000000034 method Methods 0.000 claims description 20
- 241000235058 Komagataella pastoris Species 0.000 claims description 17
- 241000588724 Escherichia coli Species 0.000 claims description 15
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 14
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 12
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 10
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
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- 101100460704 Aspergillus sp. (strain MF297-2) notI gene Proteins 0.000 description 1
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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- A—HUMAN NECESSITIES
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12Y110/03002—Laccase (1.10.3.2)
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Abstract
The invention discloses laccase EcLac and application thereof, and laccase gene EcLac and application thereof; the amino acid sequence of laccase EcLac is shown as SEQ ID NO: 1. Compared with the original laccase Lac, the activity of the laccase EcLac is improved by about 3.9 times, so that the laccase EcLac has the advantages of high yield and high activity, is low in manufacturing cost, is suitable for industrial production, and solves the problems of low yield, low activity and high cost of the existing laccase.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to laccase EcLac and application thereof, and laccase gene EcLac and application thereof.
Background
Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase. Raw lacquer is a natural resource with long history in the eastern. Yoshida in 1883 first found that the main ingredient that promoted the setting of raw lacquer was a specific protein. Bertrand in 1894 again confirmed that this functional protein is a key enzyme for lacquer-making and formally named laccase; after that, the characteristics of laccase are gradually studied in depth and the application potential of laccase is developed, and the laccase has been used for more than one hundred thirty years so far; laccase can be obtained by obtaining O 2 Catalyzing oxidation of substances such as polyphenol, polyaminobenzene and the like; statistics find that it can catalyze the oxidation of more than about three hundred different types of substrates; has great application potential in a series of industries such as degradation of refractory compounds, paper decolorization, washing, mycotoxin degradation and the like.
Laccase is widely distributed and can be classified into plant laccase, insect laccase, fungal laccase and bacterial laccase according to sources; research on fungal laccase has been carried out for a long time, but with increasing demands on laccase, the disadvantages of low yield, high cost and the like of fungal laccase lead to difficulty in meeting the demands of modern industrial industry; compared with other laccase, the bacterial laccase has the advantages of wide source, short growth period, good thermal stability, wide pH range and the like, and is valued by people; bacterial laccase belongs to copper-containing protein, laccase molecule has three structural domains, there is loop connection between every structural domain; the bacterial laccase contains 4 conserved copper atom areas to form an active center of a bacterial laccase molecule; coli (Escherichia coli) laccases exist in the periplasmic space of Escherichia coli, and their structure has a conserved active center typical of bacterial laccases and are therefore considered to be a member of the bacterial laccase family.
Bacterial laccase has been reported in some documents, but the researches on food safety, mycotoxin degradation and the like are not reported aiming at the influence of the structural domain of the laccase in the escherichia coli; although researches on structural differences of bacterial laccase are reported, at present, the key problems to be solved still exist in the industrial application of laccase: 1) Limited sources of enzymes; 2) Under natural conditions, the activity of the enzyme is low; 3) The yield is lower and the economic cost is higher.
The disadvantages described above have severely limited the industrial application of laccase, especially in the field of food processing and food safety, and there is a need for efficient laccase for degradation of mycotoxins. Under natural conditions, laccase has limited activity and yield, and the conventional breeding method is difficult to meet the requirement of industrial production. Therefore, the genetic engineering technology is utilized to carry out necessary molecular transformation on laccase, so that the capability of the recombinant expression body for secreting exogenous proteins can be effectively improved, and the activity and degradation efficiency of laccase can be improved rationally and pertinently; compared with the traditional method, the novel characteristic of the enzyme is accurately and efficiently given, so that the requirement of industrial production is better met.
Disclosure of Invention
The invention mainly aims to provide laccase EcLac and application thereof, and laccase gene EcLac and application thereof, and aims to solve the problems of low yield, low activity and high cost of the existing laccase.
In order to achieve the above purpose, the amino acid sequence of laccase EcLac provided by the invention is shown as SEQ ID NO: 1.
The invention also provides a laccase gene EcLac, which is used for encoding the laccase EcLac, and the base sequence of the laccase gene EcLac is shown in SEQ ID NO:2 or the base sequence of the laccase gene eclac is identical with that of SEQ ID NO:2 has a nucleotide sequence having an identity of 95% or more.
The invention also provides a recombinant expression vector comprising the laccase gene eclac.
