CN115725548B - A κ-carrageenase Pcar16 mutant and preparation method thereof - Google Patents

Abstract

The present invention provides a κ-carrageenase Pcar16 mutant and a preparation method thereof, the method comprising the following steps: using the disulfide bond design module in Discovery Studio, predicting κ-carrageenase, selecting 4 mutation sites, screening out the mutant N205C-G239C with improved enzyme thermal stability and enzyme activity; mutating the wild-type κ-carrageenase at its N205C-G239C mutation site to produce a κ-carrageenase Pcar16 mutant. The method can improve the thermal stability of the enzyme, the enzyme activity of the mutant enzyme is increased by about 330%, and after being treated at 50 and 55°C for 30min, the residual enzyme activity of the mutant enzyme is 1.70 and 1.76 times that of the wild-type enzyme, respectively, providing a theoretical basis for the better application of carrageenase in the preparation of carrageenan oligosaccharides.

Description

A κ-carrageenase Pcar16 mutant and preparation method thereof

Technical Field

The invention belongs to the technical field of bioengineering, and specifically relates to a kappa-carrageenase Pcar16 mutant and a preparation method thereof.

Background technique

Carrageenan is a hydrocolloid composed of 1,3-β-D-galactose and 1,4-α-D-galactose alternately connected. According to the different sulfate ester binding forms, carrageenan can be divided into κ type, ι type and λ type. Carrageenan has a wide range of sources and can be extracted from certain specific types of red algae, such as Chondrus, Eucheuma and Gelidium. It is mainly used as a thickener and gelling agent, and is widely used in the food industry. Studies have shown that carrageenan oligosaccharides formed after carrageenan degradation have multiple biological activities such as anti-tumor, anti-viral, immunomodulatory and anticoagulant. Therefore, carrageenan oligosaccharide preparations are expected to be used in medicine, food and cosmetics. The methods for degrading carrageenan mainly include acid hydrolysis, ultrasonic degradation and enzymatic hydrolysis. Acid hydrolysis is the most popular. Compared with it, the enzymatic method has mild conditions, strong specificity, and can protect the biological activity of carrageenan oligosaccharides. Therefore, enzymatic degradation has become the preferred method for preparing carrageenan oligosaccharides.

κ-carrageenase (EC 3.2.1.83) belongs to glycoside hydrolase, which is an enzyme that degrades carrageenan by hydrolyzing the β-1,4 glycosidic bonds inside κ-carrageenan. The colloidal viscosity of carrageenan decreases with increasing temperature. κ-carrageenanase with excellent thermal stability and catalytic activity can process κ-carrageenan at higher temperatures, effectively reduce the viscosity of the colloidal solution, and improve the efficiency of enzymatic hydrolysis. At present, the production of carrageenanase is mostly prepared in the form of direct fermentation of strains selected from nature, but the carrageenanase directly isolated and purified from nature has the disadvantages of low yield, low activity and poor stability. In recent years, with the development of protein engineering technology, wild-type carrageenanase has been molecularly modified in order to improve the enzymatic properties of carrageenanase such as thermal stability and enzyme activity, expand the application range of carrageenanase, and promote the high-value utilization of carrageenan.

Summary of the invention

The present invention aims to solve at least one of the technical problems in the above-mentioned technology to a certain extent. To this end, the present invention proposes a κ-carrageenase Pcar16 mutant and a preparation method thereof, which can improve the thermal stability and catalytic activity of the enzyme, and provide a theoretical basis for the better application of κ-carrageenase in the preparation of carrageenan oligosaccharides.

To this end, in a first aspect of the present invention, a method for preparing a κ-carrageenase Pcar16 mutant is provided, comprising the following steps:

Using the disulfide bond design module in Discovery Studio, we predicted κ-carrageenase, stayed away from the κ-carrageenase catalytic triad, selected four mutation sites, and screened out the mutant N205C-G239C with improved enzyme thermal stability and enzyme activity;

The wild-type κ-carrageenase was mutated at its mutation site N205C-G239C to generate the κ-carrageenase Pcar16 mutant.

