CN111424027B - 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-directed mutagenesis and application thereof, belonging to the technical field of enzyme engineering. The alginate lyase mutant of the invention is obtained by replacing glutamic acid at position 212 of the alginate lyase with histidine and/or arginine at position 222 of the alginate lyase with lysine, wherein the amino acid sequence of the alginate lyase is shown as SEQ ID NO. 1. The invention determines the active center amino acid of the alginate lyase through molecular docking simulation, changes the amino acid residue near the active site of the protein molecule through site-specific saturation mutagenesis, improves the catalytic efficiency of the alginate lyase and further improves the yield of the alginate lyase. The recombinant escherichia coli with the enhanced algin lyase secretion capability constructed by the invention can improve the algin lyase enzyme activity by 2.83 times compared with the original strain. The modified genetically engineered bacteria have obviously improved enzyme production capability, the enzyme activity of the alginate lyase produced by shake flask fermentation reaches 15000U/mL, the genetically engineered bacteria are more suitable for industrial application, the production cost can be reduced, and the production efficiency can be improved.
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 three kinds of algae, and has main components of algin, mannitol and laminarin. Among them, the related research on the utilization of mannitol and laminarin is mature, so how to efficiently degrade algin and realize industrial application is a hotspot of the current research. Algin is a linear polysaccharide composed of β -D-mannuronic acid (M) and α -L-guluronic acid (G), and the degradation product alginate oligosaccharide thereof has important physiological activities, such as oxidation resistance, tumor resistance, plant root cell growth promotion, and the like, and thus has received wide attention.
The alginate lyase (Aly) can degrade the alginate by a beta-elimination reaction mechanism, and form unsaturated carbon-carbon double bonds at the non-reducing end of the degradation product, thereby generating a special absorption peak under the ultraviolet of 235nm and being used for product detection. According to the substrate specificity of Aly, it can be divided into polyM, polyG and bifunctional type alginate lyase; according to the enzyme digestion mode, the method can be divided into endo-type (endo-type) and exo-type (exo-type) alginate lyase, the end product of degrading the alginate by the endo-type Aly is Alginate Oligosaccharide (AOs), the polymerization degree of the common product is 2-6, the product of the exo-type Aly is single, the end product is monosaccharide, but disaccharide is reported as the minimum enzyme digestion unit; according to the classification of the CAZY database, alginate lyase belongs to Polysaccharide Lyase (PL), and is specifically classified into seven families, PL-5, PL-6, PL-7, PL-14, PL-15, PL-17 and PL-18.
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 invention determines the active center amino acid of the alginate lyase through molecular docking simulation, changes the amino acid residue near the active site of the protein molecule through site-specific saturation mutagenesis, improves the catalytic efficiency of the alginate lyase and further improves the yield of the alginate lyase. The recombinant escherichia coli with the enhanced algin lyase secretion capability constructed by the invention can improve the algin lyase enzyme activity by 2.83 times compared with the original strain. The modified genetically engineered bacteria have obviously improved enzyme production capability, the enzyme activity of the alginate lyase produced by shake flask fermentation reaches 15000U/mL, the genetically engineered bacteria are more suitable for industrial application, the production cost can be reduced, and the production efficiency can be improved.
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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:
LB liquid medium: 10.0g/L of peptone, 5.0g/L of yeast powder and 5.0g/L of NaCl.
TB fermentation medium: peptone 12.0g/L, yeast powder 24.0g/L, glycerol 4.0g/L, KH2PO417mmol/L, K2HPO 472 mmol/L.
Measuring the enzyme activity of the alginate lyase:
a slight adjustment is made in the improved DNS method. Enzyme activity determination conditions: adding 0.1mL of enzyme solution into 0.9mL of 1% sodium alginate solution, uniformly mixing, reacting at 45 ℃ for 20min, adding 1mL of DNS into the system, stopping the reaction in a boiling water bath for 3min, quickly metering to 10mL, sucking 200 μ L of enzyme solution, adding into a 96-well plate, and measuring the light absorption value at 520 nm. The control group used deionized water instead of the crude enzyme solution. 1 enzyme activity unit (U) definition: under the above conditions, 1mL of the enzyme solution produced 1. mu.g of reducing sugar per minute. Preparing glucuronic acid solutions with different concentrations, adding 1mL of DNS reagent, reacting, and measuring the light absorption values of the solutions with different concentrations at 520 nm. And calculating the enzyme activity according to the 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
Using the reported alginate lyase AlyA5 alginate lyase (PDB code: 4BE3) as a template (crystal structure of the exolytic PL7 alginate lyase AlyA5 from Zobellia galactantivorans, published in 2013) (the amino acid similarity between the two is 38.2%), the crystal structure of E.coli-derived alginate lyase was simulated using molecular simulation docking software. Example 1 protein 3D structure-mimic docking was followed by selection of the active sites of alginate lyase by selecting the 212 th glutamic acid and 222 th arginine near the catalytic domain of alginate lyase.
Example 2: influence of active site mutation of alginate lyase on enzyme activity expression of alginate lyase
Using site-directed mutagenesis kit (TaKaRa) to design primers 212A-F,212A-R, 222A-F and 222A-R (shown in table 1), using constructed pET-28a (+) as a template to perform PCR, respectively replacing 212 th glutamic acid and 222 th arginine of alginate lyase with alanine, and performing PCR under the conditions that: 5min at 98 ℃, 34 cycles (30S at 98 ℃, 60S at 57.6 ℃ and 1.5min at 72 ℃) and 10min at 72 ℃. PCR amplification System: template 1. mu.L, upstream and downstream primers 1. mu.L each, Prime Star (Premix) DNA 20. mu.L, ddH2O17. mu.L. And purifying and recovering the PCR product by using a gel recovery kit, and carrying out electrophoresis test on the concentration of the recovered product. The PCR products were recovered by ligation using T4 ligase by digestion with BamHI and Mlu I fast cutter, transformed into competent E.coil JM109, spread on ampicillin LB plates, and positive colonies were picked. After shaking overnight culture at 37 ℃, plasmids are extracted and then transferred into escherichia coli E.