The invention also provides a preparation method of the recombinant expression vector, which comprises the following preparation steps:
introducing two ends of laccase gene EcLac into enzyme cutting sites EcoR I and Not I, and carrying out double enzyme cutting by restriction enzyme EcoR I and Not I to obtain laccase gene EcLac fragments;
the pichia pastoris expression vector pPICZA is respectively subjected to double enzyme digestion by restriction enzymes EcoR I and Not I to obtain a vector pPICZ alpha A fragment;
and connecting the laccase gene eclac fragment and the vector pPICZ alpha A fragment through T4DNA ligase to obtain a recombinant expression vector.
The invention also provides a recombinant expression strain which comprises the recombinant expression vector.
Alternatively, the host cell of the recombinant expression strain is pichia pastoris.
The invention also provides a preparation method of the recombinant expression strain, which comprises the following preparation steps:
obtaining a linearized laccase recombinant expression vector pPICZ alpha A-eclac by using the enzyme tangential laccase recombinant expression vector pPICZ alpha A-eclac;
and (3) introducing the linearized laccase recombinant expression vector pPICZ alpha A-eclac into a host cell to obtain a recombinant expression strain.
The invention also provides a preparation method of laccase EcLac, which comprises the following preparation steps:
culturing the recombinant expression strain to obtain a culture;
laccase EcLac is obtained from the culture.
In addition, the invention also provides application of laccase EcLac in degradation of mycotoxin.
The laccase EcLac provided by the invention is prepared by modifying and optimizing wild laccase by molecules; based on wild type Escherichia coli (Escherichia coli) laccase Lac (GenBank accession number: EFB 2856373.1), the modified laccase has higher activity in host cells by artificial rational design, mutation of aspartic acid at 112 th site into glutamic acid (D112E), mutation of valine at 270 th site into tyrosine (V270Y), mutation of glutamic acid at 506 th site into arginine (E506R), and site-directed mutagenesis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the effect of signal peptide on laccase expression in example 1 of the invention;
FIG. 2 is a schematic representation of the wild-type laccase gene lac in example 2 of the invention;
FIG. 3 is a schematic representation of the site of laccase EcLac mutation in example 2 of the invention;
FIG. 4 is a graph showing the comparison of codon usage of wild-type laccase gene lac and modified laccase gene eclac in example 3 of the invention;
FIG. 5 is a schematic diagram showing the results of the cleavage assay of example 4 of cloning the wild-type laccase gene lac and the molecularly modified laccase gene ecla into the recombinant expression vector pPICZ alpha A;
FIG. 6 is a schematic diagram showing the SDS-PAGE test result of supernatant after fermentation of the recombinant expression strain of wild-type laccase gene lac and the SDS-PAGE test result of supernatant after fermentation of the recombinant expression strain of redesigned laccase gene ecla in example 5 of the invention;
FIG. 7 is a graph showing the enzymatic activity of the supernatant of the recombinant expression strain modified in example 6 of the present invention under the condition of the fermenter;
FIG. 8 shows the hydrolysis curve of laccase EcLac against aflatoxin in example 7 of the invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Currently, the industrial application of laccase still has the key problems to be solved: 1) Limited sources of enzymes; 2) Under natural conditions, the activity of the enzyme is low; 3) The yield is lower and the economic cost is higher.
The disadvantages described above have severely limited the industrial application of laccase, especially in the food processing and food safety fields, and there is a need for efficient laccase for degradation of mycotoxins. Under natural conditions, laccase has limited activity and yield, and the conventional breeding method is difficult to meet the requirement of industrial production. Therefore, the genetic engineering technology is utilized to carry out necessary molecular transformation on laccase, so that the capability of the recombinant expression body for secreting exogenous proteins can be effectively improved, and the activity and degradation efficiency of laccase can be improved rationally and pertinently; compared with the traditional method, the novel characteristic of the enzyme is accurately and efficiently given, so that the requirement of industrial production is better met.
In view of the above, the invention provides laccase EcLac which has higher output, higher activity and low cost, and the amino acid sequence of the laccase EcLac is shown as SEQ ID NO: 1.
