CN112646799B - Ulva lactuca polysaccharide lyase mutant and application thereof - Google Patents
Ulva lactuca polysaccharide lyase mutant and application thereof Download PDFInfo
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Abstract
The invention provides an ulva polysaccharide lyase mutant and application thereof, belonging to the field of genetic engineering. The activity of the enzyme provides a degradation function in ulvan. The ulva polysaccharide lyase mutant is characterized in that on the basis of the amino acid sequence of wild ulva polysaccharide lyase, the 35 th position of the mutant is mutated from Asn to Pro, the optimum temperature and the optimum pH of the mutant and the wild enzyme are both 40 ℃ and 9.0, and the specific activity of the mutant is about 1.35 times that of the wild enzyme. Under the optimal reaction condition, the catalytic efficiency of the mutant is improved by about 2 times compared with that of the wild type. When ulva powder is used as a substrate, the content of reducing sugar produced by the catalytic reaction of the mutant enzyme is 1.27 times that of a wild degradation substrate, so that the mutant enzyme is obviously improved in enzyme activity and can be used for reducing the economic cost of industrial application.
Description
Technical Field
The invention relates to an ulva polysaccharide lyase mutant and application thereof, belonging to the field of genetic engineering.
Background
China is a big country for seaweed production and consumption. As an important marine resource, the seaweed also has wide application prospects in the aspects of biological medicines, chemical fibers, energy sources, cosmetics, pesticides and the like besides being traditionally used as food, chemical raw materials, aquatic feeds, seaweed fertilizers and the like. The research of the algae resources in China is integrated, and the application value of the algae resources is mainly limited to the development of the food field and is not fully utilized in other industries. With the continuous development of the industrialized society, the global climate change, seawater pollution and eutrophication are increased, which leads to the complication and even deterioration of the marine environment and the occurrence of the green tide phenomenon. Therefore, reasonable utilization of such resources is crucial to the alleviation of energy crisis, and the acceleration of utilization and conversion of algal biomass becomes a key. Furthermore, examples of the production of biofuels such as bioethanol using algal biomass have been reported for a long time.
Ulva lactuca is a common green alga in coastal areas of China, and has the effects of treating heatstroke, gastroenteritis and the like as a medicinal material. Ulvan is a main functional component in ulva, is sulfated water-soluble polysaccharide extracted from ulva cell walls, and is mainly composed of repeated disaccharide units composed of sulfated rhamnose, glucuronic acid, iduronic acid and part of xylose. Ulvan has been demonstrated to have a variety of pharmacological properties, such as antioxidant, anticoagulant, antitumor, antihyperlipidemic, antiviral activity. However, the biological activity of ulvan is limited by the influence of molecular weight and high viscosity, and low molecular weight ulvan oligosaccharide has higher solubility, lower viscosity, easier absorption and easier exposure of active groups, compared to high molecular weight ulvan. At present, the depolymerization of ulva polysaccharide is mostly a physical and chemical method, but the methods have the defects of sulfate group loss, complex product, low oligosaccharide yield and the like. In contrast, enzymatic degradation of polysaccharides by biological enzymes is a highly efficient method, and therefore, it is important to develop a biological enzyme having high catalytic activity, which can degrade ulva polysaccharides.
Ulva polysaccharide lyase (EC4.2.2-, Ulva polysaccharide lyase) belongs to the family of Polysaccharide Lyase (PL), the polysaccharide lyase is a powerful tool for analyzing a repeating unit of a complex polysaccharide structure, Ulva polysaccharides with different molecular weights can be efficiently and mildly prepared by using the enzyme, the structure-activity relationship of the polysaccharides can be conveniently researched, and the Ulva polysaccharide lyase has the characteristics of specificity, high efficiency and no damage to a substituent. The ulva polysaccharide lyase has a similar cracking mechanism to other polysaccharide lyase families, and an ulva polysaccharide chain containing uronic acid is cracked through a beta-elimination mechanism, so that a structural unit of micromolecule ulva oligosaccharides such as disaccharide, trisaccharide and the like containing unsaturated hexuronic acid residues and a new reducing end is generated. The ulva polysaccharide is cracked into small-molecular ulva oligosaccharide by ulva polysaccharide lyase by adopting a biological enzymolysis technology, the catalytic degradation mechanism of the ulva polysaccharide lyase on the ulva polysaccharide is researched, and the method has important significance for preparing and extracting the small-molecular oligosaccharide. Therefore, the research and development of ulva polysaccharide lyase with high catalytic activity can reduce the production cost of industrial scale and improve the economic benefit.
