CN115725548B - Kappa-carrageenan Pcar mutant and preparation method thereof - Google Patents
Kappa-carrageenan Pcar mutant and preparation method thereof Download PDFInfo
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides a kappa-carrageenan Pcar mutant and a preparation method thereof, wherein the method comprises the following steps: predicting kappa-carrageenan enzyme by using a disulfide bond design module in Discovery Studio, selecting 4 mutation sites, and screening out mutants N205C-G239C with improved enzyme thermal stability and enzyme activity; the wild-type kappa-carrageenan enzyme is mutated at the mutation site of N205C-G239C to generate kappa-carrageenan enzyme Pcar mutant. The method can improve the heat stability of the enzyme, the enzyme activity of the mutant enzyme is improved by about 330%, and after the mutant enzyme is treated for 30min at 50 and 55 ℃, the residual enzyme activity of the mutant enzyme is 1.70 times and 1.76 times that of the wild type enzyme respectively, thereby providing a theoretical basis for better application of the carrageenan enzyme in the preparation of carrageenan oligosaccharides.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a kappa-carrageenan Pcar mutant and a preparation method thereof.
Background
The carrageenan is a hydrocolloid formed by alternately connecting 1, 3-beta-D-galactose and 1, 4-alpha-D-galactose, and can be classified into kappa type, iota type and lambda type according to the combination form of sulfuric acid ester. The carrageenan has wide sources, can be extracted from red algae of certain specific types, such as carrageenan, eucheuma, agar and the like, is mainly used as a thickening agent, a gelling agent and the like, and is widely applied to the food industry. Research shows that the carrageenan oligosaccharide formed after the carrageenan is degraded has various biological activities such as anti-tumor, antivirus, immunoregulation, anticoagulation and the like, so that the carrageenan oligosaccharide preparation is expected to be applied to the fields of medicines, foods, cosmetics and the like. The method for degrading the carrageenan mainly comprises an acid hydrolysis method, an ultrasonic degradation method and an enzymolysis method, wherein the acid hydrolysis method is most popular, and compared with the acid hydrolysis method, the enzyme method has mild conditions and strong specificity, and can protect the bioactivity of the carrageenan oligosaccharide, so that the enzyme degradation method becomes the preferable method for preparing the carrageenan oligosaccharide.
Kappa-carrageenan enzyme (EC 3.2.1.83) belongs to glycoside hydrolase, is an enzyme that degrades carrageenan by hydrolyzing beta-1, 4 glycosidic bonds inside kappa-carrageenan. The colloid viscosity of the carrageenan is reduced along with the temperature rise, and the kappa-carrageenan enzyme with excellent thermal stability and catalytic activity can process kappa-carrageenan at a higher temperature, so that the viscosity of a colloid solution is effectively reduced, and the enzymolysis efficiency is improved. At present, the production of carrageenase is mostly carried out in a form of directly fermenting strains obtained by breeding in nature, but carrageenase directly separated and purified from nature has the defects of low yield, low activity and poor stability. In recent years, along with the development of protein engineering technology, wild-type carrageenase is subjected to molecular transformation so as to improve the thermal stability, enzyme activity and other enzymatic properties of carrageenase, expand the application range of carrageenase and promote the high-value utilization of carrageenase.
Disclosure of Invention
The present invention aims to solve at least to some extent one of the technical problems in the above-described technology. Therefore, the invention provides a kappa-carrageenan Pcar enzyme mutant and a preparation method thereof, and the method can improve the thermal stability and catalytic activity of the enzyme and provide a theoretical basis for better application of the kappa-carrageenan enzyme to the preparation of carrageenan oligosaccharides.
To this end, in a first aspect of the invention, there is provided a method for preparing a mutant of kappa-carrageenan enzyme Pcar, comprising the steps of:
Predicting kappa-carrageenan enzyme by using a disulfide bond design module in a Discovery Studio, selecting 4 mutation sites far away from a kappa-carrageenan enzyme catalytic triplet, and screening out an enzyme thermal stability and enzyme activity improvement mutant N205C-G239C;
the wild-type kappa-carrageenan enzyme is mutated at the mutation site of N205C-G239C to generate kappa-carrageenan enzyme Pcar mutant.
