CN112430611A

CN112430611A - Optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant thallus, surface display system and application

Info

Publication number: CN112430611A
Application number: CN202011381340.5A
Authority: CN
Inventors: 陈书容; 潘力; 王斌
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-03-02

Abstract

The invention discloses an optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant bacteria, a surface display system and application. The nucleotide sequence of the zearalenone degrading enzyme coding gene is shown as SEQ ID No. 1. The coding gene can realize high-efficiency heterologous expression in escherichia coli. The recombinant zearalenone degrading enzyme has the advantages of high enzyme activity, wide temperature and pH application range, good tolerance and the like, can efficiently degrade Zearalenone (ZEN), and has a good industrial application prospect. According to the invention, zearalenone degrading enzyme ZHD-P is displayed on the cell surface of escherichia coli BL21 in a positioning manner, and the detection of the activity of the whole-cell enzyme shows that the display expression of the zearalenone degrading enzyme ZHD-P on the cell surface is successfully realized, so that the whole-cell catalyst which is not required to be purified, can be repeatedly utilized and has low cost is obtained, and the whole-cell catalyst is used for biodegradation of zearalenone and has a good industrial application prospect.

Description

Optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant thallus, surface display system and application

Technical Field

The invention relates to the field of genetic engineering, in particular to an optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant bacteria, a surface display system and application.

Background

Zearalenone (ZEN, ZEA), also known as F-2 toxin, is an estrogen mycotoxin with a dihydroxybenzoic acid lactone structure, has strong heat resistance, and can be completely destroyed after being treated at 110 ℃ for 1 hour. Research shows that ZEN enters a food chain through polluted grain agricultural and sideline products and feed, and after being absorbed by animals, the ZEN causes the estrogen comprehensive symptoms of livestock, causes the phenomena of infertility, abortion and stillbirth in the bodies of the livestock, has strong carcinogenicity, and seriously harms the health of the livestock and human beings.

ZEN is a mycotoxin with the widest pollution range in the world, and fusarium capable of producing ZEN comprises fusarium graminearum, fusarium trilinear, fusarium flavum, fusarium kluyverum and the like; fusarium can infect various grain crops, so the ZEN is detected in grains such as wheat, barley, corn, oat, sorghum, sesame, millet, rice and the like. Currently, worldwide crop and animal husbandry losses due to ZEN contamination are as high as millions of dollars each year, and zearalenone is detected in grains in most countries of the world. The pollution condition of zearalenone in China is also not optimistic, the climate in many areas of China is humid, and grains stored, stored and processed are very easily polluted by ZEN. In the investigation of the Wang Ruojun and the like on 109 feeds and ZEN in feed raw materials in North China, China and south China, the detection rate of ZEN in complete feed and corn feed reaches 100 percent and respectively exceeds 21.4 percent and 30.8 percent. After researching the pollution condition of mycotoxin in feed and feed raw materials in China, the AoShi steel and the like discover that ZEN becomes one of the mycotoxins with the highest overproof rate and detection level in the complete feed and feed raw materials in China. Therefore, in China, the pollution of ZEN has become a considerable problem in the agricultural production process, and more effort is required to research the pollution.

The study of ZEN detoxification methods and techniques is the main measure to cope with ZEN contamination. The main methods for detoxifying ZEN at present are a physical method, a chemical method and a biological method. The detoxification efficiency by a physical method is low, and the nutritional ingredients of the grains are damaged, so that the taste of the food is influenced; the chemical method can effectively detoxify, but how to remove chemical components for detoxification from grain food is difficult, and secondary pollution is easy to cause. And the biological degradation can efficiently convert ZEN into low-toxicity or non-toxic products, is environment-friendly and safe, and cannot damage the nutrient substances of grains, so that the development of the detoxification technology of ZEN has great significance. The biological technology for degrading and detoxifying ZEN and derivatives thereof is a main method for solving the problem in the future.

The ZEN biodegradation mainly comprises a live bacteria degradation method and an enzyme degradation method. The live bacteria degradation method is to utilize the transformation of ZEN by microorganisms so as to reduce the toxicity of ZEN to animals. However, live bacteria detoxification has the problems of long strain culture time, low degradation activity, difficulty in removing the strain in the later period and the like, and even some strains are pathogenic bacteria. Therefore, the research on the structure and the property of the ZEN degrading enzyme and the degradation mechanism has important significance, and the development of the ZEN degrading enzyme is the development trend of the future ZEN degradation. The enzyme degradation not only can efficiently convert toxin into a non-toxic product, is safe and environment-friendly, but also has strong specificity of enzyme catalytic reaction and high degradation efficiency, and can not damage the nutrient substances of grains.

At present, relatively few zearalenone degrading enzymes have been found. In addition, most of the ZEN degrading enzymes have the defects of low activity and relatively limited applicable range of temperature and pH, thereby limiting the application. If an enzyme capable of degrading ZEN more efficiently is obtained, a feasible technology and a feasible process are provided for the biodegradation of ZEN, and the method has important significance on feed safety and food safety.

The basic principle of microbial cell surface display technology is to introduce foreign protein or polypeptide gene and host specific anchoring protein gene into host through DNA recombination technology, and to utilize the positioning effect of the anchoring protein to express the foreign protein or polypeptide onto the cell surface. The zearalenone degrading enzyme is displayed on the cell surface in a positioning manner by utilizing a microbial cell surface display technology, compared with immobilization operation, the free enzyme can realize repeated recycling of the enzyme, a series of complex operations such as ultrasonic crushing, chromatography purification and the like are not needed, the cost is reduced, and the method has certain operation stability and cost benefit and is potential for industrial scale production.

