A catalase mutant, its production method and application

Abstract

The application discloses a catalase mutant and a production method and application thereof. The catalase mutant is obtained by replacing asparagine at the 100th position in the amino acid sequence of a wild-type catalase with serine and replacing glutamic acid at the 215th position with arginine, and the amino acid sequence is shown as SEQ ID NO: 2. The catalase mutant provided by the application has the characteristics of high temperature resistance, can maintain 90% of enzyme activity at 60 DEG C, alkali resistance, can maintain > 85% of enzyme activity in the range of pH 4.0-10.0, and wide application range, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to an enzyme engineering technology, in particular to a catalase mutant and a production method and application thereof. BACKGROUND

[0002] Catalase (EC 1.11.1.6) is an antioxidant enzyme widely present in organisms, which can catalyze the decomposition of hydrogen peroxide (H2O2) into water and oxygen, and has important applications in the fields of medicine, food processing, textile bleaching, wastewater treatment, etc. However, in the industrial field, there are often extreme environments such as strong acid, strong base, high temperature, etc. Therefore, it is a hot research topic to find catalase with strong stability and high catalytic activity.

[0003] There are also some reports about catalase mutants in the prior art, for example, Chinese patent CN103451163A reports a high-temperature-resistant catalase mutant, in which the lysine K at position 114 of the catalase is mutated into tyrosine Y, valine V, methionine M or isoleucine I, which has good enzyme activity at 60℃. However, it depends on a specific Bacillus subtilis, and is difficult to be applied on a large scale. In addition, existing immobilization technologies (such as carrier crosslinking method) may cause loss of enzyme activity. SUMMARY

[0004] The first technical problem to be solved by the present application is to provide a catalase mutant, which has high high-temperature resistance and alkali resistance.

[0005] The second technical problem to be solved by the present application is to provide a DNA molecule.

[0006] The third technical problem to be solved by the present application is to provide an expression vector.

[0007] The fourth technical problem to be solved by the present application is to provide a production method of the catalase mutant.

[0008] The fifth technical problem to be solved by the present application is to provide an application of the catalase mutant in textile wastewater treatment.

[0009] The technical solution adopted by the present application to solve the first technical problem is a catalase mutant, the amino acid sequence of the catalase is shown as SEQ ID NO: 1, and the amino acid sequence of the catalase mutant is that the asparagine at position 100 is replaced by serine, and the glutamic acid at position 215 is replaced by arginine, and the amino acid sequence is shown as SEQ ID NO: 2.

[0010] The technical solution adopted by the present application to solve the second technical problem is a DNA molecule for encoding the catalase mutant.

[0011] Further, the DNA molecule is a coding gene of the catalase mutant, or a DNA sequence containing the coding gene, which can express the amino acid sequence shown in SEQ ID NO. 2.

[0012] As an example, in one specific embodiment of the present application, the DNA molecule is expressed by E. coli, at which time the nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 4.

[0013] The technical scheme adopted by the present application to solve its third technical problem is an expression vector for expressing the above-mentioned DNA molecule; the expression vector is pET28a.

[0014] The technical scheme adopted by the present application to solve its fourth technical problem is a production method of the above-mentioned catalase mutant, which is obtained by transforming the above-mentioned expression vector into a genetically engineered bacterium for expression.

[0015] Further, the genetically engineered bacterium is E. coli BL21.

[0016] Further, the production method further comprises: using high-density fermentation, adding 0.1 mM IPTG for induction of expression, controlling the dissolved oxygen content to be ≥ 30%; and using nickel column affinity chromatography combined with ultrafiltration concentration to obtain the catalase mutant △CAT-N100S / E215R with a purity ≥ 95%.