According to the embodiment of the invention, disulfide bonds are introduced into the structure of kappa-carrageenan Pcar by a rational design method to improve the heat stability and the enzyme activity of the enzyme; by adopting a site-directed mutagenesis technology and taking a gene of kappa-carrageenan Pcar as a template, an N205C-G239C mutant is constructed, compared with wild type kappa-carrageenan (WT), the enzyme activity of mutant N205C-G239C is improved by about 330%, the thermal stability of mutant N205C-G239C is obviously enhanced, and after 30min of treatment at 50 and 55 ℃, the residual enzyme activity of N205C-G239C is 1.70 and 1.75 times that of the wild type enzyme respectively; the enzyme property research of the mutant enzyme shows that the enzyme has an optimal reaction temperature of 55 ℃, can specifically degrade kappa-carrageenan, has an optimal pH of 8.0 and has improved pH stability; structural analysis shows that disulfide bonds connect two folded sheets, so that the rigidity of enzyme molecules is improved, and the thermal stability of the enzyme is improved. Compared with WT, the hydrophobic interaction between mutant enzyme and kappa-carrageenan tetrasaccharide substrate is enhanced, which contributes to the improvement of enzyme activity and thermal stability. The rational design strategy based on the structure can provide thought for the improvement of the heat stability and the enzyme activity of industrial enzyme, and has important significance for the high-value utilization of kappa-carrageenan.
In a second aspect of the invention, the kappa-carrageenan enzyme Pcar-Pcar mutant prepared by the preparation method is characterized in that the amino acid sequence of the kappa-carrageenan enzyme Pcar mutant is shown as SEQ ID NO. 1.
In a third aspect of the invention, there is provided a gene encoding the above-described mutant kappa-carrageenan enzyme Pcar, the nucleotide sequence of which is shown as SEQ ID NO. 2.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a colony PCR identification of a mutant kappa-carrageenan enzyme recombinant plasmid according to an embodiment of the present invention;
FIG. 2 is an SDS-PAGE analysis of mutant kappa-carrageenan enzymes according to an embodiment of the present invention;
FIG. 3 is an analysis of the viability of mutant kappa-carrageenan enzymes according to an embodiment of the present invention;
FIG. 4 is a graph showing the thermostability of a mutant kappa-carrageenan enzyme according to an embodiment of the present invention;
FIG. 5 is an optimum reaction temperature for a mutant kappa-carrageenan enzyme according to an embodiment of the present invention;
FIG. 6 is an optimal reaction pH for a mutant kappa-carrageenan enzyme according to an embodiment of the present invention;
FIG. 7 is a pH stability of a mutant kappa-carrageenan enzyme according to an embodiment of the present invention;
FIG. 8 is a substrate specificity analysis of mutant kappa-carrageenan enzymes according to an embodiment of the present invention;
FIG. 9 shows molecular docking of WT (A) and mutant (B) with substrate according to an embodiment of the invention;
FIG. 10 shows hydrophobic interaction residues between WT (A) and mutant (B) and substrate according to an embodiment of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The following disclosure provides many different embodiments, or examples, for implementing different embodiments of the invention. In order to simplify the present disclosure, specific embodiments or examples are described below. Of course, they are merely examples and are not intended to limit the invention. In addition, one of ordinary skill in the art will recognize the applicability of other processes and/or the use of other materials, as examples of the various specific processes and materials provided by the present invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques in the fields of chemistry, molecular biology, etc., which are within the ability of a person skilled in the art. In addition, unless otherwise indicated, herein, nucleic acids are written in a 5 'to 3' direction from left to right, and amino acid sequences are written in an amino-to carboxy-terminal direction from left to right.
It should be noted that:
LB liquid medium: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of NaCl, distilled water to a certain volume, and sterilizing at high temperature and high pressure for later use.
LB solid medium: 2% agar powder is added into LB liquid culture medium, and the solid culture medium is obtained after high-temperature high-pressure sterilization.
The invention is described below by way of illustrative specific examples, which are not intended to limit the scope of the invention in any way. Specifically described are: the reagents used in the present invention are commercially available unless otherwise specified.
EXAMPLE 1 construction of recombinant expression engineering Strain of mutant kappa-Carrageenan
Selection of kappa-carrageenan enzyme Pcar mutation site: the disulfide bond design module in Discovery Studio was used to predict kappa-carrageenan Pcar (encoded by the sequence shown in WP_ 138553941.1), away from the kappa-carrageenan catalytic triad (E162-D164-E167), and R44C-T292C, S C-R248C, T263C-F270C, N C-G239C was selected as the mutation site. The primer sequences were designed as shown in Table 1, with reference to the instructions of the Mut Express II Fast Mutagenesis Kit V site-directed mutagenesis kit from Norpran.
