CN114891757B - A directed evolution method of laccase and mutant laccase gene, expression vector, recombinant strain and its application in detoxifying aflatoxin - Google Patents

Abstract

The invention relates to the technical field of bioengineering and discloses a laccase directed evolution method, a mutant laccase gene, an expression vector, a recombinant strain and its application in detoxifying aflatoxin. The amino acid sequence of the laccase is as SEQ ID No: 1 As shown, the amino acid at position 235 in SEQ ID No: 1 is changed from Ser to Pro to obtain a mutant laccase, and the amino acid sequence of the mutant laccase is as shown in SEQ ID No: 3. The beneficial effect of the present invention is that: based on the laccase heterologously expressed in Pichiapastoris, the present invention constructs a "small and precise" laccase through heterologous expression of the mutated laccase gene in P. pastoris through site-directed saturation mutation. Saturated mutant library; finally, a recombinant Pichia pastoris mutant strain was obtained through screening. The efficiency of detoxifying AFB1, the enzyme protein produced by fermentation, was increased to 1.34 times. The mutant protein can be used in dry distiller's grains and its production during the production of bioethanol. Simultaneous detoxification of AFB1 in soluble matter (DDGS).

Description

A directed evolution method of laccase and mutant laccase gene, expression vector and recombinant strain and its application in detoxifying aflatoxins

Technical field

The invention relates to the field of bioengineering technology, and in particular to a laccase directed evolution method, mutant laccase gene, expression vector, recombinant strain and its application for detoxifying aflatoxin.

Background technique

Aflatoxins (AFs) are a type of mycotoxin produced by Aspergillus flavus , Aspergillus nominus , and Aspergillus paraciticus . They mainly contaminate some crops, such as peanuts, corn, and wheat. etc., causing great economic losses to agricultural production in various countries around the world.

AFB1 can be degraded through physical, chemical and biological methods, but physical methods are time-consuming and cannot guarantee that AFB1 is completely removed. Although the use of chemical substances can significantly reduce the concentration of AFB1, it also leads to the loss of nutrients and reduces the quality of food or feed; Directly using microorganisms to degrade AFB1 will also damage the sensory properties of the product, and the safety of the degradation process cannot be guaranteed.

Biological detoxification, as one of the detoxification methods, is deeply favored by people because of its green, environmental protection, safety and other characteristics. Among them, enzymes have the characteristics of substrate specificity, high catalytic efficiency, and environmental friendliness, and are widely used in the food and feed industry. Therefore, the safest and most effective method is to use specific enzymes to degrade AFB1.

Laccase (ρ-diphenol oxidase, EC 1.10.3.2) is a copper-containing polyphenol oxidase with a broad substrate spectrum. In 2009, Alberts et al. first discovered that fungal laccase can be used to detoxify aflatoxins. However, there are currently few studies on directed evolution to improve the efficiency of laccase detoxification of AFB1. Therefore, research on directed evolution to improve the efficiency of laccase detoxification of AFB1 has great research significance and practical application value.

Contents of the invention

The technical problem to be solved by the present invention is how to provide a directed evolution method of laccase and mutant laccase genes, expression vectors, recombinant strains and their applications for detoxifying aflatoxin, which utilizes site-directed saturation mutation technology to achieve the target laccase. Through targeted transformation, the mutant laccase obtained can improve the efficiency of laccase detoxification of AFB1, and has great practical application value in the simultaneous detoxification of aflatoxin in dry distiller's grains and its solubles during the production of bioethanol.

The present invention solves the above technical problems through the following technical means:

A first aspect of the present invention proposes a mutant laccase. The amino acid sequence of the laccase is as shown in SEQ ID No: 1. The amino acid at position 235 in SEQ ID No: 1 is changed from Ser to Phe to obtain a mutant laccase. The amino acid sequence of the mutant laccase is shown in SEQ ID No: 3.

Beneficial effects: The present invention utilizes fixed-site saturation mutation technology to achieve directional transformation of the target laccase, and obtains a mutant laccase with improved detoxification efficiency for AFB1. Its detoxification efficiency is increased to 1.34 times, and can be used for aflatoxin removal. poison.