The literature reports that the zearalenone degrading enzymes which are characterized are Zhd101, ZENJJM and ZLhy-6, the original genes of the zearalenone degrading enzymes are all derived from Gliocladium roseum, and the properties of the zearalenone degrading enzymes are basically consistent. According to the report of Delfina Popiel et al, the original gene containing zearalenone degrading enzyme ZHD-P invades Trichoderma, the efficiency of live bacteria degrading ZEN is very low, the ZEN degradation rate is less than 15% after 4 days of culture, and the ZEN degradation rate is about 50% after 6 days of culture. The method has the advantages that the method utilizes the Trichoderma inhabiting containing the zearalenone degrading enzyme ZHD-P original gene to detoxify live bacteria, has the problems of long strain culture time, low degradation activity, difficulty in removing later strains and the like, and has little significance for practical application. At present, no experiment proves that the original gene of the zearalenone degrading enzyme ZHD-P can be heterologously expressed in other hosts, and no literature is available for carrying out extraction and purification and research on the enzymological properties of the enzyme. Therefore, if the zearalenone degrading enzyme ZHD-P gene can be optimized to realize heterologous high-efficiency expression and purify the zearalenone degrading enzyme, the method has important significance for the research and the application of the property and the degradation mechanism of the enzyme.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide an optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant bacteria, a surface display system and application.

The invention provides an optimized encoding gene of recombinant zearalenone degrading enzyme ZHD-P capable of realizing heterologous expression in escherichia coli BL21, a recombinant expression vector containing the gene, a recombinant strain containing the gene and a genetic engineering method for preparing the recombinant zearalenone degrading enzyme ZHD-P. The invention further provides a surface display vector pET22b (+) -pgsa-zhd-P of the optimized zearalenone degrading enzyme ZHD-P coding gene, a surface display system containing the surface display vector, a method for preparing the recombinant strain and application of the recombinant strain.

The purpose of the invention is realized by at least one of the following technical solutions.

The zearalenone degrading enzyme ZHD-P original gene is derived from Trichoderma aggresivum.

The invention provides an optimized zearalenone degrading enzyme ZHD-P coding gene nucleotide sequence (zearalenone lactohydrolase-DNA (optimized)) shown as SEQ ID NO.1, and the coding gene can realize heterologous high-efficiency expression in escherichia coli BL 21.

The optimized zearalenone degrading enzyme ZHD-P coding gene has the total length of 795 bp.

The nucleotide sequence of the optimized zearalenone degrading enzyme ZHD-P coding gene provided by the invention is shown in SEQ ID No. 1. The coding gene can be heterologously expressed in escherichia coli BL21 to obtain recombinant zearalenone degrading enzyme ZHD-P.

The zearalenone degrading enzyme ZHD-P (zearalenone lactohydrolase-PRT) provided by the invention has an amino acid sequence shown in SEQ ID NO. 2.

The recombinant expression vector provided by the invention comprises a pET22b (+) vector and an optimized zearalenone degrading enzyme ZHD-P encoding gene as shown in SEQ ID NO. 1. The optimized sequence of the zearalenone degrading enzyme ZHD-P coding gene is inserted between proper restriction sites of an expression vector pET22b (+), so that the nucleotide sequence of the optimized sequence is connected with other expression elements to obtain a recombinant expression vector pET22b (+) -zhd-P.

The recombinant strain provided by the invention contains the recombinant expression vector; the host cell of the recombinant strain is Escherichia coli BL 21.

The optimized zearalenone degrading enzyme ZHD-P coding gene and the application of the recombinant expression vector in heterologous high-efficiency expression of the recombinant zearalenone degrading enzyme ZHD-P in large intestine BL21 are provided by the invention.

The optimized zearalenone degrading enzyme ZHD-P coding gene and the application of the recombinant expression vector in heterologously and efficiently expressing recombinant zearalenone degrading enzyme ZHD-P in large intestine BL21 provided by the invention comprise the following steps:

(1) constructing a recombinant expression vector of zearalenone degrading enzyme ZHD-P;

(2) transforming host cells by using the recombinant expression vector constructed in the step (1) to obtain a recombinant strain;

(3) culturing a recombinant strain, and inducing the recombinant strain to express the recombinant zearalenone degrading enzyme ZHD-P by IPTG;

(4) recovering and purifying the recombinant zearalenone degrading enzyme ZHD-P (zearalenone lactohydrolase-PRT).

The recombinant strain provided by the invention is applied to preparation of recombinant zearalenone degrading enzyme ZHD-P. The recombinant zearalenone degrading enzyme ZHD-P can be applied to degrading mycotoxin Zearalenone (ZEN).

The invention inserts the encoding anchoring protein gene pgsa and the optimized zearalenone degrading enzyme ZHD-P encoding gene in series between proper restriction enzyme sites of an expression vector pET22b (+) to ensure that the nucleotide sequence of the encoding anchoring protein gene pgsa and the optimized zearalenone degrading enzyme ZHD-P encoding gene is connected with other expression elements to obtain a recombinant surface display vector pET22b (+) -pgsa-zhd-P.