The laccase EcLac provided by the invention is prepared by modifying and optimizing wild laccase by molecules; based on wild type Escherichia coli laccase Lac (GenBank accession number: EFB 2856373.1), the modified laccase has higher activity in host cells by artificial rational design, mutation of aspartic acid at 112 th site into glutamic acid (D112E), mutation of valine at 270 th site into tyrosine (V270Y) and mutation of glutamic acid at 506 th site into arginine (E506R); meanwhile, the expression quantity of the laccase gene eclac in host cells is obviously improved after codon optimization; compared with the original laccase Lac, the activity of the laccase EcLac is improved by about 3.9 times, so that the laccase EcLac has the advantages of high yield and high activity, and the laccase EcLac is low in manufacturing cost and suitable for industrial production.
The laccase EcLac is modified by wild Escherichia coli (Escherichia coli) laccase Lac (GenBank accession number: EFB 2856373.1) through artificial rational design; wherein, the amino acid sequence of the wild laccase Lac is shown in SEQ ID NO: 3.
Specifically, in some embodiments, the laccase EcLac can be obtained by: removing a wild type signal peptide sequence in laccase Lac, which influences laccase secretory expression, to obtain laccase Lac without the wild type signal peptide sequence, mutating 112 th aspartic acid in laccase Lac without the wild type signal peptide sequence into glutamic acid (D112E) by a site-directed mutagenesis method, mutating 270 th valine into tyrosine (V270Y), mutating 506 th glutamic acid into arginine (E506R), and obtaining laccase EcLac; in this embodiment, the site-directed mutagenesis is performed by using conventional procedures in the art, and will not be described in detail herein.
The invention also provides a laccase gene EcLac, which is used for encoding the laccase EcLac, and the base sequence of the laccase gene EcLac is shown in SEQ ID NO:2 or the base sequence of the laccase gene eclac is identical with that of SEQ ID NO:2 has a nucleotide sequence having an identity of 95% or more.
As a preferred embodiment of the present embodiment, the base sequence of laccase gene eclac is shown in SEQ ID NO: 2.
In some embodiments, the laccase gene eclac is artificially designed and optimized based on the laccase amino acid sequence; the optimized core nucleotide sequence of laccase gene eclac contains SEQ ID NO:2, a nucleotide sequence shown in seq id no; compared with the original laccase gene lac, the laccase gene EcLac provided by the invention is redesigned according to the codon bias of Pichia pastoris; the codon with higher or highest use frequency in pichia pastoris is used for replacing the codon used at low frequency, so that the expression quantity of the artificially designed laccase gene ecla in pichia pastoris cells is obviously improved; wherein the sequence of the original laccase gene lac is shown as SEQ ID NO:4 is shown in the figure; according to the amino acid sequence of original laccase, a brand new laccase gene sequence is designed artificially, and the laccase gene fragment is obtained by an artificial synthesis method; before protein domain optimization, the highest activity of the expression strain is 2760U/mL; after optimization, the highest activity is 10700U/mL, and the enzyme activity is improved by 3.9 times compared with the front and back.
The invention provides a recombinant expression vector, which comprises the laccase gene eclac. In addition, the recombinant expression vector also includes other functional units.
In some embodiments, in the case of determination of the amino acid sequence of laccase EcLac and the nucleotide sequence of laccase gene EcLac, suitable recombinant expression vectors and other functional units may be selected according to the actual situation, in particular, in one embodiment, pichia pastoris expression vectors are selected as recombinant expression vectors.
The invention also provides a preparation method of the recombinant expression vector, which is characterized by comprising the following preparation steps:
s1, introducing two ends of a laccase gene EcLac into enzyme cutting sites EcoR I and Not I, and carrying out double enzyme cutting by restriction enzyme enzymes EcoR I and Not I to obtain a laccase gene EcLac fragment;
s2, respectively carrying out double enzyme digestion on a pichia pastoris expression vector pPICZ alpha A by using restriction enzymes EcoR I and Not I to obtain a vector pPICZ alpha A fragment;
and S3, connecting the laccase gene eclac fragment and the vector pPICZ alpha A fragment through T4DNA ligase to obtain a recombinant expression vector.
The laccase gene EcLac fragment obtained in the step S3 has a cohesive end, and the vector ppiczαa fragment obtained in the step S2 has a cohesive end; in some embodiments, the expression vector is a pichia expression vector ppiczαa; in other embodiments, other Pichia expression vectors may be used as the expression vector.
The method for obtaining the recombinant expression vector is not particularly limited, and the recombinant expression vector can be prepared according to the preparation method of the recombinant expression vector or by referring to the conventional technical means in the field.
The invention also provides a recombinant expression strain which comprises the recombinant expression vector.