The enzyme activity is generally not high and the stability is poor by combining the related research on ulva polysaccharide lyase at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel ulva polysaccharide lyase and application thereof, and the ulva polysaccharide lyase is modified mainly by using a site-specific mutagenesis technology to obtain an ulva polysaccharide lyase mutant with improved enzyme activity, so that the economic cost of industrial application is reduced.
One of the purposes of the invention is to provide an ulva polysaccharide lyase mutant, wherein the amino acid sequence of the ulva polysaccharide lyase mutant is shown as SEQ ID NO: 1 is shown.
The other purpose of the invention is to provide a coding gene of the ulva polysaccharide lyase mutant.
In a specific embodiment of the invention, the nucleotide sequence of the coding gene is as shown in SEQ ID NO: 2, respectively.
It is a further object of the present invention to provide a vector comprising the encoding gene. The vector may be one of vectors for producing a protein by gene recombination, such as an expression vector, known to those skilled in the art.
In one embodiment of the invention, the vector is pQE 80L.
In another aspect, the present invention provides a host cell comprising said encoding gene or vector. The host cell may be any host suitable for producing the ulvan lyase mutant of the present invention from the gene or vector of the present invention, for example, escherichia coli.
In a specific embodiment of the invention, the host cell is E.coli BL 21.
In another embodiment of the present invention, the host cell is Escherichia coli JM 109.
The fifth purpose of the invention is to provide a specific primer pair for reverse PCR amplification of the coding gene.
In a specific embodiment of the present invention, the nucleotide sequence of the specific primer pair is as shown in SEQ ID NO: 5 and SEQ ID NO: and 6.
The sixth purpose of the invention is to provide application of the ulva polysaccharide lyase mutant in producing ulva oligosaccharides by splitting ulva polysaccharide.
The seventh purpose of the invention is to provide a method for preparing the ulvan lyase mutant, wherein the method utilizes the coding gene of the ulvan lyase mutant or the vector of the invention to carry out gene recombination and expression. Genetic recombination methods and expression hosts known to those skilled in the art can be used, and media and culture conditions suitable for expression by the host are selected. The method may further comprise a step of recovering the ulvan lyase mutant, which may involve a step of isolating or purifying the ulvan lyase mutant from a culture or an expression product of the host, and may be performed using any method known to those skilled in the art.
An eighth object of the present invention is to provide a method for degrading ulvan, which comprises the step of contacting ulvan with the ulvan lyase mutant of the present invention or with the host cell of the present invention under a condition capable of degrading ulvan by enzymatic catalysis of the ulvan lyase mutant or the host cell.
The invention has the beneficial effects that:
under the same condition, the specific activity of the ulva polysaccharide lyase mutant is 1.35 times that of wild enzyme, and the improvement of the enzyme activity can effectively reduce the economic cost of industrial application. K of wild-type enzymecat/KmThe value was 51.15 mL. mg-1·min-1The mutant of the invention is 163.83 mL. mg-1·min-1The catalytic efficiency of the mutant is improved by about 2 times compared with that of the wild type.
Drawings
FIG. 1 shows the PCR verification of the transformants of the wild type and mutant of ulva polysaccharide lyase in the example, wherein M is 1kb Marker; 1, a wild-type transformant; 2, transformant of mutant.
FIG. 2 shows the enzyme activities of the wild type and the mutant of ulva polysaccharide lyase under different temperature conditions at pH 9.0.
FIG. 3 shows the enzyme activities of the wild type and the mutant of ulva polysaccharide lyase at 40 ℃ and different pH values.
FIG. 4 is the kinetic analysis of wild type and mutant of ulva polysaccharide lyase in the examples.
