Algal toxin-degrading enzyme mutants and uses thereof

Abstract

The application discloses an algal toxin degrading enzyme mutant and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The algal toxin degrading enzyme mutant is obtained by carrying out unit point mutation on the 32th position of the wild-type algal toxin degrading enzyme amino acid sequence shown in SEQ ID No. 2. The gene engineering bacteria of the mutant algal toxin degrading enzyme obtained by the application can degrade algal toxin, greatly improves the activity and service life of the cell, and meets the requirements of current industrial application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a microcystin-degrading enzyme mutant and application thereof. BACKGROUND

[0002] Due to eutrophication and global climate change, the frequency and extent of cyanobacterial blooms have increased in different degrees around the world, which seriously threatens the ecosystem and human health. Microcystins (MCs) produced by cyanobacterial blooms can cause gastrointestinal symptoms and acute liver injury, and are also related to non-alcoholic fatty liver disease, chronic kidney disease, cancer and other diseases, and are strong liver cancer inducers. MCs can be transferred along the food chain, posing a health threat to aquatic organisms, wildlife, livestock, and even humans.

[0003] MCs are cyclic heptapeptides, and more than 250 MC variants have been identified, among which MC-LR is the most common and the most toxic. The World Health Organization and the Ministry of Health of China have stipulated that the safe limit of MC-LR in drinking water is 1 μg / L. The stability of cyclic MCs is extremely strong, and they cannot be degraded by high temperature, extreme pH, sunlight, common enzymes, etc. Existing MCs degradation strategies include activated carbon adsorption, chemical oxidation, photocatalysis, electrolysis and biodegradation. Due to the advantages of high efficiency, low cost and environmental friendliness, biodegradation has become the development trend of MCs degradation. Species reported to have MCs biodegradation include bacteria, fungi, aquatic plants and protozoa, among which bacteria have the strongest degradation ability (Jieming L, et al. Current research scenario for microcystins biodegradation-A review on fundamental knowledge, application prospects and challenges. Sci Total Environ, 2017, 595: 615-632.).

[0004] So far, more than 50 kinds of bacteria degrading MCs have been screened from nature, and the process and molecular mechanism of bacterial degradation of MCs have been studied at the genetic level (Qin D, et al. Multiple pathways for the anaerobic biodegradation of microcystin-LR in the enriched microbial communities from Lake Taihu. Environ Pollut, 2022, 297: 118787.). The currently recognized aerobic degradation pathway of MCs was proposed by Bourne et al. They obtained the gene cluster of Sphingomonas sp. ACM-3962 degrading MC-LR through library screening, including four genes mlrA, mlrB, mlrC and mlrD (Bourne DG, et al. Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR. Environ Toxicol, 2001, 16: 523-534.). MlrA is the starting step and key control point of aerobic degradation of MCs. After the cyclic MC-LR is hydrolyzed by MlrA to linear MC-LR, its toxicity can be reduced by 2100 times, basically becoming a non-toxic product.

[0005] Mlr gene clusters or gene sequences homologous to mlrA derived from 32 kinds of bacteria have been reported, among which Sphingopyxis sp. strain a7 is the highest degrading efficiency of MC-LR strain reported so far, with a degrading efficiency of 3.33 mg / L / h (Zhang J, et al. A Novel and Native Microcystin-Degrading Bacterium of Sphingopyxis sp. Isolated from Lake Taihu. Int J Environ Res Public Health, 2017, 14(10)). The similarity of its mlrA gene sequence with that of Sphingopyxis sp. USTB-05 is more than 99%. However, the stability of the reported MlrA is poor, and its continuous degradation ability in water body is poor, which greatly limits its working efficiency. Therefore, how to improve the stability of MlrA is a problem urgently needed to be solved for biodegradation of algal toxins.

[0006] With the development of protein engineering technology and molecular biology, using the means of directed evolution and rational / semi-rational design to artificially evolve and transform enzyme molecules has become a hotspot in the field of enzyme engineering. Among them, error-prone PCR, DNA shuffling, semi-rational design and the like have become common means in the transformation of enzyme molecules, which greatly accelerates the evolution process of proteins. SUMMARY

[0007] In view of the problems in the prior art, the purpose of the present application is to provide a technical solution of an algal toxin degrading enzyme mutant and its application.

[0008] The present application is specifically implemented by the following technical solutions:

[0009] The present application provides an algal toxin degrading enzyme mutant in the first aspect, which is obtained by unit point mutation of the 32nd position of the wild-type algal toxin degrading enzyme amino acid sequence shown in SEQ ID No. 2.

