CN111424027A - Site-directed mutagenesis modified alginate lyase mutant and application thereof - Google Patents
Site-directed mutagenesis modified alginate lyase mutant and application thereof Download PDFInfo
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Abstract
The invention discloses an alginate lyase mutant modified by site-specific mutagenesis and application thereof, belonging to the technical field of enzyme engineering.A 212 th glutamic acid of the alginate lyase with an amino acid sequence shown as SEQ ID NO.1 is replaced by histidine and/or a 222 th arginine is replaced by lysine.
Description
Technical Field
The invention relates to an alginate lyase mutant modified by site-directed mutagenesis and application thereof, belonging to the technical field of enzyme engineering.
Background
Brown algae is one of the three major algae, and the main components are algin, mannitol and laminarin, wherein, the utilization of the mannitol and the laminarin is relatively mature, so that how to efficiently degrade the algin and realize the industrial application is the focus of the current research, the algin is a linear polysaccharide composed of β -D-mannuronic acid (M) and α -L-guluronic acid (G), and the degradation product of the algin has important physiological activity, such as oxidation resistance, tumor resistance, plant root cell growth promotion and the like, so the algin has received wide attention.
The algin lyase (Aly) can degrade algin through a β -elimination reaction mechanism, and forms unsaturated carbon-carbon double bonds at the non-reducing end of a degradation product, thereby generating a special absorption peak under ultraviolet 235nm and being used for product detection, according to the substrate specificity of Aly, the algin lyase can be divided into polyM, polyG and bifunctional algin lyase, according to the enzyme cutting mode, the algin lyase can be divided into endo-type (endo-type) and exo-type (exo-type) algin lyase, the final product of the alginate degradation by the endo-type Aly is Alginate Oligosaccharide (AOs), the polymerization degree of the product is 2-6, the product of the exo-type Aly is single, the final product is monosaccharide, but the report that disaccharide is the minimum enzyme cutting unit, according to the classification of a CAZY database, the algin lyase belongs to polysaccharide lyase (P L), and is specifically divided into seven algin 73725-5, P733-6, P L-7, P L-14, P L-15, P-8517-8217 and P73717 families.
At present, the method for preparing the alginate oligosaccharide is mainly a chemical method such as acidolysis, the preparation conditions are severe and environmental pollution is easily caused, and in contrast, the method for degrading the alginate polysaccharide by a biological method is mild in conditions, easy to control and environment-friendly, so that the development of the alginate lyase which is efficient, high in stability and easy to prepare is necessary. The current research shows that the genetic engineering is the main means for improving the enzyme activity of the alginate lyase. In addition, the conventional methods for increasing the expression level of recombinant proteins include replacement of strong promoters, signal peptides with high secretion ability, and the like.
Different classification modes are provided according to different properties of the alginate lyase: different alginate lyase enzymes according to the specificity of the brown degradation substrate can be divided into three categories: an enzyme for specifically hydrolyzing a mannuronic acid segment (PolyM), an enzyme for specifically degrading a guluronic acid segment (Poly G) and a bifunctional lyase in which both segments can be cleaved. However, the performance of the existing alginate lyase needs to be further improved to meet the requirement of industrial application.
Disclosure of Invention
In order to solve the problems, the invention obtains the mutant with obviously improved catalytic activity and substrate preference through PCR site-specific saturation mutagenesis, and lays a foundation for further industrial application.
The invention aims to provide an alginate lyase mutant, wherein the alginate lyase mutant is obtained by replacing glutamic acid at position 212 of the alginate lyase with histidine and/or replacing arginine at position 222 of the alginate lyase with lysine, the amino acid sequence of which is shown as SEQ ID NO. 1.
Furthermore, when the 212 th glutamic acid of the alginate lyase with the amino acid sequence shown as SEQ ID NO.1 is replaced by histidine, the amino acid sequence of the mutant is shown as SEQ ID NO. 2.
Furthermore, when the 222 th arginine of the alginate lyase with the amino acid sequence shown as SEQ ID NO.1 is replaced by lysine, the amino acid sequence of the mutant is shown as SEQ ID NO. 3.
Further, when the 212 th glutamic acid of the alginate lyase with the amino acid sequence shown as SEQ ID NO.1 is replaced by histidine and the 222 th arginine is replaced by lysine, the amino acid sequence of the mutant is shown as SEQ ID NO. 4.
It is a second object of the present invention to provide a gene encoding the mutant.
The third purpose of the invention is to provide an expression plasmid carrying the gene.
Furthermore, the plasmid takes pET-28a (+) as a vector.