TABLE 1 primer sequences
Site-directed mutagenesis: the operations were performed with reference to the mutation kit Mut Express II Fast Mutagenesis Kit II 2 (Nanjinouzan Biotechnology Co., ltd.).
(1) The recombinant plasmid pET-28a-Pcar containing wild enzyme gene fragment is used as a template, and the PCR technology is utilized to amplify the target plasmid. The amplification reaction system and reaction conditions are shown in tables 2 and 3, wherein the gene-specific primers were purchased from platinum biotechnology limited.
TABLE 2 amplification reaction System
Tables 1 to 4 amplification reaction conditions
(2) Digestion reaction. 1. Mu.L of DpnI was added to the amplified product obtained in the previous step, and the mixture was allowed to react at a constant temperature of 37℃for 1.5 hours and then cooled on ice.
(3) And (3) carrying out recombination reaction. The reaction conditions were that the temperature was 37℃for 30min and then lowered to 4℃and the reaction system was shown in Table 4.
TABLE 4 recombination reaction System
After transformation of E.coli DH 5. Alpha. Competent cells, 10. Mu.L of the product after the recombination reaction was plated on LB plates (containing 50. Mu.g/mL kanamycin) and incubated at 37℃for 18h. The single clone is selected and inoculated into 5mL LB liquid culture medium (containing 50 mug/mL kanamycin), after being cultured at 37 ℃ and 180rpm overnight, the bacterial liquid is sent to platinum biotechnology limited company for sequencing, and the sequencing result shows that the target sequence of the inserted gene is consistent with the theoretical sequence.
EXAMPLE 2 heterologous expression of mutant kappa-Carrageenan Gene in E.coli BL21
Recombinant plasmid extraction of mutant enzyme-containing genes was performed with reference to the instructions of TIANGEN's bacterial plasmid minikit, and E.coli BL21 competent cells were transformed with the plasmids, and the cells were plated onto LB plates (containing 50. Mu.g/mL kanamycin) and cultured at 37℃for 18h. Single colonies on the above LB plates were selected, inoculated into 5mL of LB liquid medium (containing 50. Mu.g/mL kanamycin), cultured overnight at 37℃and 180rpm, and positive transformants were verified by colony PCR, and the reaction system and the reaction conditions were as shown in Table 5 and Table 6, respectively.
TABLE 5 colony PCR reaction System
TABLE 6 colony PCR reaction conditions
The PCR products were detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 1. In the figure, M, DNA molecular weight standard DL2000;1-5, the mutant kappa-carrageenan enzyme gene has bright bands around 800bp according to colony PCR results of the strains 1-5.
Induction expression of mutant kappa-carrageenan enzyme: e.coli BL21 containing the mutant enzyme gene was inoculated into 50mL of LB liquid medium and cultured at 37℃and 180rpm for 18 hours. The bacterial liquid is transferred into 300mL LB liquid culture medium according to the inoculation amount of 1%, and the shaking culture is continued at 37 ℃ until the bacterial OD 600 is between 1.0 and 1.2. To the bacterial liquid, 30. Mu.L of IPTG (0.5 mol/L) was added, and the mixture was cultured at 16℃for 24 hours at 180 rpm.
Affinity chromatography purification and analysis of mutant kappa-carrageenan enzyme: recombinant proteins were purified with reference to Qiagen's Ni-NTA instructions. Glycerol was added to the purified sample to 20%, and after mixing, stored at-20 ℃. Protein concentration was determined by Bradford method. A suitable amount of sample was taken for SDS-PAGE analysis and the results are shown in FIG. 2. In the figure, M, protein molecular mass standard; 1-5, purified wild-type kappa-carrageenan Pcar, N205C-G239C, R C-T292C, S C-R248C, T C-F270C; the molecular weight of the mutant kappa-carrageenase sample band is between 30 kDa and 40kDa, which is consistent with the molecular weight of the wild-type enzyme protein of 32 kDa.
EXAMPLE 3 study of enzymatic Properties
Determination of kappa-carrageenase Activity: taking 490 mu L of 50mmol/L NaH 2PO4-Na2HPO4 buffer (pH 8.0) containing 0.5% kappa-carrageenan, adding 10 mu L of enzyme solution, reacting at 40 ℃ for 15min, adding 500 mu L of DNS reagent, cooling after boiling water bath for 10min, measuring the absorbance at 520nm wavelength, and determining the content of reducing sugar by using a galactose standard curve. Kappa-carrageenase activity is defined as: under the above conditions, the amount of enzyme required to release 1. Mu. MoL of reducing sugar (in terms of galactose) per minute was 1 enzyme activity unit (U). As shown in FIG. 3, the enzyme activities of the other 3 mutants are greatly improved, particularly the enzyme activities of the N205C-G239C mutants are improved by about 330%, except that the enzyme activities of the mutants S146C-R248C are remarkably reduced compared with the wild-type kappa-carrageenan enzyme (WT).
