CN116064429B - Method for efficiently expressing laccase by recombinant escherichia coli - Google Patents
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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
- C12N9/0004—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)
-
- 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
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for efficiently expressing laccase by recombinant escherichia coli, which comprises the following steps: s1, extracting an escherichia coli SDB2 genome according to an existing method, designing a PCR primer, and performing PCR amplification by taking the escherichia coli SDB2 genome as a template; s2, connecting a target PCR product with a prokaryotic expression vector pET28a to construct a recombinant plasmid pET28a-Lac; s3, converting the recombinant plasmid pET28a-Lac into competent cells of the escherichia coli BL21 for culture, and screening out recombinant strains BL21/pET28a-Lac; s4, inoculating and culturing the recombinant strain BL21/pET28a-Lac, and inoculating the obtained bacterial liquid into an induction culture medium to induce laccase gene expression. According to the invention, the laccase gene is obtained by screening the wild escherichia coli SDB2 through the inventor, and the laccase gene can be accurately and efficiently expressed in recombinant bacteria by combining an original induction culture method, so that a foundation is laid for the commercial production of the functional recombinant laccase applied to the feed field.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for efficiently expressing laccase by recombinant escherichia coli.
Background
The compound polyphenols are anti-nutritional factors widely existing in plant feed resources, and can reduce the digestion and absorption of nutrients by forming complexes with proteins, microelements and the like; the toxic degradation products can also influence animal health and breeding stock reproductive performance, reduce animal product quality and the like, so that the dosage of the feed additive in industrial feeds is limited. Laccase (laccase) can improve the feeding value of plant feed resources by degrading polyphenol substances and the dosage of the plant feed resources in industrial feeds, thereby achieving the purposes of fully utilizing unconventional feed resources and reducing the production cost of feeds. Laccase is widely used in plants, animals, fungi and bacteria, can catalyze and oxidize more than 250 organic matters including polyphenols, arylamines and carboxylic acids to generate oxidation, polymerization and other reactions, and is mainly applied to the fields of dye decolorization and detoxification, paper pulp, textile bleaching and the like at present.
In the process of researching escherichia coli, the inventor screens out a wild escherichia coli SDB2, and researches prove that the wild escherichia coli SDB2 can efficiently secrete laccase, but the method is unfavorable for the industrial production of laccase due to the defects of undefined safety, poor specificity and the like, so that the safe and efficient heterologous expression of laccase genes of the bacteria becomes urgent.
In recent years, in research of laccase heterologous expression, bacterial laccase with poor thermal stability, need of translation modification and long production period, high stability, no need of glycosylation modification and short production period has attracted extensive attention from researchers. According to the escherichia coli SDB2 laccase gene, constructing a genetic engineering strain for heterologous expression of laccase is an effective way for realizing industrial production of laccase. Coli BL21 is the earliest strain developed and suitable for prokaryotic protein expression, and the expressed exogenous protein has high stability and can maintain the original natural structure of the target protein. The strain SDB2 and BL21 screened by the inventor belong to escherichia coli, so that the heterologous expression of the SDB2 laccase protein can be accurately realized by taking BL21 as a host strain for the SDB2 laccase gene. However, conventional heterologous expression methods still face the problems of low yield, unstable product quality and the like, and in order to realize efficient and stable expression, intensive research on other expression methods is required.
Chinese patent CN114032189A discloses a lignin degrading fusion strain R3, which is obtained by fusing parent strains X1 and X19 by using a PEG induction method, wherein the lignin is degraded by secreting various enzymes such as peroxidase, manganese peroxidase, laccase and the like by the R3 fusion strain, but the degradation degree of lignin by a single individual enzyme is not compared, and the action value and high-efficiency expression of the laccase are not studied deeply.
Chinese patent CN101736026B discloses a method for efficiently expressing soluble bacterial laccase in escherichia coli, which realizes efficient expression of maltose binding protein-bacterial laccase fusion protein in recombinant escherichia coli, and finally obtains maltose binding protein-bacterial laccase instead of bacterial laccase monomer.
Chinese patent CN103320453A discloses a Bacillus pumilus laccase gene and expression and application thereof, wherein the laccase gene is derived from Bacillus pumilus, an adopted expression vector is pColdII, an induction culture medium for constructing recombinant escherichia coli expression laccase comprises IPTG (induction reagent), tryptone, yeast extract and sodium chloride, the obtained recombinant laccase is applied to azo dye and anthraquinone dye decolorization, and the patent does not carry out intensive study on efficient expression of laccase.