The invention provides a construction method of an optimized surface display vector pET22b (+) -pgsa-zhd-P of a zearalenone degrading enzyme ZHD-P encoding gene, which comprises the following steps:

(1) carrying out PCR amplification to obtain a target gene fragment zhd-p, wherein the nucleotide sequence of the target gene fragment is shown as SEQ ID NO. 1; performing PCR amplification on an anchor protein pgsA gene from the bacillus subtilis, and recovering to obtain a target fragment pgsA, wherein the nucleotide sequence of the target fragment pgsA is shown as SEQ ID NO. 5;

(2) carrying out double enzyme digestion on the unloaded pET22b (+) by NdeI and XhoI, and recovering a linearized vector fragment;

(3) connecting the amplified target gene segment zhd-p and the anchored protein PgsA gene segment with a linearized universal expression vector pET22(b) +, transforming the connection product into escherichia coli DH5 alpha, selecting a transformant for bacterial liquid verification, selecting a positive clone, carrying out NdeI and XhoI double-enzyme digestion identification and sequencing identification, and constructing a surface display vector pET22b (+) -PgsA-zhd-p;

(4) transforming a surface display vector pET22b (+) -pgsa-zhd-P into escherichia coli BL21, carrying out bacterial liquid verification on a transformant, selecting positive clones, inoculating the positive clones into 10mL of LB liquid culture medium (containing 100mg/L Amp), culturing at 37 ℃ for 8 hours, taking 1mL of the positive clones, transferring the positive clones into 100mL of LB liquid culture medium (containing 100mg/L Amp), and carrying out fermentation culture to obtain the zearalenone degrading enzyme ZHD-P surface display system.

The invention provides a surface display vector pET22b (+) -pgsa-zhd-P of an optimized zearalenone degrading enzyme ZHD-P coding gene prepared by the construction method.

The invention provides an application of a surface display carrier of an optimized zearalenone degrading enzyme ZHD-P coding gene in degrading mycotoxin zearalenone.

The zearalenone degrading enzyme ZHD-P surface display system provided by the invention comprises host escherichia coli and a surface display expression vector pET22b (+) -pgsa-zhd-P.

The host of the zearalenone degrading enzyme ZHD-P surface display system provided by the invention is escherichia coli BL 21.

The anchoring protein gene pgsa, the optimized zearalenone degrading enzyme ZHD-P encoding gene, the recombinant surface display expression vector and the application thereof in preparing the recombinant surface display strain.

Further, the application of the recombinant surface display strain preparation comprises the following steps:

(1) constructing a recombinant expression vector;

(2) transforming host cells by using the recombinant expression vector constructed in the step (1) to obtain a recombinant surface display strain;

(3) culturing the recombinant surface display strain, and inducing and culturing the recombinant strain by IPTG to obtain the recombinant surface display strain.

The invention provides a construction method of a zearalenone degrading enzyme ZHD-P surface display system, which comprises the following steps: the surface display vector pET22b (+) -pgsa-zhd-p is transformed into escherichia coli BL21, 1m L activated bacterial liquid is taken to be transferred into 100m L LB liquid culture medium containing aminobenzyl resistance, the culture is carried out for about 2 hours at 37 ℃ and 200r/min, when the culture exceeds 0.7A (not more than 0.8A), isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to the final concentration of 1mmol/L and the culture lasts for 16-22 hours at 22 ℃ and 200 rpm.

The invention provides an application of a zearalenone degrading enzyme ZHD-P surface display system in degrading mycotoxin Zearalenone (ZEN).

The invention provides a surface display vector pET22b (+) -pgsa-zhd-P of the optimized zearalenone degrading enzyme ZHD-P coding gene. The invention provides a zearalenone degrading enzyme ZHD-P surface display system, which comprises a host escherichia coli BL21 and a recombinant surface display expression vector pET22b (+) -pgsa-zhd-P.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the characterized zearalenone degrading enzymes are Zhd101, ZENJJM and ZLhy-6, the original genes of the zearalenone degrading enzymes are all derived from Gliocladium roseum, and the properties of the zearalenone degrading enzymes are basically consistent; the zearalenone degrading enzyme ZHD-P original gene is derived from Trichoderma, the amino acid consistency of the gene with Zhd101 is 97%, the zearalenone degrading enzyme is a brand new source, and the zearalenone degrading enzyme has research significance;

(2) according to the report of Delfina Popiel et al, the original gene containing zearalenone degrading enzyme ZHD-P invades trichoderma, the efficiency of degrading ZEN by live bacteria is very low, the degradation rate of ZEN is less than 15% after 4 days of culture, and the degradation rate of ZEN is about 50% after 6 days of culture; the recombinant zearalenone degrading enzyme ZHD-P realizes heterologous expression in escherichia coli BL21, and the specific enzyme activity of the purified pure enzyme reaches 95.99U/mg; 5 mu L of pure enzyme (0.3473mg/mL), 5.0 mu L of 1.0mg/mL ZEN and 240.0 mu L of 50mmol/L Tris-HCl (p H7.5.5), and reacting for 30min at 45 ℃ after uniform mixing, so that the ZEN can be completely degraded; the recombinant zearalenone degrading enzyme ZHD-P can degrade ZEN efficiently and quickly, and has a good industrial application prospect;