[0010] Further, the unit point mutation is to mutate proline at the 32nd position to tryptophan.

[0011] Further, the amino acid sequence of the algal toxin degrading enzyme mutant is shown in SEQ ID No. 1.

[0012] The present application provides a gene encoding the above-mentioned algal toxin degrading enzyme mutant in the second aspect.

[0013] The present application provides a recombinant vector containing the above-mentioned coding gene in the third aspect.

[0014] The present application provides a host cell containing the above-mentioned recombinant vector in the fourth aspect.

[0015] The present application provides an application of the above-mentioned algal toxin degrading enzyme mutant in degrading algal toxins in the fifth aspect.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1. An algal toxin degrading enzyme mutant is obtained by error-prone PCR and site-directed mutagenesis technology, and the amino acid sequence thereof is shown in SEQ ID No. 1, which is encoded by the nucleotide sequence shown in SEQ ID No. 3.

[0018] 2. A new recombinant vector is obtained by genetic engineering technology, which contains a DNA molecule with a nucleotide sequence shown in SEQ ID No. 3.

[0019] 3. A new host cell containing a recombinant vector comprising a DNA molecule as shown in SEQ ID No. 3 is obtained by genetic engineering technology, and the host cell can express the mutant algal toxin degrading enzyme.

[0020] 4. The wild type algal toxin degrading enzyme is mutated from proline at position 32 to tryptophan, which greatly improves the activity and thermal stability of the enzyme.

[0021] 5. A genetically engineered bacterium expressing the mutant algal toxin degrading enzyme of the present application is obtained by genetic engineering technology, and the use of the genetically engineered bacterium to degrade algal toxins greatly improves the activity and service life of the cell, meeting the requirements of current industrial applications. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Effect of temperature on the activity of wild type and mutant MlrA enzymes.

[0023] Figure 2 Thermal stability results of wild type and mutant MlrA enzymes. DETAILED DESCRIPTION

[0024] In the following examples of the present application, the detection methods are as follows:

[0025] (1) Enzyme activity determination method: in a PBS buffer at pH 7.0, 1 mg / mL of MC-LR is added, a certain amount of enzyme solution is added, and the reaction is carried out at 40℃ for 10 min, 10 μL of 1% trifluoroacetic acid is added to terminate the reaction, after the sample is cooled, centrifugation is carried out at 12000 rpm for 10 min, the supernatant is taken for HPLC analysis, and the content of residual MC-LR in the reaction solution is determined. Under the above reaction conditions, the degrading MC-LR enzyme activity is defined as relative activity 100%.

[0026] (2) Detection method of MC-LR content: refer to the standard DB32 / T 1481-2009.

[0027] (3) Protein concentration determination method: use the improved Bradford protein concentration determination kit (Shanghai Genechem Biotech Co., Ltd.).

[0028] The present application is further described in conjunction with the following examples, it should be pointed out that the examples are only used to explain the present application, and are not limited to the scope of the present application.

[0029] Example 1: Synthesis of algal toxin degrading enzyme MlrA gene and construction of genetically engineered bacteria

[0030] According to the mlrA gene enzyme coding gene (GenBank Accession No. HM245411) of Sphingopyxis sp. USTB-05, the nucleotide sequence of which is shown as SEQ ID No. 4, the gene sequence was synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., the nucleotide sequence of which is shown as SEQ ID No. 2, which was inserted into the pGEX-4T-1 vector using the BamHI and Xho I restriction endonuclease sites, a GST fusion tag was added to the N terminus of the protein, and E. coli DH5α was transformed. Then, the genetically engineered bacteria containing the Sphingopyxis MlrA enzyme were obtained from Shengong Bioengineering (Shanghai) Co., Ltd.

[0031] Example 2: Construction of MlrA enzyme random mutation library by error-prone PCR method

[0032] (1) The above-mentioned microcystin-degrading enzyme MlrA gene was randomly mutated using the GeneMorph II Random Mutagenesis kit (purchased from Agilent, Code No. 200550). The primers used were pGEX 5' and pGEX 3', the nucleotide sequences of which are shown as SEQ ID No. 5 and SEQ ID No. 6. The reaction conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, a total of 25 cycles, and 1% agarose gel electrophoresis at the end of PCR and recovery of the gene fragment using a gel recovery kit (purchased from Sangon, Code No. B518131).