Chinese patent CN104593312A discloses a recombinant Escherichia coli for expressing bacillus subtilis laccase and an induction method for realizing the secretory expression of laccase by the strain, and the induction culture medium prepared by the invention comprises IPTG and CuSO 4 LB (tryptone, yeast extract, sodium chloride), the obtained genetically engineered strain is applied to decolorization in synthetic dyes, and accordingly, the patent does not carry out intensive research on efficient expression of laccase.
Disclosure of Invention
The invention aims at: aiming at the problems existing in the prior art, the invention provides a method for efficiently expressing laccase by recombinant escherichia coli, the recombinant laccase gene is obtained by screening wild escherichia coli SDB2 by the inventor, and the laccase gene can be accurately and efficiently expressed in recombinant bacteria by combining an original induction culture method, so that the defects existing in the prior art are overcome.
The technical scheme adopted by the invention is as follows: a method for efficiently expressing laccase by recombinant escherichia coli comprises the following steps:
s1, extracting an escherichia coli SDB2 genome according to an existing method, designing a PCR primer, and performing PCR amplification by taking the escherichia coli SDB2 genome as a template to obtain a target PCR product; wherein the target PCR product is laccase gene, and the nucleotide sequence of the laccase gene is SEQ ID NO.1;
s2, recovering and purifying a target PCR product, and connecting the target PCR product with a prokaryotic expression vector pET28a to construct a recombinant plasmid pET28a-Lac;
s3, converting the recombinant plasmid pET28a-Lac into competent cells of the escherichia coli BL21 for culture, and screening out recombinant strains BL21/pET28a-Lac which are identified as positive by PCR;
s4, inoculating the recombinant strain BL21/pET28a-Lac into a liquid culture medium for culture, and inoculating the obtained bacterial liquid into an induction culture medium for inducing laccase gene expression;
s5, centrifugally collecting thalli after the induction culture is finished, and suspending, crushing and centrifuging thalli to obtain laccase protein.
In the invention, the induction medium has the following composition: IPTG at 0.2-1 mM/L, cuCl at 1-10 mM/L 2 MnSO of 1-10 mM/L 4 FeSO of 1-10 mM/L 4 CaCl of 1-10 mM/L 2 0.1-2 g/L of rapeseed meal juice, 0.1-2 g/L of white mustard seed juice and 0.1-2 g/L of cottonseed meal juice. The specific dosage is adjusted according to the actual requirement.
Preferably, the induction medium has a composition of: 1mM/L IPTG, 6mM/L CuCl 2 MnSO of 6mM/L 4 FeSO at 4mM/L 4 CaCl 2mM/L 2 The concentration of (1 g/L) vegetable pulp juice, 1g/L white mustard seed juice and 1g/L cotton seed pulp juice.
Further, the PCR primer includes a YfiH-F primer and a YfiH-R primer, and the sequences of the YfiH-F primer and the YfiH-R primer are as follows:
YfiH-F：CACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGA GCAAACTGATTGTTCCGCAGTGGCCGCT；
YfiH-R：AGCCGGATCTCAGTGGTGGT GGTGGTGGTGCTCGAGTTAAATCAGC CAAATAAAGCTGGCCATGC。
further, the liquid medium is a liquid medium containing 30+ -5 μg/ml of calicheamicin.
In step S1, two rounds of PCR amplification reaction are carried out by taking the escherichia coli SDB2 genome as a template, and the amplified product is subjected to 0.8w% agarose gel electrophoresis to obtain a target PCR product.
Further, in step S2, the purified target PCR product was digested with Nde l and Xho l, and then ligated with the expression vector pET-28a (+) digested with Nde l and Xho l by DNA ligase to construct a recombinant plasmid pET28a-Lac.
Further, in step S5, tris-NaCl buffer is added into the collected thalli for suspension, ultrasonic crushing is carried out, and a supernatant sample is collected by centrifugation, thus obtaining laccase protein.