According to an embodiment of the present invention, a disulfide bond is introduced into the structure of κ-carrageenase Pcar16 by a rational design method to improve the thermal stability and enzyme activity of the enzyme; a N205C-G239C mutant is constructed using the site-directed mutagenesis technique and the gene of κ-carrageenase Pcar16 as a template. Compared with the wild-type κ-carrageenase (WT), the enzyme activity of the mutant enzyme N205C-G239C is increased by about 330%, and the thermal stability of the mutant N205C-G239C is significantly enhanced. After being treated at 50 and 55°C for 30 minutes, the residual enzyme activity of N205C-G239C is 1.70 and 1.75 times that of the wild-type enzyme, respectively; the enzymatic properties of the mutant enzyme are studied, and it is found that the optimal reaction temperature of the enzyme is 55°C, it can specifically degrade κ-carrageenan, the optimal pH is 8.0, and the pH stability is improved; structural analysis shows that the disulfide bond connects the two layers of folded sheets, which improves the rigidity of the enzyme molecule, thereby improving the thermal stability of the enzyme. Compared with the WT, the mutant enzyme has enhanced hydrophobic interactions with the κ-carrageenan tetrasaccharide substrate, which contributes to the improvement of enzyme activity and thermal stability. The rational design strategy based on structure can provide ideas for the modification of thermal stability and enzyme activity of industrial enzymes, and is of great significance for the high-value utilization of κ-carrageenan.

In the second aspect of the present invention, the κ-carrageenase Pcar16 mutant is prepared by the above-mentioned preparation method, and the amino acid sequence of the κ-carrageenase Pcar16 mutant is shown in SEQ ID NO.1.

In the third aspect of the present invention, a gene encoding the above-mentioned κ-carrageenase Pcar16 mutant is provided, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a colony PCR identification of a mutant κ-carrageenase recombinant plasmid according to an embodiment of the present invention;

FIG2 is an SDS-PAGE analysis of mutant κ-carrageenase according to an embodiment of the present invention;

FIG3 is an activity analysis of mutant κ-carrageenase according to an embodiment of the present invention;

FIG4 is a graph showing the thermal stability of mutant κ-carrageenase according to an embodiment of the present invention;

FIG5 is the optimal reaction temperature of mutant κ-carrageenase according to an embodiment of the present invention;

FIG6 shows the optimal reaction pH of mutant κ-carrageenase according to an embodiment of the present invention;

FIG. 7 shows the pH stability of mutant κ-carrageenase according to an embodiment of the present invention;

FIG8 is a substrate specificity analysis of mutant κ-carrageenase according to an embodiment of the present invention;

FIG9 is a molecular docking diagram of WT (A) and mutants (B) with substrates according to an embodiment of the present invention;

FIG. 10 shows the hydrophobic interaction residues between WT (A) and mutants (B) and substrates according to an embodiment of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

The disclosure below provides many different embodiments or examples to realize different implementation methods of the present invention. In order to simplify the disclosure of the present invention, specific embodiments or examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the examples of various specific processes and materials provided by the present invention, and those of ordinary skill in the art can be aware of the applicability of other processes and/or the use of other materials. Unless otherwise stated, the implementation of the present invention will adopt the traditional techniques in the fields of chemistry, molecular biology, etc. within the capabilities of those skilled in the art. In addition, unless otherwise stated, in this article, nucleic acids are written from left to right in the direction of 5' to 3', and amino acid sequences are written from left to right in the direction of amino terminal to carboxyl terminal.

It should be noted:

LB liquid culture medium: 10 g/L tryptone, 5 g/L yeast powder, 10 g/L NaCl, distilled water to volume, sterilize at high temperature and high pressure for use.

LB solid culture medium: Add 2% agar powder to LB liquid culture medium and sterilize it at high temperature and high pressure to make a solid culture medium.

The present invention is described below by means of illustrative specific examples, which are not intended to limit the scope of the present invention in any way. It is particularly noted that the reagents used in the present invention are commercially available unless otherwise specified.

Example 1 Construction of mutant κ-carrageenase recombinant expression engineering strain

Selection of mutation sites of κ-carrageenase Pcar16: Using the disulfide bond design module in Discovery Studio, κ-carrageenase Pcar16 (encoded by the sequence shown in WP_138553941.1) was predicted, away from the κ-carrageenase catalytic triad (E162-D164-E167), and R44C-T292C, S146C-R248C, T263C-F270C, and N205C-G239C were selected as mutation sites. Referring to the instructions of the Mut ExpressⅡFast Mutagenesis Kit V2 site-directed mutagenesis kit of Novazon, the primer sequences were designed as shown in Table 1.