Preferably, the nucleotide sequence encoding the laccase amino acid is shown in SEQ ID No: 2; the nucleotide sequence encoding the mutant laccase amino acid is shown in SEQ ID No: 4.

A second aspect of the present invention proposes a recombinant expression vector containing the above mutant laccase gene.

Preferably, the recombinant expression vector is the pPIC9K- mlcc5 expression vector obtained by connecting the mutant laccase gene to pPIC9K.

The third aspect of the present invention proposes a recombinant strain containing the above recombinant expression vector.

Preferably, the recombinant strain is P. pastoris -pPIC9K- mlcc5 obtained by transforming the recombinant expression vector into Pichia pastoris.

The fourth aspect of the present invention proposes a method for obtaining the above-mentioned recombinant strain, including the following steps:

(1) Extract laccase gene template: Cultivate and collect Escherichia coli Trans1 T1-pPIC9K- lcc5 , and extract the pPIC9K- lcc5 plasmid;

(2) Cloning the mutant gene: Using the pPIC9K- lcc5 plasmid as a template, perform saturation mutations on the potential beneficial mutation sites obtained by molecular dynamics simulation and sequence conservation analysis, and use PCR amplification to obtain the mutant mlcc5 gene;

(3) Construct a recombinant expression vector: insert the mutant mlcc5 gene into the downstream of pPIC9K promoter AOX1 through enzyme digestion ligation method to construct a pPIC9K- mlcc5 expression vector;

(4) Obtain the recombinant strain: transform the pPIC9K- mlcc5 expression vector into Trans1 T1 competent cells to obtain Trans1 T1-pPIC9K- mlcc5 ;

Restriction endonuclease Sac I was used to linearize the pPIC9K- mlcc5 plasmid, and the linearized expression vector was transferred into the host Pichia pastoris through electroporation transformation to obtain P. pastoris -pPIC9K- mlcc5 ;

(5) Screen the saturated mutation library: use P. pastoris -pPIC9K and wild-type P. pastoris -pPIC9K- lcc 5 as the control group, and transfer the control group and the P. pastoris -pPIC9K- mlcc5 to BMGY-containing medium respectively Cultured in a 96-well plate, and then added BMM medium to induce the expression of mutant laccase; and used AFB1 as a substrate to determine the detoxification efficiency, and screened the mutant laccase P. pastoris -pPIC9K- mlcc5 whose AFB1 detoxification efficiency was better than that of the control group.

Beneficial effects: The present invention is based on the laccase heterologously expressed in Pichia pastoris , through site-directed saturation mutation, and heterologously expresses the mutated laccase gene in P. pastoris to construct a "small but refined" saturated mutant. Library; Finally, a recombinant Pichia pastoris mutant strain was obtained through screening. The efficiency of detoxifying AFB1 in the enzyme protein produced by its fermentation was increased to 1.34 times. The mutant protein can be used in dry distiller's grains and its solubles in the production of bioethanol. Synchronized detoxification of AFB1 in (DDGS).

Preferably, the enzyme digestion and ligation method in step (3) includes: using two restriction endonucleases, Sna B Ⅰ and Not Ⅰ, to double-enzyme digest the mutant mlcc5 gene and pPIC9K empty vector respectively, and then ligate through T4 Enzymes link the vector to the mutated gene.

The fifth aspect of the present invention proposes an application of the above recombinant strain in aflatoxin detoxification, which includes the following steps: using AFB1 as a substrate, adding ethanol, mutant laccase fermentation broth, and citric acid-Na with pH=4.0 thereto ₂ HPO ₄ buffer, react at 30~32 ℃, 800~1000 rpm for 20~24 hours; among them, the final concentration of AFB1 is 0.1~0.15 μg/mL, the final concentration of ethanol is 15~20%, and the final concentration of enzyme solution is 0.2 ~0.25 U, citric acid-Na ₂ HPO ₄ buffer to make up the balance.

The sixth aspect of the present invention proposes the application of the above-mentioned recombinant strain in the simultaneous detoxification of aflatoxin in dried distiller's grains and its solubles during the preparation of bioethanol.