Enzyme thermostability analysis: after the enzymes were left at different temperatures (45, 50, 55, 60 ℃) for 30min, the residual activities of the enzymes were measured and the thermostability of the enzymes was investigated. The enzyme activity without heat treatment is 100%. As a result, as shown in FIG. 4, after 30min of treatment at 45, 50 and 55 ℃, mutant N205C-G239C retained 91%, 74% and 56% of residual enzyme activity, respectively, while wild-type enzyme retained 79%, 44% and 32% of residual enzyme activity, respectively. Wherein the residual enzyme activities of the mutant N205C-G239C after 50 and 55℃treatment were 1.70 and 1.75 times that of the wild-type enzyme, respectively. From this, the mutant N205C-G239C has significantly improved thermostability compared to WT. And selecting a mutant N205C-G239C with improved heat stability for subsequent enzymatic property research.
Determination of the optimum temperature of the enzyme: the activity of kappa-carrageenan enzyme is detected at different temperatures (35, 40, 45, 50, 55 and 60 ℃), and the optimal reaction temperature of the enzyme is researched by taking the highest enzyme activity as 100%. As a result, as shown in FIG. 5, the optimal reaction temperature of the mutant N205C-G239C was 55℃higher than that of WT. When the mutant enzyme is at a lower temperature, the enzyme activity is lower, and the relative enzyme activity gradually rises with the gradual rise of the temperature and reaches the highest at 55 ℃; after which the temperature continues to rise and the enzyme activity begins to decline.
Determination of optimal pH of enzyme: the enzyme activity was measured under different pH conditions, and the optimum reaction pH of the enzyme was studied with the highest enzyme activity being 100%. The 50mmol/L buffer used was NaH 2PO4-Na2HPO4 buffer (pH 6.0-8.0), tris-HCl (pH 8.0-9.0), gly-NaOH (pH 9.0-10.0), respectively. As a result, as shown in FIG. 6, the mutant kappa-carrageenan enzyme had the highest activity at pH 8.0, and the same pH as that of the wild-type enzyme for the optimal reaction.
Analysis of pH stability of enzyme: the enzyme is placed in buffer solutions with different pH values, after being subjected to warm bath for 30min at 25 ℃, the residual activity of the enzyme is measured, and the pH stability of the enzyme is studied. The untreated enzyme activity was 100%. As a result, as shown in FIG. 7, the mutant N205C-G239C had a higher pH stability than the wild-type enzyme, the residual enzyme activity in NaH 2PO4-Na2HPO4 buffer at pH 7.0 and 8.0 was about 30% higher than that of the wild-type enzyme, and the residual enzyme activity in Gly-NaOH buffer at pH 9.0 was 4.2 times that of the wild-type enzyme, and the residual enzyme activity after treatment with Gly-NaOH buffer at pH 10.0 was still higher than that of the wild-type enzyme.
Substrate specificity analysis of enzyme: solutions containing different substrates (kappa-carrageenan, iota-carrageenan and lambda-carrageenan) with the concentration of 0.5% (w/v) are respectively prepared, the same amount of enzyme is respectively added, the activity of the enzyme is measured, and the substrate specificity of the enzyme is studied. As a result, as shown in FIG. 8, the substrate specificity of the enzyme was not greatly changed after the mutation. The mutant has strong degradation capability on kappa-type carrageenan, and does not hydrolyze iota-type carrageenan and lambda-type carrageenan.
Structure of enzyme: the structure of mutant N205C-G239C was constructed by PyMOL Wizard Mutagenesis Protein module using the kappa-carrageenan Pcar structure as template. Flexible docking of substrate with enzyme using Discovery Studio 2019, the docking sphere radius is set toLigplot is used to show the hydrophobic amino acid residues in the state of enzyme binding to kappa-carrageenan tetrasaccharide. The results are shown in FIG. 9, where WT (A) and mutant (B) are shown. Mutant N205C-G239C mainly comprises a plurality of layers of bent beta-sheet which are connected by alpha-spiral, and a cavity formed at the bent part is the joint of enzyme and substrate; the disulfide bond formed between amino acid residues Cys239 and Cys205 in the mutant is located in the β -sheet region of the protein molecule. The introduction of disulfide bonds into proteins can increase the rigidity of the molecules, which is beneficial to maintaining the spatial structure of the proteins. Thus, in mutant N205C-G239C, disulfide bonds connect the two folded sheets, increasing the rigidity of the enzyme molecule and thus increasing its thermostability.
