阅读说明：本技术 一种耐碱κ-卡拉胶酶及其应用 (Alkali-resistant kappa-carrageenase and application thereof ) 是由 朱艳冰 黄小艺 胡青松 倪辉 姜泽东 杜希萍 李志朋 郑明静 李清彪 杨远帆 李 于 2021-08-20 设计创作，主要内容包括：本发明提供了一种耐碱κ-卡拉胶酶及其应用,该耐碱κ-卡拉胶酶是由如SEQ ID NO.1所示序列编码的野生型κ-卡拉胶酶经过其第237位氨基酸突变而产生的突变体。与野生型κ-卡拉胶酶相比,该突变体的耐碱性得到了一定的提高,野生型酶在pH为11.0下处理30min后仅保留19.9％的相对酶活力,而突变酶E237R保留了36.0％的相对酶活力,从而使该突变κ-卡拉胶酶在碱性条件下水解κ-卡拉胶的工业生产中具有良好的应用前景。(The invention provides alkali-resistant kappa-carrageenase and application thereof, wherein the alkali-resistant kappa-carrageenase is a mutant generated by mutating 237 th amino acid of wild kappa-carrageenase coded by a sequence shown as SEQ ID NO. 1. Compared with wild kappa-carrageenase, the alkali resistance of the mutant is improved to a certain extent, the wild enzyme only retains 19.9 percent of relative enzyme activity after being treated for 30min at the pH of 11.0, and the mutant enzyme E237R retains 36.0 percent of relative enzyme activity, so that the mutant kappa-carrageenase has good application prospect in the industrial production of hydrolyzing kappa-carrageenase under the alkaline condition.)

1. An alkali-resistant kappa-carrageenase, which is characterized in that a mutant is generated by mutating 237 th amino acid of wild kappa-carrageenase coded by a sequence shown as SEQ ID NO. 1.

2. The alkali-resistant kappa-carrageenase of claim 1, wherein the 237 th amino acid of the wild-type kappa-carrageenase is changed from Glu to Arg.

3. The alkali-resistant kappa-carrageenase of claim 1, wherein the mutation is a site-directed mutation.

4. The gene for encoding the alkali-resistant kappa-carrageenase according to claim 1, characterized in that the nucleotide sequence of the gene is shown as SEQ ID No. 2.

5. A construct comprising the gene of claim 4.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to alkali-resistant kappa-carrageenase and application thereof.

Background

The carrageenan is linear sulfated polysaccharide formed by alternately connecting alpha-1, 3-D-galactose and beta-1, 4-D-galactose which are used as basic skeletons. Carrageenan oligosaccharide is a degradation product of carrageenan, has lower molecular weight and higher solubility compared with polysaccharide, is easier to be absorbed by human body, and gradually becomes a research hotspot in the fields of food, medicine and the like. The carrageenin enzyme is used for degrading the carrageenin, has the characteristics of mild reaction conditions and high substrate specificity, and is considered as a promising method for preparing the carrageenin oligosaccharide. At the same time, carrageenases have also found use in other areas, such as the textile industry, components of detergents, saccharifying agents in bioethanol production, prevention of red algae outbreaks and reuse of algal biomass.

In industrial production, enzymes are often applied in extreme environments, and the enzymes are required to have excellent thermal stability, acid and alkali resistance, salt tolerance and the like. The reported kappa-carrageenase has poor alkali resistance, how to reasonably improve the kappa-carrageenase and optimize the alkali resistance of the enzyme, so that the kappa-carrageenase has good application prospect to be researched in the industrial production of hydrolyzing the kappa-carrageenase under the alkaline condition.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the art described above. Therefore, the invention provides the alkali-resistant kappa-carrageenase, which has certain improvement on alkali resistance and good application potential in the treatment of the industrial waste residue of the carrageenase.

To this end, in a first aspect of the invention, an alkali-resistant kappa-carrageenase is provided, which is a mutant produced by mutating the 237 th amino acid of a wild-type kappa-carrageenase encoded by the sequence shown in SEQ ID No. 1.