The fourth purpose of the invention is to provide a recombinant bacterium for expressing the mutant.
Furthermore, the recombinant bacterium takes bacteria, fungi or animal and plant cells as hosts.
The fifth purpose of the invention is to provide the application of the recombinant strain in the production of products containing alginate lyase.
The invention has the beneficial effects that:
the recombinant escherichia coli with enhanced secretion capacity of the alginate lyase constructed by the invention can improve the enzyme activity of the alginate lyase by 2.83 times compared with that of a starting strain, obviously improve the enzyme production capacity of the transformed genetically engineered bacteria, and the enzyme activity of the alginate lyase produced by shake flask fermentation reaches 15000U/m L, so that the recombinant escherichia coli is more suitable for industrial application, can reduce the production cost and improve the production efficiency.
Drawings
FIG. 1 is a nucleic acid gel electrophoresis of mutated alginate lyase; m, marker; 1 to are respectively: alg212A, Alg222A, Alg212H, Alg222K, Alg 212H-222K;
FIG. 2 is a gel electrophoresis diagram of alginate lyase proteins in a mutant strain; m, marker; 1 to 3 are respectively: alg212H, Alg222K, Alg 212H-222K;
FIG. 3 shows the activity of alginate lyase when the site-directed mutation is alanine;
FIG. 4 shows the activity of alginate lyase in the case of 212-point mutation;
FIG. 5 shows the activity of alginate lyase in the case of 222-site mutation;
FIG. 6 shows the activity of alginate lyase before and after combined mutation.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Culture medium:
l B liquid culture medium, peptone 10.0 g/L, yeast powder 5.0 g/L5.0.0 g/L.
TB fermentation medium, peptone 12.0 g/L, yeast powder 24.0 g/L, glycerol 4.0 g/L2 PO417 mmol/L, K2HPO 472 mmol/L.
Measuring the enzyme activity of the alginate lyase:
the enzyme activity measuring condition is that 0.1m L enzyme liquid is added into 0.9m L1 percent sodium alginate solution, the mixture is evenly mixed and reacts for 20min at 45 ℃, then 1m L DNS is added into the system, boiling water bath is carried out for 3min to stop the reaction, the volume is quickly determined to 10m L, 200 mu L is absorbed and added into a 96-hole plate, the light absorption value is measured at 520nm, a contrast group adopts deionized water to replace crude enzyme liquid, 1 enzyme activity unit (U) is defined, under the condition, 1m L enzyme liquid produces 1 mu g of enzyme amount required by reducing sugar per minute, glucose aldehyde acid solutions with different concentrations are prepared, 1m L DNS reagent is added for reaction, the light absorption values of the solutions with different concentrations are measured at 520nm, and the enzyme activity is calculated according to a standard curve.
Specific enzyme activity (U/mg) ═ enzyme activity/protein mass
TABLE 1 primer sequences
Example 1: crystal structure simulation of escherichia coli-derived alginate lyase
The 212 th glutamic acid and 222 th arginine near the catalytic domain of alginate lyase are selected as the active sites of the alginate lyase in the study after the simulated docking of the protein 3D structure in example 1.
Example 2: influence of active site mutation of alginate lyase on enzyme activity expression of alginate lyase
Using site-directed mutagenesis kit (TaKaRa), primers 212A-F,212A-R, 222A-F and 222A-R (shown in Table 1) were designed, and pET-28a (+) constructed was used as a template to perform PCR, wherein 212 th glutamic acid and 222 th arginine of alginate lyase were replaced with alanine, respectively, and the PCR reaction conditions were 98 ℃ for 5min, 34 cycles (98 ℃ for 30S, 57.6 ℃ for 60S, 72 ℃ for 1.5min), 72 ℃ for 10min, PCR amplification system, template 1. mu. L, upstream and downstream primers for 1. mu. L, Prime Star (Premix) DNA 20. mu. L, ddH2O17 mu L, purifying and recovering the PCR product by using a gel recovery kit, carrying out electrophoresis test on the concentration of the recovered product, carrying out enzyme digestion under the action of Bam HI and Mlu I fast cutting enzymes, trying to connect the PCR recovered product by using a T4 ligase method, converting the product into a competent E.coil JM109, coating an ampicillin L B plate, selecting a positive colony, extracting a plasmid after overnight culture in a shaking table at 37 ℃, and transferring the plasmid into Escherichia coli E.coli Rosetta to obtain the recombinant strain with site-directed saturation mutation.