The invention also comprises a recombinant strain, wherein the recombinant strain is the recombinant strain BL21/pET28a-Lac prepared by the method, and the recombinant strain is used for degrading polyphenol substances in plants.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. the escherichia coli SDB2 screened by the inventor can degrade polyphenol by utilizing laccase secreted by the inventor, and in order to selectively obtain the functional laccase, the invention introduces an SDB2 laccase gene into escherichia coli BL21 to construct a recombinant genetic engineering strain BL21/pET28a-Lac for specifically expressing the laccase protein, and the recombinant strain can efficiently degrade polyphenol substances in polyphenol plant feed, effectively improve the application value of plant feed resources and fill the blank of related technologies in the feed field;
2. the invention designs a special induction culture medium for recombinant strain BL21/pET28a-Lac, the induction culture medium adopts polymetallic ions to cooperate with nutrient sources rich in polyphenols, and the recombinant strain is jointly induced to efficiently secrete and express SDB2 laccase, thus laying a foundation for commercial production of functional recombinant laccase applied to the field of feed.
Drawings
FIG. 1 is a diagram showing the cleavage verification of recombinant plasmid pET28a-Lac of the invention;
FIG. 2 is a SDS-PAGE graph showing the results of laccase expression at different induction temperatures;
FIG. 3 is a SDS-PAGE plot of the results of laccase expression at different IPTG concentrations;
FIG. 4 is a SDS-PAGE graph showing the results of laccase expression at different metal ion concentrations;
FIG. 5 is a SDS-PAGE graph of the results of laccase expression of rapeseed meal juice, white mustard seed juice, cottonseed meal juice;
FIG. 6 is a SDS-PAGE of laccase expression results from different induction media;
FIG. 7 is a graph showing the effect of recombinant strains on the relative concentration of polyphenols at various incubation times.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
1. Preparation of recombinant E.coli
1. Cloning of E.coli SDB2 laccase Gene
The escherichia coli SDB2 genome is extracted as a template, primers YfiH-F and YfiH-R are designed, and restriction enzyme sites Nde l and Xho l are respectively introduced into the upstream and downstream primers.
The PCR primer YfiH-F: CACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGA GCAAACTGATTGTTCCGCAGTGGCCGCT;
YfiH-R：AGCCGGATCTCAGTGGTGGT GGTGGTGGTGCTCGAGTTAAATCAGC CAAATAAAGCTGGCCATGC。
the amplification template is escherichia coli SDB2 genome DNA, and the reaction conditions of two rounds of amplification are shown in tables 1 and 2:
TABLE 1 first round PCR amplification reaction conditions
TABLE 2 first round PCR amplification reaction conditions
The PCR amplified product is subjected to agarose gel electrophoresis with the concentration of 0.8% to obtain 741bp bands (namely laccase genes), sequencing is carried out to prove that the bands are SDB2 laccase genes, the SEQ ID NO.1 is shown, the amino acid sequence of the coded protein is SEQ ID NO.2, the first 20 amino acids of the SEQ ID NO.2 are common tag amino acids, and the amino acid sequence of the coded protein is actually from the 21 st amino acid.
2. And (3) recovering the target PCR product by using a DNA gel recovery kit, and purifying.
3. The recovered and purified SDB2 laccase gene PCR product is subjected to double digestion by Nde l and Xho l, and then is connected with an expression vector pET-28a (+) subjected to double digestion by Nde l and Xho l by DNA ligase, so that a recombinant plasmid pET28a-Lac is constructed.
4. Recombinant plasmid pET28a-Lac was transformed into competent cells of E.coli BL21, and after heat shock at 42℃was plated onto plates containing 30. Mu.g/mL kanamycin, and incubated at 37℃for 12h. Selecting positive transformant, extracting transformant plasmid, and making single and double enzyme cutting verification to define the constructed strain as target recombinant strain BL21/pET28a-Lac. As shown in FIG. 1, the recombinant plasmid was digested with Nde l and Xho l to obtain a band size of about 5163bp (gene of plasmid pET-28a (+) digested) and 741bp (laccase gene), which indicates that the recombinant plasmid pET28a-Lac containing laccase gene was successfully obtained.
2. Preparation method of recombinant escherichia coli high-efficiency expression laccase
In the invention, because the temperature and the composition formula of the induction culture medium can influence the laccase gene expression result, different experimental design methods are adopted to verify the influence degree of each factor one by one, so that the optimal induction condition is established, and a set of method for efficiently expressing laccase by recombinant escherichia coli is created.
The induction temperature was chosen at 15 ℃, 25 ℃,37 ℃ at three different levels to verify the effect on laccase gene expression.
Three different levels of IPTG concentration in the induction medium were chosen, 0.2mM/L, 0.5mM/L, 1.0mM/L, to verify the effect on laccase gene expression.