The advantages of the present invention are:

1. This application uses site-directed saturation mutation technology to achieve targeted transformation of the target laccase;

2. This application obtained a mutant laccase that improves the detoxification efficiency of AFB1, and its detoxification efficiency is increased to 1.34 times;

3. The mutant laccase obtained in this application can be used for aflatoxin detoxification.

Description of the drawings

Figure 1 is a diagram showing the amplification results of detecting saturated mutant genes using agarose gel electrophoresis in Example 1 of the present application.

Figure 2 is a schematic diagram of the construction of the mutant gene expression vector in Example 1 of the present application.

Figure 3 is a diagram of substrate screening results in Example 1 of the present application.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the present invention. Examples, not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

The test materials and reagents used in the following examples can all be obtained from commercial sources unless otherwise specified.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in literature in the field can be followed or the product instructions can be followed.

Example 1

This embodiment provides a laccase directed evolution method, mutant laccase gene, expression vector, recombinant strain and its application for detoxifying aflatoxin, including the following steps:

1. Extract laccase gene template

Take out the Escherichia coli Trans1 T1-pPIC9K- lcc5 from the Anhui Provincial Key Laboratory of Modern Biomanufacturing, streak it on the LB plate medium (containing AMP), and culture it in an incubator at 37°C for 8 to 12 hours; then pick Take a single clone, inoculate it into a test tube containing LB liquid culture medium (containing AMP), and culture it on a shaking table at 37°C overnight; collect the bacterial cells, and extract the pPIC9K- lcc5 plasmid according to the instructions of the plasmid extraction kit to obtain the laccase gene template.

After determination, the amino acid sequence of laccase is shown in SEQ ID No: 1, and the nucleotide sequence encoding the laccase amino acid is shown in SEQ ID No: 2.

2. Clone mutant genes

Using the lcc5 plasmid as the laccase gene template, SnapGene software was used to design primers. The primer sequences are shown in Table 1. The codons preferred by Pichia pastoris were selected, and the potential beneficial mutation sites obtained by molecular dynamics simulation and sequence conservation analysis were separately analyzed. Saturation mutation. The mutated mlcc5 gene was obtained by PCR amplification. For the PCR amplification operation, refer to the instructions of PrimeSTAR® HSDNA Polymerase. The reaction conditions of each reaction program are shown in Table 2. The dosage of each component in the reaction system is shown in Table 3.

Table 1 Primer design

Table 2 PCR reaction conditions

Table 3 PCR reaction system (50 μL)

After PCR amplification, agarose gel electrophoresis was used to detect the amplification of the saturated mutant gene. The results are shown in Figure 1. Figure 1A shows the amplification results of the upstream and downstream fragments of the mutation site. Figure 1B shows the amplification results of the full-length mutant gene. The amplification result showed that the size of the amplified band was correct.

3. Construct recombinant expression vector

① As shown in Figure 2, two restriction endonucleases, Sna B Ⅰ and Not Ⅰ, were used to double-digest the mutant mlcc5 gene and pPIC9K empty vector obtained by PCR amplification. Each group in the double-digestion reaction system The dosage is shown in Table 4. Enzyme digestion was carried out at 37°C for 60 minutes. After the enzyme digestion is completed, the product is purified and recovered using 1% nucleic acid gel.

Table 4 Double enzyme digestion reaction system (50 μL)

② Under the action of T4 ligase, insert the digested mutant mlcc5 gene into the downstream of pPIC9K empty promoter AOX1 at a molar ratio of 1:6, and ligate at 22°C for 60 minutes to construct pPIC9K- mlcc5 expression vector.

4. Obtain recombinant strains

①Transform the constructed pPIC9K- mlcc5 expression vector into Trans1 T1 competent cells to obtain Trans1T1-pPIC9K- mlcc5 .

② Cultivate Trans1 T1-pPIC9K- mlcc5 at 37°C for 8~12 hours, and extract the pPIC9K- mlcc5 plasmid; as shown in Figure 2, then use restriction endonuclease Sac I to linearize the pPIC9K- mlcc5 plasmid, the conditions are: React at 37°C for 60 minutes; then transfer the linearized expression vector into the host P. pastoris ( P. pastoris ) through electroporation transformation method to obtain P. pastoris -pPIC9K- mlcc5 .