(3) the optimum reaction temperature of the characterized zearalenone degrading enzyme Zhd101 is 37 ℃, and the optimum pH is 9.5; the optimum temperature of the zearalenone degrading enzyme ZHD-P in the invention is 45 ℃, and the optimum pH is in the range of 7.5-9.0, so that the zearalenone degrading enzyme does not require an environment with high alkalinity; in addition, the reaction is carried out within the range of pH6.0-10.0, and the degradation rate is more than 63% of the highest degradation rate; the reaction is carried out at the temperature of 25-50 ℃, and the degradation rate still keeps more than 82% of the highest degradation rate; this demonstrates the significant advantage of ZHD-P obtained by the present invention;

(4) the method is characterized in that a microbial cell surface display technology is utilized for the first time, and zearalenone degrading enzyme is subjected to surface display; the whole cell activity detection shows that zearalenone degrading enzyme ZHD-P is successfully positioned and displayed on the cell surface of escherichia coli BL21, recombinant surface display thalli (obtained by centrifuging a mixture after 3mL fermentation culture), 40.0 muL of 1.0mg/mL ZEN and 460.0 muL of 50mmol/L Tris-HCl (P H7.5.5) are mixed uniformly and react for 30min at the temperature of 45 ℃, and then the ZEN can be completely degraded; compared with free enzyme, the recombinant surface display system can realize the repeated recycling of the enzyme, does not need a series of complex operations such as ultrasonic crushing, chromatography purification and the like, reduces the cost, has certain operation stability and cost benefit, and is potential for industrial scale production.

Drawings

FIG. 1 is a diagram of Ni column affinity chromatography purification elution peak of recombinant zearalenone degrading enzyme ZHD-P;

FIG. 2 is an SDS-PAGE electrophoresis of recombinant zearalenone degrading enzyme ZHD-P protein before and after purification; lane 1 shows the purified enzyme solution, lane 2 shows the crude enzyme solution, and lane M shows Thermo Scientific Protein Ladders No. 26616.

FIG. 3 is a temperature optimum diagram of recombinant zearalenone degrading enzyme ZHD-P.

FIG. 4 is a diagram showing the optimum pH of the recombinant zearalenone degrading enzyme ZHD-P.

FIG. 5 is a graph showing the effect of the activity of recombinant zearalenone degrading enzyme ZHD-P on the stability at different temperatures.

FIG. 6 is a graph showing the effect of the activity of the recombinant zearalenone degrading enzyme ZHD-P on the stability at different pH.

FIG. 7 is a graph showing the effect of the activity of the recombinant zearalenone degrading enzyme ZHD-P on different metal ions.

FIG. 8a is an HPLC check chart of zearalenone standard;

FIG. 8b is a HPLC check of a mixture of control bacteria and zearalenone;

FIG. 8c is a HPLC check chart of a mixed solution of a zearalenone degrading enzyme ZHD-P surface display system and zearalenone.

Detailed Description

The following description of the embodiments of the present invention is provided in connection with the accompanying drawings and examples, but the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.

The literature reports that the zearalenone degrading enzymes which have been characterized are Zhd101, ZENJJM and ZLhy-6, and the original genes of the zearalenone degrading enzymes are all derived from Gliocladium roseum and have basically consistent properties (CN 201710516347.5). The invention searches for a sequence with high homology by a database comparison method of Zhd101 gene of gliocladium roseum, hopes to screen a zearalenone gene with a brand-new source so as to obtain an enzyme with better performance, and finally screens zearalenone degrading enzyme gene from Trichoderma. According to the report of Delfina Popiel et al, the original gene containing zearalenone degrading enzyme ZHD-P invades Trichoderma, the efficiency of live bacteria degrading ZEN is very low, the ZEN degradation rate is less than 15% after 4 days of culture, and the ZEN degradation rate is about 50% after 6 days of culture. However, no experiment proves that the original gene of the zearalenone degrading enzyme ZHD-P can be heterologously expressed in other hosts, and no literature exists for carrying out extraction and purification and enzymological property research on the enzyme.

Optimizing a nucleotide sequence of a target gene (zearalenone degrading enzyme gene) and an amino acid sequence (zearalenone degrading enzyme) according to the target gene (zearalenone degrading enzyme gene) in a database (National Center for Biotechnology Information Search database, National Center for Biotechnology Information) so as to realize high-efficiency expression in the large and medium intestine BL21, and artificially synthesizing the optimized nucleotide sequence (zearalenone lactohydrolase-DNA (optimized) in vitro, wherein the nucleotide sequence is shown as SEQ ID NO. 1); designing a primer to amplify the target gene segment by taking the synthesized target gene nucleotide sequence as a template. The amplified target gene fragment was ligated with a linearized universal expression vector pET22(b) + to construct an expression vector containing the target gene fragment. And (3) transforming the expression vector into escherichia coli BL21, carrying out sequencing verification on the transformant, selecting positive clones, inoculating liquid LB for culture, finally inoculating the positive clones into a fermentation culture medium for fermentation for 16-22h, finally obtaining crude enzyme, and carrying out Ni column affinity chromatography purification to obtain purified enzyme. The purified pure enzyme has the advantages of high enzyme activity, wide temperature and pH application range, good tolerance and the like, can efficiently and quickly degrade Zearalenone (ZEN), and has good industrial application prospect.

Example 1 construction of recombinant expression vector

1. Artificial synthesis of gene sequences

The optimized nucleotide sequence shown in SEQ ID NO.1 is entrusted to Nanjing Kingsrey Biotech Co., Ltd to carry out gene artificial synthesis according to the conventional technology in the field, and the gene is inserted into a plasmid vector pUC57 and stored for later use.