Induction of metal ions Cu in culture media 2+ （CuCl 2 Form), mn 2+ （MnSO 4 Form), fe 2+ （FeSO 4 Form, ca 2+ （CaCl 2 Form) was added to verify the effect on laccase gene expression using a four-factor three-level orthogonal design (see table 3 below).
TABLE 3 orthogonal test one factor level table
The added concentrations of rapeseed meal juice, white mustard seed juice and cottonseed meal juice in the induction medium were designed in a three-factor two-level orthogonal manner (see table 4 below) to verify the effect on laccase gene expression.
TABLE 4 orthogonal test two factor level table
Test one: effect of Induction temperature on laccase expression
Example 1
S1, inoculating a recombinant strain BL21/pET28a-Lac into a liquid culture medium containing 30 mug/mL of calicheamicin, and culturing at 35 ℃ overnight for 16 hours;
s2, inoculating the bacterial liquid obtained in the S1 into an induction culture medium, and inducing efficient expression of the SDB2 laccase gene at 15 ℃;
s3, centrifuging at 4000rpm for 10min after the induction culture is finished, and collecting thalli;
s4, adding Tris-NaCl buffer into the collected thalli for suspension, performing ultrasonic crushing, and centrifugally collecting supernatant for sample preparation;
s5, verifying the recombinant laccase protein by SDS-PAGE.
Example 2
Example 2 is identical to example 1, except that the induction temperature in example 2 is 25 ℃.
Example 3
Example 3 is identical to example 1, except that the induction temperature in example 3 is 37 ℃.
The results of the experiment showed that the optimal induction temperature was 37 ℃ (example 3), and as shown in fig. 2, only one specific band (laccase) of about 28KD protein molecular weight appeared under the induction conditions of 37 ℃ (example 1) and no specific band appeared at 15 ℃ (example 2).
And (2) testing II: effect of IPTG concentration on laccase expression
Example 4
S1, inoculating a recombinant strain BL21/pET28a-Lac into a liquid culture medium containing 30 mug/mL of calicheamicin, and culturing at 35 ℃ overnight for 16 hours;
s2, inoculating the bacterial liquid obtained in the S1 into an induction culture medium, and inducing efficient expression of the SDB2 laccase gene at 37 ℃;
s3, centrifuging at 4000rpm for 10min after the induction culture is finished, and collecting thalli;
s4, adding Tris-NaCl buffer into the collected thalli for suspension, performing ultrasonic crushing, and centrifugally collecting supernatant for sample preparation;
s5, verifying the recombinant laccase protein by SDS-PAGE.
In the induction medium, the concentration of IPTG was 0.2mM/L.
Example 5
Example 5 is identical to example 4 except that the IPTG concentration in the special induction medium of example 5 is 0.5mM/L.
Example 6
Example 6 is the same as example 4 except that the IPTG concentration in the special induction medium of example 6 is 1.0mM/L.
The results of the experiment showed that the optimal IPTG concentration was 1.0mM/L (example 6), and as shown in FIG. 3, a specific band (laccase) of about 28KD protein molecular weight appeared at the IPTG concentration of 0.2mM/L (example 4), 0.5mM/L (example 5) and 1.0mM/L (example 6), wherein the laccase expression level of example 6 was significantly higher than that of examples 4, 5 and 3.
And (3) test III: effect of different Metal ion concentrations on laccase expression
Example 7
S1, inoculating a recombinant strain BL21/pET28a-Lac into a liquid culture medium containing 30 mug/mL of calicheamicin, and culturing at 35 ℃ overnight for 16 hours;
s2, inoculating the bacterial liquid obtained in the S1 into a special induction culture medium to induce the efficient expression of the SDB2 laccase gene at 37 ℃;
s3, centrifuging at 4000rpm for 10min after the induction culture is finished, and collecting thalli;
s4, adding Tris-NaCl buffer into the collected thalli for suspension, performing ultrasonic crushing, and centrifugally collecting supernatant for sample preparation;
s5, verifying the recombinant laccase protein by SDS-PAGE.
In S2, the concentration of IPTG in the induction medium was 1.0mM/L, cu 2+ At a concentration of 2mM/L, mn 2+ At a concentration of 6mM/L, fe 2+ At a concentration of 6mM/L, ca 2+ The concentration was 6mM/L.
Example 8
Example 8 is the same as example 7 except that the induction medium of example 8 has an IPTG concentration of 1.0mM/L, cu 2+ At a concentration of 4mM/L, mn 2+ At a concentration of 4mM/L, fe 2+ At a concentration of 6mM/L, ca 2+ The concentration was 2mM/L.