Table 1 Primer sequences

Site-directed mutagenesis: The operation was performed according to the instructions of the mutation kit Mut ExpressⅡFast Mutagenesis KitⅡ2 (Nanjing Novogene Biotech Co., Ltd.).

(1) Using the recombinant plasmid pET-28a-Pcar16 containing the wild-type enzyme gene fragment as a template, the target plasmid was amplified using PCR technology. The amplification reaction system and reaction conditions are shown in Tables 2 and 3, where the gene-specific primers were purchased from Platinum Biotechnology Co., Ltd.

Table 2 Amplification reaction system

Table 1-4 Amplification reaction conditions

(2) Digestion reaction: Add 1 μL of DpnⅠ to the amplified product obtained in the previous step, react at 37°C for 1.5 h, and then cool on ice.

(3) Recombination reaction. The reaction conditions were: 37°C for 30 min and then cooled to 4°C. The reaction system is shown in Table 4.

Table 4 Recombination reaction system

Take 10 μL of the product after the recombination reaction, transform E. coli DH5α competent cells, spread the cells on LB culture plates (containing 50 μg/mL kanamycin), and culture at 37°C for 18 hours. Pick a single clone and inoculate it into 5 mL LB liquid culture medium (containing 50 μg/mL kanamycin), culture it overnight at 37°C and 180 rpm, and then send the bacterial solution to Platinum Biotechnology Co., Ltd. for sequencing. The sequencing results show that the target sequence of the inserted gene is consistent with the theoretical sequence.

Example 2 Heterologous expression of mutant κ-carrageenase gene in E. coli BL21

The recombinant plasmid containing the mutant enzyme gene was extracted by referring to the instructions of the bacterial plasmid mini-extraction kit of TIANGEN Company, and the plasmid was transformed into E. coli BL21 competent cells, and the cells were spread on LB culture plates (containing 50 μg/mL kanamycin) and cultured at 37°C for 18 hours. The single colony on the above LB plate was selected and inoculated into 5 mL LB liquid culture medium (containing 50 μg/mL kanamycin). After overnight culture at 37°C and 180 rpm, the positive transformants were verified by colony PCR. The reaction system and reaction conditions are shown in Tables 5 and 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 Figure 1. In the figure, M, DNA molecular weight standard DL2000; 1-5, colony PCR results of strain 1-5, the mutant κ-carrageenase gene has a bright band at about 800 bp.

Induced expression of mutant κ-carrageenase: Inoculate E. coli BL21 containing the mutant enzyme gene into 50 mL LB liquid medium and culture at 37°C and 180 rpm for 18 hours. Transfer the bacterial solution to 300 mL LB liquid medium at a 1% inoculum and continue shaking culture at 37°C until the bacterial OD ₆₀₀ is between 1.0 and 1.2. Add 30 μL IPTG (0.5 mol/L) to the bacterial solution and culture at 16°C and 180 rpm for 24 hours.

Affinity chromatography purification and analysis of mutant κ-carrageenase: Purify the recombinant protein according to the Ni-NTA instruction manual of Qiagen. Add glycerol to the purified sample to 20%, mix well, and store at -20°C. Determine the protein concentration by Bradford method. Take an appropriate amount of sample for SDS-PAGE electrophoresis analysis, and the results are shown in Figure 2. In the figure, M, protein molecular weight standard; 1-5, purified wild-type κ-carrageenase Pcar16, N205C-G239C, R44C-T292C, S146C-R248C, T263C-F270C; the molecular weight of the mutant κ-carrageenase sample band is between 30-40kDa, which is consistent with the protein molecular weight of the wild-type enzyme of 32kDa.

Example 3 Enzyme properties study

Activity determination of κ-carrageenase: 490 μL of 50 mmol/L NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer (pH 8.0) containing 0.5% κ-carrageenan was taken, 10 μL of enzyme solution was added, and the reaction was carried out at 40°C for 15 minutes, and then 500 μL of DNS reagent was added, and the absorbance at a wavelength of 520 nm was measured after boiling water bath for 10 minutes, and the reducing sugar content was determined using the galactose standard curve. The activity of κ-carrageenase is defined as: under the above conditions, the amount of enzyme required to release 1 μmoL reducing sugar (in terms of galactose) per minute is 1 enzyme activity unit (U). The results are shown in Figure 3. Compared with the wild-type κ-carrageenase (WT), except for the mutant S146C-R248C, whose enzyme activity was significantly reduced, the enzyme activities of the other three mutants were greatly improved, especially the enzyme activity of the N205C-G239C mutant was increased by about 330%.