The recombinant expression vector pPICZ alpha A-ecLac has the expression product of laccase ecLac; in the case of amino acid sequence determination of laccase EcLac, a person skilled in the art is able to obtain suitable recombinant expression strains, which are not described in detail here.
Generally, the host cell may be E.coli, bacillus, aspergillus, yeast, or other cell types, such as animal and plant cells. Preferably, the host cell of the recombinant expression strain is pichia pastoris; meanwhile, the laccase EcLac provided by the invention adopts a strategy more suitable for transcription and translation expression of pichia pastoris through optimization, and improves the expression quantity of laccase gene EcLac in pichia pastoris cells.
The invention also provides a preparation method of the recombinant expression strain, which is characterized by comprising the following preparation steps:
s10, obtaining a linearized laccase recombinant expression vector pPICZ alpha A-eclac through an enzyme tangential laccase recombinant expression vector pPICZ alpha A-eclac;
s20, introducing the linearized laccase recombinant expression vector pPICZ alpha A-eclac into a host cell to obtain a recombinant expression strain.
In particular, in some embodiments, the pichia pastoris is pichia pastoris X-33; in other embodiments, other strains of Pichia may be used according to the actual situation, and will not be described in detail herein.
The method for obtaining the recombinant expression strain is not particularly limited, and the recombinant expression strain can be prepared according to the preparation method of the recombinant expression strain, and can also be prepared by referring to the conventional technical means in the field.
The invention discloses a preparation method of laccase EcLac, which comprises the following preparation steps:
step S100, culturing recombinant expression strains to obtain cultures;
step S200, obtaining laccase EcLac from the culture.
In some embodiments, in step S200, in order to obtain laccase EcLac with better performance, a purification method is generally adopted, and specific purification steps are referred to in a conventional manner in the art, and are not described in detail herein; in other embodiments, the fermentation supernatant may also be treated directly as laccase product without purification.
Specifically, in performing step S100, the following steps may be performed: inoculating laccase recombinant expression strain into YPD culture medium with 30mL, culturing to OD600 of about 3.0-6.0h, centrifuging and collecting thallus; then 30mL of culture medium BMMY is added for continuous culture, methanol is added every 24h to make the final concentration be 1%, and laccase expression is induced to 96h.
In some embodiments, laccase EcLac may also be produced by means of a fermenter, in particular by the following steps: inoculating the recombinant expression strain into YPD liquid culture medium to culture and prepare seed liquid; inoculating into 30L of BSM fermentation medium at a ratio of 1:10 for culturing; controlling the dissolved oxygen to be more than 10% at the stage, gradually increasing the rotating speed from 200rpm to 500rpm, and carrying out ventilation and dissolved oxygen linkage; then, transferring to a methanol induction stage, wherein the time lasts for 120 hours, and mixing and inducing are carried out in the whole process according to the ratio of methanol to glycerol of 8:1; the induction amount is between 2 and 4mL/Lh, the temperature is controlled at 28+/-0.5 ℃, the pH value of fermentation liquor is=5.0, and the dissolved oxygen DO is more than 10%; samples were taken every 24h and the activity of laccase and protein content in the fermentation supernatant were determined.
In addition, the invention also provides application of laccase EcLac in degrading mycotoxin. In the actual use process, the method can be carried out by the following steps: contacting the laccase with a mycotoxin-containing material. In some embodiments, the mycotoxin-containing material may be aflatoxin.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1
This example is presented to illustrate the removal of wild-type laccase signal peptide sequences to increase laccase expression.
This example is based on the wild-type laccase Lac (GenBank accession number: EFB 2856373.1) from Escherichia coli; the enzyme comprises two parts of a signal peptide and a mature enzyme, wherein the amino acid sequence of the wild type escherichia coli laccase pre-Lac containing the signal peptide is shown as SEQ ID NO:5, the pre-lac gene sequence is shown as SEQ ID NO: shown at 6. The amino acid sequence of the wild type escherichia coli laccase Lac without the signal peptide is shown as SEQ ID NO:3, the lac gene sequence is shown as SEQ ID NO:4 is shown in the figure; FIG. 1 shows the results of the enzyme activity measurement of pre-Lac and Lac in example 1 of the present invention.
From fig. 1, it can be derived that: the enzyme activity of pre-Lac containing the wild type signal peptide is only 1250U/mL, and the Lac activity without the wild type signal peptide is obviously improved and is 2760U/mL. Thus, the activity of laccase can be increased by removing the wild-type laccase signal peptide sequence to increase laccase.