[0017] The technical scheme adopted by the present application to solve its fifth technical problem is the application of the above-mentioned catalase mutant in textile wastewater treatment.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The catalase mutant provided by the present application still maintains > 85% of the initial activity under high temperature (60℃) and strong alkali (pH 10.0) conditions, while the activity of the wild-type catalase is lost by more than 70% under the same conditions, and the remaining enzyme activity is less than 30% of the initial activity. The thermal stability half-life of the mutant is prolonged to 11.7 times that of the wild-type catalase (t 1 / 2 : 3.5h vs 0.3h), significantly reducing the enzyme supplement frequency in industrial applications. The pH application range is expanded to 4.0-10.0 (the wild-type catalase is 6.0-8.0). In textile wastewater treatment applications, the catalase mutant improves the H202 decomposition efficiency by more than 40%. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1SDS-PAGE gel electrophoresis diagram of the catalase mutant and wild-type catalase provided by the present application.

[0021] Figure 2 Half-life analysis diagram of the catalase mutant and wild-type catalase provided by the present application. DETAILED DESCRIPTION

[0022] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details set forth in this description. In other instances, well-known methods have not been described in detail in order to avoid obscuring the present application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0024] The term "expression" refers to the process by which DNA is transcribed into messenger RNA (mRNA) and then translated into a protein.

[0025] The term "expression vector" has the ability to incorporate and express a heterologous polynucleic acid fragment in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. It is within the knowledge of the skilled person to select a suitable expression vector.

[0026] The present application will be further described in conjunction with the accompanying drawings and examples. The reagents used in the examples are all commercially available.

[0027] Example 1 Mutant gene synthesis and expression

[0028] The wild-type catalase (the amino acid sequence of which is shown in SEQ ID NO. 1) was subjected to sequence design and high-throughput screening, and finally a heat-resistant and alkali-resistant catalase mutant (the amino acid sequence of which is shown in SEQ ID NO. 2) was obtained. The two amino acid sequences contain two different sites (N100S, E215R), i.e., the amino acid sequence of the catalase mutant is that the 100th asparagine of the wild-type catalase amino acid sequence is replaced by serine and the 215th negatively charged glutamic acid is replaced by positively charged arginine.

[0029] 1. Preparation of catalase mutant

[0030] 1.1 Construction of recombinant cells

[0031] The coding genes of wild-type catalase and catalase mutant were taken as target genes, and the nucleotide sequences of the target genes were synthesized by Beijing Genki Biotechnology Co., Ltd. and inserted into the expression vector pET28a (+) respectively.

[0032] The nucleotide sequence of the coding gene of wild-type catalase is shown in SEQ ID NO. 3, and the nucleotide sequence of the coding gene of the heat-resistant and alkali-resistant catalase mutant is shown in SEQ ID NO. 4.

[0033] Specifically, the plasmid pET28a (+) was inserted to obtain the corresponding recombinant plasmids pET28a-cat and pET28a-△cat. Then the recombinant plasmids were transformed into the chassis cell E. coli BL21 (DE3), thereby constructing the E. coli strains pET28a-cat / BL21 and pET28a-△cat / BL21 containing different recombinant plasmids.

[0034] 1.2 Expression and purification of catalase mutant

[0035] The recombinant bacteria were inoculated in liquid LB medium (5 g yeast extract, 10 g peptone, 10 g NaCl) and cultured at 37℃ on a shaking table (at a speed of 220 r / min) until the OD 600 value was 1.0-1.2, then IPTG was added to a concentration of 0.1 mmol / L, and the temperature was adjusted to 16℃ for overnight induction at low temperature. The high-density fermentation broth was collected by centrifugation at 4000 g for 10 min, and the bacterial cells were resuspended in 10% volume of buffer NTA (50 mM Tris-Cl, 300 mM NaCl, pH = 8.0). The bacterial cells were broken by ultrasonic, and centrifuged at 12000 r / min for 30 min. The supernatant was the crude enzyme solution.

[0036] The crude enzyme solution was subjected to affinity chromatography by Ni 2+ Resin, because the target protein expressed at the C-terminal has a histidine tag which can be combined on the resin. The resin was washed with buffer NTA and imidazole buffer, and then eluted with buffer NTA. The resulting solution was dialyzed to remove imidazole, and the dialysis bag had a cutoff volume of 1 kDa. The dialyzed solution was concentrated by ultrafiltration tube until the wild-type catalase CAT and the catalase mutant △CAT-N100S / E215R with a purity ≥95% were obtained.