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), designing primers 212H-F, 212H-R and 222K-F,222K-R (shown in Table 1), using the constructed pET-28a (+) -Alg212A as a template to perform PCR, mutating and replacing glutamic acid at 212 th position or arginine at 222 th position, and the PCR reaction conditions are as follows: 5min at 98 ℃, 34 cycles (30S at 98 ℃, 60S at 57.8 ℃ and 1.5min at 72 ℃) and 10min at 72 ℃. PCR amplification System: template 1. mu.L, upstream and downstream primers 1. mu.L each, Prime Star (Premix) DNA 20. mu.L, ddH2O17. mu.L. And purifying and recovering the PCR product by using a gel recovery kit, and carrying out electrophoresis test on the concentration of the recovered product. The enzyme digestion is carried out under the action of Bam HI and Mlu I fast cutting enzymes, and the ligation treatment PCR is carried out by using a T4 ligase method to recover a product, and the product is transformed into a competent E.coil JM109 was plated on ampicillin LB plates, and positive colonies were picked. After overnight shake culture at 37 ℃ the plasmid was extracted to obtain site-directed mutant strains.
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, the enzyme activity of the genetically engineered alginate lyase is significantly improved when the 222 th arginine of the alginate lyase is point-mutated to lysine (as shown in fig. 5), is 8000U/ml, is 1.51 times of the enzyme activity of the original strain, is named as pET-28a (+) -Alg222K, and is 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
Using a site-directed mutagenesis kit (TaKaRa) to design primers 222K-F and 222K-R (shown in table 1), using the constructed pET-28a (+) -Alg212H as a template to perform PCR, mutating and replacing arginine at position 222, wherein the PCR reaction conditions are as follows: 5min at 98 ℃, 34 cycles (30S at 98 ℃, 60S at 57.8 ℃ and 1.5min at 72 ℃) and 10min at 72 ℃. PCR amplification System: template 1. mu.L, upstream and downstream primers 1. mu.L each, Prime Star (Premix) DNA 20. mu.L, ddH2O17. mu.L. And purifying and recovering the PCR product by using a gel recovery kit, and carrying out electrophoresis test on the concentration of the recovered product. The PCR products were recovered by ligation using T4 ligase by digestion with BamHI and Mlu I fast cutter, transformed into competent E.coil JM109, spread on ampicillin LB plates, and positive colonies were picked. After shaking overnight at 37 ℃, extracting a plasmid named as pET-28a (+) -Alg212H-222K, and transferring into Escherichia coli EColi 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
Transformants in examples 2, 3 and 4 were selected and inoculated into 96-well plates containing LB liquid medium, cultured at 37 ℃ for 6h, and inoculated into fermentation medium in an inoculum size of 2% for 30 h. Collecting fermentation supernatant, and detecting the enzyme activity of the fermentation supernatant. And selecting strains with obviously changed yield to carry out shake flask fermentation. The results of detecting the activity of the alginate lyase are shown in FIG. 2. And selecting the 212 th site and the 222 th site with larger influence of the mutation sites to carry out protein gel electrophoresis verification. The protein was electrophoretically shown (fig. 2) to have no significant difference in expression levels. Subsequently, 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 results of free energy analysis showed that the mutant was derived fromThe energy can be respectively increased 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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<210> 6
<211> 32
<212> DNA
<213> (Artificial sequence)
<400> 6
tcttcgtgta gttccagaac accgagccag tg 32
<210> 7
<211> 32
<212> DNA
<213> (Artificial sequence)
<400> 7
tcccaaagca acggatgtca gctatgcggt at 32
<210> 8
<211> 32
<212> DNA
<213> (Artificial sequence)
<400> 8
catccgtcgt tttgggatct tccttggcga ga 32
<210> 9
<211> 36
<212> DNA
<213> (Artificial sequence)
<400> 9
ctggaactac gaaaagaatc tcgccaagga agatcc 36
<210> 10
<211> 32
<212> DNA
<213> (Artificial sequence)
<400> 10
tcttttcgta gttccagaac accgagccag tg 32
<210> 11
<211> 32
<212> DNA
<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 bacterium according to claim 8, wherein the recombinant bacterium is Escherichia coliE. coliRosetta is the host.
10. The use of the recombinant bacterium of claim 8 in the production of a product comprising alginate lyase.
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| CN112458073B (en) * | 2020-12-01 | 2021-12-10 | 北京化工大学 | H-protein mutant and application thereof |
| CN112980821B (en) * | 2021-03-02 | 2022-04-12 | 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 |
| 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 |
| CN113223617B (en) * | 2021-04-27 | 2024-06-18 | 集美大学 | Method for screening salt-activated PL7 family algin lyase |
| CN113481187B (en) * | 2021-05-18 | 2023-07-25 | 江南大学 | Algin 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 |
| CN114774400B (en) * | 2022-04-13 | 2023-05-12 | 齐鲁工业大学 | High-activity mutant pectin lyase and application thereof |
| CN115948373B (en) * | 2022-11-11 | 2023-07-25 | 深圳润康生态环境股份有限公司 | Algin 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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