Example 2
The present example is to illustrate the modification of wild-type laccase Lac by artificial rational design to obtain novel laccase EcLac.
Aspartic acid at position 112 is mutated to glutamic acid (D112E), valine at position 270 is mutated to tyrosine (V270Y), and glutamic acid at position 506 is mutated to arginine (E506R) by means of site-directed mutagenesis. Wherein, the amino acid sequence of the wild laccase Lac is shown as SEQ ID NO:3, the base sequence of the wild laccase gene lac is shown as SEQ ID NO:4 is shown in the figure; the amino acid sequence of the modified laccase EcLac is shown as SEQ ID NO: 1.
FIGS. 2 and 3 are three amino acid positions (D112E), (V270Y) and (E506R) subjected to site-directed mutagenesis in example 2 of the present invention; in FIG. 2, the amino acid sequences of the wild-type laccase Lac mutation points are D112, V270 and E506, respectively; in FIG. 3, the amino acid sequences of the mutant laccase EcLac mutation points are E112, Y270 and R506, respectively.
Example 3
This example is presented to demonstrate the optimization of laccase gene domains to obtain the engineered gene eclac.
The embodiment utilizes bioinformatics software DNA2.0 to assist in manually redesign the nucleotide sequence of laccase gene eclac based on the mutated laccase amino acid sequence; the method specifically comprises the following steps: adapting the laccase gene sequence to the expression logic of the host cell and the bias of codon usage, and replacing low-frequency codons with high frequency of usage in the host cell; meanwhile, the complexity of the secondary structure of mRNA coded by laccase gene eclac is reduced, the repression effect of the secondary structure on laccase translation is reduced, a novel sequence of laccase gene eclac is obtained, and laccase gene eclac fragments are obtained by an artificial synthesis method.
The embodiment designs a brand new laccase gene sequence artificially, and obtains the laccase gene fragment by an artificial synthesis method; the base sequence of laccase gene eclac is shown in SEQ ID NO:2, the laccase gene eclac codes laccase with the amino acid sequence shown in SEQ ID NO:1 is shown in the specification; FIG. 4 is a graph showing the codon usage frequency of the wild-type laccase gene lac and the optimized laccase eclac of example 3 of the invention for the first 100 amino acids.
From fig. 4, it can be derived that: the codon usage frequency of the EcLac after optimization is obviously higher than that of Lac.
Example 4
This example is presented to illustrate the construction of recombinant expression strains of the modified laccase gene eclac and the original laccase gene lac.
(1) Respectively introducing enzyme cutting sites EcoR I and Not I at two ends of laccase gene ecla, wherein the fragment is cut by EcoR I and Not I, and the vector pPICZ alpha A is also cut by EcoR I and Not I; wherein, the enzyme digestion system is as follows: mu.g of DNA, 1.5. Mu.L of EcoR I, 1.5. Mu.L of Not I, 20. Mu.L of Buffer H were added with water to a volume of 200. Mu.L, and the mixture was digested for 4 hours at 37 ℃.
(2) Connecting laccase gene eclac enzyme fragments and pPICZ alpha A enzyme fragments through T4DNA ligase to obtain a recombinant expression vector pPICZ alpha A-eclac; wherein, the connection system is: 100ng of eclac, 50ng of pPICZαA, 1 μL of T4 buffer, 1 μL of T4DNA ligase, and water to 10 μL; the results are shown in FIG. 5 (in the figure, M is DL5000 DNA Marker, lanes 1 and 2 are laccase gene lac and optimized gene eclac, respectively, and lanes 3 and 4 are recombinant expression vectors pPICZαA-lac and pPICZαA-lac, respectively, and double digestion verification (EcoRI, notI) is performed.
(3) The recombinant expression vector pPICZαA-eclac was transformed into Pichia pastoris X-33, and then screened on a plate containing YPD medium to obtain a recombinant expression strain. Wherein, the YPD medium comprises the following components: yeast powder 1%, peptone 2% and D-glucose 2%. Standing the mixture in an incubator at 28 ℃ for 3d on a YPD plate, and observing the growth condition of thalli on the plate at any time to obtain laccase recombinant expression strains containing lac and eclac genes.
From fig. 5, it can be derived that: the present example successfully constructs laccase expression plasmids.
Example 5
The embodiment is used for the operation of expressing laccase in shake flasks by wild type and laccase recombinant expression strains subjected to molecular transformation.