An alkali-resistant kappa-carrageenase according to an embodiment of the present invention is mutated at amino acid position 237 of a wild-type kappa-carrageenase. Compared with wild kappa-carrageenase, the alkali resistance of the mutant is improved to a certain extent, the wild enzyme only retains 19.9 percent of relative enzyme activity after being treated for 30min at the pH of 11.0, and the mutant enzyme E237R retains 36.0 percent of relative enzyme activity, so that the mutant kappa-carrageenase has an application prospect in the treatment of industrial waste residues of carrageenase.

Optionally, the 237 th amino acid of the wild-type kappa-carrageenase is changed from Glu to Arg.

Optionally, the mode of mutation is site-directed mutation.

In a second aspect of the invention, a gene encoding the alkali-resistant kappa-carrageenase is provided, and the nucleotide sequence of the gene is shown as SEQ ID No. 2.

In a third aspect of the invention, there is provided a construct comprising a gene encoding the above alkali-resistant kappa-carrageenase.

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 an SDS-PAGE analysis of wild type and mutant kappa-carrageenases;

FIG. 2 is a graph of pH stability of wild-type and mutant kappa-carrageenases under alkaline conditions;

FIG. 3 is a graph of the ability of wild-type and mutant kappa-carrageenases to degrade kappa-carrageenase under alkaline conditions.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The following disclosure provides many different embodiments, or examples, for implementing different embodiments of the invention. To simplify the disclosure, specific embodiments or examples are described below. Of course, they are merely examples and are not intended to limit the present invention. In addition, the present invention provides examples of various specific processes and materials, and one of ordinary skill in the art will recognize the applicability of other processes and/or the use of other materials. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, and the like, which are within the capabilities of persons skilled in the art. In addition, unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction and amino acid sequences are written from left to right in the amino terminus to carboxy terminus direction herein.

The invention is described below by way of illustrative specific examples, which do not limit the scope of the invention in any way. Specifically, the following are mentioned: the reagents used in the present invention are commercially available unless otherwise specified.

EXAMPLE 1 construction of alkali-resistant kappa-Carrageenase mutants

Construction of mutant enzyme recombinant expression vector:

site-directed Mutagenesis is carried out by taking a vector carrying a wild type kappa-carrageenase coding gene (SEQ ID NO.1) as a template to construct a mutant enzyme recombinant expression vector, and a site-directed Mutagenesis primer is designed according to the design requirement of a mutation Kit Mut Express II Fast Mutagenesis Kit V2 primer, wherein the Mutagenesis primer is shown in Table 1.

TABLE 1 mutant primer Table

Site-directed Mutagenesis was performed with reference to the instructions of the Mutagenesis Kit Mut Express ii Fast Mutagenesis Kit V2. Comprises three steps of target plasmid amplification, amplification product Dpn I digestion and recombination reaction; the coding gene of mutant enzyme E237R obtained by mutation is shown in SEQ ID NO. 2.

Amplification of the target plasmid:

the reagents are added in sequence, mixed evenly and then subjected to PCR. PCR reaction parameters: 30s at 95 deg.C, 15s at 62 deg.C, 2min30 s at 72 deg.C, 30 cycles, 5min at 72 deg.C, and 4 deg.C.

Digestion of the amplification product Dpn I:

DpnⅠ 1μL

amplification product 40-50 μ L

Adding 1uL of Dpn I into the PCR reaction solution, mixing uniformly, and reacting for 1h 30min at 37 ℃.

And (3) recombinant cyclization reaction:

and (4) lightly sucking and uniformly mixing by using a pipette gun, and collecting the reaction solution to the bottom of the tube by short-time centrifugation. Reacting at 37 deg.C for 30min, cooling to 4 deg.C or immediately cooling on ice.

Transforming the recombinant product into an expression host:

(1) coli BL21 competent cells were thawed on ice.