2. Amplification of gene sequences and linearization of expression vectors

The nucleotide sequence shown in SEQ ID NO.1 was amplified using a Primer zhd-p-F (shown in SEQ ID NO.3, Primer means Primer, the same shall apply hereinafter) and a Primer zhd-p-R (shown in SEQ ID NO. 4), and it was named zhd-p DNA fragment.

Plasmid pET22b (+) was double-digested with NdeI and XhoI, and the digested product was recovered by agarose electrophoresis, thereby obtaining linearized expression vector pET22b (+).

3. Construction of recombinant expression vectors

Zhd-p and a linearized expression vector pET22b (+) were linked by infusion PCR using a NEBuilder HiFi DNA Assembly Cloning Kit to form a circular plasmid (see the NEBuilder HiFi DNA Assembly Cloning Kit instructions). And transforming the connecting product into escherichia coli Match1T1 competence, culturing for 12h at 37 ℃, then picking the transformant in a liquid LB + Amp (final concentration is 100 mu g/ml, antibiotic ampicillin) culture medium, culturing for 12h at 37 ℃ and at the rotating speed of a shaker of 200rpm, performing bacterial liquid electrophoresis, and preliminarily screening out a positive transformant. And extracting plasmids from the positive transformants obtained by the electrophoretic screening of the bacterial liquid, carrying out enzyme digestion verification, and selecting 3 positive transformants with correct plasmid size and correct enzyme digestion verification and sending the transformants to a sequencing company for sequencing. As a result, a zhd-p DNA fragment including nucleotides 1 to 792 from the 5' end of SEQ ID NO.2 was inserted between the NdeI and XhoI cleavage sites of pET22b (+) in the correct direction, and the recombinant plasmid was named pET22b (+) -zhd-p.

4. Preparation of engineering bacteria

The recombinant plasmid pET22b (+) -zhd-p is transformed into Escherichia coli BL21, then spread on LB plate containing 100 mug/ml ampicillin, and cultured for 8-12h at 37 ℃ to obtain engineering bacteria containing the plasmid pET22b (+) -zhd-p, and the engineering bacteria are expressed as BL21/pET22b (+) -zhd-p.

Coli BL21 was transformed with pET22b (+) instead of pET22b (+) -zhd-p, and a recombinant bacterium containing pET22b (+) was obtained as a control bacterium in the same manner as above. The positive recombinant strain transformed into BL21 was designated BL21/pET22b (+).

Example 2 expression and purification of the protein of interest

1. IPTG induced expression of recombinant bacteria

The positive recombinant bacterium BL21/pET22b (+) -zhd-p prepared in step 4 of example 1 was inoculated into 10mL of LB liquid medium (containing 100mg/L Amp), cultured at 37 ℃ for 8-10 hours, and 1mL of the recombinant bacterium was inoculated into 100mL of LB liquid medium (containing 100mg/L Amp). Culturing at 37 deg.C and 200rpm for 2h, measuring OD600, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) to final concentration of 1mmol/L when 0D600 exceeds 0.7A (not more than 0.8A), culturing at 22 deg.C and 200rpm for 16-22 h. The control bacteria prepared in step 4 of example 1 were cultured in the same manner.

2. Preparation of crude enzyme solution

After the induction is finished, the fermentation liquor is placed in two 100mL centrifuge tubes, the temperature is 4 ℃, the rpm is 8000, the centrifugation is carried out for 5min, the supernatant is poured off, 20mL TE buffer is added, after the thalli are resuspended, the centrifugation is carried out for 5min at the temperature of 4 ℃, the rpm is 8000, the supernatant is poured off, and the washing is repeated once. The pellet was then centrifuged once more and the remaining liquid was aspirated off with a pipette. After the bacteria washing is finished, 30mL of Buffer A is added into each deposited bacteria, the two tubes are combined into one tube, and an ultrasonic crusher is adopted to crush cells under the conditions that: 35% power, 3s work, 3s intermittence, crushing for 30 min. After the cells are broken, centrifuging for 15min at 4 ℃ and 10000rpm, and collecting supernatant to obtain crude enzyme solution. And (4) preparing the reference bacteria prepared in the step (4) by adopting the same steps, and taking the obtained solution as a reference crude enzyme solution.

TE buffer: 1mM EDTA, 20mM Tris, adjusted to pH 8.0 with HCl solution.

Buffer a solution: 0.5M NaCl, 20Mm Tris, adjusted to pH 8.0 with HCl solution.

3. Purification of proteins of interest

The crude enzyme solution prepared in step 2 was filtered through a 0.22 μm water filter head. And placing the filtered crude enzyme solution on ice, and performing affinity chromatography purification by using a Ni column according to the fact that the recombinase contains a His-tag label.

1) Before column loading, the column is flushed to a baseline level (A, B pumps each 50%) with 20% ethanol at a flow rate of 1.0mL/min by volume; 2) column packing; 3) rinsing with ultrapure water at a flow rate of 1.0mL/min to baseline level (A, B pump 50% each); 4) flushing to a baseline level (0% of A pump and 100% of B pump) by using a Buffer B with the flow rate of 1.0 mL/min; 5) flushing to a baseline level (100% for A pump and 0% for B pump) with a flow of 1.0mL/min Buffer A; 6) injecting samples at the flow rate of 1.0mL/min (100 percent of A pump and 0 percent of B pump); 7) flushing to a baseline level (100% for A pump and 0% for B pump) with a flow of 1.0mL/min Buffer A; 8) the elution was carried out with a Buffer B gradient with 10% as a gradient, and a peak was observed.