In example 2, the enzyme activities of the genetically engineered alginate lyase are both greatly reduced when the 212 th glutamic acid and the 222 th arginine of the alginate lyase are site-directed mutated to alanine (as shown in fig. 3), and are 1200U/ml and 900U/ml respectively, which are 0.22 times and 0.17 times of the enzyme activity of the original strain, and are named as pET-28a (+) -Alg212A and pET-28a (+) -Alg222A, and then transformed into E.coli Rosetta to obtain site-directed mutated mutant strains E.coli Rosetta-pET-28a (+) -Alg212A and E.Coli Rosetta-pET-28a (+) -Alg 222A.
The two sites are proved to have great influence on the enzyme activity of the genetic engineering alginate lyase.
Example 3: influence of single-point mutation of active site of alginate lyase on enzyme activity expression of alginate lyase
Using site-directed mutagenesis kit (TaKaRa), primers 212H-F, 212H-R and 222K-F,222K-R (shown in Table 1) were designed, PCR was performed using the constructed pET-28a (+) -Alg212A as a template, and the 212 nd glutamic acid was mutatedOr arginine at position 222, under PCR conditions of 98 deg.C for 5min, 34 cycles (98 deg.C for 30S, 57.8 deg.C for 60S, 72 deg.C for 1.5min), 72 deg.C for 10min, 1 μ L for template, 1 μ L for upstream and downstream primers, 20 μ L for Prime Star (Premix) DNA, and ddH2And O17 mu L, purifying and recovering the PCR product by using a gel recovery kit, carrying out electrophoresis test on the concentration of the recovered product, carrying out enzyme digestion under the action of Bam HI and Mlu I fast cutting enzymes, trying to connect the PCR recovered product by using a T4 ligase method, transforming the product into competent E.coil JM109, coating an ampicillin L B plate, selecting a positive colony, carrying out shake culture at 37 ℃ overnight, and extracting plasmid to obtain the mutant strain with site-directed mutation.
In example 3, when the 212 th glutamic acid point of the genetically engineered alginate lyase is mutated into histidine, the enzyme activity of the alginate lyase is obviously improved (as shown in figure 4), is 7300U/ml, is 1.38 times of the enzyme activity of the original strain, is named as pET-28a (+) -Alg212H, and is then transferred into E.coli Rosetta, so as to obtain the site-directed mutated mutant strain E.coli Rosetta-pET-28a (+) -Alg 212H. Proves that the enzyme activity of the algin lyase can be obviously changed by carrying out point mutation on the 212 th site of the algin lyase.
In example 3, when the 222 th arginine of the genetically engineered alginate lyase is point-mutated to lysine, the activity of the alginate lyase is significantly improved (as shown in fig. 5), 8000U/ml, which is 1.51 times of the activity of the original strain, is named as pET-28a (+) -Alg222K, and then transformed into E.coli Rosetta, so as to obtain the site-directed mutated mutant strain E.coli Rosetta-pET-28a (+) -Alg 222K. Proves that the enzyme activity of the alginate lyase can be obviously changed by carrying out point mutation on the 222 th site of the alginate lyase.
Example 4: influence of combined mutation of active sites of alginate lyase on enzyme activity expression of alginate lyase
The site-directed mutagenesis kit (TaKaRa) was used to design primers 222K-F and 222K-R (shown in Table 1), PCR was performed using the constructed pET-28a (+) -Alg212H as a template to mutate and replace arginine at position 222, and the PCR reaction conditions were 98 ℃ for 5min, 34 cycles (98 ℃ for 30S, 57.8 ℃ for 60S, and 72 ℃ for 1.5min), and 72 ℃ for 10min, and the PCR amplification system consisted of 1. mu. L for the template, 1. mu. L for each of the upstream and downstream primers, and 20. mu. L for Prime Star (Premix) DNA, and d for the ddH2O17 mu L, purifying and recovering the PCR product by adopting a gel recovery kit, and carrying out electrophoresis test on the concentration of the recovered product, carrying out enzyme digestion under the action of Bam HI and Mlu I fast cutting enzymes, trying to connect the PCR recovered product by using a T4 ligase method, transforming the product into a competent E.coil JM109, coating an ampicillin L B plate, picking out positive colonies, carrying out shaking table overnight culture at 37 ℃, extracting a plasmid, and then transferring the plasmid into Escherichia coli E.coli Rosetta to obtain a site-specific mutant strain E.coli Rosetta-pET-28a (+) -Alg 212H-222K.