[0037] The genetically engineered bacterial cells of wild-type catalase (CAT) and heat-resistant and alkali-resistant catalase mutant (△CAT-N100S / E215R) were subjected to SDS-PAGE electrophoresis analysis of the expression products, and the detection results are shown in Figure 1 Figure 1 ​It can be seen that the molecular weight of the catalase mutant and the wild type catalase is about 38 kDa, which is consistent with the expected value (37.6 kD).

[0038] 1.3 Catalase activity detection

[0039] The catalase enzyme activity determination is carried out at 40 DEG C. 100 μL of enzyme solution is added into 3 mL of reaction solution (50 mmol / L potassium phosphate buffer with pH = 8.0, 15 mmol / L H2O2), and the change of absorbance at 240 nm within 60 s is determined. The enzyme activity unit: the amount of enzyme required for degrading 1 μmol of hydrogen peroxide at 37 DEG C is defined as 1 enzyme activity unit.

[0040] 1.4 Protein quantitative detection

[0041] According to the Bradford method, the protein content is determined as follows: (1) the bovine serum albumin (BSA) standard solution is mixed with PBS at different proportions, and then the Coomassie brilliant blue staining solution is added to restore the room temperature, and the absorbance at 595 nm is determined to prepare a standard curve; (2) 200 μl of Coomassie brilliant blue staining solution is added to each 96-well plate, and then 20 μl of enzyme solution diluted to a suitable concentration with PBS is added, and then mixed thoroughly, and reacted for 5 min, and then the absorbance at 595 nm is determined by using a full-wavelength enzyme marker, and the protein content of each sample is converted according to the standard curve.

[0042] 2, Heat and alkali resistance detection of catalase mutant

[0043] 2.1 Thermal stability analysis of catalase mutant

[0044] In the thermal stability experiment, the pure enzyme solution of natural catalase and catalase mutant is placed under the condition of pH 8.0, and after being incubated at 30-70 DEG C for 40 min, the residual hydrogen peroxide specific activity is determined according to the standard method, and the specific activity of the catalase mutant and the wild type catalase is compared (see Table 1).

[0045] Table 1 Comparison results of temperature stability

[0046]

[0047] As shown in Table 1, the activity of the catalase mutant in the range of 50-70 DEG C is balanced, and 75% activity is still retained at 70 DEG C. The activity of the wild type catalase is sharply attenuated after 50 DEG C, and only 40% is left at 60 DEG C. It can be seen that the thermal stability of the catalase mutant of the present application is significantly improved, and the industrial application temperature window is widened.

[0048] 2.2 Half-life analysis of catalase mutant

[0049] Pure enzyme solutions of natural hydrogen peroxide and catalase mutant were placed at pH 8.0 and 60℃, respectively, and samples were taken at different times. The remaining hydrogen peroxide activity was determined according to standard methods, and the half-life (half-inactivation time) of catalase mutant and wild-type catalase was compared (see...). Figure 2 ).

[0050] Depend on Figure 2 It can be seen that the half-life (t) of the catalase mutant is... 1 / 2 The lifespan reaches 3.5 hours (compared to only 0.3 hours for wild-type catalase). Therefore, the immobilized catalase mutant of this invention can extend its lifespan by more than 10 times in continuous industrial reactions.

[0051] 2.3 Analysis of alkali tolerance of catalase mutants

[0052] In the alkali resistance test, the pure enzyme solutions of natural hydrogen peroxide and catalase mutant were placed at 40℃ and pH 6.0-10.0 for 40 min. The specific enzyme activity of the remaining hydrogen peroxide was measured according to the standard method, and the specific enzyme activity of catalase mutant and wild-type catalase was compared (see Table 2).

[0053] Table 2. Comparison results of pH tolerance

[0054]

[0055] As shown in Table 2, the catalase mutant maintained >85% activity within a pH range of 4.0-10.0, with only a 12% loss of activity in an alkaline environment (pH 10). In contrast, the wild-type catalase exhibited a sharp decline in activity at pH >8.0.