Under the condition that the Buffer B gradient is 40%, the recombinase ZHD-P has a single and obvious absorption peak, and the sample collected under the peak is presumed to be the recombinant zearalenone degrading enzyme ZHD-P. No absorption peak was observed for the control enzyme.

Buffer a solution: 0.5M NaCl, 20Mm Tris, adjusted to pH 8.0 with HCl.

Buffer B solution: 0.5M imidazole, 0.5M NaCl, 20Mm Tris, adjusted to pH 8.0 with HCl.

4. SDS-PAGE (sodium dodecyl sulfate-PAGE) electrophoretic analysis of recombinant ZHD-P crude enzyme solution and purified enzyme solution

Taking the crude enzyme solution of the recombinant ZHD-P enzyme obtained in the step (2) and the purified enzyme of the recombinant ZHD-P enzyme obtained in the step (3), analyzing by SDS-PAGE electrophoresis, and staining by Coomassie brilliant blue. The electrophoresis result is shown in FIG. 2, wherein lane 1 in FIG. 2 is the purified enzyme solution, lane 2 is the crude enzyme solution, lane M is Thermo Scientific Protein Ladders No.26616, the recombinant zearalenone degrading enzyme ZHD-P can realize heterologous expression in Escherichia coli BL21, a distinct Protein band (30kDa position) appears, and other hetero-proteins are substantially removed by the purified zearalenone degrading enzyme ZHD-P.

Example 3 test of degradation of zearalenone by recombinant zearalenone degrading enzyme ZHD-P

The protein concentration of the recombinant zearalenone ZHD-P purified enzyme solution in step 4 of example 2 was determined to be 1.3891 mg/mL. The pure enzyme solution of recombinant zearalenone degrading enzyme ZHD-P in step 4 of example 2 was diluted 4-fold with Buffer A solution (0.5M NaCl, 20Mm Tris, pH adjusted to 8.0 with HCl solution) to give a protein concentration of 0.3473 mg/mL. And (4) carrying out enzyme activity determination on the diluted enzyme solution. The diluted enzyme solution was recorded as a diluted enzyme solution.

The degradation rate of the zearalenone degrading enzyme ZHD-P to the substrate is measured by taking the zearalenone as the substrate. The determination method comprises the following steps: mu.L of the diluted enzyme solution, 5.0. mu.L of 1.0mg/ml ZEN and 240.0. mu.L of 50mmol/L Tris-HCl (p H7.5.5) were mixed, reacted at 45 ℃ for 30min, and then 250. mu.L of methanol was added to terminate the reaction. The amount of substrate degradation of the reacted product was measured using High Performance Liquid Chromatography (HPLC).

The HPLC analysis conditions were as follows: the detector is an ultraviolet detector, the chromatographic column is a reversed-phase C18 chromatographic column (250mm multiplied by 4.6mm, 5 μm), the ultraviolet absorption wavelength is 236nm, and the mobile phase is methanol: water 80:20(V/V), flow rate 1.0mL/min, sample size 30 μ L. In addition, a standard curve of zearalenone content versus peak area will be plotted. The method comprises the steps of preparing zearalenone standard products with different concentration gradients, filtering the zearalenone standard products through a filter membrane with the aperture of 0.22 mu m, and then loading the sample to detect the residual amount of the zearalenone.

1. Optimum temperature

The temperature was set at 25 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃ and 60 ℃ for a total of 8 levels. Reaction system 250.0 μ L: 5 μ L of diluted enzyme solution, 5.0 μ L of 1.0mg/ml ZEN and 240.0 μ L of 50mmol/L Tris-HCl (p H7.5.5), mixing, reacting at different temperature gradients for 30min, and adding 250 μ L methanol to stop the reaction. The amount of substrate degradation after the reaction was measured by High Performance Liquid Chromatography (HPLC), and the results are shown in FIG. 3.

FIG. 3 shows that recombinant zearalenone degrading enzyme ZHD-P has an activity of degrading zearalenone with high efficiency. Under the condition of 45 ℃, the zearalenone degrading enzyme has the highest enzyme activity, and can completely degrade 20 mu g/m L ZEN within 30 min; the ZEN degradation rate is still kept to be more than 90 percent within the range of 25-45 ℃; under the condition of higher temperature of 50 ℃, the degradation rate of the ZEN is still over 82 percent.

The above experiment was carried out using a protein obtained from the control strain BL21/pET22b (+) (referred to as a control enzyme solution), and as a result, the control enzyme solution had no activity of degrading zearalenone under any temperature condition.

2. Optimum pH value

Preparing phosphate buffer solution, adjusting pH to 3, 4, 5 and 6 respectively, preparing 50mmol/L Tris-HCl buffer solution, adjusting pH to 7.0, 7.5, 8.0, 8.5 and 9.0 respectively, preparing glycine buffer solution, adjusting pH to 10.0 and 11.0. Reaction system 250.0 μ L: mu.L of diluted enzyme solution, 5.0. mu.L of 1.0mg/ml ZEN and 240.0. mu.L of 50mmol/L Tris-HCl (p H7.5.5), mixing, reacting at different pH gradients for 30min, and adding 250. mu.L methanol to stop the reaction. The amount of substrate degradation after the reaction was measured by High Performance Liquid Chromatography (HPLC), and the results are shown in FIG. 4.