Analysis of enzyme thermal stability: After the enzyme was placed at different temperatures (45, 50, 55, 60°C) for 30 minutes, the residual activity of the enzyme was measured to study the thermal stability of the enzyme. The activity of the enzyme without heat treatment was 100%. The results are shown in Figure 4. After treatment at 45, 50, and 55°C for 30 minutes, the mutant N205C-G239C retained 91%, 74%, and 56% of the residual enzyme activity, respectively, while the wild-type enzyme retained 79%, 44%, and 32% of the residual enzyme activity, respectively. Among them, after treatment at 50 and 55°C, the residual enzyme activity of the mutant N205C-G239C was 1.70 and 1.75 times that of the wild-type enzyme, respectively. It can be seen that compared with WT, the thermal stability of the mutant N205C-G239C is significantly improved. The mutant N205C-G239C with improved thermal stability was selected for subsequent enzymatic property studies.

Determination of the optimal temperature of the enzyme: The activity of κ-carrageenase was detected at different temperatures (35, 40, 45, 50, 55, 60°C), and the optimal reaction temperature of the enzyme was studied with the highest enzyme activity as 100%. The results are shown in Figure 5. The optimal reaction temperature of the mutant N205C-G239C is 55°C, which is higher than that of WT. When the mutant enzyme is at a lower temperature, the enzyme activity is low. As the temperature gradually increases, the relative enzyme activity gradually increases and reaches the highest at 55°C; after that, the temperature continues to rise and the enzyme activity begins to decrease.

Determination of the optimal pH of the enzyme: The activity of the enzyme was determined under different pH conditions, and the optimal reaction pH of the enzyme was studied with the highest enzyme activity as 100%. The 50mmol/L buffers used were _{NaH2PO4 - Na2HPO4} buffer (pH 6.0-8.0), Tris-HCl (pH 8.0-9.0), and Gly-NaOH (pH 9.0-10.0). The results are shown in Figure 6. The mutant κ _- carrageenase has the highest activity at pH 8.0, which is the same as the optimal reaction pH of the wild-type enzyme.

Analysis of enzyme pH stability: The enzyme was placed in buffers with different pH values, and after being warmed at 25°C for 30 minutes, the residual activity of the enzyme was measured to study the pH stability of the enzyme. The activity of the untreated enzyme was taken as 100%. As shown in Figure 7, the pH stability of the mutant N205C-G239C was higher than that of the wild-type enzyme. The residual enzyme activity in NaH ₂ PO ₄ -Na ₂ HPO ₄ buffers at pH 7.0 and 8.0 was about 30% higher than that of the wild-type enzyme. The residual enzyme activity in Gly-NaOH buffer at pH 9.0 was 4.2 times that of the wild-type enzyme. After being treated with Gly-NaOH buffer at pH 10.0, the residual enzyme activity was still higher than that of the wild-type.

Analysis of enzyme substrate specificity: Prepare solutions containing 0.5% (w/v) concentration of different substrates (κ-carrageenan, ι-carrageenan, λ-carrageenan), add the same amount of enzyme, measure the activity of the enzyme, and study the substrate specificity of the enzyme. The results are shown in Figure 8. After the mutation, the substrate specificity of the enzyme did not change significantly. The mutant has a strong ability to degrade κ-type carrageenan, and does not hydrolyze ι-type and λ-type carrageenan.

Enzyme structure: Using the structure of κ-carrageenase Pcar16 as a template, the structure of mutant N205C-G239C was constructed using the PyMOL Wizard MutagenesisProtein module. Discovery Studio 2019 was used to perform flexible docking of the substrate and enzyme, and the docking sphere radius was set to Ligplot was used to display the hydrophobic amino acid residues in the state of enzyme binding to κ-carrageenan tetrasaccharide. The results are shown in Figure 9, in which WT (A) and mutant (B). The mutant N205C-G239C is mainly composed of multiple layers of curved β-pleated sheets connected by α-helices, and the cavity formed at the bend is the binding site of the enzyme and the substrate; the disulfide bond formed between the amino acid residues Cys239 and Cys205 in the mutant is located in the β-pleated region of the protein molecule. The introduction of disulfide bonds in proteins can increase the rigidity of the molecule and help maintain the spatial structure of the protein. Therefore, in the mutant N205C-G239C, the disulfide bond connects the two layers of folded sheets, which increases the rigidity of the enzyme molecule and thus improves its thermal stability.