[0056] Example 2: Application of catalase mutant in textile wastewater treatment

[0057] 1. Preparation of sodium alginate-silica composite carrier

[0058] Tetraethyl orthosilicate (TEOS) was added dropwise to a 5% (w / v) sodium alginate (SA) solution (volume ratio 1:4) at pH 5.0 and 40℃, and magnetically stirred for 2 h to form a homogeneous sol. Then, 0.1 MCaCl2 solution (final concentration 0.5%) was added to initiate cross-linking of the sodium alginate. The mixture was injected into a mold (spherical / microsphere) and allowed to stand at 4℃ for 12 h to form a gel network. Supercritical CO2 drying (conditions: 35℃, 10 MPa, 3 h) yielded a porous support. Support characteristics: average pore size 20 μm, specific surface area 180 m² / g. 2 / g, silicon content 18.5wt%.

[0059] 2. Carrier activation

[0060] The carrier was soaked in a toluene solution (5% v / v, 60°C, 4h) of 3-aminopropyltriethoxysilane (APTES). After washing, the aldehyde group-activated surface was formed by cross-linking with 2.5% glutaraldehyde (GA) (25°C, 2h).

[0061] 3. Enzyme immobilization

[0062] The wild-type catalase and catalase mutant were dissolved in 0.1M PBS (pH 7.0) to form a 10mg / mL solution, and the activated carrier was added (enzyme: carrier = 1:5, w / w), and adsorbed by oscillation at 4°C for 12h. 1% polyethyleneimine (PEI) was added to assist cross-linking (25°C, 1h) to enhance the enzyme-carrier binding force. The residual aldehyde group was blocked with 0.1M glycine solution (30min). The residual enzyme protein in the supernatant was determined by the BCA method to determine the immobilization efficiency; the immobilized enzyme activity recovery rate of the sodium alginate-silica composite carrier was measured by comparing the free enzyme activity at pH 8.0 and 60°C. The immobilization effects of different catalases were compared. The immobilized wild-type catalase and catalase mutant were respectively filled in the reaction column in the same way, and the textile wastewater containing 1000ppm H2O2 was treated at pH 8.0 and 60°C, and the application effects of different immobilized catalases in decomposing H2O2 within 30min were compared (see Table 3).

[0063] Table 3 Comparison of immobilization and application effects of different catalases

[0064]

[0065] As can be seen from Table 3, the immobilization effect, enzyme activity recovery rate, and H2O2 decomposition rate in textile wastewater of the catalase mutant are obviously superior to those of the wild-type catalase.

[0066] Therefore, the catalase mutant obtained by the present application has heat and alkali resistance, and its immobilization effect and application effect are the best compared with the wild-type catalase and the existing commercially available catalase.

[0067] The above only describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above-described embodiments. Any technical solution falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary researchers in the technical field, some improvements and refinements without departing from the principle of the present application shall also be considered as the protection scope of the present application.

Claims

1. A catalase mutant, characterized in that, The amino acid sequence of wild-type catalase is shown in SEQ ID NO:

1. The catalase mutant is formed by replacing asparagine at position 100 with serine and glutamic acid at position 215 with arginine in the amino acid sequence of wild-type catalase, and its amino acid sequence is shown in SEQ ID NO:

2.

2. A DNA molecule, characterized in that, The DNA molecule is the encoding gene of the catalase mutant as described in claim 1.

3. The DNA molecule as described in claim 2, characterized in that, The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:

4.

4. An expression vector for expressing the DNA molecule as described in claim 2 or 3; wherein the expression vector is pET28a.

5. A method for producing a catalase mutant, characterized in that, The expression vector described in claim 4 is transferred into genetically engineered bacteria for expression.

6. The method for producing a catalase mutant according to claim 5, characterized in that, The genetically engineered bacterium is Escherichia coli BL21(DE3).

7. A method for producing a catalase mutant according to claim 5 or 6, characterized in that, The production method further includes: high-density fermentation, adding 0.1 mM IPTG to induce expression, and controlling dissolved oxygen content ≥30%; nickel column affinity chromatography combined with ultrafiltration concentration to obtain catalase mutant with a purity ≥95%.

8. The application of the catalase mutant as described in claim 1 in the treatment of textile wastewater.

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