FIG. 4 shows that the recombinant zearalenone degrading enzyme ZHD-P has the characteristic of wide pH application range when degrading zearalenone. Under the condition of pH 7.5-9.0, zearalenone degrading enzyme has the highest enzyme activity, and can completely degrade ZEN of 20 microgram/ml within 30 min; the ZEN degradation rate is still kept above 63% within the pH range of 6.0-10.0.

The above experiment was carried out using a protein obtained from the control strain BL21/pET22b (+) (referred to as a control enzyme solution), and as a result, the control enzyme solution had no activity of degrading zearalenone under any pH condition.

3. Temperature stability

The purified enzyme solution is respectively placed at the temperature conditions of 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 60 ℃, and after each temperature gradient is placed for 1h, the enzyme solution reacts with ZEN immediately. Reaction system 250.0 μ L: mu.L of the diluted enzyme solution, 5.0. mu.L of 1.0mg/ml ZEN and 240.0. mu.L of 50mmol/L Tris-HCl (p H8.0.0) were mixed, reacted at 45 ℃ for 30min, and then 250. mu.L of methanol was added to terminate the reaction. The amount of substrate degradation after the reaction was measured by High Performance Liquid Chromatography (HPLC), and the results are shown in FIG. 5.

FIG. 5 shows that the activity of the recombinant zearalenone degrading enzyme ZHD-P is still stable after being placed for 1h at the temperature of 25 ℃, 30 ℃, 35 ℃ and 40 ℃, and 20 microgram/m L of ZEN can be completely degraded within 30 min; the activity of the zearalenone degrading enzyme ZHD-P is greatly influenced at the temperature of 45 ℃ and above.

4. Stability of pH

The diluted enzyme solution is respectively placed in buffer solutions with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0 and 11.0 at the temperature of 4.0 ℃ for 12 hours, and then the zearalenone is used as a substrate to determine the residual enzyme activity. Reaction system 250.0 μ L: mu.L of diluted enzyme solution, 5.0. mu.L of 1.0mg/ml ZEN and 240.0. mu.L buffer solution, mixing uniformly, reacting at 45 ℃ for 30min, and adding 250. mu.L of methanol to terminate the reaction. The amount of substrate degradation after the reaction was measured by High Performance Liquid Chromatography (HPLC), and the results are shown in fig. 6.

FIG. 6 shows that more than 60% of degradation rate still remained under the pH of 6.0-9.0. Indicating that the enzyme has good pH tolerance.

5. Effect of Metal ions on enzyme Activity

Adding Ba into Tris-HCl buffer solution system respectively²⁺、Ca²⁺、Co²⁺、Cu²⁺、K⁺、Mg²⁺、Mn²⁺、Na⁺、Ni²⁺、Zn² ⁺And (5) waiting for metal ions to ensure that the concentration of the metal ions in the system reaches 5.0 mmol/L. Reaction system 250.0 μ L: mu.L of pure enzyme (diluted 4 times), 5.0. mu.L of 1.0mg/ml ZEN and 240.0. mu.L of 50mmol/L Tris-HCl (p H7.5.5), mixed well, reacted at 45 ℃ for 30min, and then 250. mu.L methanol was added to terminate the reaction. The amount of substrate degradation after the reaction was measured by High Performance Liquid Chromatography (HPLC), and the results are shown in FIG. 7.

FIG. 7 shows that Ba²⁺、K⁺、Mg²⁺、Na⁺Has little influence on zearalenone degrading enzyme ZHD-P, while Ca²⁺、Co²⁺、Cu²⁺、Mn²⁺、Ni²⁺、Zn²⁺Has stronger inhibiting effect on the degradation activity of zearalenone degrading enzyme ZHD-P.

Example 4 construction of recombinant surface display expression vector

1. Amplification of gene sequences and linearization of expression vectors

The nucleotide sequence shown in SEQ ID NO.1 was amplified using the primer pgsa-zhd-p-F (shown in SEQ ID NO. 6) and the primer pgsa-zhd-p-R (shown in SEQ ID NO. 7) and named zhd-p DNA fragment.

The nucleotide sequence shown in SEQ ID NO.9 was amplified using the Bacillus subtilis genome as a template and the primers pgsa-F (shown in SEQ ID NO. 8) and pgsa-R (shown in SEQ ID NO. 9) and named as pgsa DNA fragment.

2. Construction of recombinant expression vectors

The anchoring protein gene pgsa, the target gene zhd-p and the linearized expression vector pET22b (+) were linked by infusion PCR using a NEBuilder HiFi DNA Assembly Kit to form a circular plasmid (see the NEBuilder HiFi DNA Assembly Kit instructions for details). And transforming the connecting product into the competence of escherichia coli Match1T1, culturing for 12h at 37 ℃, then selecting transformants in a liquid LB + Amp (final concentration of 100 mu g/ml) culture medium, culturing for 12h at 37 ℃ and at the rotating speed of a shaker of 200rpm, performing bacterial liquid electrophoresis, and preliminarily screening out positive transformants. And extracting plasmids from the positive transformants obtained by the electrophoretic screening of the bacterial liquid, carrying out enzyme digestion verification, and selecting 3 positive transformants with correct plasmid size and correct enzyme digestion verification and sending the transformants to a sequencing company for sequencing. As a result, the pgsa DNA fragment and the zhd-p DNA fragment were inserted in tandem between the NdeI and XhoI cleavage sites of pET22b (+), which includes the nucleotides 1 to 1140 of SEQ ID NO.5 and the nucleotides 4 to 792 of SEQ ID NO.1 from the 5' end, in the correct insertion direction, and the recombinant surface display plasmid was named pET22b (+) -pgsa-zhd-p.