FIG. 5 shows the reducing sugar content of wild type and mutant of ulva polysaccharide lyase in the example of degrading ulva powder.
Detailed Description
The invention is further described below by means of specific embodiments. Technical means, materials and the like to which the following embodiments refer may be known to those skilled in the art, and appropriate ones may be selected among known means and materials capable of solving the respective technical problems, unless otherwise specified. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The following definitions are used in the present invention:
1. amino acid residues are identified using the IUPAC nomenclature, either in the form of three letter abbreviations or single letter symbols. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of mutants: "amino acid substituted by original amino acid position" is used to indicate mutated amino acid in ulva polysaccharide lyase mutant. E.g. N35P, indicating a substitution of the amino acid at position 35 from asparagine Asn to proline Pro of the parent ulvan lyase, the numbering of the positions corresponding to SEQ ID NO: 3, numbering the amino acid sequence of ulva polysaccharide lyase.
In a specific embodiment of the present invention, a method for obtaining the ulva polysaccharide lyase mutant by using a site-directed mutagenesis technology is provided, which mainly comprises the following steps:
(6) and respectively synthesizing nucleotide sequences shown as SEQ ID NO: 4, performing double enzyme digestion on the wild type gene and an expression vector pQE80L by using two restriction enzymes of BamH I and Hind III, and then connecting to construct a recombinant vector containing a wild type ulva polysaccharide lyase coding gene, wherein the recombinant vector takes escherichia coli Jm109 as a cloning host;
(7) and (2) taking the recombinant vector in the step (1) as a template, taking an oligonucleotide sequence with a mutation site as a primer pair, and performing reverse PCR amplification to obtain a primer containing the nucleotide sequence shown in SEQ ID NO: 2, and 2, a PCR product of the nucleotide sequence shown in the figure;
(8) connecting the PCR products obtained in the step (2) by using ligase, transferring the PCR products into escherichia coli Jm109, and culturing to obtain a recombinant vector containing the coding gene of the ulva polysaccharide lyase mutant;
(9) transforming the recombinant vector in the step (3) into competent cells of escherichia coli BL21 to obtain host cells containing ulva polysaccharide lyase mutant genes;
(10) culturing the host cell in the step (4) under a proper condition, inducing expression, collecting ulvan lyase in an expression product, and purifying by an affinity chromatography method to obtain the ulvan lyase active protein.
Preferably, the amplification is performed by PrimerStar enzyme during reverse PCR amplification.
Preferably, the ligase in the step (3) is T4 polyphosphate kinase and Ligation high ligase.
Example 1:
preparing an ulva polysaccharide lyase mutant.
(1) Constructing a recombinant plasmid pQE80L-NuPL 28: a recombinant vector containing a coding gene of wild ulva polysaccharide lyase.
In this example, the amino acid sequence of the mature peptide of wild type ulva polysaccharide lyase (shown in SEQ ID NO: 3) was obtained by reference to GenBank: KEZ94336.1, and the amino acid sequence was codon-optimized using E.coli as an expression host, and the optimized nucleotide sequence was shown in SEQ ID NO: 4, and then synthesizing the optimized gene. The synthesized gene and the vector pQE80L are cut by two restriction enzymes of BamH I and Hind III, and are transformed into a cloning host escherichia coli Jm109 after being connected by ligase. The correctly sequenced plasmid was designated pQE80L-NuPL 28.
(2) Constructing a recombinant plasmid pQE80L-NuPL 28-N35P: a recombinant vector containing the coding gene of the ulva polysaccharide lyase mutant.
In the embodiment, the recombinant plasmid pQE80L-NuPL28 in the step (1) is used as a template, and an oligonucleotide sequence with a mutation site is used as a primer pair (shown as SEQ ID No.5 and SEQ ID No. 6) to carry out reverse PCR, so that a PCR product with a mutation gene is obtained through amplification; the PCR products were ligated using ligase, transferred to e.coli Jm109 competent cells, plated on solid LB medium containing kanamycin resistance, and cultured overnight in a 37 ℃ incubator. And (4) selecting a single colony on the culture medium for sequencing verification to obtain a transformant which is verified correctly. Then extracting the recombinant plasmid in the transformant, namely the recombinant plasmid containing the ulva polysaccharide lyase mutant is named as pQE80L-NuPL 28-N35P.