In example 4, when the 212 th glutamic acid and 222 th arginine of the genetically engineered alginate lyase are site-specifically mutated into histidine and lysine, the enzyme activity of the alginate lyase is greatly improved (as shown in fig. 6), is 15000U/ml, is 2.83 times of that of the original strain, is named as pET-28a (+) -Alg212H-222K, and is transformed into E.coli Rosetta, so as to obtain a site-specifically mutated strain E.coli Rosetta-pET-28a (+) -Alg 212H-222K. The combination mutation of the two sites is proved to obviously improve the enzyme activity of the genetic engineering alginate lyase.
Example 5: verification of alginate lyase production strain
Selecting the transformants in examples 2, 3 and 4, inoculating the transformants into a 96-well plate filled with L B liquid culture medium, culturing for 6h at 37 ℃, transferring the transformants into a fermentation culture medium with the inoculum size of 2%, culturing for 30h, collecting fermentation supernatant, detecting the activity of the fermentation supernatant, selecting strains with obviously changed yield, performing shake flask fermentation, detecting the activity of alginate lyase, and obtaining a result shown in figure 2, selecting the 212 th site and the 222 th site with larger mutation sites, performing protein gel electrophoresis verification, displaying on protein electrophoresis (figure 2), wherein the expression level has no obvious difference, and then, using Ni2+And purifying the protein by using an affinity column to obtain the high-purity protein.
The analytical enzymology properties are shown in the following table 2, and kinetic parameter analysis shows that the affinity of the mutant to the algin is obviously improved, and the catalytic efficiency is also obviously increased. K compared with the wild enzymemRespectively reduced by 2.1-2.5 times, KcatThe value is not substantially changed, and the catalytic efficiency (K)cat/Km) The improvement is 2.7-3.2 times of that of wild enzyme. In contrast, the specific enzyme activity of the mutant with improved yield is also obviously improved, wherein the specific enzyme activity of Alg212H-222K is improved by 2.7 times compared with that of the wild enzyme and reaches 382U/mg. Meanwhile, stability analysis shows that the optimal reaction temperature of the mutant is not obviously changed (16 ℃), and the half-life period of the mutant (t) is shown by detecting the half-life period of the mutant at 16 DEG C1/2) The improvement is respectively 261 min and 288min, which is 4.5 times higher than that of wild enzyme. The analysis of the free energy result shows that the free energy of the mutant is respectively improved by 3.63 kJ/mol. The results show that the substrate binding capacity, the catalytic efficiency and the thermal stability of the alginate lyase can be obviously improved after continuous mutation, modification and replacement.
TABLE 2 enzymatic Properties of the mutants
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
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<213> (Artificial sequence)
<400>11
tcccaaagaa acggatgtca gctatgcggt at 32
<210>12
<211>32
<212>DNA
<213> (Artificial sequence)
<400>12
catccgtttc tttgggatct tccttggcga ga 32
<210>13
<211>36
<212>DNA
<213> (Artificial sequence)
<400>13
ctggaactac aaaaagaatc tcgccaagga agatcc 36
<210>14
<211>32
<212>DNA
<213> (Artificial sequence)
<400>14
tctttttgta gttccagaac accgagccag tg 32
<210>15
<211>32
<212>DNA
<213> (Artificial sequence)
<400>15
tcccaaaaaa acggatgtca gctatgcggt at 32
<210>16
<211>32
<212>DNA
<213> (Artificial sequence)
<400>16
catccgtttt tttgggatct tccttggcga ga 32
<210>17
<211>36
<212>DNA
<213> (Artificial sequence)
<400>17
ctggaactac cataagaatc tcgccaagga agatcc 36
<210>18
<211>32
<212>DNA
<213> (Artificial sequence)
<400>18
tcttatggta gttccagaac accgagccag tg 32
<210>19
<211>32
<212>DNA
<213> (Artificial sequence)
<400>19
tcccaaacat acggatgtca gctatgcggt at 32
<210>20
<211>32
<212>DNA
<213> (Artificial sequence)
<400>20
catccgtatg tttgggatct tccttggcga ga 32
Claims (10)
1. The alginate lyase mutant modified by site-directed mutagenesis is characterized in that the alginate lyase mutant is obtained by replacing glutamic acid at the 212 th site of the alginate lyase with histidine and/or replacing arginine at the 222 th site with lysine, wherein the amino acid sequence of the alginate lyase is shown as SEQ ID NO. 1.
2. The mutant alginate lyase as claimed in claim 1, wherein when the amino acid sequence of the alginate lyase shown as SEQ ID No.1 is replaced by histidine at the 212 th glutamic acid position, the amino acid sequence of the mutant is shown as SEQ ID No. 2.