[0033] (2) The recovered fragment was digested with BamHI and Xho I, and then ligated with the same enzyme-digested pGEX-4T-1 vector (ampicillin resistance) to perform a ligation reaction, the reaction conditions of which were: the vector and the fragment were mixed in a ratio of 1:3, 1 μL of T4 ligase (purchased from Thermo, Code No. EL0014) was added, and the ligation was performed at 16°C overnight.

[0034] (3) The above-mentioned ligation fragment was transformed into E. coli DH5α competent cells, which were plated on LB solid plates (10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, 15 g / L agar, pH 7.0) resistant to ampicillin (concentration of 50 μg / mL) and incubated at 37°C for 16 h under inversion, to obtain a mutant library.

[0035] (4) The mutant strains on the above flat plate were inoculated into 96-well plates containing 150 μL LB medium (10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, pH 7.0) containing 50 μg / mL ampicillin, and after 4 h of culture at 37°C with shaking in a plate shaker, 1 mM isopropylthio-β-D-galactoside was added, and then the culture was shaken for 12 h to obtain a mutant expression library.

[0036] (5) 50 μL of the bacterial solution from the mutant expression library was taken into another new 96-well plate, and 40 μL of PBS buffer (pH 7.0), 10 μL of 10 mg / mL MC-LR, and 1 μL of 1% triton X-100 were added, and the mixture was shaken at 50°C for 10 min. Then 10 μL of 1% trifluoroacetic acid was added to each well to terminate the reaction, and the supernatant was centrifuged and subjected to HPLC analysis to determine the content of MC-LR in the reaction solution. The wild-type MlrA enzyme was used as a control, and mutant strains with good thermal stability and high enzyme activity were selected and sequenced to determine the mutation site.

[0037] Thus, one mutant strain was obtained by screening, in which the codon (CCG) encoding proline at position 32 in the mutant P32W was mutated to the codon (TGG) encoding tryptophan, and the nucleotide sequence is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 1.

[0038] Example 3: Expression and purification of wild-type and mutant MlrA enzymes

[0039] The engineered bacteria in Example 1 and the P32W mutant strain in Example 2 were inoculated into LB medium containing 50 μg / mL ampicillin, respectively, and when the bacterial concentration (OD600) reached 0.6-0.8, the culture was induced with 1 mM IPTG and cultured at 37°C for 4 h. 600When the concentration reaches 0.4-0.6, add 0.2 mM isopropyl thio-β-D-galactoside and incubate at 37°C with shaking for 12 h. Collect bacterial cells by centrifugation at 5000 rpm for 10 min at 4℃. After washing three times with physiological saline, add lysis buffer (50 mmol / L Tris-HCl, 0.15 mol / L NaCl, 10% glycerol, pH 8.0) at a ratio of 5 ml per gram of bacterial cells. Perform pre-ultrasonic lysis on ice, centrifuge at 8000 rpm for 30 min at 4℃, collect the supernatant, and load it onto a GST-agarose affinity column (purchased from Xi'an Qiyue Biotechnology Co., Ltd.). Wash away impurities with washing buffer (50 mmol / L Tris-HCl, 0.15 mol / L NaCl, pH 8.0), and elute the target protein with elution buffer (50 mmol / L Tris-HCl, 0.15 mol / L NaCl, 15 mmol / L reduced glutathione, pH 8.0). Incubate the collected enzyme solution at 4℃ with 50 mmol / L PBS buffer (pH 8.0). The protein was dialyzed thoroughly in 7.0, concentrated with polyethylene glycol, and then glycerol was added to a final concentration of 50%, and stored at -20°C. The purification effect was detected by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the purified protein had a single band, consistent with the theoretical molecular weight.

[0040] Example 4: Activity and thermostability analysis of wild-type and mutant MlrA enzymes

[0041] The wild-type and P32W mutants purified in Example 3 were subjected to enzyme activity assays at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 65°C, respectively, according to the above-described detection method. The activity of the wild-type enzyme measured at 40°C was defined as 100%. The results showed (see...) Figure 1 Under different temperature conditions, the specific activity of the mutant MlrA enzyme was higher than that of the wild type. The optimal temperature for both wild-type and mutant MlrA enzymes was 40℃. At the optimal temperature of 40℃, the activity of the mutant MlrA enzyme was approximately 1.2 times that of the wild type.