3. Preparation of engineering bacteria

The recombinant surface display plasmid pET22b (+) -pgsa-zhd-p is transformed into Escherichia coli BL21, then spread on an LB plate containing 100 mu g/ml ampicillin, and cultured for 8-12h at 37 ℃ to obtain the engineering bacteria containing the plasmid pET22b (+) -pgsa-zhd-p, and the plasmid BL21/pET22b (+) -pgsa-zhd-p.

Escherichia coli BL21 was transformed with pET22b (+) -pgsa instead of pET22b (+) -pgsa-zhd-p, and a recombinant strain containing pET22b (+) -pgsa was obtained as a control strain in the same manner as above. The negative recombinant bacterium transformed into BL21 was designated BL21/pET22b (+) -pgsa.

Example 5 inducible expression of recombinant surface display System and preparation and application of thallus

1. IPTG inducible expression of recombinant surface display system

The positive recombinant bacterium pET22b (+) -pgsa-zhd-p prepared in the step 3 is inoculated into 10mL of LB liquid culture medium (containing 100mg/L Amp), cultured at 37 ℃ for 8-10h, and then 1mL of the bacterium is transferred into 100mL of LB liquid culture medium (containing 100mg/L Amp). Culturing at 37 deg.C and 200rpm for about 2h, measuring OD600, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) to final concentration of 1mmol/L when 0D600 exceeds 0.7A (not more than 0.8A), and culturing at 22 deg.C and 200rpm for 16-22 h. And (4) culturing the control bacteria prepared in the step (3) by adopting the same steps.

2. Preparation and application of recombinant surface display engineering bacteria

After the induction is finished, 3mL (divided into two times) of fermentation liquor is taken to be put into a 1.5mL EP tube, the temperature is 4 ℃, the rpm is 8000, the centrifugation is carried out for 5min, and the supernatant is poured out, thus obtaining the recombinant surface display engineering bacteria. And (4) preparing the control bacteria prepared in the step (3) by adopting the same steps, and taking the obtained solution as the control bacteria.

And (3) detecting the activity of the whole-cell enzyme of the engineering bacteria obtained in the step (a), wherein a reaction system is 500 (mu L): the recombinant surface-displayed cells (obtained by centrifuging 3mL of the mixture after fermentation culture), 40.0. mu.L of 1.0mg/mL ZEN and 460.0. mu.L of 50mmol/L Tris-HCl (p H7.5.5) were mixed, reacted at 45 ℃ for 30min, and then 500. mu.L methanol was added to terminate the reaction. After the reaction, the amount of substrate degradation was measured by High Performance Liquid Chromatography (HPLC), and as a result, the reaction time was 30min as shown in fig. 8a, 8b, and 8 c.

Fig. 8a, 8b and 8c show that recombinant surface display engineered bacteria can completely degrade ZEN at a system ZEN concentration of 40 μ L/mL. The above experiments were carried out with control bacteria and did not have zearalenone degrading activity.

The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.

Sequence listing

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Claims

1. An optimized zearalenone degrading enzyme ZHD-P encoding gene is characterized in that a nucleotide sequence is shown as SEQ ID No. 1.

2. A recombinant expression vector comprising a pET22b (+) vector and the nucleotide sequence set forth in SEQ ID No.1 of claim 1.

3. A recombinant strain comprising the recombinant expression vector of claim 3; the recombinant strain is escherichia coli BL 21.

4. The optimized zearalenone degrading enzyme ZHD-P encoding gene of claim 1 and the use of the recombinant expression vector of claim 2 for the heterologous high-efficiency expression of recombinant zearalenone degrading enzyme ZHD-P in large intestine BL 21.

5. Use according to claim 4, characterized in that it comprises the following steps:

(1) constructing a recombinant expression vector of the zearalenone degrading enzyme ZHD-P;

(3) culturing the recombinant strain, and inducing the recombinant strain to express the zearalenone degrading enzyme ZHD-P by IPTG;

(4) recovering and purifying the zearalenone degrading enzyme ZHD-P.

6. Use of the recombinant strain of claim 3 for preparing recombinant zearalenone degrading enzyme ZHD-P.

7. A construction method of an optimized surface display carrier of zearalenone degrading enzyme ZHD-P coding genes is characterized by comprising the following steps:

(4) and (3) transforming the surface display vector pET22b (+) -pgsa-zhd-P into escherichia coli BL21, carrying out bacterial liquid verification on the transformant, selecting positive clones, inoculating liquid LB for culture, finally inoculating the positive clones into a fermentation culture medium for fermentation, and obtaining the surface display system of zearalenone degrading enzyme ZHD-P.

8. The method for constructing the optimized zearalenone degrading enzyme ZHD-P encoding gene surface display vector of claim 7, wherein the time for fermentation of the inoculation into the fermentation medium in step (4) is 16-22 h.

9. A surface display vector of the optimized zearalenone degrading enzyme ZHD-P-encoding gene prepared by the construction method of any one of claims 7 to 8.

10. Use of the surface display vector of the optimized zearalenone degrading enzyme ZHD-P encoding gene of claim 9 for degrading the mycotoxin zearalenone.