Example 9
Example 9 is the same as example 7 except that the induction medium in example 9 has an IPTG concentration of 1.0mM/L, cu 2+ At a concentration of 6mM/L, mn 2+ At a concentration of 6mM/L, fe 2+ At a concentration of 4mM/L, ca 2+ The concentration was 2mM/L.
The test results show that: optimal metal ion combination Cu 2+ At a concentration of 6mM/L, mn 2+ At a concentration of 6mM/L, fe 2+ At a concentration of 4mM/L, ca 2+ At a concentration of 2mM/L (example 9), as shown in FIG. 4, a specific band (laccase) of about 28KD protein molecular weight appears in each of example 7, example 8 and example 9 in FIG. 4, wherein the laccase expression level of example 9 is higher than that of example 7, example 8 and example 6.
And (3) testing four: influence of rapeseed meal juice, white mustard seed juice and cottonseed meal juice on laccase expression
Example 10
S1, inoculating a recombinant strain BL21/pET28a-Lac into a liquid culture medium containing 30 mug/mL of calicheamicin, and culturing at 35 ℃ overnight for 16 hours;
s2, inoculating the bacterial liquid obtained in the S1 into a special induction culture medium to induce the efficient expression of the SDB2 laccase gene at 37 ℃;
s3, centrifuging at 4000rpm for 10min after the induction culture is finished, and collecting thalli;
s4, adding Tris-NaCl buffer into the collected thalli for suspension, performing ultrasonic crushing, and centrifugally collecting supernatant for sample preparation;
s5, verifying the recombinant laccase protein by SDS-PAGE;
in S2, the concentration of IPTG in the induction medium was 1.0mM/L, cu 2+ At a concentration of 6mM/L, mn 2+ At a concentration of 6mM/L, fe 2+ At a concentration of 4mM/L, ca 2+ The concentration is 2mM/L, the concentration of the rapeseed meal juice is 0.5g/L, the concentration of the white mustard juice is 0.5g/L, and the concentration of the cottonseed meal juice is 0.5g/L.
Example 11
Example 11 is the same as example 10 except that the concentration of rapeseed meal juice in the induction medium of example 11 is 0.5g/L, the concentration of white mustard juice is 1g/L, and the concentration of cottonseed meal juice is 1g/L.
Example 12
Example 12 is the same as example 10 except that the concentration of rapeseed meal juice in the induction medium of example 12 is 1g/L, the concentration of white mustard juice is 0.5g/L, and the concentration of cottonseed meal juice is 1g/L.
Example 13
Example 13 is the same as example 10 except that the concentration of rapeseed meal juice in the induction medium of example 13 is 1g/L, the concentration of white mustard juice is 1g/L, and the concentration of cottonseed meal juice is 0.5g/L.
The test results show that: the optimal combination concentration is 1g/L of rapeseed meal juice, 1g/L of white mustard seed juice and 0.5g/L of cottonseed meal juice (example 13), as shown in FIG. 4, a specific band (laccase) with a molecular weight of about 28KD protein appears in each of examples 10, 11, 12 and 13, wherein the laccase expression level of example 13 is higher than that of examples 10, 11, 12 and 9.
Test five: effect of different Induction Medium on laccase expression
Example 14
S1, inoculating a recombinant strain BL21/pET28a-Lac into a liquid culture medium containing 30 mug/mL of calicheamicin, and culturing at 35 ℃ overnight for 16 hours;
s2, inoculating the bacterial liquid obtained in the S1 into a special induction culture medium to induce the efficient expression of the SDB2 laccase gene at 37 ℃;
s3, centrifuging at 4000rpm for 10min after the induction culture is finished, and collecting thalli;
s4, adding Tris-NaCl buffer into the collected thalli for suspension, performing ultrasonic crushing, and centrifugally collecting supernatant for sample preparation;
s5, verifying the recombinant laccase protein by SDS-PAGE;
in S2, the concentration of IPTG in the induction medium was 1.0mM/L, cu 2+ At a concentration of 6mM/L, mn 2+ At a concentration of 6mM/L, fe 2+ At a concentration of 4mM/L, ca 2+ The concentration is 2mM/L, the concentration of the vegetable pulp juice is 1g/L, the concentration of the white mustard juice is 1g/L, and the concentration of the cotton seed pulp juice is 0.5g/L.