The gene mutation sites of the mutant are as follows:
(3) constructing a genetically engineered bacterium pQE80L-NuPL28-N35P E. coli BL 21: an expression host containing ulva polysaccharide lyase mutant genes.
In this example, the recombinant plasmid pQE80L-NuPL28-N35P described in step (2) was transformed into competent cells of E.coli BL21, spread on a solid LB medium containing kanamycin resistance, and cultured at 37 ℃. The single clone transformant was selected to verify the target gene, and the result is shown in FIG. 1. And if the correct transformant is verified, the transformant is the engineering bacterium pQE80L-NuPL28-N35P E. coli BL21 containing the ulva polysaccharide lyase mutant gene.
Example 2:
and (3) measuring the expression and enzyme activity of the ulva polysaccharide lyase mutant.
(1) Expression of genetically engineered bacteria
The screened genetic engineering bacteria pQE80L-NuPL28-N35P E. coli BL21 are selected and placed in a 5mL LB liquid culture medium for culture overnight at 37 ℃, then transferred to a 250mL LB culture medium, cultured for 2-2.5 h at 37 ℃, when OD600 is 0.6-0.8, IPTG is added and placed in a constant temperature shaking table at 16 ℃ for culture for 18h, and the expression of the target protein is induced. Then centrifuging at 8000r/min for 10min to collect cells, re-suspending the cells with buffer solution and crushing the cells, centrifuging at 12000r/min for 30min to collect crushed supernatant, namely crude enzyme extract expressed in cells, and filtering with a water system membrane with the pore diameter of 0.22 μm. With Ni2+The protein was purified on a chelating agarose resin column, and the eluate was collected and then used to replace the buffer with a 10kDa ultrafiltration tube using MilliQwater. And (3) identifying the purity of the target protein in the purification process by SDS-PAGE electrophoresis, determining the protein concentration of the target protein by using a BCA kit, determining the enzyme activity and calculating the specific activity. Except for the mutation point, the genetic background of the wild bacteria without site-directed mutagenesis is completely the same.
(2) Enzyme activity assay
Definition of enzyme activity (U): the amount of protein required to produce 1. mu. mol unsaturated uronic acid per minute.
The enzyme activity determination method comprises the following steps: ulva polysaccharide is used as a substrate, the reaction time is 5min, and the change of the light absorption value is measured at the wavelength of 232 nm.
Measurement of optimum temperature: the wild type and the mutant are respectively tested for enzyme activity at different temperatures under the condition of pH 9.0, and the specific activity is calculated according to the protein concentration. As shown in FIG. 2, the optimum temperature was 40 ℃ for both the wild type and the mutant, but the specific activity of the mutant was about 1.35 times that of the wild type.
Determination of optimum pH: and (3) measuring the enzyme activity in different pH buffer solution systems at the optimal temperature of 40 ℃ and calculating the specific activity. As can be seen from FIG. 3, under all pH conditions of the assay, the enzyme activity of the mutant is greater than that of the wild type, the enzyme activity is highest in a buffer system with pH 9, and the specific activity of the mutant is about 1.35 times that of the wild type.
Under the conditions of the optimal temperature of 40 ℃ and the optimal pH value of 9.0, the specific activity of the wild enzyme is 17.83U/mg, the specific activity of the mutant enzyme is 24.14U/mg, which is 1.35 times of the specific activity of the wild enzyme, and the relative enzyme activity is calculated by taking the enzyme activity of the wild enzyme as 100% of the other enzyme activities.
(3) Enzymatic kinetic analysis
Performing enzyme kinetic analysis on wild type and mutant by using ulva polysaccharide with different concentrations (0.3, 0.6, 0.9, 1.2, 1.5, 2.0, 3.0mg/mL) as substrate under the conditions of optimum temperature of 40 ℃ and optimum pH of 9.0 (the results are shown in Table 1 and figure 4), and obtaining k of wild type enzymecat/KmThe value was 51.15 mL. mg-1·min-1The mutant is 163.83 mL. mg-1·min-1The catalytic efficiency of the mutant is improved by about 2 times compared with that of the wild type.