3. The mutant alginate lyase as claimed in claim 1, wherein when arginine at position 222 of the alginate lyase with an amino acid sequence shown in SEQ ID No.1 is replaced by lysine, the amino acid sequence of the mutant is shown in SEQ ID No. 3.
4. The mutant alginate lyase as claimed in claim 1, wherein when the amino acid sequence of the alginate lyase shown as SEQ ID No.1 is replaced by histidine at the 212 th glutamic acid position and lysine at the 222 th arginine position, the amino acid sequence of the mutant is shown as SEQ ID No. 4.
5. A gene encoding the mutant alginate lyase described in any one of claims 1 to 4.
6. An expression plasmid carrying the gene of claim 5.
7. The expression plasmid of claim 6, wherein the plasmid is pET-28a (+).
8. A recombinant bacterium expressing the mutant according to any one of claims 1 to 4.
9. The recombinant strain of claim 8, wherein the recombinant strain is E.coli Rosetta as a host.
10. The use of the recombinant bacterium of claim 8 in the production of a product comprising alginate lyase.
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| CN112921020A (en) * | 2021-03-02 | 2021-06-08 | 中国科学院青岛生物能源与过程研究所 | Algin lyase mutant for relieving divalent metal ion dependence and application thereof |
| CN112980821A (en) * | 2021-03-02 | 2021-06-18 | 中国科学院青岛生物能源与过程研究所 | Alginate lyase mutant capable of specifically recognizing substrate and application thereof |
| CN113223617A (en) * | 2021-04-27 | 2021-08-06 | 集美大学 | Method for screening salt-activated PL7 family alginate lyase |
| CN113481187A (en) * | 2021-05-18 | 2021-10-08 | 江南大学 | Alginate lyase mutant and application thereof |
| CN113549608A (en) * | 2021-05-24 | 2021-10-26 | 中国农业科学院麻类研究所 | Pectin lyase mutant delta PelG403 and encoding gene, preparation method and application thereof |
| CN114134135A (en) * | 2020-12-01 | 2022-03-04 | 北京化工大学 | H-protein mutant |
| CN114774400A (en) * | 2022-04-13 | 2022-07-22 | 齐鲁工业大学 | High-activity mutant pectin lyase and application thereof |
| CN115948373A (en) * | 2022-11-11 | 2023-04-11 | 深圳润康生态环境股份有限公司 | Alginate lyase mutant Pl7AaM and application thereof |
| CN117821432A (en) * | 2023-12-19 | 2024-04-05 | 江南大学 | Method for improving thermal stability of algin lyase and application thereof |
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| CN114134135B (en) * | 2020-12-01 | 2023-11-28 | 北京化工大学 | H-protein mutant |
| CN114134135A (en) * | 2020-12-01 | 2022-03-04 | 北京化工大学 | H-protein mutant |
| CN112921020B (en) * | 2021-03-02 | 2022-04-08 | 中国科学院青岛生物能源与过程研究所 | Algin lyase mutant for relieving divalent metal ion dependence and application thereof |
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| CN113223617A (en) * | 2021-04-27 | 2021-08-06 | 集美大学 | Method for screening salt-activated PL7 family alginate lyase |
| CN113481187B (en) * | 2021-05-18 | 2023-07-25 | 江南大学 | Algin lyase mutant and application thereof |
| CN113481187A (en) * | 2021-05-18 | 2021-10-08 | 江南大学 | Alginate lyase mutant and application thereof |
| CN113549608B (en) * | 2021-05-24 | 2022-03-08 | 中国农业科学院麻类研究所 | Pectin lyase mutant delta PelG403 and encoding gene, preparation method and application thereof |
| CN113549608A (en) * | 2021-05-24 | 2021-10-26 | 中国农业科学院麻类研究所 | Pectin lyase mutant delta PelG403 and encoding gene, preparation method and application thereof |
| CN114774400A (en) * | 2022-04-13 | 2022-07-22 | 齐鲁工业大学 | High-activity mutant pectin lyase and application thereof |
| CN114774400B (en) * | 2022-04-13 | 2023-05-12 | 齐鲁工业大学 | High-activity mutant pectin lyase and application thereof |
| CN115948373A (en) * | 2022-11-11 | 2023-04-11 | 深圳润康生态环境股份有限公司 | Alginate lyase mutant Pl7AaM and application thereof |
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| CN117821432A (en) * | 2023-12-19 | 2024-04-05 | 江南大学 | Method for improving thermal stability of algin lyase and application thereof |
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