The optimized laccase recombinant expression strain or the wild laccase recombinant expression strain obtained in the example 4 is inoculated in a 30mL YPD medium and subjected to constant temperature shaking culture at 28 ℃ and 200 r/min. When the OD600 is about 3.0-6.0, the thalli are collected by centrifugation at 10000 rpm; then adding 30mL of culture medium BMMY for suspension and continuing culture; in the medium, the BMMY medium comprises the following components: yeast powder 1%, peptone 2%, biotin 0.05%; adding methanol every 24 hours to make the final concentration of the laccase be 1%, and inducing laccase to express for 96 hours; proteins in the fermentation broth were detected by SDS-PAGE.
According to the procedure described in this example, the wild-type laccase Lac and the modified laccase EcLac recombinant expression strain were subjected to enzyme-producing fermentation in shake flasks; FIG. 6 shows the SDS-PAGE test of the supernatant of the recombinant expression strain of the wild-type laccase in example 6 of the invention; wherein M is a protein Marker, and lanes 1-4 are wild laccase Lac of different strains; lanes 5-8 are the different optimized laccase EcLac.
From fig. 6, it can be derived that: the output of the laccase EcLac after optimization and transformation is obviously higher than that of the wild laccase Lac.
Example 6
The example is used to illustrate the enzyme productivity assessment experiment of the laccase EcLac after optimization and modification in a 50-L small-sized fermentation tank.
(1) Inoculating a recombinant expression strain containing the laccase gene ecla after molecular modification to 50mL of YPD liquid culture medium, and shake culturing for 15h at 28 ℃ to obtain primary seed liquid; and then 1:10 is transferred to a 3-L YPD liquid culture medium for 15h to be used as a secondary seed liquid; preparing 30-L BSM basic salt for culture; wherein the BSM medium comprises: KH 175g 2 PO 4 3.5g of CaSO 4 21g (NH) 4 ) 2 SO 4 49g of MgSO 4 K of 60g 2 SO 4 140g of glycerol/glucose, 2mL/L of biotin solution, 2mL/L of trace elements, 56g of peptone and distilled water to 30L; sterilizing in situ, and regulating pH to 5.5 with ammonia water; and transferring the 3L seed solution into a fermentation tank for culture.
(2) The fermentation of laccase recombinant strain is mainly divided into two stages, namely a nutrition growth stage and a methanol induction stage; the time lasts for 20 hours in the nutrition growth stage, the dissolved oxygen is controlled to be more than 10 percent, the rotating speed is gradually increased from 200rpm to 500rpm, and ventilation and dissolved oxygen are linked; in the vegetative growth stage, the fresh weight of the cells reaches 200g/L; in the methanol induction stage, the time lasts for 120 hours, and the whole process mixing induction is carried out according to the ratio of methanol to glycerol of 8:1; the induction amount is 3mL/Lh, the temperature is controlled at 28+/-0.5 ℃ in the process of mixed feeding methanol induced fermentation, the pH value of fermentation liquor is=5.0, and the dissolved oxygen DO is more than 10%; samples were taken every 24h and the activity of laccase in the fermentation supernatant was determined.
Determination of laccase Activity: the sample and control groups were each filled with 500. Mu.L of buffer (pH=5.5), 10. Mu.L of ABTS solution (0.1 mol/L concentration) was added, mixed well and placed at 70℃for 2min, 2. Mu.L of laccase solution was added, reacted for 5min, and 500. Mu.L of methanol was added to terminate the reaction. 200. Mu.L was added to a 96-well plate, and the absorbance OD was read with an ELISA reader at the wavelength of OD 420. The enzyme activities were calculated from the OD420 values of the sample and control groups.
Wherein, the enzyme activity calculation formula is:
wherein Δod is the absorbance change before and after the reaction; ζ is 36000M -1 ·cm -1 The method comprises the steps of carrying out a first treatment on the surface of the l is 0.58cm measured using a microplate reader; t is time in minutes; [ E]Protein content in μg/mL; v (V) Enzymes The volume of the amount of enzyme; v (V) Reaction system Is the size and volume of the whole reaction system.
According to the operation procedure of the embodiment, the laccase EcLac gene recombinant expression strain is subjected to enzyme production fermentation in a fermentation tank; FIG. 7 shows the enzyme yields of the supernatant after fermentation of the recombinant expression strain of laccase EcLac in example 6 of the invention.