TABLE 1 enzymatic kinetic analysis of Ulva lactuca lyase
Example 3:
research on the catalytic application of ulva polysaccharide lyase to ulva polysaccharide.
Reaction system: reacting 20mg/mL ulva powder serving as a substrate and 0.1mg/mL wild enzyme or mutant enzyme (all enzymes are ulva polysaccharide lyase) prepared by the invention serving as a catalyst in 20mM Tris-HCl buffer solution with pH of 9.0 for 48 hours at 30 ℃ and 220 rpm. The control group did not add ulva polysaccharide lyase, and the other reaction conditions were consistent. After the reaction is finished, the sample is boiled for 10min to finish the reaction of the system, the sample is centrifuged at 12000r/min for 10min, and the content of reducing sugar in the supernatant is measured by using a DNS method.
In the application research of ulva polysaccharide lyase, compared with the wild type, the mutant can obviously improve the degradation efficiency of the substrate (refer to the attached figure 5). After reacting for 48 hours by taking ulva powder as a substrate, the content of reducing sugar generated by the action of wild enzyme on the substrate is 0.51mg/mL, and the content of mutant enzyme is 0.65mg/mL which is 1.27 times of that of the wild enzyme.
Although the present invention has been disclosed in the form of preferred embodiments, it is not intended to limit the present invention, and those skilled in the art may make various changes, modifications, substitutions and alterations in form and detail without departing from the spirit and principle of the present invention, the scope of which is defined by the appended claims and their equivalents.
SEQUENCE LISTING
<110> Tianjin science and technology university
Ulva lactuca polysaccharide lyase mutant and application thereof
<130> 2020
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<170> PatentIn version 3.5
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tgtacaatca acaacagggc ctgtat 26
Claims (8)
1. An ulva polysaccharide lyase mutant is characterized in that the amino acid sequence of the ulva polysaccharide lyase mutant is shown as SEQ ID NO: 1 is shown.
2. The ulva polysaccharide lyase mutant of claim 1, which encodes a gene.
3. A recombinant vector comprising the coding gene of claim 2.
4. A host cell comprising the coding gene of claim 2 or the recombinant vector of claim 3.
5. The host cell of claim 4, wherein the host cell is Escherichia coli BL21 or Escherichia coli JM 109.
6. Use of the ulva polysaccharide lyase mutant of claim 1 for producing ulva oligosaccharides by cleaving ulva polysaccharides.
7. A method for preparing the ulva polysaccharide lyase mutant of claim 1, comprising the following steps:
(1) and respectively synthesizing nucleotide sequences shown as SEQ ID NO: 4 and expression vector pQE80LBamH I andHind III, performing double enzyme digestion on two restriction enzymes, and then connecting to construct a recombinant vector containing a wild ulva polysaccharide lyase coding gene, wherein the recombinant vector takes escherichia coli JM109 as a cloning host;
(2) taking the recombinant vector in the step (1) as a template, and utilizing the nucleotide sequence shown as SEQ ID NO: 5 and SEQ ID NO: 6, obtaining a primer pair containing the nucleotide sequence shown in SEQ ID NO: 2, and 2, a PCR product of the nucleotide sequence shown in the figure;
(3) connecting the PCR products obtained in the step (2) by using ligase, transferring the PCR products into escherichia coli JM109, and culturing to obtain a recombinant vector containing the coding gene of the ulva polysaccharide lyase mutant;
(4) transforming the recombinant vector in the step (3) into competent cells of escherichia coli BL21 to obtain host cells containing ulva polysaccharide lyase mutant genes;
(5) culturing the host cell in the step (4) under a proper condition, inducing expression, collecting ulvan lyase in an expression product, and purifying by an affinity chromatography method to obtain the ulvan lyase active protein.
8. A method of degrading ulvan, the method comprising the step of contacting ulvan with the ulvan lyase mutant of claim 1 under conditions capable of degrading ulvan by enzymatic catalysis of the ulvan lyase mutant.
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