Example 15
Example 15 is the same as example 14 except that the induction medium of example 15 does not contain rapeseed meal, white mustard seed, and cotton seed meal, but contains peptone at a concentration of 2.5 g/L (i.e., total concentration of rapeseed meal, white mustard seed, and cotton seed meal).
Example 16
Example 16 is the same as example 14 except that in the induction medium of example 16, the metal salt contains only CuCl 2 And Cu is 2+ Is 18mM/L (i.e., the total concentration of each metal ion of example 14).
Example 17
Example 17 is the same as example 14 except that the induction medium of example 17 is a modified LB medium having the following composition: IPTG at a concentration of 1.0mM/L and CuCl at a concentration of 18mM/L 2 Peptone at a concentration of 2.5 g/L and yeast extract at a concentration of 1g/L.
The test results are shown in FIG. 6:
(1) When the rapeseed meal juice, the white mustard seed juice and the cotton seed meal juice were replaced with peptone (example 15), the laccase expression level was significantly lower than in example 14.
(2) When the metal salt is CuCl only 2 In this case (example 16), the laccase expression level was significantly lower than in example 14.
(3) When the induction medium was a modified LB medium (example 17), the laccase expression level was significantly lower than in example 14.
3. Application effect of recombinant strain BL21/pET28a-Lac in degrading polyphenol
Control group: untransformed blank strain (e.coli BL 21);
test group: recombinant strain BL21/pET28a-Lac;
two strains of bacteria of the control group and the test group were inoculated into the induction medium prepared in example 14, and cultured at 37℃for 48 hours on an air shaker. The initial concentration of the two groups of polyphenols is the same and is recorded as 100%, the content of the polyphenols in the two groups of culture solutions of 8h, 16h, 24h, 32h, 40h and 48h after culture is measured and converted into the relative concentration, and the degradation effect of the recombinant strain on the polyphenols at different culture times is analyzed, and is shown in figure 6.
As shown in FIG. 6, the recombinant strain BL21/pET28a-Lac with high laccase yield obtained by the invention can be cultured for 48 hours only to reduce the relative concentration of polyphenol to 38%, which shows that the recombinant strain BL21/pET28a-Lac has good enzyme source characteristics.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (5)
1. A method for efficiently expressing laccase by recombinant escherichia coli is characterized by comprising the following steps:
s1, connecting a laccase gene with a prokaryotic expression vector pET28a to construct a recombinant plasmid pET28a-Lac, wherein the nucleotide sequence of the laccase gene is SEQ ID NO.1;
s2, converting the recombinant plasmid pET28a-Lac into competent cells of the escherichia coli BL21 for culture, and screening out recombinant strains BL21/pET28a-Lac which are identified as positive by PCR;
s3, inoculating the recombinant strain BL21/pET28a-Lac into a liquid culture medium for culture, and inoculating the obtained bacterial liquid into an induction culture medium for inducing laccase gene expression; wherein, the composition of the induction culture medium is as follows: IPTG at 0.2-1 mM/L, cuCl at 1-10 mM/L 2 MnSO of 1-10 mM/L 4 FeSO of 1-10 mM/L 4 CaCl of 1-10 mM/L 2 0.1-2 g/L of rapeseed meal juice, 0.1-2 g/L of white mustard seed juice and 0.1-2 g/L of cottonseed meal juice.
S4, centrifugally collecting thalli after the induction culture is finished, and suspending, crushing and centrifuging thalli to obtain laccase protein.
2. The method for efficiently expressing laccase by recombinant escherichia coli according to claim 1, wherein the liquid culture medium is a liquid culture medium containing 30+/-5 mug/ml of calicheamicin.
3. The method for efficiently expressing laccase by recombinant E.coli according to claim 1, wherein in the step S1, the laccase gene is subjected to double digestion by Nde l and Xho l, and then is connected with an expression vector pET-28a (+) subjected to double digestion by Nde l and Xho l by DNA ligase, so as to construct a recombinant plasmid pET28a-Lac.
4. The method for efficiently expressing laccase by recombinant escherichia coli according to claim 1, wherein in the step S4, tris-NaCl buffer is added into the collected thalli for suspension, ultrasonic crushing is carried out, and a supernatant sample is collected by centrifugation, so that laccase protein is obtained.
5. A recombinant strain is characterized in that the recombinant strain is a recombinant strain BL21/pET28a-Lac prepared by the method of any one of claims 1-4, and the recombinant strain is used for degrading polyphenols in plants.
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