5. Screen saturation mutation library

①Transfer a single clone of P. pastoris -pPIC9K- mlcc5 from the storage tube into a 96-well plate containing 100 mL BMGY medium, and culture the bacteria at 28°C and 800 rpm for 24 hours.

② Use P. pastoris-pPIC9K and wild-type P. pastoris -pPIC9K- lcc 5 as the control group. Transfer the cultured control group and P. pastoris -pPIC9K- mlcc5 to 96 wells containing 800 μL BMGY medium using printed membranes. Culture in plates, make three parallel samples of each group of samples, try to ensure that the inoculation amount of each plate is consistent, and culture at 28°C and 800 rpm for 24 h.

③Centrifuge the above cultured samples at 600 rpm for 15 min, remove the supernatant, then add 800 μL BMM medium to each well, induce culture for 24 h, and express the control strain and mutant laccase.

④ Use aflatoxin B1 (AFB1) as the substrate, add ethanol, the induced control group or mutant laccase fermentation broth diluted 1 to 5 times, and citric acid-Na ₂ HPO ₄ buffer with pH=4.0, and react. The total volume of the system is 200 μL, in which the final concentration of AFB1 is 0.1 μg/mL, the final concentration of ethanol is 15%, the final concentration of the enzyme solution is 0.2 U, and the citric acid-Na ₂ HPO ₄ buffer is used to make up the volume to 200 μL.

Each reaction system was reacted at 32°C and 800 rpm for 24 hours, and the detoxification efficiency of AFB1 in each reaction system was compared with the wild-type detoxification rate multiple. The multiple can be expressed by the formula: detoxification rate multiple = mutant laccase detoxification rate Toxicity rate/wild-type laccase detoxification rate, the measurement results are shown in Figure 3.

According to Figure 3, P. pastoris -pPIC9K- mlcc5, which has a 1.34-fold improvement in AFB1 detoxification efficiency compared to the control group, was selected as the target mutant laccase . After determination, the amino acid sequence of the screened mutant laccase is shown in SEQ ID No: 3, and the nucleotide sequence encoding the amino acid of the mutant laccase is shown in SEQ ID No: 4.

6. Application of recombinant strains in the simultaneous detoxification of AFB1 in distillers dried grains and solubles (DDGS) during the production of bioethanol

Using DDGS in the bioethanol preparation process as the substrate, ethanol, the screened mutant laccase culture solution, and citric acid- _{Na 2} HPO ₄ buffer with pH = 4.0 were added to it. The total volume of the reaction system was 200 μL. Among them, the final concentration of AFB1 is 0.1 μg/mL ^-1 , the final concentration of ethanol is 15%, the final concentration of enzyme solution is 0.2 U, and the citric acid-Na ₂ HPO ₄ buffer is added to the volume to 200 μL. The reaction system was reacted at 32°C and 800 rpm for 24 h, and the detoxification efficiency of AFB1 in the reaction system was determined to be 16%.

Example 2

This embodiment provides a laccase directed evolution method, mutant laccase gene, expression vector, recombinant strain and its application for detoxifying aflatoxin, including the following steps:

Steps 1-5 are the same as in Example 1.

6. Application of recombinant strains in the simultaneous detoxification of AFB1 in distillers dried grains and solubles (DDGS) during the production of bioethanol

Using DDGS in the bioethanol preparation process as the substrate, ethanol, the screened mutant laccase culture solution, and citric acid- _{Na 2} HPO ₄ buffer with pH = 4.0 were added to it. The total volume of the reaction system was 200 μL. Among them, the final concentration of AFB1 is 0.13 μg/mL ^-1 , the final concentration of ethanol is 18%, the final concentration of enzyme solution is 0.23 U, and the citric acid-Na ₂ HPO ₄ buffer is added to the volume to 200 μL. The reaction system was reacted at 31°C and 900 rpm for 22 h, and the detoxification efficiency of AFB1 in the reaction system was determined to be 16.2%.