[0042] The wild-type and P32W mutant enzymes purified in Example 3 were incubated at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, and 80℃ for 30 min, respectively. Then, at 40℃, the activities of the wild-type and mutant MlrA enzymes were measured according to the above detection method, and the residual activity was calculated based on the control group. The control group consisted of MlrA enzymes that were not incubated and were directly measured at 40℃; the corresponding enzyme activity was defined as 100%. The results showed (see...).Figure 2 ), the residual activity of wild-type MlrA enzyme was 73.2% after incubation at 40℃ for 30 min, and then the residual activity decreased rapidly, only 61.1% of the activity remained after incubation at 50℃ for 30 min, and only 35.1% of the activity remained after incubation at 60℃ for 30 min. Subsequently, the activity decreased rapidly until the activity was completely lost. However, the activity of mutant P32W did not change substantially after incubation at 40℃ for 30 min. After incubation at 50℃ and 60℃ for 30 min, the residual activity was still 83.2% and 68.2%, respectively, which was significantly higher than that of the wild type. After incubation at the optimum temperature of 40℃ for 30 min, the residual activity of mutant P32W increased by about 32%.

[0043] This shows that the temperature stability of the mutant MlrA enzyme is significantly improved compared with the wild type. The P32W mutant MlrA enzyme described in the present application has an amino acid sequence as shown in SEQ ID NO. 1, which is obtained by expressing a recombinant vector containing a nucleotide sequence as shown in SEQ ID NO. 3 in a host cell; the recombinant vector of the present application contains a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 3; the host cell of the present application contains a recombinant vector containing a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 3.

[0044] Example 5: Degradation of algal toxins by wild-type and mutant genetically engineered bacterial cells according to the present application

[0045] The method for degrading algal toxins according to the present application is: adding algal toxin degrading enzyme MlrA (wild-type and / or mutant) and / or genetically engineered bacteria (wild-type and / or mutant) to a solution containing algal toxins or algal sludge containing algal toxins to degrade the algal toxins in the solution or algal sludge. The algal toxins involved in the present application include but are not limited to MC-LR, MC-RR, MC-YR, etc. The following is an example:

[0046] Take 1g of wild type and mutant genetically engineered bacteria cells induced by isopropylthio-beta-D-galactoside respectively, and put them in 100ml 50mg / L MC-LR or MC-RR or MC-YR solution respectively, and shake at 30 DEG C, and detect the content of algal toxin in the solution every 2h. In the degradation process, we detect the enzyme activity and stability of wild type and mutant genetically engineered bacteria by the content of algal toxin in the solution. When the wild type genetically engineered bacteria completely degrade 100ml solution containing 50mg / L MC-LR, MC-RR and MC-YR respectively, it needs 20h, 32h and 40h respectively. Under the same condition, when the mutant genetically engineered bacteria completely degrade 1000ml solution containing 100mg / L MC-LR, MC-RR and MC-YR respectively, it only needs 8h, 12h and 16h respectively. In addition, take 1g of wild type and mutant genetically engineered bacteria cells induced by isopropylthio-beta-D-galactoside respectively, and mix them with 1000g algal mud containing algal toxin (MC-LR content is 3.2mg / L), and place them at room temperature, and turn them every 1h, and detect the content of algal toxin in the algal mud every 2h. The wild type and mutant genetically engineered bacteria completely degrade the algal toxin, and it needs 10h and 6h respectively. This shows that the mutant genetically engineered bacteria not only has high enzyme activity, but also has good stability, which is significantly higher than that of the wild type genetically engineered bacteria. The mutant genetically engineered bacteria in the application contains a recombinant vector, and the recombinant vector contains a DNA molecule shown as SEQ ID NO. 3, and the DNA molecule encodes a protein shown as SEQ ID NO. 1, and the protein has algal toxin degradation enzyme activity.

Claims

1. A mutant algal toxin-degrading enzyme, characterized in that, This algal toxin degrading enzyme mutant was obtained by a single point mutation at position 32 of the amino acid sequence of the wild-type algal toxin degrading enzyme shown in SEQ ID No.

2. The single point mutation was to mutate proline at position 32 to tryptophan. The amino acid sequence of this algal toxin degrading enzyme mutant is shown in SEQ ID No.

1.

2. A gene encoding a mutant of the algal toxin-degrading enzyme of claim 1.

3. A recombinant vector containing the encoding gene of claim 2.

4. A host cell containing the recombinant vector of claim 3.

5. The application of the algal toxin-degrading enzyme mutant of claim 1 in the degradation of algal toxins.

Images