(2) Add 10. mu.L of the recombinant product into 100. mu.L of the competent cells, flick the tube wall and mix well, and keep standing on ice for 30 min.

(3) And (3) after the heat shock is carried out for 45s in a water bath at the temperature of 42 ℃, immediately placing on ice for cooling for 2-3 min.

(4) 900. mu.L of LB liquid medium (without antibiotics) was added and shaken at 37 ℃ for 1h (200 rpm).

(5) Centrifuge at 5000rpm for 5min at 4 deg.C, and discard 900. mu.L of supernatant. The cells were resuspended in the remaining medium.

(6) The thalli are coated on an LB solid medium plate (containing 50 mu g/mL kanamycin) and are inversely cultured in a 37 ℃ incubator for 12-16 h.

Identification of the alkali-resistant mutant enzyme positive recon:

and (3) PCR system:

the reagents are added in sequence, mixed evenly and then subjected to PCR. PCR reaction parameters: 3min at 95 ℃, 15s at 60 ℃, 45s at 72 ℃, 30 cycles, 5min at 72 ℃ and 4 ℃ storage. The PCR products were detected using 1% agarose gel electrophoresis. DNA sequence analysis of the PCR-positive recombinants confirmed the DNA sequence of the mutant enzyme.

Induced expression and purification of kappa-carrageenase:

the genetically engineered bacterium solution containing the mutant or wild kappa-carrageenase gene is inoculated into 50mL LB liquid medium (containing 50 ug/mL kanamycin) according to the inoculation amount of 1 percent, cultured for 12h at 37 ℃ and 180rpm, and the activation of the strain is carried out. The activated bacterial solution was inoculated into 200mL of LB liquid medium (containing 50. mu.g/mL kanamycin) at an inoculum size of 1%, and cultured at 37 ℃ and 180rpm to OD₆₀₀0.6 to 0.8. Adding isopropyl-beta-D-thiogalactoside (IPTG) to a final concentration of 0.5mmol/L, and performing low-temperature induced expression at 16 ℃ for 18-24 h. Centrifuging the induced expression bacteria solution at 5000rpm and 4 deg.C for 15min, removing supernatant, and adding 15mL precooled buffer (50mmol/L NaH) to the bacteria₂PO₄300mmol/L NaCl, 15mmol/L imidazole, pH 8.0). And (3) carrying out ultrasonic crushing under the ice bath condition, centrifuging the crushed lysate for 20min at 4 ℃ and 10000rpm, and obtaining supernatant, namely the crude enzyme solution. The recombinant protein was isolated and purified by affinity chromatography according to the instructions for use of metal nickel-chelating sepharose 6FF from GE Healthcare Life Sciences. The purified mesh was analyzed by SDS-PAGEMolecular weight and purity of the target protein.

Single protein bands of the wild-type enzyme and the mutant enzyme were obtained by affinity chromatography, and SDS-PAGE analysis is shown in FIG. 1. The mutant enzyme had a molecular mass consistent with that of the wild-type enzyme and was approximately 48.8kDa in size.

Example 2 partial enzymatic determination of alkaline-resistant kappa-Carrageenase

mu.L of the enzyme solution (obtained in example 1) was mixed with 490. mu.L of a kappa-carrageenan solution (50mmol/L Na) having a concentration of 0.5% (w/v)₂HPO₄-NaH₂PO₄Buffer solution, pH 8.0) at different temperatures (40, 45, 50, 55, 60 deg.C) for 15min, adding 500 μ L DNS reagent, boiling in water bath for 10min, cooling to room temperature, and measuring absorbance at 520 nm. The temperature when the enzyme activity is highest is the optimum reaction temperature, and the enzyme activity under the temperature is 100 percent.