Example 3

This embodiment provides a laccase directed evolution method, mutant laccase gene, expression vector, recombinant strain and its application for detoxifying aflatoxin, including the following steps:

Steps 1-5 are the same as in Example 1.

6. Application of recombinant strains in the simultaneous detoxification of AFB1 in distillers dried grains and solubles (DDGS) during the production of bioethanol

Using DDGS in the bioethanol preparation process as the substrate, ethanol, the screened mutant laccase culture solution, and citric acid- _{Na 2} HPO ₄ buffer with pH = 4.0 were added to it. The total volume of the reaction system was 200 μL. Among them, the final concentration of AFB1 is 0.15 μg/mL ^-1 , the final concentration of ethanol is 20%, the final concentration of enzyme solution is 0.25 U, and the citric acid-Na ₂ HPO ₄ buffer is added to the volume to 200 μL. The reaction system was reacted at 30°C and 1000 rpm for 20 h, and the detoxification efficiency of AFB1 in the reaction system was determined to be 16.5%.

Comparative example 1

In this comparative example, P. pastoris bacteria containing empty pPIC9K were used to simultaneously detoxify AFB1 in distillers dried grains and solubles (DDGS) during the production of bioethanol.

Use DDGS as the substrate in the bioethanol preparation process, add ethanol, P. pastoris strain containing pPIC9K empty, citric acid-Na ₂ HPO ₄ buffer with pH=4.0, and the total volume of the reaction system is 200 μL. Among them, the final concentration of AFB1 is 0.1 μg/mL ^-1 , the final concentration of ethanol is 15%, the final concentration of enzyme solution is 0.2 U, and the citric acid-Na ₂ HPO ₄ buffer is added to the volume to 200 μL. The reaction system was reacted at 32°C and 800 rpm for 24 h, and the detoxification efficiency of AFB1 in the reaction system was determined to be 0%.

Comparative example 2

In this comparative example, the wild-type P. pastoris -pPIC9K- lcc 5 strain was used to simultaneously detoxify AFB1 in distillers dried grains and solubles (DDGS) during bioethanol preparation.

Use DDGS in the bioethanol preparation process as the substrate, add ethanol, wild-type P. pastoris -pPIC9K- lcc 5 strain liquid, and citric acid-Na ₂ HPO ₄ buffer with pH=4.0. The total volume of the reaction system is 200 μL. . Among them, the final concentration of AFB1 is 0.1 μg/mL ^-1 , the final concentration of ethanol is 15%, the final concentration of enzyme solution is 0.2 U, and the citric acid-Na ₂ HPO ₄ buffer is added to the volume to 200 μL. The reaction system was reacted at 32°C and 800 rpm for 24 h, and the detoxification efficiency of AFB1 in the reaction system was determined to be 12%.

The implementation principle of this application is: the present invention is based on the laccase heterologously expressed in Pichia pastoris , through site-directed saturation mutation, and heterologously expresses the mutated laccase gene in P. pastoris to construct a "small but precise" A saturated mutant library; a recombinant Pichia pastoris mutant strain was finally obtained through screening. The efficiency of detoxifying AFB1, an enzyme protein produced by fermentation, was increased to 1.34 times. The mutant protein can be used in dry distiller's grains and in the production of bioethanol. Simultaneous detoxification of AFB1 in its soluble form (DDGS).

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the foregoing embodiments. The recorded technical solutions may be modified, or some of the technical features thereof may be equivalently replaced; however, these modifications or substitutions shall not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of each embodiment of the present invention.