Performing an interaction analysis of kappa-carrageenan enzyme and substrate, wherein acting force of mutant N205C-G239C and kappa-carrageenan tetrasaccharide comprises: trp94 forms Pi-Sulfur type force with the substrate, arg259 forms ATTRACTIVE CHARGE type force with the substrate, trp94, arg259, asn268, glu167, gly257, ser255, trp143, gln170 forms hydrogen bond with the substrate. Table 7 counts the differences in the types and amounts of interactions formed between WT and mutant and substrate, and it can be found that: the ATTRACTIVE CHARGE type of force was unchanged during the action of WT and mutant with substrate, but other types of force were changed: pi-Anion type force 1, pi-Sulfur type force 4, pi-Sigma type force 1 and hydrogen bond force 8 exist in the process of docking the WT with the substrate, but Pi-Anion type force disappears, pi-Sulfur type force only 1, pi-Sigma type force disappears and hydrogen bond force increases to 10 in the process of docking the mutant with the substrate.
TABLE 7 interaction of WT and N205C-G239C with substrates
Analysis of the hydrophobic interactions of mutant enzymes with substrates showed that 11 residues total of Trp265, phe270, asp164, ala141, trp143, trp66, tyr63, gly257, phe93, asn268, gin 269 formed hydrophobic interactions with kappa-carrageenan tetrasaccharides (fig. 10B), whereas only 10 residues of WT formed hydrophobic interactions with substrates (fig. 10A). The reason for the improved thermostability and catalytic activity of the mutant N205C-G239C may be the introduction of additional hydrophobic interactions.
In summary, according to embodiments of the present invention, disulfide bonds are introduced into the kappa-carrageenan Pcar enzyme Pcar structure by rational design to increase the thermal stability and catalytic activity of the enzyme. 4 mutants (R44C-T292C, S C-R248C, T263C-F270C and N205C-G239C) are constructed by using a fixed point mutation technology and using a gene of kappa-carrageenan Pcar as a template, and after induced expression is carried out in E.coli BL21, enzyme activity and thermal stability analysis is carried out, mutant kappa-carrageenan with improved thermal stability and catalytic activity is screened, and enzymatic property analysis is carried out. The results show that compared with the wild-type kappa-carrageenan enzyme, the enzyme activities of the 3 mutants except the mutant S146C-R248C are obviously improved, wherein the enzyme activity of the mutant N205C-G239C is improved by about 330 percent. The thermal stability of the mutant N205C-G239C is obviously enhanced, and the residual enzyme activities of the N205C-G239C are respectively 1.70 times and 1.75 times that of the wild type enzyme after being treated at 50 ℃ and 55 ℃ for 30 min. The enzyme property research of the mutant enzyme shows that the enzyme has an optimal reaction temperature of 55 ℃, can specifically degrade kappa-carrageenan, has an optimal pH of 8.0 and has improved pH stability. Structural analysis shows that disulfide bonds connect two layers of folding sheets, which may increase the rigidity of the enzyme molecule, thereby increasing the thermal stability thereof. The increased hydrophobic interactions of mutant enzymes with kappa-carrageenan tetrasaccharide substrates, compared to WT, may contribute to an increase in enzyme activity and thermostability. In the embodiment, the rational design strategy based on the structure can provide thought for the heat stability modification of industrial enzyme and has important significance for the high-value utilization of kappa-carrageenan.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (3)
1. A method for preparing a kappa-carrageenan Pcar mutant, which is characterized by comprising the following steps:
Predicting wild kappa-carrageenan enzyme coded by a sequence shown as WP_138553941.1 by using a disulfide bond design module in a Discovery Studio, and screening out an enzyme heat stability and enzyme activity improvement mutant N205C-G239C;
the wild-type kappa-carrageenan enzyme is mutated at the mutation site of N205C-G239C to generate kappa-carrageenan enzyme Pcar mutant.
2. The kappa-carrageenan enzyme Pcar mutant prepared by the preparation method of claim 1, which is characterized in that the amino acid sequence of the kappa-carrageenan enzyme Pcar mutant is shown as SEQ ID NO. 1.
3. A gene encoding a mutant of kappa-carrageenan enzyme Pcar as defined in claim 2, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 2.
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