Respectively using 50mmol/L NaH₂PO₄-Na₂HPO₄Buffer (pH 6.0, 7.0, 8.0), Tris-HCl buffer (pH 8.0, 9.0) and glycine-NaOH buffer (pH 9.0, 10.0, 11.0) were used to prepare kappa-carrageenan solutions of 0.5% (w/v) concentration. Taking 490 mu L substrate solution, adding 10 mu L enzyme solution, reacting at optimum temperature for 15min, adding 500 mu L DNS reagent, boiling water bath for 10min, cooling to room temperature, and measuring 520nm light absorption value. The pH value when the enzyme activity is highest is the optimum reaction pH value, and the enzyme activity under the pH value is 100 percent. The results of the optimum reaction temperature and optimum reaction pH for the wild type and mutant kappa-carrageenases are shown in Table 2.

TABLE 2 optimum reaction temperature and optimum reaction pH for kappa-Carrageenase

mu.L of enzyme solution (obtained in example 1, protein content 400ng) was treated with 90. mu.L of 50mmol/L Gly-NaOH buffer (pH 8.0, 9.0, 10.0, 11.0, 12.0) at 25 ℃ for 30min, immediately placed on ice, 400. mu.L of carrageenan substrate solution (pH 8.0) containing 0.5% (w/v) concentration was added, the residual activity of the enzyme was determined, and the stability of the enzyme at different pH values was investigated, taking the enzyme activity without buffer treatment as 100%.

After wild type enzyme and mutant enzyme are respectively treated for 30min at the pH value of 8.0-12.0, the residual enzyme activity is shown in figure 2. The wild enzyme has the pH value of 11.0, and only retains 19.9 percent of relative enzyme activity after 30min of treatment, while the mutant enzyme E237R retains 36.0 percent of relative enzyme activity. It can be seen that the mutant enzyme has a certain improvement in pH stability under alkaline conditions as compared with the wild-type enzyme. The alkali-resistant kappa-carrageenase has good application prospect in the industrial production of hydrolyzing kappa-carrageenase under the alkaline condition.

Example 3 degradation of kappa-Carrageenan by kappa-Carrageenan in alkaline conditions

A0.5% (w/v) kappa-carrageenan substrate solution was prepared using 50mmol/L glycine-NaOH buffer (pH 8.0, 9.0, 10.0, respectively). Taking 490 μ L substrate, adding 10 μ L enzyme solution (protein content 400ng), reacting at 45 deg.C for 15, 30, 60, 90, 120min respectively, adding 500 μ L DNS reagent, cooling in boiling water bath for 10min, and measuring absorbance at 520 nm. And calculating the reducing sugar production amount of the wild type and the mutant enzyme according to the galactose standard.

The change in reducing sugar content after reacting the wild-type enzyme and the mutant enzyme with substrates of different pH at 45 ℃ for 2h is shown in FIG. 3. FIG. A, B, C represents kappa-carrageenan substrate solutions at pH 8.0, 9.0, and 10.0, respectively. When the pH value of the carrageenan substrate solution is 8.0, the degradation rate of the wild enzyme to the kappa-carrageenan is higher than that of the mutant enzyme; when the pH value is 9.0, after mutant enzyme hydrolyzes kappa-carrageenan for 90min, the content of the obtained reducing sugar is 57 mu g/mL, which is obviously higher than that of wild enzyme. This was also true at pH 10.0, indicating that the mutant enzymes had a higher ability to degrade kappa-carrageenan in alkaline environments than the wild-type enzyme.

In conclusion, according to the embodiment of the invention, the wild-type enzyme is subjected to site-directed mutagenesis by using the site-directed mutagenesis technology, and the 237 th amino acid of the wild-type enzyme is changed from Glu to Arg, so that compared with the wild-type enzyme, the alkali resistance of the mutant enzyme is improved to a certain extent, the degradation capability of the mutant enzyme on kappa-carrageenan is improved under an alkaline condition, and the advantages enable the mutant enzyme to have good application prospects in industrial production of hydrolyzing kappa-carrageenan under the alkaline condition.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer 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. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

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Alkali-resistant kappa-carrageenase and application thereof

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