SEQUENCE LISTING

<110> Anhui University

<120> A laccase directed evolution method and mutant laccase gene, expression vector, recombinant strain and detoxified yellow yeast

Mycotoxin applications

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<400> 4

atcattggca accaagatac tatgaccatc tctaatatta atgctggccc agatggcttc 60

accagacctg tcatcgcggt caatggcgaa ttcccttctc ccctcgttag ggccaacaag 120

ggcgacgact ttcgaattaa cgtcgtgaac aatttggacg atgacactat gcttcgtcaa 180

accagtgttc actggcacgg tgtcttccag caccagtctg cttgggctga tggcccggat 240

ggtgttatactc agtgtcccat tccccaatca ggccaagagt tcgaatatgc attcaatgca 300

ggacaagagg caggcacatt ctggtaccat tctcattacg gtactcaata ctgcgatggc 360

ctccgtggtc ctttggtcat ctacgatccc gaagatcctc atcaggatct ctacgatgtc 420

gacgacgaga atactattat caccttggcc gattggtacc atctccaagc gccttccatc 480

caaggccctg ctgtctctca ggcgaccctc attaatggaa agggccgacg acctggcagc 540

accgaaggcg acatcgccgt cgtgaacgtc gagaaggact cgaggtaccg attccgcatc 600

gtttcccttt cctgcgaccc cgactacacg ttctcgattg ataaccacac catgaccatc 660

attgaagctg acgggcaaaa caccaagccc ctcgaggtcg agtttatcag agtcttcgct 720

ggacaaaggt actctgtcgt cgtgaacgct gaccaggcta tcggcaacta ctggatccgt 780

gctgagccca acatcggtga cactggactg gttggaacca gcggaggtgg tgtcaactcc 840

gccatcctgc gctatgccac cgccgacgag gtcgagcccg atacaccaag gcttaccaac 900

cgacccgcct tgcaagagtc gaacctccgc gcattgactt ccggtgtccc aggaggcgat 960

ggtcctgctg atattacctt caccttcaac ctcggcctca acttcgcaac tggcacattc 1020

tccatgaacc ctggcgagag ctgggtccac cctgacactc ctgtcatggt ccagatcatg 1080

aatggcgttc ctgccgagga tctcgttccc gctgagtccc tccatactat tactcgcggc 1140

caagttgtgg aagttgtcat cccacccttc ggtatcgctg gacctcatcc tttccatctc 1200

cacggtcacg ccttcagcgt tatcaagagc gccggtggtt ctcccaactt cgtcgaccca 1260

gtccgccgtg acgtggtcgc tgttggtact gaagctggac agggagacac tattatccga 1320

ttcgttgctg ataacccagg accgtggttc ttccattgcc acatcgagtt ccaccttgtc 1380

accggtttgg ctgctgtctt catggaagcc cccgacgaga tcgccagcag caacccccct 1440

cctccctcgt gggatgccct gtgccctgct ttctctgctc ttcctccctc ggccacgagc 1500

atccgcattg tccccaccccc tactccctaa 1530

Claims (8)

1. A mutant laccase, characterized in that: the amino acid sequence of the mutant laccase is shown in SEQ ID No: 3. 2. A gene encoding a mutant laccase as claimed in claim 1, characterized in that: the nucleotide sequence encoding the amino acid of the mutant laccase is as shown in SEQ ID No: 4. 3. A recombinant expression vector containing the mutant laccase gene of claim 2. 4. The recombinant expression vector according to claim 3, characterized in that: the recombinant expression vector is the pPIC9K- mlcc5 expression vector obtained by connecting the mutant laccase gene described in claim 3 to pPIC9K. 5. A recombinant strain containing the recombinant expression vector according to claim 3. 6. The recombinant strain according to claim 5, characterized in that: the recombinant strain is P. pastoris -pPIC9K- mlcc5 obtained by transforming the recombinant expression vector described in claim 3 into Pichia pastoris. 7. The application of a recombinant strain in aflatoxin detoxification as claimed in claim 5, characterized in that: comprising the following steps: Use AFB1 as the substrate, add ethanol, mutant laccase fermentation broth, and citric acid-Na ₂ HPO ₄ buffer with pH=4.0, and react at 30~32°C and 800~1000 rpm for 20~24 h; among them, AFB1 The final concentration is 0.1~0.15 μg/mL, the final concentration of ethanol is 15~20%, the final concentration of enzyme solution is 0.2~0.25 U, and citric acid-Na ₂ HPO ₄ buffer is used to make up the balance. 8. Application of a recombinant strain according to claim 5 in the simultaneous detoxification of aflatoxin in dried distiller's grains and its solubles during the preparation of bioethanol.