From fig. 7, it can be derived that: after 168 hours of fermentation, laccase activity reached 10700U/mL.
Example 7
This example illustrates the degradation of aflatoxin by laccase EcLac.
(1) The degradation substrate aflatoxin is dissolved by adopting a mixture of acetonitrile and water, so that the final concentration is 120 mug/L. Taking 500 mu L of buffer solution (pH=5.5) containing 10 mu L of ABTS (concentration is 0.1 mol/L) of laccase solution, uniformly mixing, culturing at a constant temperature of 40 ℃ for 12 hours, and periodically sampling;
(2) And (3) measuring the content of aflatoxin by using an enzyme-linked immunosorbent assay kit: 200 mu L of the sample is added into a 96-well plate, and the value of absorbance OD is read under the light wavelength of OD420 by an enzyme-labeling instrument; and calculating the degradation rate and the residual rate of the aflatoxin according to the OD420 values of the sample group and the control group.
According to the procedure described in this example, the degradation of aflatoxin by laccase EcLac was determined. Fig. 8 shows the test results in example 7 of the present invention.
From fig. 8, it can be derived that: the degradation rate of laccase EcLac after optimization on aflatoxin at 3h is over 95%.
In conclusion, the original laccase is redesigned and modified, and the laccase gene codons are optimized, so that novel laccase EcLac and laccase gene EcLac are successfully obtained; the gene is transferred into pichia pastoris for expression after being connected with an expression vector pPICZ alpha A; obtaining a high-efficiency laccase recombinant expression strain; the redesign and the reconstruction of laccase, and the optimization of laccase gene codons obviously improves laccase activity and protein expression; the laccase is induced to be expressed for 150 hours in a fermentation tank, and the enzyme activity of laccase in supernatant fluid is 10700U/mL; the application of the enzyme shows that the enzyme can hydrolyze aflatoxin with high efficiency and has good application potential.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. A laccase EcLac is characterized in that,
the laccase EcLac is transformed from a wild escherichia coli laccase Lac, and the transformation method is as follows:
removing a wild type signal peptide sequence affecting laccase secretion expression in a wild type escherichia coli laccase Lac to obtain a laccase Lac without the wild type signal peptide sequence, mutating 112 th aspartic acid in the laccase Lac without the wild type signal peptide sequence into glutamic acid by a site-directed mutagenesis method, mutating 270 th valine into tyrosine, mutating 506 th glutamic acid into arginine, and obtaining laccase EcLac;
wherein GenBankaccession number of the wild type E.coli laccase Lac is EFB2856373.1.
2. A laccase gene EcLac, characterized in that it encodes a laccase EcLac according to claim 1, which replaces codons of low frequency with codons of high frequency used in the host cell; the host cell is Pichia pastoris.
3. A recombinant expression vector comprising the laccase gene eclac of claim 2.
4. A method of preparing the recombinant expression vector of claim 3, comprising the steps of:
introducing two ends of laccase gene EcLac into enzyme cutting sites EcoR I and Not I, and carrying out double enzyme cutting by restriction enzymes EcoR I and Not I to obtain laccase gene EcLac fragments;
cutting a pichia pastoris expression vector pPICZA by restriction enzymes EcoRI and Not I respectively to obtain a vector pPICZA fragment;
and connecting the laccase gene eclac fragment and the vector pPICZA fragment through T4DNA ligase to obtain a recombinant expression vector.
5. A recombinant expression strain comprising the recombinant expression vector of claim 3, wherein the host cell of the recombinant expression strain is pichia pastoris.
6. A method for producing a recombinant expression strain according to claim 5, comprising the steps of:
obtaining a linearized laccase recombinant expression vector pPICZA-eclac by using the enzyme tangential laccase recombinant expression vector pPICZA-eclac;
and introducing the linearized laccase recombinant expression vector pPICZA-eclac into a host cell to obtain a recombinant expression strain.
7. The method for preparing laccase EcLac according to claim 1, comprising the following steps:
culturing the recombinant expression strain of claim 5 to obtain a culture;
laccase EcLac is obtained from the culture.
8. Use of the laccase EcLac of claim 1 for degrading mycotoxins.
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| Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities;Takao Kajikawa et al.;《Biochemical and Biophysical Research Communications》;第152-156页 * |
| The functional roles of the three copper sites associated with the methionine-rich insert in the multicopper oxidase CueO from E. coli;Laura Cortes et al.;《Metallomics》;第776-785页 * |
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