The interaction analysis between κ-carrageenase and substrate was performed, among which the interaction forces between mutant N205C-G239C and κ-carrageenan tetrasaccharide included: Trp 94 formed a Pi-Sulfur type interaction force with the substrate, Arg 259 formed an Attractivecharge type interaction force with the substrate, and Trp94, Arg259, Asn268, Glu167, Gly257, Ser255, Trp143, and Gln170 formed hydrogen bonds with the substrate. Table 7 summarizes the differences in the types and quantities of interaction forces formed between WT and mutants and the substrate. It can be found that the attractive charge type force does not change during the interaction between WT and mutants and the substrate, but other types of forces change: in the docking process between WT and the substrate, there is 1 Pi-Anion type force, 4 Pi-Sulfur type forces, 1 Pi-Sigma type force and 8 hydrogen bond forces, but in the docking process between the mutant and the substrate, the Pi-Anion type force disappears, there is only 1 Pi-Sulfur type force, the Pi-Sigma type force disappears, and the hydrogen bond force increases to 10.

Table 7 Interactions of WT and N205C-G239C with substrates

The results of hydrophobic interaction analysis between the mutant enzyme and the substrate showed that 11 residues, including Trp265, Phe270, Asp164, Ala141, Trp143, Trp66, Tyr63, Gly257, Phe93, Asn268, and Gln269, formed hydrophobic interactions with κ-carrageenan tetrasaccharide (Figure 10B), while only 10 residues of WT formed hydrophobic interactions with the substrate (Figure 10A). The improved thermal stability and catalytic activity of the mutant N205C-G239C may be due to the introduction of additional hydrophobic interactions.

In summary, according to the embodiments of the present invention, a disulfide bond is introduced into the structure of κ-carrageenase Pcar16 by a rational design method to improve the thermostability and catalytic activity of the enzyme. Using site-directed mutagenesis technology, four mutants (R44C-T292C, S146C-R248C, T263C-F270C and N205C-G239C) were constructed using the gene of κ-carrageenase Pcar16 as a template, and after induced expression in E.coli BL21, enzyme activity and thermostability analysis were performed to screen mutant κ-carrageenase with improved thermostability and catalytic activity, and enzymatic properties were analyzed. The results showed that compared with the wild-type κ-carrageenase, except for the mutant S146C-R248C, the enzyme activities of the other three mutants were significantly improved, among which the enzyme activity of the mutant enzyme N205C-G239C was increased by about 330%. The thermal stability of the mutant N205C-G239C was significantly enhanced. After being treated at 50 and 55°C for 30 minutes, the residual enzyme activity of N205C-G239C was 1.70 and 1.75 times that of the wild-type enzyme, respectively. The enzymatic properties of the mutant enzyme were studied, and it was found that the optimal reaction temperature of the enzyme was 55°C, it could specifically degrade κ-carrageenan, the optimal pH was 8.0, and the pH stability was improved. Structural analysis showed that the disulfide bonds connected the two layers of folded sheets, which may have increased the rigidity of the enzyme molecule and thus improved its thermal stability. Compared with WT, the hydrophobic interaction between the mutant enzyme and the κ-carrageenan tetrasaccharide substrate was enhanced, which may have contributed to the improvement of the enzyme's activity and thermal stability. In this embodiment, the rational design strategy based on structure can provide ideas for the thermal stability modification of industrial enzymes, and is of great significance for the high-value utilization of κ-carrageenan.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (3)

1. A method for preparing a κ-carrageenase Pcar16 mutant, comprising the following steps: The disulfide bond design module in Discovery Studio was used to predict the wild-type κ-carrageenase encoded by the sequence shown in WP_138553941.1, and the mutant N205C-G239C with improved enzyme thermal stability and enzyme activity was screened out; The wild-type κ-carrageenase was mutated at its mutation site N205C-G239C to generate the κ-carrageenase Pcar16 mutant. 2. The κ-carrageenase Pcar16 mutant prepared by the preparation method of claim 1, characterized in that the amino acid sequence of the κ-carrageenase Pcar16 mutant is shown in SEQ ID NO.1. 3. A gene encoding the κ-carrageenase Pcar16 mutant according to claim 2, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO.2.